WO2003045990A2 - Protein-protein interactions involving transforming growth factor beta signalling - Google Patents

Protein-protein interactions involving transforming growth factor beta signalling Download PDF

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WO2003045990A2
WO2003045990A2 PCT/EP2002/013866 EP0213866W WO03045990A2 WO 2003045990 A2 WO2003045990 A2 WO 2003045990A2 EP 0213866 W EP0213866 W EP 0213866W WO 03045990 A2 WO03045990 A2 WO 03045990A2
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protein
cells
tgfβ
seq
sid
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PCT/EP2002/013866
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WO2003045990A3 (en
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Pierre Legrain
Jean-Michel Gauthier
Frédéric COLLAND
Xavier Jacq
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Hybrigenics
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Publication of WO2003045990A3 publication Critical patent/WO2003045990A3/en

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition.
  • protein-protein interactions are involved, for example, in the assembly of enzyme subunits, in antibody-antigen recognition, in the formation of biochemical complexes, in the correct folding of proteins, in the metabolism of proteins, in the transport of proteins, in the localization of proteins, in protein turnover, in first translation modifications, in the core structures of viruses and in signal transduction.
  • General methodologies to identify interacting proteins or to study these interactions have been developed. Among these methods are the two-hybrid system originally developed by Fields and co-workers and described, for example, in U.S. Patent Nos. 5,283,173, 5,468,614 and 5,667,973, which are hereby incorporated by reference.
  • the earliest and simplest two-hybrid system which acted as basis for development of other versions, is an in vivo assay between two specifically constructed proteins.
  • the first protein known in the art as the "bait protein” is a chimeric protein which binds to a site on DNA upstream of a reporter gene by means of a DNA-binding domain or BD.
  • the binding domain is the DNA-binding domain from either Gal4 or native E. coli LexA and the sites placed upstream of the reporter are Gal4 binding sites or LexA operators, respectively.
  • the second protein is also a chimeric protein known as the "prey" in the art.
  • This second chimeric protein carries an activation domain or AD.
  • This activation domain is typically derived from Gal4, from VP16 or from B42.
  • Another advantage of the two-hybrid plus one system is that it allows or prevents the formation of the transcriptional activator since the third partner can be expressed from a conditional promoter such as the methionine-repressed Met25 promoter which is positively regulated in medium lacking methionine.
  • the presence of the methionine-regulated promoter provides an excellent control to evaluate the activation or inhibition properties of the third partner due to its "on" and "off' switch for the formation of the transcriptional activator.
  • the three-hybrid method is described, for example in Tirode ef al., The Journal of Biological Chemistry, 272, No. 37 pp. 22995-22999 (1997) incorporated herein by reference.
  • the first recombinant yeast cell or the second recombinant yeast cell also contains at least one detectable reporter gene that is activated by a polypeptide including a transcriptional activation domain.
  • the method described in W099/42612 permits the screening of more prey polynucleotides with a given bait polynucleotide in a single step than in the prior art systems due to the cell to cell mating strategy between haploid yeast cells. Furthermore, this method is more thorough and reproducible, as well as sensitive. Thus, the presence of false negatives and/or false positives is extremely minimal as compared to the conventional prior art methods.
  • TGF ⁇ Transforming growth factor ⁇
  • BMP Bone Morphologenetic Proteins
  • Smad proteins Ten mammalian Smad proteins have been identified and divided into three classes.
  • the first includes pathway-restricted proteins such as Smadl , Smad5 and Smad ⁇ which are specifically involved in BMP signaling and Smad2 and Smad3 which are restricted to TGF ⁇ /activin pathway.
  • the second class contains the common-mediator Smad4 implicated in both BMP and TGF ⁇ /activin pathways.
  • the third class contains the inhibitory Smads, Smad6 and Smad7. At least Smad2 and Smad3 are retained in the cytoplasm by binding to the SARA protein.
  • pathway-restricted Smads form heteromeric complexes with Smad4 and then translocate to the nucleus where they control expression of diverse genes involved in various biological processes such as control of cellular proliferation and differentiation, regulation of the immune system and regulation of the extracellular matrix formation.
  • proteins such as TGIF, Ski, SnoN, SNIP1 and CBP have been identified as Smad transcriptional co-regulators and shown to modulate the transcriptional ability of Smad proteins by direct interactions.
  • proteins such as Smurfl and Smurf2 are involved in degradation of Smad proteins by the proteasome machinery.
  • Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition.
  • protein-protein interactions are involved, for example, in the assembly of enzyme subunits, in antibody-antigen recognition, in the formation of biochemical complexes, in the correct folding of proteins, in the metabolism of proteins, in the transport of proteins, in the localization of proteins, in protein turnover, in first translation modifications, in the core structures of viruses and in signal transduction.
  • SID® polypeptides It is still another aspect of the present invention to identify selected interacting domains of the polypeptides, called SID® polypeptides. It is still another aspect of the present invention to identify selected interacting domains of the polynucleotides, called SID® polynucleotides. It is still another aspect of the present invention to provide a diagnostic kit to test for deficiencies in the transforming growth factor ⁇ super-family of cytokines transduction pathway.
  • nucleic acids of the present invention via gene therapy. It is yet another aspect of the present invention to provide protein chips or protein microarrays.
  • the present invention relates to a complex of interacting proteins of columns 1 and 4 of Table 2.
  • the present invention provides SID® polynucleotides and SID® polypeptides of Table 3, as well as a PIM® involved in transforming growth factor ⁇ -mediated disorders and/or diseases.
  • the present invention also provides antibodies to the protein-protein complexes involved in transforming growth factor ⁇ -mediated disorders and/or diseases.
  • the present invention provides a method for screening drugs for agents that modulate the protein-protein interactions and pharmaceutical compositions that are capable of modulating protein-protein interactions.
  • the present invention provides protein chips or protein microarrays.
  • the present invention provides a report in, for example, paper, electronic and/or digital forms.
  • Fig. 1 is a schematic representation of the pB6 plasmid.
  • Fig. 2 is a schematic representation of the pB20 plasmid.
  • Fig. 3 is a schematic representation of the pP6 plasmid.
  • Fig. 4 is a schematic representation of vectors expressing the T25 fragment.
  • Fig. 5 is a schematic representation of vectors expressing the T18 fragment.
  • Fig. 6 is a schematic representation of various vectors of pCmAHLI , pT25 and pT18.
  • Fig. 7 is a schematic representation identifying the SID®'s of proteins of the present invention.
  • the "Full-length prey protein” is the Open Reading Frame (ORF) or coding sequence (CDS) where the identified prey polypeptides are included.
  • ORF Open Reading Frame
  • CDS coding sequence
  • Interaction Domain is determined by the commonly shared polypeptide domain of every selected prey fragment.
  • Fig. 8 is a protein map (PIM®).
  • Fig. 9 is a schematic representation of the pB27 plasmid.
  • Fig. 10 is a schematic representation of the pB28 plasmid.
  • Fig. 11 is a schematic representation of a protein interaction map around the newly functionally characterized proteins described in the present invention. These 10 proteins are highlighted by the symbol "*".
  • the Predicted Biological Score (PBS) is represented by a code on each line and classified from A to E (Rain et al., 2001 ).
  • PP1ca is also named PPP1CA.
  • MADH5 and MADH6 correspond to Smad ⁇ and Smad9, respectively.
  • hMAD-2 and h-MAD-3 correspond to Smad2 and Smad3, respectively.
  • MAN1 is the orthologous of SANE, a protein recently identified as involved in the BMP pathway (Raju et al., 2002)
  • Fig. 12 is a schematic representation of a protein interaction map between ZNF8 and Smad proteins. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, 2NF8 was shown to interact with Smadl (A), Smad4 (B), Smad5 (C) and Smad9 (D). Amino-acid position are indicated.
  • Fig. 13 A, B and C are graphs showing that ZNF8 siRNA represses TGF ⁇ - and BMP- dependent luciferase reporter activities.
  • HepG2 cells were transiently transfected in 24 well- plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC) 12 -MLP-Luc (A & B) or the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc (C). All experiments included pRL-TK as an internal transfection control.
  • a T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF ⁇ pathway.
  • a mutated version of the T ⁇ RI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 4 and 40nM.
  • Co- transfection of ZNF8-targeting siRNA duplex was tested in cells treated or not with 50ng/ml BMP7 (A), 50ng/ml BMP6 (B) or 5 ng/ml TGF ⁇ l (C) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10 ⁇ l of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
  • Fig. 14A, B and C are graphs showing that ZNF8 siRNA specifically represses BMP- dependent markers.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or ZNF8-targeting siRNA duplex.
  • Cells were treated or not with 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed either at 0.5nM and 2.5nM (A & B) or at 4 and 40nM (C) of duplex. Cells were harvested and lysed 48 hours after transfection.
  • RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the BMP pathway markers junB (A) and alkaline phosphatase (B& C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using either GAPDH (A & B) or hGUS (C).
  • Fig. 15 A and B are graphs showing that ZNF8 siRNA does not repress BMP- independent markers.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or ZNF8- targeting siRNA duplex.
  • RNA samples were treated or not with 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed either at 0.5nM and 2.5nM (A) or at 4 and 40nM (B) of duplex. Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGF ⁇ pathway marker PAI-1 (PAI-1 hereinafter Plasminogen Activator inhibitor I) (A) and an unrelated marker, hGUS (B). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using either GAPDH (A) or relative levels (B).
  • PAI-1 Plasminogen Activator inhibitor I
  • Fig. 16 is a schematic representation of an Interaction between LAPTm ⁇ and Smurf2.
  • the full-length proteins are represented in grey and black boxes correspond to the interaction domains.
  • Smurf2 and LAPTm ⁇ were found in both directions.
  • Smurf2 was shown to interact with the C-terminal domain of LAPTm ⁇ .
  • Fig. 17 A and B are graphs showing that LAPTm ⁇ specifically inhibits the TGF ⁇ pathway.
  • TGF-RE TGF ⁇ responsive element
  • BMP-RE p(GC) 12 -MLP-Luc
  • an unrelated reporter pGL3 control
  • Fig. 18 A and B are graphs showing that LAPTm ⁇ expression is up-regulated by TGF ⁇
  • the endogenous level of LAPTm ⁇ mRNA was determined in several cell lines by Q-PCR experiments using the LAPTm ⁇ probe (see Materials & Methods). Ct levels of LAPTm ⁇ mRNA is given for each cell lines (A). The endogenous level of mRNA was determined in HepG2 cells in the presence or absence of TGF ⁇ (10 ng/ml) with or without a T ⁇ RI-targeting siRNA duplex (B) (T ⁇ RI hereinafter Transforming Growth Factor ⁇ Receptor I.
  • Fig. 19 A and B are graphs showing that LAPTm ⁇ siRNA up-regulates BMP and TGF ⁇ - dependent reporter activities.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the TGF ⁇ reponsive luciferase reporter, p(GTCT) B -MLP-Luc (A) or the BMP responsive luciferase reporter, p(GC) ⁇ 2 -MLP-Luc (B). All experiments included pRL-TK as an internal transfection control.
  • a T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF ⁇ pathway.
  • a mutated version of the T ⁇ RI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 4 and 40nM.
  • Co-transfection of LAPTm ⁇ -targeting siRNA duplex was tested in cells treated or not with ⁇ ng/ml recombinant human TGF ⁇ (A), ⁇ Ong/ml recombinant human BMP7 (B) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10 ⁇ l of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
  • Fig. 20 A, B, C and D are graphs showing that LAPTm ⁇ siRNA up-regulates BMP and TGF ⁇ -dependent markers.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or LAPTm ⁇ -targeting siRNA duplex.
  • Cells were treated or not with ⁇ ng/ml of recombinant human TGF ⁇ l or 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed at 40nM of duplex (A, B, C & D). Cells were harvested and lysed 48 hours after transfection.
  • RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGF ⁇ pathway markers PAI-1 and junB (A & B, respectively) and a BMP pathway marker, alkaline phosphatase (C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A, B & C). Relative levels of hGUS in the same experiment are also shown (D).
  • Fig. 21 is a schematic representation of an Interaction between RNF11 Smurfl, Smurf2 and SARA.The full-length proteins are represented in grey and black boxes correspond to the interaction domains.
  • Fig. 22 is a gel showing that RNF11 is involved in regulating SARA protein levels.
  • Fig. 23 is a schematic diagram showing the Interaction between KIAA1196 and Smadl .
  • the full-length proteins are represented in grey and black boxes correspond to the interaction domains.
  • KIAA1196 was shown to interact with Smadl .
  • Fig. 24 A and B are graphs showing that KIAA1196 siRNA specifically represses TGF ⁇ -dependent markers in HepG2 cells.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc (A) or the BMP reponsive luciferase reporter, p(GC) ⁇ 2 -MLP-Luc (B). All experiments included pRL-TK as an internal transfection control.
  • a T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF ⁇ pathway.
  • T Rl-targeting siRNA duplex A mutated version of the T Rl-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 4 and 40nM.
  • Co- transfection of KIAA1196-targeting siRNA duplex was tested in cells treated or not with 5ng/ml recombinant human TGF ⁇ (A) and 50ng/ml recombinant human BMP6 (B) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10 ⁇ l of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
  • Fig. 25 is a graph showing that KIAA1196 siRNA specifically represses TGF ⁇ - dependent reporter activity in HEK293 cells.
  • HEK 293 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc. All experiments included pRL-TK as an internal transfection control.
  • a T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF ⁇ pathway.
  • a mutated version of the T ⁇ RI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 30nM.
  • Fig. 26 A, B, C and D are graphs showing that KIAA1196 siRNA specifically represses TGF ⁇ -dependent markers.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or KIAA1196-targeting siRNA duplex.
  • Cells were treated or not with 5 ng/ml of recombinant human TGF ⁇ l or 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed at 40nM of duplex (A, B, C & D). Cells were harvested and lysed 48 hours after transfection.
  • RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGF ⁇ pathway markers PAI-1 and junB (A & B, respectively) and a BMP pathway marker, alkaline phosphatase (C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A, B & C). Relative levels of hGUS in the same experiment are also shown (D).
  • TGF ⁇ pathway markers PAI-1 and junB A & B, respectively
  • C alkaline phosphatase
  • Fig. 27 is a schematic representation showing the Interaction between LM04 and Smad9. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, LM04 was shown to interact with Smad9.
  • Fig. 28 A, B and C are graphs showing that LM04 siRNA specifically repress a BMP- dependent luciferase reporter. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC) 12 - MLP-Luc (A) or the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc (B).
  • T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF pathway.
  • a mutated version of the T ⁇ RI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 4 and 40nM.
  • Co-transfection of LM04-targeting siRNA duplex was tested in cells treated or not with 50ng/ml recombinant human BMP7 or BMP6 (A & B, respectively) and 5ng/ml recombinant human TGF ⁇ (C) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • Fig. 29 A and B are graphs showing that LM04 siRNA specifically represses BMP- induced markers in BMP7-treated HepG2 cells.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or LM04-targeting siRNA duplex.
  • Cells were treated or not with 50ng/ml of recombinant human BMP7 for 18 hours in ceils pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed at O. ⁇ or 2. ⁇ nM of duplex (A) and 4 or 40nM of duplex (B). Cells were harvested and lysed 48 hours after transfection.
  • RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the BMP pathway marker alkaline phosphatase (A & B). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A, B).
  • Fig. 30 A, B and C are graphs showing that LM04 siRNA does not repress BMP- independent markers in BMP7-treated HepG2 cells.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3- targeting siRNA) or LM04-targeting siRNA duplex.
  • Cells were treated or not with ⁇ Ong/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed at 4 or40nM of duplex (A, B) and 0.5 or 2.5nMof duplex (C). Cells were harvested and lysed 48 hours after transfection.
  • RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGF ⁇ and BMP pathways marker junB (A) and a TGF ⁇ pathway marker, PAI-1 (C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A) or using GAPDH (C). Relative levels of hGUS in the same experiment are also shown (B).
  • Fig. 31 is a schematic diagram showing the interaction between PPIca and SARA. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, PPIca was shown to interact with SARA.
  • TGF ⁇ 10 ng/ml
  • BMP7 60 ng/ml
  • This study was performed with 0, 10, 60 or 200 ng of pV3- PPIca in HepG2 cells (A) or in HEK293 cells (B).
  • the specific Luciferase activity was normalized using the pRL-TK vector. Experiments were performed in triplicate.
  • Fig. 33 A, B and C are graphs showing that PPIca stimulates PAI-1 mRNA expression.
  • Baculoviruses containing the Smad3 or PPIca genes under the control of the CMV promoter were generated and used to infect HepG2 cells (see Materials & Methods). The over- expression level was checked and quantified by Q-PCR (A).
  • the endogenous PAI-1 mRNA levels were measured by Q-PCR 24 hours post infection with Smad3 or PP1ca-containing baculoviruses in the presence or absence of TGF ⁇ (10 ng/ml). The value 1 is attributed to the mRNA amount of PAI-1 in the absence of TGF ⁇ and in the absence of infection (B).
  • Fig. 34 is a schematic diagram showing the Interaction between HYPA and Smad4. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, HYPA was shown to interact with Smad4.
  • Fig. 3 ⁇ A, B and C are graphs showing that HYPA siRNA specifically represses BMP- dependent reporter activity. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC) 12 - MLP-Luc (A & B) or the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc (C).
  • T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF ⁇ pathway.
  • a mutated version of the T ⁇ RI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 4 and 40nM.
  • Co-transfection of HYPA-targeting siRNA duplex was tested in cells treated or not with ⁇ Ong/ml recombinant human BMP7 or BMP6 (A & B, respectively) and ⁇ ng/ml recombinant human TGF ⁇ (C) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • Fig. 36 is a graph showing that HYPA siRNA represses BMP-dependent markers.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or HYPA-targeting siRNA duplex.
  • Cells were treated or not with ⁇ Ong/ml of recombinant human BMP7 for 18 hours in cells pre- starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed at 0.5 or 2.5nM of duplex. Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the BMP pathway marker alkaline phosphatase. Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using GAPDH.
  • Fig. 37 is a schematic diagram showing the Interaction between FLJ20037 and SARA.
  • Fig. 38 A, B and C are graphs showing that FLJ20037 stimulates PAI-1 mRNA expression.
  • Baculoviruses containing the Smad3 or FLJ20037 genes under the control of the CMV promoter were generated and used to infect HepG2 cells (see Materials & Methods). The over-expression level was checked and quantified by Q-PCR (A).
  • the endogenous PAI- 1 mRNA levels were measured by Q-PCR 24 hours post containing baculoviruses in the presence or absence of TGF ⁇ (10 ng/mL). The value 1 is attributed to the mRNA amount of PAI-1 in the absence of TGF ⁇ and in the absence of infection (B).
  • FIG. 39 is a graph showing that FLJ20037 siRNA down-regulates TGF ⁇ -dependent markers.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or FLJ20037-targeting siRNA duplex.
  • Cells were treated or not with ⁇ ng/ml of recombinant human TGF ⁇ for 18 hours in cells pre-starved for 2 hours in serum-free culture medium.
  • SiRNA transfections were performed at O. ⁇ or 2. ⁇ nM of duplex. Cells were harvested and lysed 48 hours after transfection.
  • Fig. 40 is a schematic diagram showing the Interaction between PTPN12 and Smad ⁇ .
  • the full-length proteins are represented in grey and black boxes correspond to the interaction domains.
  • PTPN12 was shown to interact with Smad ⁇ . Amino-acid positions are indicated.
  • Fig. 41 A and B are graphs showing that PTPN12 siRNA up-regulates BMP and TGF ⁇ - dependent reporter activities.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC) 12 - MLP-Luc (A) or the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc (B). All experiments included pRL-TK as an internal transfection control.
  • a T ⁇ RI-targeting siRNA duplex was used as a positive control for disruption of the TGF pathway.
  • T ⁇ RI-targeting siRNA duplex 2 mismatches versus consensus sequence
  • SiRNA transfections were performed at 4 and 40nM.
  • Co-transfection of PTPN12-targeting siRNA duplex was tested in cells treated or not with 50ng/ml recombinant human BMP6 (A) and 5ng/ml recombinant human TGF ⁇ (B) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10 ⁇ l of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
  • Fig. 42 A and B are schematic diagrams showing the Interaction between HIPK3, SnoN and SNIP1.
  • the full-length proteins are represented in grey and black boxes correspohd to the interaction domains.
  • HIPK3 was shown to interact with the N-terminal domains of SNIP1 (A) and SnoN (B). Amino-acid positions are indicated.
  • Fig. 43 A and B are graphs showing that HIPK3 siRNA specifically up-regulates BMP- dependent reporter activities.
  • HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC) ⁇ 2 -MLP-Luc (A) or the TGF ⁇ responsive luciferase reporter, p(GTCT) 8 -MLP-Luc (B). All experiments included pRL-TK as an internal transfection control.
  • a T Rl-targeting siRNA duplex was used as a positive control for disruption of the TGF pathway.
  • a mutated version of the T ⁇ RI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control.
  • SiRNA transfections were performed at 4 and 40nM.
  • Co-transfection of HIPK3-targeting siRNA duplex was tested in cells treated or not with ⁇ Ong/ml recombinant human BMP6 (A) and ⁇ ng/ml recombinant human TGF ⁇ (B) for 18 hours in cells pre-starved for 2 hours in serum- free culture medium. Cells were harvested 48 hours after transfection and 10 ⁇ l of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
  • polynucleotides As used herein the terms “polynucleotides”, “nucleic acids” and “oligonucleotides” are used interchangeably and include, but are not limited to RNA, DNA, RNA/DNA sequences of more than one nucleotide in either single chain or duplex form.
  • the polynucleotide sequences of the present invention may be prepared from any known method including, but not limited to, any synthetic method, any recombinant method, any ex vivo generation method and the like, as well as combinations thereof.
  • polypeptide means herein a polymer of amino acids having no specific length.
  • peptides, oligopeptides and proteins are included in the definition of “polypeptide” and these terms are used interchangeably throughout the specification, as well as in the claims.
  • polypeptide does not exclude post-translational modifications such as polypeptides having covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like. Also encompassed by this definition of "polypeptide” are homologs thereof.
  • orthologs structurally similar genes contained within a given species
  • orthologs are functionally equivalent genes from a given species or strain, as determined for example, in a standard complementation assay.
  • a polypeptide of interest can be used not only as a model for identifying similiar genes in given strains, but also to identify homologs and orthologs of the polypeptide of interest in other species.
  • the orthologs for example, can also be identified in a conventional complementation assay.
  • orthologs can be expected to exist in bacteria (or other kind of cells) in the same branch of the phylogenic tree, as set forth, for example, at fr ⁇ ://ftp.cme.msu.edu/pub/rdp/SSIJ-rR A/SSU Prok. ⁇ hylo.
  • prey polynucleotide means a chimeric polynucleotide encoding a polypeptide comprising (i) a specific domain; and (ii) a polypeptide that is to be tested for interaction with a bait polypeptide.
  • the specific domain is preferably a transcriptional activating domain.
  • a "bait polynucleotide” is a chimeric polynucleotide encoding a chimeric polypeptide comprising (i) a complementary domain; and (ii) a polypeptide that is to be tested for interaction with at least one prey polypeptide.
  • the complementary domain is preferably a DNA-binding domain that recognizes a binding site that is further detected and is contained in the host organism.
  • complementary domain is meant a functional constitution of the activity when bait and prey are interacting; for example, enzymatic activity.
  • specific domain is meant a functional interacting activation domain that may work through different mechanisms by interacting directly or indirectly through intermediary proteins with RNA polymerase II or Ill-associated proteins in the vicinity of the transcription start site.
  • complementary means that, for example, each base of a first polynucleotide is paired with the complementary base of a second polynucleotide whose orientation is reversed.
  • the complementary bases are A and T (or A and U) or C and G.
  • sequence identity refers to the identity between two peptides or between two nucleic acids. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of sequence identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acid sequences that are shared between these sequences.
  • two polypeptides may each (i) comprise a sequence (i.e., a portion of a complete polynucleotide sequence) that is similar between two polynucleotides, and (ii) may further comprise a sequence that is divergent between two polynucleotides
  • sequence identity comparisons between two or more polynucleotides over a "comparison window" refers to the conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference nucleotide sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino acid sequence or a first nucleic acid sequence for optimal alignment with the second amino acid sequence or second nucleic acid sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.
  • sequences can be the same length or may be different in length.
  • Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 91 (2) pgs. 370-380 (1981 ), by the homology alignment algorithm of Needleman and Wunsch, J. Miol. Biol., 48(3) pgs. 443-463 (1972), by the search for similarity via the method of Pearson and Lipman, PNAS, USA, 85( ⁇ ) pgs. 2444-2448 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetic Computer Group, 675, Science Drive, Madison, Wisconsin) or by inspection.
  • sequence identity means that two polynucleotide sequences are identical
  • the term "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) and multiplying the result by 100 to yield the percentage of sequence identity.
  • the same process can be applied to polypeptide sequences.
  • sequence similarity means that amino acids can be modified while retaining the same function. It is known that amino acids are classified according to the nature of their side groups and some amino acids such as the basic amino acids can be interchanged for one another while their basic function is maintained.
  • isolated means that a biological material such as a nucleic acid or protein has been removed from its original environment in which it is naturally present. For example, a polynucleotide present in a plant, mammal or animal is present in its natural state and is not considered to be isolated. The same polynucleotide separated from the adjacent nucleic acid sequences in which it is naturally inserted in the genome of the plant or animal is considered as being “isolated.”
  • isolated is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with the biological activity and which may be present, for example, due to incomplete purification, addition of stabilizers or mixtures with pharmaceutically acceptable excipients and the like.
  • purified means at least one order of magnitude of purification is achieved, preferably two or three orders of magnitude, most preferably four or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term “purified” as utilized herein does not mean that the material is 100% purified and thus excludes any other material.
  • variants when referring to, for example, polynucleotides encoding a polypeptide variant of a given reference polypeptide are polynucleotides that differ from the reference polypeptide but generally maintain their functional characteristics of the reference polypeptide.
  • a variant of a polynucleotide may be a naturally occurring alleiic variant or it may be a variant that is known naturally not to occur.
  • Such non-naturally occurring variants of the reference polynucleotide can be made by, for example, mutagenesis techniques, including those mutagenesis techniques that are applied to polynucleotides, cells or organisms.
  • Variants of polynucleotides according to the present invention include, but are not limited to, nucleotide sequences which are at least 95% identical after alignment to the reference polynucleotide encoding the reference polypeptide. These variants can also have 96%, 97%, 98% and 99.999% sequence identity to the reference polynucleotide. Nucleotide changes present in a variant polynucleotide may be silent, which means that these changes do not alter the amino acid sequences encoded by the reference polynucleotide.
  • Substitutions, additions and/or deletions can involve one or more nucleic acids. Alterations can produce conservative or non-conservative amino acid substitutions, deletions and/or additions.
  • Variants of a prey or a SID® polypeptide encoded by a variant polynucleotide can possess a higher affinity of binding and/or a higher specificity of binding to its protein or polypeptide counterpart, against which it has been initially selected.
  • variants can also loose their ability to bind to their protein or polypeptide counterpart.
  • fragment of a polynucleotide or “fragment of a SID® polynucleotide” is meant that fragments of these sequences have at least 12 consecutive nucleotides, or between 12 and 5,000 consecutive nucleotides, or between 12 and 10,000 consecutive nucleotides, or between 12 and 20,000 consecutive nucleotides.
  • fragment of a polypeptide or “fragment of a SID® polypeptide” is meant that fragments of these sequences have at least 4 consecutive amino acids, or between 4 and 1 ,700 consecutive amino acids, or between 4 and 3,300 consecutive amino acids, or between 4 and 6,600 consecutive amino acids.
  • anabolic pathway is meant a reaction or series of reactions in a metabolic pathway that synthesize complex molecules from simpler ones, usually requiring the input of energy.
  • An anabolic pathway is the opposite of a catabolic pathway.
  • a "catabolic pathway” is a series of reactions in a metabolic pathway that break down complex compounds into simpler ones, usually releasing energy in the process.
  • a catabolic pathway is the opposite of an anabolic pathway.
  • drug metabolism is meant the study of how drugs are processed and broken down by the body. Drug metabolism can involve the study of enzymes that break down drugs, the study of how different drugs interact within the body and how diet and other ingested compounds affect the way the body processes drugs.
  • metabolic means the sum of all of the enzyme-catalyzed reactions in living cells that transform organic molecules.
  • second metabolism is meant pathways producing specialized metabolic products that are not found in every cell.
  • SID® means a Selected Interacting Domain and is identified as follows: for each bait polypeptide screened, selected prey polypeptides are compared. Overlapping fragments in the same ORF or CDS define the selected interacting domain.
  • PIM® means a protein-protein interaction map. This map is obtained from data acquired from a number of separate screens using different bait polypeptides and is designed to map out all of the interactions between the polypeptides.
  • the affinity of a SID® polypeptide of the present invention or a variant thereof for its polypeptide counterpart can be assessed, for example, on a BiacoreTM apparatus marketed by Amersham Pharmacia Biotech Company such as described by Szabo et al. (Curr Opin Struct Biol 5 pgs. 699-705 (1995)) and by Edwards and Leartherbarrow (Anal. Biochem 246 pgs. 1-6 (1997)).
  • the phrase "at least the same affinity" with respect to the binding affinity between a SID® polypeptide of the present invention to another polypeptide means that the Ka is identical or can be at least two-fold, at least three-fold or at least five fold greater than the Ka value of reference.
  • modulating compound means a compound that inhibits or stimulates or can act on another protein which can inhibit or stimulate the protein-protein interaction of a complex of two polypeptides or the protein-protein interaction of two polypeptides.
  • the present invention comprises complexes of polypeptides or polynucleotides encoding the polypeptides composed of a bait polypeptide, or a bait polynucleotide encoding a bait polypeptide and a prey polypeptide or a prey polynucleotide encoding a prey polypeptide.
  • the prey polypeptide or prey polynucleotide encoding the prey polypeptide is capable of interacting with a bait polypeptide of interest in various hybrid systems.
  • Protein-protein interactions can also be detected using complementation assays such as those described by Pelietier et al. at http://www.abrf.org/JBT/ATticles/JBT0012/ibtO012.htmI. WO 00/07038 and WO98/34120.
  • the present invention is not limited to detecting protein-protein interactions using yeast, but also includes similar methods that can be used in detecting protein-protein interactions in, for example, mammalian systems as described, for example in Takacs et al. (Proc. Natl. Acad. Sci., USA, 90 (21 ): 10375-79 (1993)) and Vasavada et al. (Proc. Natl. Acad. Sci., USA, 88 (23): 10686-90 (1991)), as well as a bacterial two-hybrid system as described in Karimova et al. (1998), W099/28746, WO00/66722 and Legrain et al. (FEBS Letters, 480 pgs. 32-36 (2000)).
  • suitable cells include, but are not limited to, VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines such as ATCC No. CCL61 , COS cells such as COS-7 cells and ATCC No. CRL 1650 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361 , A549, PC12, K662 cells, 293 cells, Sf9 cells such as ATCC No. CRL1711 and Cv1 cells such as ATCC No. CCL70.
  • suitable cells include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5- ⁇ ), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus.
  • suitable cells include yeast cells such as those of Saccharomyces such as Saccharomyces cerevisiae.
  • the bait polynucleotide, as well as the prey polynucleotide can be prepared according to the methods known in the art such as those described above in the publications and patents reciting the known method perse.
  • the bait and the prey polynucleotide of the present invention is obtained from transforming growth factor ⁇ cDNA, or variants of cDNA fragment from a library of transforming growth factor ⁇ , and fragments from the genome or transcriptome of transforming growth factor ⁇ cDNA ranging from about 12 to about 5,000, or about 12 to about 10,000 or from about 12 to about 20,000.
  • the prey polynucleotide is then selected, sequenced and identified.
  • a transforming growth factor ⁇ super-family of cytokines prey library is prepared from the transforming growth factor ⁇ cDNA and constructed in the specially designed prey vector pP6 as shown in Figure 3 after ligation of suitable linkers such that every cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcription activation domain of a reporter gene.
  • Any transcription activation domain can be used in the present invention. Examples include, but are not limited to, Gal4,YP16, B42, His and the like.
  • Toxic reporter genes such as CAT R , CYH2, CYH1 , URA3, bacterial and fungi toxins and the like can be used in reverse two-hybrid systems.
  • prey polypeptides encoded by the nucleotide inserts of the transforming growth factor ⁇ prey library thus prepared are termed "prey polypeptides" in the context of the presently described selection method of the prey polynucleotides.
  • the bait polynucleotides can be inserted in bait plasmid pB27 or pB28 as illustrated in Figure 8 and Figure 9.
  • the bait polynucleotide insert is fused to a polynucleotide encoding the binding domain of, for example, the Gal4 DNA binding domain and the shuttle expression vector is used to transform cells.
  • the bait polynucleotides used in the present invention are described in Table 1.
  • any cells can be utilized in transforming the bait and prey polynucleotides of the present invention including mammalian cells, bacterial cells, yeast cells, insect cells and the like.
  • the present invention identifies protein-protein interactions in yeast.
  • a prey positive clone is identified containing a vector which comprises a nucleic acid insert encoding a prey polypeptide which binds to a bait polypeptide of interest.
  • the method in which protein-protein interactions are identified comprises the following steps: i) mating at least one first haploid recombinant yeast cell clone from a recombinant yeast cell clone library that has been transformed with a plasmid containing the prey polynucleotide to be assayed with a second haploid recombinant yeast cell clone transformed with a plasmid containing a bait polynucleotide encoding for the bait polypeptide; ii) cultivating diploid cell clones obtained in step i) on a selective medium; and iii) selecting recombinant cell clones which grow on the selective medium.
  • This method may further comprise the step of: iv) characterizing the prey polynucleotide contained in each recombinant cell clone which is selected in step iii).
  • Escherichia coli is used in a bacterial two-hybrid system, which encompasses a similar principle to that described above for yeast, but does not involve mating for characterizing the prey polynucleotide.
  • mammalian cells and a method similar to that described above for yeast for characterizing the prey polynucleotide are used.
  • the prey polynucleotide that has been selected by testing the library of preys in a screen using the two-hybrid, two plus one hybrid methods and the like encodes the polypeptide interacting with the protein of interest.
  • the present invention is also directed, in a general aspect, to a complex of polypeptides, polynucleotides encoding the polypeptides composed of a bait polypeptide or bait polynucleotide encoding the bait polypeptide and a prey polypeptide or prey polynucleotide encoding the prey polypeptide capable of interacting with the bait polypeptide of interest.
  • complexes are identified in Table 2.
  • the present invention relates to a complex of polynucleotides consisting of a first polynucleotide, or a fragment thereof, encoding a prey polypeptide that interacts with a bait polypeptide and a second polynucleotide or a fragment thereof.
  • This fragment has at least 12 consecutive nucleotides, but can have between 12 and 5,000 consecutive nucleotides, or between 12 and 10,000 consecutive nucleotides or between 12 and 20,000 consecutive nucleotides.
  • the present invention relates to an isolated complex of at least two polypeptides encoded by two polynucleotides wherein said two polypeptides are associated in the complex by affinity binding and are depicted in columns 1 and 4 of Table 2.
  • the present invention relates to an isolated complex comprising at least a polypeptide as described in column 1 of Table 2 and a polypeptide as described in column 4 of Table 2.
  • the present invention is not limited to these polypeptide complexes alone but also includes the isolated complex of the two polypeptides in which fragments and/or homologous polypeptides exhibit at least 95% sequence identity, as well as from 96% sequence identity to 99.999% sequence identity.
  • Also encompassed in another embodiment of the present invention is an isolated complex in which the SID® of the prey polypeptides encoded by SEQ ID N°27 to 64 in Table 3 form the isolated complex.
  • nucleic acids coding for a Selected Interacting Domain (SID®) polypeptide or a variant thereof or any of the nucleic acids set forth in Table 3 can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • transcription elements include a regulatory region and a promoter.
  • the nucleic acid which may encode a marker compound of the present invention is operably linked to a promoter in the expression vector.
  • the expression vector may also include a replication origin.
  • useful expression vectors include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col El, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith et al (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM989, as well as other phage DNA such as M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2m plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in
  • both non-fusion transfer vectors such as, but not limited to pVL941 (BamHI cloning site Summers), pVL1393 (BamHI, Smal, Xba ⁇ ,
  • pAc700 BamHI and pnl cloning sites, in which the SamHI recognition site begins with the initiation codon; Summers), pAc701 and pAc70-2 (same as pAc700, with different reading frames), pAc360 (BamHI cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen (1995)) and pBlueBacHisA, B, C (three different reading frames with BamHI, BglW, Pst ⁇ , ⁇ col and Hind ⁇ l ⁇
  • Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase promoters, any expression vector with a DHFR expression cassette or a DHFR/methotrexate co-amplification vector such as pED (Psfl, Sail, Sbal, Smal and EcoRI cloning sites, with the vector expressing both the cloned gene and DHFR; Kaufman, 1991).
  • inducible promoters such as the dihydrofolate reductase promoters
  • any expression vector with a DHFR expression cassette or a DHFR/methotrexate co-amplification vector such as pED (Psfl, Sail, Sbal, Smal and EcoRI cloning sites, with the vector expressing both the cloned gene and DHFR; Kaufman, 1991).
  • glutamine synthetase/methionine sulfoximine co-amplification vector such as pEE14 (Hindlll, Xball, Smal, Sbal, EcoRI and Bell cloning sites in which the vector expresses glutamine synthetase and the cloned gene; Celltech).
  • a vector that directs episomal expression under the control of the Epstein Barr Virus (EBV) or nuclear antigen (EBNA) can be used such as pREP4 (BamHI, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull and Kpnl cloning sites, constitutive RSV- LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHI, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull and Kpnl cloning sites, constitutive hCMV immediate early gene promoter, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, Hindlll, Notl, Xhol, Sfil, BamHI cloning sites, inducible methallothionein Ha gene promoter, hygromycin selectable marker, Invitrogen), pREP
  • Selectable mammalian expression vectors for use in the invention include, but are not limited to, pRc/CMV (Hindlll, BstXl, Notl, Sbal and Apal cloning sites, G418 selection, Invitrogen), pRc/RSV (Hindll, Spel, BstXl, Notl, Xbal cloning sites, G418 selection, Invitrogen) and the like.
  • Vaccinia virus mammalian expression vectors include, but are not limited to, pSC11 (Smal cloning site, TK- and ⁇ -gal selection), pMJ601 (Sail, Smal, Afll, Na ⁇ , SspMII, BamHI, Apal, Nhel, Sac l, Kpnl and Hindlll cloning sites; TK- and ⁇ -gal selection), pTKgptFIS (EcoRI, Psfl, Sa/ll, Accl, Hindll, Sbal, BamHI and Hpa cloning sites, TK or XPRT selection) and the like.
  • Yeast expression systems that can also be used in the present include, but are not limited to, the non-fusion pYES2 vector (X ⁇ al, Sp ⁇ l, Shol, Notl, GstXl, EcoRI, BsfXI, BamHI, Sacl, Kpnl and Hindlll cloning sites, Invitrogen), the fusion pYESHisA, B, C (Xball, Sphl, Shol, Notl, BstXl, EcoRI, BamHI, Sacl, Kpnl and Hindlll cloning sites, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), pRS vectors and the like.
  • the non-fusion pYES2 vector X ⁇ al, Sp ⁇ l, Shol, Notl, GstXl, EcoRI, BsfXI, BamHI, Sacl, Kpnl and Hindlll cloning sites,
  • mammalian and typically human cells as well as bacterial, yeast, fungi, insect, nematode and plant cells an used in the present invention and may be transfected by the nucleic acid or recombinant vector as defined herein.
  • suitable cells include, but are not limited to, VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines such as ATCC No. CCL61, COS cells such as COS-7 cells and ATCC No. CRL 1660 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361,
  • suitable cells include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5- ⁇ ), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus.
  • prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5- ⁇ ), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus.
  • yeast cells such as those of Saccharomyces such as Saccharomyces cerevisiae.
  • yeast cells such as those of Saccharomyces such as Saccharomyces cerevisiae.
  • the present invention relates to and also encompasses SID® polynucleotides.
  • SID® polynucleotides of the present invention are represented by the shared nucleic acid sequences of SEQ ID N° 27 to 64 encoding the SID® polypeptides of SEQ ID N° 6 ⁇ to 102 in columns ⁇ and 7 of Table 3, respectively.
  • the present invention is not limited to the SID® sequences as described in the above paragraph, but also includes fragments of these sequences having at least 12 consecutive nucleic acids, between 12 and ⁇ ,000 consecutive nucleic acids and between 12 and 10,000 consecutive nucleic acids and between 12 and 20,000 consecutive nucleic acids, as well as variants thereof.
  • the fragments or variants of the SID® sequences possess at least the same affinity of binding to its protein or polypeptide counterpart, against which it has been initially selected.
  • this variant and/or fragments of the SID® sequences alternatively can have between 95% and 99.999% sequence identity to its protein or polypeptide counterpart.
  • variants of polynucleotide or polypeptides can be created by known mutagenesis techniques either in vitro or in vivo. Such a variant can be created such that it has altered binding characteristics with respect to the target protein and more specifically that the variant binds the target sequence with either higher or lower affinity.
  • Polynucleotides that are complementary to the above sequences which include the polynucleotides of the SID®'s, their fragments, variants and those that have specific sequence identity are also included in the present invention.
  • polynucleotide encoding the SID® polypeptide, fragment or variant thereof can also be inserted into recombinant vectors which are described in detail above.
  • the present invention also relates to a composition
  • a composition comprising the above-mentioned recombinant vectors containing the SID® polynucleotides in Table 3, fragments or variants thereof, as well as recombinant host cells transformed by the vectors.
  • the recombinant host cells that can be used in the present invention were discussed in greater detail above.
  • the compositions comprising the recombinant vectors can contain physiological acceptable carriers such as diluents, adjuvants, excipients and any vehicle in which this composition can be delivered therapeutically and can include, but is are not limited to sterile liquids such as water and oils.
  • the present invention relates to a method of selecting modulating compounds, as well as the modulating molecules or compounds themselves which may be used in a pharmaceutical composition.
  • modulating compounds may act as a cofactor, as an inhibitor, as antibodies, as tags, as a competitive inhibitor, as an activator or alternatively have agonistic or antagonistic activity on the protein-protein interactions.
  • the activity of the modulating compound does not necessarily, for example, have to be
  • the modulating compound can be selected according to a method which comprises:
  • said first vector comprises a polynucleotide encoding a first hybrid polypeptide having a DNA binding domain
  • said second vector comprises a polynucleotide encoding a second hybrid polypeptide having a transcriptional activating domain that activates said toxic reporter gene when the first and second hybrid polypeptides interact
  • the present invention relates to a modulating compound that inhibits the protein- protein interactions of a complex of two polypeptides of columns 1 and 4 of Table 2.
  • the present invention also relates to a modulating compound that activates the protein-protein interactions of a complex of two polypeptides of columns 1 and 4 of Table 2.
  • the present invention relates to a method of selecting a modulating compound, which modulating compound inhibits the interactions of two polypeptides of columns 1 and 4 of Table 2. This method comprises:
  • said first vector comprises a polynucleotide encoding a first hybrid polypeptide having a first domain of an enzyme
  • said second vector comprises a polynucleotide encoding a second hybrid polypeptide having an enzymatic transcriptional activating domain that activates said toxic reporter gene when the first and second hybrid polypeptides interact; (b) selecting said modulating compound which inhibits or permits the growth of said recombinant host cell.
  • the present invention provides a kit for screening a modulating compound.
  • This kit comprises a recombinant host cell which comprises a reporter gene the expression of which is toxic for the recombinant host cell. The host cell is transformed with two vectors.
  • the first vector comprises a polynucleotide encoding a first hybrid polypeptide having a DNA binding domain; and the second vector comprises a polynucleotide encoding a second hybrid polypeptide having a transcriptional activating domain that activates said toxic reporter gene when the first and second hybrid polypeptides interact.
  • a kit for screening a modulating compound by providing a recombinant host cell, as described in the paragraph above, but instead of a DNA binding domain, the first vector encodes a first hybrid polypeptide containing a first domain of a protein.
  • the second vector encodes a second polypeptide containing a second part of a complementary domain of a protein that activates the toxic reporter gene when the first and second hybrid polypeptides interact.
  • the activating domain can be p42 Gal 4, YP16 (HSV) and the DNA-binding domain can be derived from Gal4 or Lex A.
  • the protein or enzyme can be adenylate cyclase, guanylate cyclase, DHFR and the like.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the modulating compounds for preventing or treating disorders and/or diseases involving members of the TGF ⁇ family of cytokines in a human or animal, most preferably in a mammal.
  • This pharmaceutical composition comprises a pharmaceutically acceptable amount of the modulating compound.
  • the pharmaceutically acceptable amount can be estimated from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range having the desired effect in an in vitro system. This information can thus be used to accurately determine the doses in other mammals, including humans and animals.
  • the therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or in experimental animals. For example, the LD50 (the dose lethal to 50% of the population) as well as the ED50 (the dose therapeutically effective in 50% of the population) can be determined using methods known in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index which can be expressed as the ratio between LD 50 and ED50 compounds that exhibit high therapeutic indexes.
  • the data obtained from the cell culture and animal studies can be used in formulating a range of dosage of such compounds which lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the pharmaceutical composition can be administered via any route such as locally, orally, systemically, intravenously, intramuscularly, mucosally, using a patch and can be encapsulated in liposomes, microparticles, microcapsules, and the like.
  • the pharmaceutical composition can be embedded in liposomes or even encapsulated.
  • any pharmaceutically acceptable carrier or adjuvant can be used in the pharmaceutical composition.
  • the modulating compound will be preferably in a soluble form combined with a pharmaceutically acceptable carrier.
  • the techniques for formulating and administering these compounds can be found in "Remington's Pharmaceutical Sciences” Mack Publication Co., Easton, PA, latest edition.
  • the mode of administration optimum dosages and galenic forms can be determined by the criteria known in the art taken into account the seriousness of the general condition of the mammal, the tolerance of the treatment and the side effects.
  • the present invention also relates to a method of treating or preventing diseases involving the trasduction pathways of members of the transforming growth factor ⁇ super- family of cytokines in a human or mammal in need of such treatment.
  • This method comprises administering to a mammal in need of such treatment a pharmaceutically effective amount of a modulating compound which binds to a targeted mammalian or human or inner ear cell protein.
  • the modulating compound is a polynucleotide which may be placed under the control of a regulatory sequence which is functional in the mammal or human.
  • the present invention relates to a pharmaceutical composition comprising a SID® polypeptide, a fragment or variant thereof.
  • the SID® polypeptide, fragment or variant thereof can be used in a pharmaceutical composition provided that it is endowed with highly specific binding properties to a bait polypeptide of interest.
  • the original properties of the SID® polypeptide or variants thereof interfere with the naturally occurring interaction between a first protein and a second protein within the cells of the organism.
  • the SID® polypeptide binds specifically to either the first polypeptide or the second polypeptide.
  • SID® polypeptides of the present invention or variants thereof interfere with protein-protein interactions between mammalian and especially human protein.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable amount of a SID® polypeptide or variant thereof, provided that the variant has the above-mentioned two characteristics; i.e., that it is endowed with highly specific binding properties to a bait polypeptide of interest and is devoid of biological activity of the naturally occurring protein.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of a polynucleotide encoding a SID® polypeptide or a variant thereof wherein the polynucleotide is placed under the control of an appropriate regulatory sequence.
  • Appropriate regulatory sequences that are used are polynucleotide sequences derived from promoter elements and the like.
  • Polynucleotides that can be used in the pharmaceutical composition of the present invention include the nucleotide sequences of SEQ ID N° 27 to 64.
  • the pharmaceutical composition of the present invention can also include a recombinant expression vector comprising the polynucleotide encoding the SID® polypeptide, fragment or variant thereof.
  • compositions can be administered by any route such as orally, systemically, intravenously, intramuscularly, intradermally, mucosally, encapsulated, using a patch and the like.
  • Any pharmaceutically acceptable carrier or adjuvant can be used in this pharmaceutical composition.
  • the SID® polypeptides as active ingredients will be preferably in a soluble form combined with a pharmaceutically acceptable carrier. The techniques for formulating and administering these compounds can be found in "Remington's Pharmaceutical Sciences" supra.
  • the amount of pharmaceutically acceptable SID® polypeptides can be determined as described above for the modulating compounds using cell culture and animal models. Such compounds can be used in a pharmaceutical composition to treat or prevent transforming growth factor ⁇ -mediated disorders and/or diseases.
  • the present invention also relates to a method of preventing or treating transforming growth factor ⁇ -mediated disorders and/or diseases in a mammal said method comprising the steps of administering to a mammal in need of such treatment a pharmaceutically effective amount of:
  • SID® polynucleotide encoding a SID® polypeptide of SEQ ID N° 6 ⁇ to 102 or a variant or a fragment thereof wherein said polynucleotide is placed under the control of a regulatory sequence which is functional in said mammal.
  • nucleic acids comprising a sequence of SEQ ID N° 27 to 64 which encodes the protein of sequence SEQ ID N° 66 to 102 and/or functional derivatives thereof are administered to modulate complex (from Table 2) function by way of gene therapy.
  • Any of the methodologies relating to gene therapy available within the art may be used in the practice of the present invention such as those described by Goldspiel et al Clin. Pharm. 12 pgs. 488- ⁇ O ⁇ (1993).
  • Delivery of the therapeutic nucleic acid into a patient may be direct in vivo gene therapy (i.e., the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or indirect ex vivo gene therapy (i.e., cells are first transformed with the nucleic acid in vitro and then transplanted into the patient).
  • direct in vivo gene therapy i.e., the patient is directly exposed to the nucleic acid or nucleic acid-containing vector
  • indirect ex vivo gene therapy i.e., cells are first transformed with the nucleic acid in vitro and then transplanted into the patient.
  • an expression vector containing the nucleic acid is administered in such a manner that it becomes intracellular; i.e., by infection using a defective or attenuated retroviral or other viral vectors as described, for example in U.S. Patent 4,980,286 or by Robbins et al, Pharmacol. Ther. , 80 No. 1 pgs. 3 ⁇ -47 (1998).
  • the various retroviral vectors that are known in the art are such as those described in
  • adenoviral vectors can be used which are advantageous due to their ability to infect non-dividing cells and such high-capacity adenoviral vectors are described in Kochanek (Human Gene Therapy, 10, pgs. 2461-2459 (1999)).
  • Chimeric viral vectors that can be used are those described by Reynolds et al. (Molecular Medecine Today, pgs. 25 -31 (1999)).
  • Hybrid vectors can also be used and are described by Jacoby et al. (Gene Therapy, 4, pgs. 1282-1283 (1997)).
  • Direct injection of naked DNA or through the use of microparticle bombardment (e.g., Gene Gun®; Biolistic, Dupont) or by coating it with lipids can also be used in gene therapy.
  • Cell-surface receptors/transfecting agents or through encapsulation in liposomes, microparticles or microcapsules or by administering the nucleic acid in linkage to a peptide which is known to enter the nucleus or by administering it in linkage to a ligand predisposed to receptor-mediated endocytosis See Wu & Wu, J. Biol. Chem., 262 pgs. 4429-4432 (1987)
  • a nucleic acid ligand compound may be produced in which the ligand comprises a fusogenic viral peptide designed so as to disrupt endosomes, thus allowing the nucleic acid to avoid subsequent lysosomal degradation.
  • the nucleic acid may be targeted in vivo for cell specific endocytosis and expression by targeting a specific receptor such as that described in WO92/06180, W093/14188 and WO 93/20221.
  • the nucleic acid may be introduced intracellularly and incorporated within the host cell genome for expression by homologous recombination (See Zijlstra et al, Nature, 342, pgs. 435-428 (1989)).
  • a gene is transferred into cells in vitro using tissue culture and the cells are delivered to the patient by various methods such as injecting subcutaneously, application of the cells into a skin graft and the intravenous injection of recombinant blood cells such as hematopoietic stem or progenitor cells.
  • Cells into which a nucleic acid can be introduced for the purposes of gene therapy include, for example, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes and blood cells.
  • the blood cells that can be used include, for example, T- lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryotcytes, granulocytes, hematopoietic cells or progenitor cells and the like.
  • the present invention relates to protein chips or protein microarrays. It is well known in the art that microarrays can contain more than 10,000 spots of a protein that can be robotically deposited on a surface of a glass slide or nylon filter. The proteins attach covalently to the slide surface, yet retain their ability to interact with other proteins or small molecules in solution. In some instances the protein samples can be made to adhere to glass slides by coating the slides with an aldehyde-containing reagent that attaches to primary amines. A process for creating microarrays is described, for example by MacBeath and Schreiber (Science, Volume 289, Number 5486, pgs, 1760-1763 (2000)) or (Service, Science, Vol, 289, Number 5485 pg.
  • the present invention also relates to the use of a SID® or an interaction or a prey to screen molecules that inhibit TGF ⁇ or a TGF ⁇ super-family of cytokines pathway, as well as molecules that inhibit TGF ⁇ or a TGF ⁇ super-family of cytokines pathway obtained by this screening method.
  • the screening can occur in mammalian or yeast cells.
  • the inhibition can be detected by fluorescence polarization, FRET, BRET, filter binding assays or radioactive techniques.
  • cDNA For mRNA sample from transforming growth factor ⁇ , random-primed cDNA was prepared from 5 ⁇ g of polyA+ mRNA using a TimeSaver cDNA Synthesis Kit (Amersham Pharmacia Biotech) and with 5 ⁇ g of random N9-mers according to the manufacturer's instructions. Following phenolic extraction, the cDNA was precipitated and resuspended in water. The resuspended cDNA was phosphorylated by incubating in the presence of T4
  • Oligonucleotide HGX931 ( ⁇ ' end phosphorylated) 1 ⁇ g/ ⁇ l and HGX932 1 ⁇ g/ ⁇ l were used.
  • Linkers were preincubated (5 minutes at 9 ⁇ °C, 10 minutes at 68°C, 1 ⁇ minutes at 42°C) then cooled down at room temperature and ligated with cDNA fragments at 16°C overnight.
  • Linkers were removed on a separation column (Chromaspin TE 400, Clontech), according to the manufacturer's protocol. 1 A3. Vector preparation
  • Plasmid pP6 (see Figure 3) was prepared by replacing the SpellXhol fragment of pGAD3S2X with the double-stranded oligonucleotide: ⁇ 'CTAGCCATGGCCGCAGGGGCCGCGGCCGCACTAGTGGGGATCCTTAATTAAGGGCC ACTGGGGCCCCC3' (SEQ ID No.105)
  • CTGCGGCCATGG3' (SEQ ID No.106)
  • the pP6 vector was successively digested with Sf1 and BamHI restriction enzymes (Biolabs) for 1 hour at 37°C, extracted, precipitated and resuspended in water. Digested plasmid vector backbones were purified on a separation column (Chromaspin TE 400, Clontech), according to the manufacturer's protocol. 1.A.4. Li ⁇ ation between vector and insert of cDNA
  • the prepared vector was ligated overnight at 15°C with the blunt-ended cDNA described in section 2 using T4 DNA ligase (Biolabs). The DNA was then precipitated and resuspended in water.
  • the DNA from section 1.A.4 was transformed into Electromax DH10B electrocompetent cells (Gibco BRL) with a Cell Porator apparatus (Gibco BRL). 1 ml SOC medium was added and the transformed cells were incubated at 37°C for 1 hour. 9 mis of
  • SOC medium per tube was added and the cells were plated on LB+ampicillin medium. The colonies were scraped with liquid LB medium, aliquoted and frozen at -80°C.
  • Saccharomyces cerevisiae strain (YHGX13 (MAT ⁇ Gal4 ⁇ Gal ⁇ O ⁇ ade2- 101::KAN R , his3, leu2-3, -112, trp1-901, ura3-62 URA3::UASGAL1-LacZ, Met) was transformed with the cDNA library.
  • the plasmid DNA contained in E. coli was extracted (Qiagen) from aliquoted E. coli frozen cells (1.A.6.). Saccharomyces cerevisiae yeast YHGX13 in YPGIu were grown.
  • Yeast transformation was performed according to standard protocol (Giest et al. Yeast, 11, 356-360, 1995) using yeast carrier DNA (Clontech). This experiment leads to 10 4 to ⁇ x 10 4 cells/ ⁇ g DNA. 2 x 10 4 cells were spread on DO-Leu medium per plate. The cells were aliquoted into vials containing 1 ml of cells and frozen at -80°C. 1.C. Construction of bait plasmids
  • bait fragments were cloned into plasmid pB27and pB28.
  • Plasmid pB27 was prepared by replacing the ampicillin resistance of pB20 with the tetracyclin resistance.
  • Plasmid pB28 was prepared by replacing the EcoRI/Pstl polylinker fragment of pB27 with the double stranded DNA fragment : ⁇ 'GAATTCGGGGCCGCAGGGGCCGCGGCCGCACTAGTGGGGATCCTTAATTAAGGGCC ACTGGGGCCCCTCGACCTGCAG 3' (SEQ ID No 109) ⁇ 'CTGCAGGTCGAGGGGCCCCAGTGGCCCTTAATTAAGGATCCCCACTAGTGCGGCCG CG CGGCCCCTGCGGCCCCGAATTC 3'(SEQ ID No 110)
  • the amplification of the bait ORF was obtained by PCR using the Pfu proof-reading Tag polymerase (Stratagene), 10 pmol of each specific amplification primer and 200 ng of plasmid DNA as template.
  • the PCR program was set up as follows :
  • the amplification was checked by agarose gel electrophoresis.
  • the PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol.
  • PCR fragments were digested with adequate restriction enzymes.
  • the PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol.
  • the digested PCR fragments were ligated into an adequately digested and dephosphorylated bait vector (pB27 or pB28) according to standard protocol (Sambrook et al.) and were transformed into competent bacterial cells. The cells were grown, the DNA extracted and the plasmid was sequenced.
  • Example 2 Screening the collection with the two-hybrid in yeast system 2. A. The mating protocol
  • the mating procedure allows a direct selection on selective plates because the two fusion proteins are already produced in the parental cells. No replica plating is required.
  • bait-encoding plasmids were first transformed into S. cerevisiae (CG1945 strain (MATa Gal4- ⁇ 42 Gal180- ⁇ 38 ade2- 101 his3 ⁇ 200, Ieu2-3,112, trpl-901 , ura3- ⁇ 2, Iys2-801 , URA3::GAL4 17mers (X3)-
  • bait-encoding plasmids were first transformed into S. cerevisiae (L40 ⁇ gal4 strain (MATa ade2, trpl-901, Ieu2 3,112, Iys2-801 , his3 ⁇ 200, LYS2::(lexAop) 4 -HIS3, ura3-62::URA3 (lexAop) 8 -LacZ, GAL4::Kan R )) according to step 1.B. and spread on DO-Trp medium. Day 1, morning : preculture
  • the cells carrying the bait plasmid obtained at step 1.C. were precultured in 20 ml DO-Trp medium and grown at 30°C with vigorous agitation. Day 1, late afternoon : culture
  • the OD 600nm of the DO-Trp pre-culture of cells carrying the bait plasmid was measured.
  • the OD 6 oon must lie between 0.1 and O. ⁇ in order to correspond to a linear measurement.
  • the OD ⁇ oonm of the DO-Trp culture was measured. It should be around 1. For the mating, twice as many bait cells as library cells were used. To get a good mating efficiency, one must collect the cells at 10 8 cells per cm 2 .
  • the amount of bait culture (in ml) that makes up 60 OD600nm units for the mating with the prey library was estimated.
  • a vial containing the library of step 1 B was thawed slowly on ice. 1.0ml of the vial was added to 20 ml YPGIu. Those cells were recovered at 30°C, under gentle agitation for 10 minutes. Mating The 50 OD600nm units of bait culture was placed into a 60 ml falcon tube.
  • step 1B culture was added to the bait culture, then centrifuged, the supernatant discarded and resuspended in 1.6ml YPGIu medium.
  • the cells were distributed onto two 15cm YPGIu plates with glass beads. The cells were spread by shaking the plates. The plate cells-up at 30°C for 4h30min were incubated. Collection of mated cells
  • Clones that were able to grow on DO-Leu-Trp-His+Tetracyclin were then selected. This medium allows one to isolate diploid clones presenting an interaction. The His+ colonies were counted on control plates.
  • the number of His+ cell clones will define which protocol is to be processed : Upon 60.10 6 Trp+Leu+ colonies :
  • the X-Gal overlay assay was performed directly on the selective medium plates after scoring the number of His * colonies. Materials
  • a waterbath was set up.
  • the water temperature should be 60°C.
  • the temperature of the overlay mix should be between 45°C and 50°C.
  • the overlay- mix was poured over the plates in portions of 10 ml. When the top layer was settled, they were collected. The plates were incubated overlay-up at 30°C and the time was noted. Blue colonies were checked for regularly. If no blue colony appeared, overnight incubation was performed. Using a pen the number of positives was marked. The positives colonies were streaked on fresh DO-Leu-Trp-His plates with a sterile toothpick.
  • PCR amplification of fragments of plasmid DNA directly on yeast colonies is a quick and efficient procedure to identify sequences cloned into this plasmid. It is directly derived from a published protocol (Wang H. et al., Analytical Biochemistry, 237, 146-146, (1996)). However, it is not a standardized protocol and it varies from strain to strain and it is dependent of experimental conditions (number of cells, Ta ⁇ f polymerase source, etc). This protocol should be optimized to specific local conditions. Materials
  • PCR mix composition was:
  • the positive colonies were grown overnight at 30°C on a 96 well cell culture cluster
  • Thermowell was placed in the thermocycler (GeneAmp 9700, Perkin Eimer) for 5 minutes at 99.9 D C and then 10 minutes at 4°C. In each well, the PCR mix was added and shaken well. The PCR program was set up as followed:
  • the quality, the quantity and the length of the PCR fragment was checked on an agarose gel.
  • the length of the cloned fragment was the estimated length of the PCR fragment minus 300 base pairs that corresponded to the amplified flanking plasmid sequences.
  • Extraction buffer 2% Triton X100, 1% SDS, 100 mM NaCl, 10 mM TrisHCI pH 8.0, 1 mM EDTA pH 8.0.
  • each patch was scraped into an Eppendorf tube, 300 ⁇ l of glass beads was added in each tube, then, 200 ⁇ l extraction buffer and 200 ⁇ l phenol:chloroform:isoamyl alcohol (25:24:1) was added.
  • the tubes were centrifuged for 10 minutes at 16,000 rpm. 180 ⁇ l supernatant was transferred to a sterile Eppendorf tube and 600 ⁇ l each of ethanol/NH 4 Ac was added and the tubes were vortexed. The tubes were centrifuged for 15 minutes at 16,000 rpm at 4°C. The pellet was washed with 200 ⁇ l 70% ethanol and the ethanol was removed and the pellet was dried. The pellet was resuspended in 10 ⁇ l water. Extracts were stored at -20°C. Electroporation
  • Electrocompetent MC1066 cells prepared according to standard protocols (Sambrook et al. supra).
  • yeast plasmid DNA-extract 1 ⁇ l was added to a pre-chilled Eppendorf tube, and kept on ice. 1 ⁇ l plasmid yeast DNA-extract sample was mixed and 20 ⁇ l electrocompetent cells was added and transferred in a cold electroporation cuvette.
  • the Biorad electroporator was set on 200 ohms resistance, 26 ⁇ F capacity; 2.6 kV.
  • the cuvette was placed in the cuvette holder and electroporation was performed.
  • the previous protocol leads to the identification of prey polynucleotide sequences.
  • a suitable software program e.g., Blastwun, available on the Internet site of the University of Washington: http://bioweb.pasteur.fr/seganal/interfaces/blastwu.html.
  • the mRNA transcript that is encoded by the prey fragment may be identified and whether the fusion protein encoded is in the same open reading frame of translation as the predicted protein or not can be determined.
  • prey nucleotide sequences can be compared with one another and those which share identity over a significant region (60nt) can be grouped together to form a contiguous sequence (Contig) whose identity can be ascertained in the same manner as for individual prey fragments described above.
  • SID® Selected Interacting Domain
  • mice are immunized with an immunogen comprising the above mentionned complexes conjugated to keyhole limpet hemocyanin using glutaraldehyde or
  • EDC as is well known in the art.
  • the complexes can also be stabilized by crosslinking as described in WO 00/37483.
  • the immunogen is then mixed with an adjuvant.
  • Each mouse receives four injections of 10 ⁇ g to 100 ⁇ g of immunogen, and after the fourth injection, blood samples are taken from the mice to determine if the serum contains antibodies to the immunogen.
  • Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.
  • Spleens are removed from immune mice and single-cell suspension is prepared
  • Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibodies for characterization and assay development.
  • Antibodies are tested for binding to bait polypeptide of column 1 of Table 2 alone or to prey polypeptide of column 4 of Table 2 alone, to determine which are specific for the protein-protein complex of columns 1 and 4 of Table 2 as opposed to those that bind to the individual proteins.
  • Monoclonal antibodies against each of the complexes set forth in columns 1 and 4 of Table 2 are prepared in a similar manner by mixing specified proteins together, immunizing an animal, fusing spleen cells with myeloma cells and isolating clones which produce antibodies specific for the protein complex, but not for individual proteins.
  • Example 7 Modulating compounds identification
  • Each specific protein-protein complex of columns 1 and 4 of Table 2 may be used to screen for modulating compounds.
  • One appropriate construction for this modulating compound screening may be:
  • Example 8 ZNF8 (hgx554)
  • the predicted ZNF8 protein (576 aa) contains 7 zinc finger domains (Lania et al., 1990).
  • mZNF8 mouse ZNF8
  • mZNF ⁇ mouse ZNF8
  • TCCCATGTCA CGGGAAGGGA AGGATTCCCG ACAGATGCTC CTTATCCCAC
  • CTGTGCACGT TATTGGGGTG GAAGAGCCTT CTGTGGGTGC TTCCATGTTA TTTGACATCA GAGAATCCAC ATAG (SEQ ID N0.113)
  • ZNF8 interacts with several members of the BMP and TGF ⁇ pathways
  • SID Nucleic sequence, SEQ ID No.27 and Proteic sequence, SEQ ID No. 65
  • SID Nucleic sequence, SEQ ID No.31 and Proteic sequence, SEQ ID No. 69
  • SID Nucleic sequence, SEQ ID N ⁇ .42 and Proteic sequence, SEQ ID No. 80 Smad9-ZNF8
  • SID Nucleic sequence, SEQ ID No.45 and Proteic sequence, SEQ ID No. 83
  • SID Nucleic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID NO: SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID No.28, 29, 30 and Proteic sequence
  • SID Nucleic sequence, SEQ ID No.38 and Proteic sequence, SEQ ID No. 76 Rebound screening experiments using ZNF8 as bait (nt 732-1301 ) on Placenta library allowed us to confirm the Smadl -ZNF8 and Smad9-ZNF8 interactions ZNF8-Smad1
  • Yeast-two-hybrid screens show that amino-acids 22-268 from Smadl (SEQ ID No.14) interact with amino-acids 364-433 from ZNF8 (SEQ ID No.114) (see . 11 A).
  • Amino-acids 1-162 from Smad4 (SEQ ID No.17) interact with amino-acids 172-441 from ZNF8 (see fig. 11B).
  • Amino-acids 1-268 from Smad ⁇ (SEQ ID No.19) interact with amino- acids 276-437 from ZNF8 (see fig. 11C).
  • amino-acids 1-233 from Smad9 SEQ ID No.20 interact with amino-acids 208-1209 from ZNF8 (see fig. 11D).
  • ZNF8 is an essential player in the TGF ⁇ and BMP pathways
  • ZNF8 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GTCT) 8 -MLP-Luc reporter and ZNF8-targeting siRNA duplex, a specific dose-dependant repression of the TGF ⁇ -dependant reporter activity was observed (see Fig. 12A) demonstrating a function for ZNF8 in the response to the TGF ⁇ pathway. The repressive effect of ZNF8-targeting siRNA duplex was observed at low concentration of siRNA duplex (4nM) and was enhanced at higher concentrations (40nM).
  • TGF ⁇ and/or BMPs in cells a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGF ⁇ or BMP-dependant markers was performed.
  • PAI-1 was a well-known target of TGF ⁇ and was strongly induced by TGF ⁇ in many cell types (Keeton et al., 1991).
  • Osteoblastic differentiation was characterized by expression of alkaline phosphatase as an early pre-osteoblastic marker and alkaline phosphatase transcription is directly controled by BMP signals (Wagner EF and Karsenty G, 2001).
  • TGF ⁇ AP1/jun expression
  • TGF ⁇ activates c-jun expression only in epithelial cells, whereas it induces junB in mesenchymal cells.
  • JunB is also an immediate early gene induced by BMP-2 (Mauviel ef al., 1996; Chalaux er a/., 1998).
  • HPRT hyperxanthine-guanine phosphoribosyltransferase, Patel et al., 1986, data not shown
  • GAPDH glycosyceraldehyde-3- phosphate dehydrogenase, Allen etal., 1987, data not shown
  • 18S ribosomal RNA Schotgen ef al., 2000, data not shown.
  • LAPTmS hgx596
  • LAPTm ⁇ has also been found to be an immediate-early gene induced by retinoic acid during granulocytic differentiation in murine retinoic acid-inducible MPRO promyelocyte cell line (Scott et al., 1996). Finally, LAPTm ⁇ was shown to be up-regulated in the Sjogren's syndrome which is a chronic autoimmune disease (Azuma ef al., 2002) and to be co-expressed with activated macrophage genes in rheumatoid arthritis (Walker et al., 2002).
  • LAPTm5-Smurf2 yeast-two-hybrid screens showed that amino-acids 234-335 from Smurf2 (SEQ ID No.22) interact with amino-acids 261-262 from LAPTm ⁇ (SEQ ID No.116) (see Fig.16). II. LAPTm ⁇ modulates the TGF ⁇ pathway
  • LAPTm ⁇ cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and LAPTm ⁇ -targeting siRNA duplex, a specific dose-dependant activation of the TGF ⁇ -dependa ⁇ t reporter activity was observed (see Fig. 18 A) demonstrating a function for LAPTm ⁇ in the response to the TGF ⁇ pathway.
  • LAPTm ⁇ -targeting siRNA duplex The activating effect of LAPTm ⁇ -targeting siRNA duplex was observed at low concentration of siRNA duplex (4nM) and was enhanced at higher concentrations (40nM). While transiently co-transfecting HepG2 cells using the p(GC)12-MLP-Luc reporter and LAPTm ⁇ -targeting siRNA duplex, a specific, dose-dependant and BMP-dependant activation of the BMP-dependant reporter activity was observed (see Fig. 18 B) demonstrating a function for LAPTm ⁇ in the response to the BMP pathway. In order to further elucidate its role on the expression of genes naturally controlled by
  • TGF ⁇ and/or BMPs in cells a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGF ⁇ or BMP-dependant markers were performed. Endogenous levels of PAI-1 , junB and alkaline phosphatase mRNA were specifically and dose-dependently increased following transient transfection of LAPTm ⁇ -targeti ⁇ g siRNA duplex in HepG2 cells treated with either TGF ⁇ (PAI-1 and junB, see Fig. 19 A & B) or BMP7 (alkaline phosphatase, see fig 19 C). Expression levels of various controls were not significantly affected following the same LAPTm ⁇ -targeting siRNA duplex transfection: hGUS (see fig 19 D) HPRT, GAPDH and 18S (data not shown).
  • LAPTm ⁇ is involved in the negative feedback of the TGF ⁇ signalling. It has been suggested by Kavsak and coll. (Kavsak ef al. 2000) that Smurf2 could address the TGF ⁇ receptors and smad7 to the lysosome for degradation.
  • LAPTm ⁇ could be a smurf2 receptor in the lysosomal membrane and could address some TGF ⁇ signaling members to the lysosomal compartment to induce their degradation.
  • Example 10 RNF11 (hgx555) Gl:7657519 Seki ef al. (1999) identified a new member of the RING finger family, named RNF11 (164 amino acids). Recently, a differential display analysis of gene expression using NIH 3T3 cells expressing the RET-MEN2A or RET-MEN2B mutant proteins was performed. These germ- line point mutations of the RET gene are responsible for multiple endocrine neoplasia (MEN) type 2A and 2B that develop medullary thyroid carcinoma and pheochromocytoma. It has been shown that RNF11 was up-regulated in these mutant cells (Watanabe ef al., 2002). In addition, GNDF was found to up-regulate RNF11 levels (Watanabe ef al., 2002). However, no function for RNF11 has been attributed yet.
  • MEN endocrine neoplasia
  • RNF11 interacts with SARA and Smurf2, proteins involved in the TGF ⁇ pathway
  • SARA the "Smad anchoring for Receptor Activation”
  • Smurf2 a E3 ubiquitin ligase known to regulate the protein level of Smadl , 2, 7, SnoN and T ⁇ RI.
  • Smurf2-RNF11 a E3 ubiquitin ligase known to regulate the protein level of Smadl , 2, 7, SnoN and T ⁇ RI.
  • SID Nucleic sequence, SEQ ID No.50, 51 , 52, 53 and Proteic sequence, SEQ ID No. 88, 89, 90, 91 SARA-RNF1 1
  • SID Nucleic sequence, SEQ ID No.54, 5 ⁇ and Proteic sequence, SEQ ID No. 92, 93
  • yeast-two-hybrid screens showed that amino-acids 239-335 from Smurf2 (SEQ ID No.22) (aa 239-335) interact with amino-acids 31-84 from RNF11 (SEQ ID No.118) (see Fig. 20 A).
  • Amino-acids 665-1323 from SARA (SEQ ID No.23) interact with amino-acids 61-154 from RNF11 (see Fig. 20B) and amino-acids 236-415 from Smurfl interact with amino-acids 31-154 from RNF11 (see Fig. 20 C).
  • RNF11 regulates the SARA protein level
  • KIAA1193 the hypothetical zinc finger protein KIAA1196 was identified. Since this putative protein contains 7 zinc fingers (C2H2 type), it has been suspected that it may function as a transcription factor. Moreover, KIAA1196 contains a leucine zipper motif in the domain that we have discovered interacting with Smadl and is predicted to be a nuclear protein, reinforcing its potential function as a transcription factor. However, no function for this protein has been attributed, yet.
  • AAACACCCAA AAAGTTTACA GGGGAGCAGC CATCCATCTC AGGGACCTTT
  • SID Nucleic sequence, SEQ ID No.35, 36, 37 and Proteic sequence, SEQ ID No. 73, 74, 75
  • TGF ⁇ binds ALK1 (which induces phosphorylation of Smadl and 5) and ALK ⁇ (which induces phosphorylation of Smad2 and 3) in transfected COS cells (Ten Dijke et al., 1994).
  • ALK1 which induces phosphorylation of Smadl and 5
  • ALK ⁇ which induces phosphorylation of Smad2 and 3
  • TGF ⁇ regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signaling (Goumans ef al., 2002). Since KIAA1196 was found interacting with Smadl, it was investigated whether KIAA1196 could be involved in the TGF ⁇ and/or BMP pathways.
  • KIAA1196 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and KIAA1196-targeting siRNA duplex, a specific, dose-dependant and TGF ⁇ -dependant repression of the luciferase reporter activity was observed (see Fig. 23 A) demonstrating a function for KIAA1196 in the TGF ⁇ pathway.
  • SiRNA-mediated KIAA1196 cellular knock-down were also performed in another cell type: HEK293 cells.
  • a specific, dose-dependant and TGF ⁇ -dependa ⁇ t repression of the p(GTCT)8-MLP-Luc reporter activity was also observed (see Fig. 24).
  • the extend of the repression of the TGF ⁇ -dependant reporter activity observed using KIAA1196-targeting siRNA duplex was almost as efficient as the repression obseved using the positive control (T ⁇ RI-targeting siRNA duplex).
  • Modulation of the TGF ⁇ luciferase reporter activity using KIAA1196 cellular knock-down demonstrated an essential implication of this putative transcription factor in the regulation of the TGF ⁇ pathway.
  • KIAA1196 In order to further elucidate KIAA1196's role on the expression of genes naturally controlled by TGF ⁇ in mammalian cells, a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGF ⁇ -dependant markers were performed. Endogenous levels of PAI-1 and junB mRNA were specifically and dose-dependently decreased following transient transfection of KIAA1196-targeting siRNA duplex in HepG2 cells treated with TGF ⁇ (see Fig. 25 A & B). As expected, endogenous alkaline phosphatase mRNA levels were not stimulated following BMP7 treatment and thus were not affected by KIAA1196-targeting siRNA (see Fig. 25 C).
  • LlM-only proteins are transcriptional regulators that function by mediating protein- protein interactions and include the T cell oncogenes LM01 and LM02.
  • T cell oncogenes LM01 and LM02. By screening expression libraries with the LIM interaction domain of NL1/CLIM2/LDB1 , Kenny ef a/. (1998) isolated and characterized LM04, a novel LlM-only gene.
  • the LM04 gene was further characterized in terms of genomic organization and comparative chromosomal mapping (Tse ef al., 1999). LM04 was found to be a candidate gene associated with prostate cancer progression since LM04 was down-regulated in prostate cancer (Mousses ef al., 2001).
  • LM04 interacts with Smad9 a protein involved in the BMP pathway
  • Smad9 a protein involved in the BMP pathway
  • yeast-two-hybrid screens showed that amino-acids 209-430 from Smad9 (SEQ ID No.20) (aa 209-430) interact with amino-acids 7-126 from LM04 (SEQ ID No.121) (see Fig. 26).
  • LM04 modulates BMP signaling
  • LM04 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC) ⁇ 2 -MLP-Luc reporter and LM04-targeting siRNA duplex, a specific, dose-dependant and BMP7-dependant repression of the BMP-dependant reporter activity was observed (see Fig. 27 A) suggesting a general function for LM04 in the response to the BMP7 pathway.
  • Protein phosphatase 1 is a major eukaryotic protein serine/threo ⁇ ine phosphatase that regulates an enormous variety of cellular functions through the interaction of its catalytic subunit (PP1c) with over fifty different established or putative regulatory subunits (see for review; Cohen, 2002). Most of these target PP1c to specific sub-cellular locations and interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif - the RVxF motif - which is often preceded by further basic residues. Recently, Bennett and Alphey (2002) showed that PP1 binds SARA and negatively regulates Dpp signaling in Drosophila melanogaster.
  • CTCAGCTCCC AACTACTGTG GCGAG1 ⁇ GA CAATGCTGGC GCCATGATGA
  • PPIca interacts with SARA, a protein involved in the TGF ⁇ pathway
  • SARA a protein involved in the TGF ⁇ pathway
  • SARA-PP1ca SID Nucleic sequence, SEQ ID No.56, 57,58, 59 and Proteic sequence, SEQ ID No. 94, 95, 96, 97.
  • yeast-two-hybrid screens showed that amino-acids 668-947 from SARA (SEQ ID No.23) interact with amino-acids 29-296 from PPI ca (SEQ ID No.123) (see Fig. 30).
  • PPIca is a regulator of the TGF ⁇ signaling
  • a Baculovirus over-expressing the smad3 protein (as positive control) and the PPI ca protein was generated.
  • This baculovirus expression system has been genetically engineered to allow infection and expression in mammalian cells (see material & methods). Both viruses were used to infect the HepG2 cells for 24 hours with or without TGF ⁇ .
  • the over-expression level of our proteins of interest by Q-PCR experiments was checked. In these conditions, the Smad3 and PPIca mRNA were shown to be over- expressed by a 350-fold and 50-fold, respectively, as compared to the endogenous mRNA level ( Figure 32 A).
  • Example 14 HYPA (hgx530) GI: 3341989 Huntington's disease, with its hallmark choreiform movements and graded loss of striatal neurons, is a dominantly inherited disorder caused by expansion of a CAG repeat in one copy of the HD gene. The HD mutation elongates an N-terminal glutamine segment in the huntingtin protein. HYPA, HYPB and HYPC were found to interact with the huntingtin protein (Faber ef al., 1998). HYPA is a protein containing a WW domain, known to bind proiin-rich peptides stretches. This protein is the human homolog of the essential pre-mRNA splicing factor PrP40 and is also called FBP11.
  • mutant huntingtin in target neurons may promote an abnormal interaction with one, or all, huntingtin's WW domain partners, perhaps altering ribonucleo-protein function with toxic consequences (Passani ef al., 2000).
  • HYPA contains a FF domain, with a structure recently determined, which is a 60 amino acid residue phosphopeptide-binding module (Allen ef al., 2002). However, no link between HYPA and the TGF ⁇ /BMP pathway was previously made.
  • CTGAGCCCGA CGATGAGGCC GGGGACGGGA GCTGAGCGTG GAGGCCTCAT
  • AACTCTTATC TAAATGCCCC TGGAAGGAAT ACAAATCAGA TTCTGGAAAG
  • HYPA interacts with Smad4, a protein involved in the TGF ⁇ / BMP pathway
  • HYPA interacts with Smad4, a protein involved in the TGF ⁇ /BMP pathway.
  • Smad4-HYPA SID Nucleic sequence, SEQ ID No.39, 40, 41 and Proteic sequence, SEQ ID No. 77, 78, 79.
  • yeast-two-hybrid screens showed that amino-acids 251-552 from Smad4 (SEQ ID No.17) interact with amino-acids 276-387 from HYPA (SEQ ID No.123) (see Fig. 33).
  • HYPA is a regulator of the TGF ⁇ signaling
  • HYPA was found interacting with Smad4, it was investigated whether HYPA could be involved in the TGF ⁇ and/or BMP pathways.
  • HYPA cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC) 8 -MLP-Luc reporter and HYPA-targeting siRNA duplex, a specific dose-dependant repression of the BMP-dependant reporter activity was observed (see Fig. 34 A) demonstrating a function for HYPA in the response to the BMP pathway.
  • Example 15 FLJ20037 (hgx594) GI: 8923041
  • C6orf37 chromosome 6 open reading frame 37
  • FLJ20037 chromosome 6 open reading frame 37
  • Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues.
  • the C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. The N-terminal part of this protein contains some glycine- rich repeats. However, no link between FLJ20037 and the TGF ⁇ /BMP pathway was previously made.
  • ACCTTATCAC CATGCTGGCT ATCCGGGTGT TAGCTGACCA AAATGTCATT
  • FLJ20037 interacts with SARA, a protein involved in the TGF ⁇ pathway
  • SARA a protein involved in the TGF ⁇ pathway
  • SARA-FLJ20037 SID Nucleic sequence, SEQ ID No.60, 61 and Proteic sequence, SEQ ID No. 98, 99.
  • yeast-two-hybrid screens showed that amino-acids 665-1323 from SARA (SEQ ID No.23) interact with amino-acids 58-253 from FLJ20037 (SEQ ID No.125) (see Fig. 36). FLJ20037 modulates the TGF ⁇ signaling
  • FLJ20037 could be involved in the TGF ⁇ and/or BMP pathways.
  • baculoviruses over-expressing the smad3 protein (as positive control) and the FLJ20037 protein were generated. Both viruses were used to infect the HepG2 cells during 24 hours, treated or not with TGF ⁇ .
  • the over-expression level of our proteins of interest by Q-PCR experiments was checked. Smad3 and FLJ20037 mRNA were shown to be over-expressed 350-fold and 200- fold, respectively, when compared to their respective endogenous mRNA levels (Figure 37 A).
  • endogenous PAI-1 and JunB mRNA levels were looked at, which were previously shown to be up-regulated by TGF ⁇ .
  • PTPs Protein tyrosine phosphatases
  • PTKs protein tyrosine kinases
  • FISH fluorescence in situ hybridization
  • PTPN12 interacts with Smad5, a protein involved in the BMP pathway
  • yeast-two-hybrid screens showed that amino-acids 1-268 from Smad ⁇ (SEQ ID No.19) interact with amino-acids 99-337 from PTPN12 (SEQ ID No.127) (see Fig. 39).
  • PTPN12 modulates the TGF ⁇ and BMP signaling
  • PTPN12 Since PTPN12 was found interacting with Smad ⁇ , it was investigated whether PTPN12 could be involved in the TGF ⁇ and/or BMP pathways.
  • PTPN12 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC) 12 -MLP-Luc reporter and PTPN12-targeting siRNA duplex, a specific BMP6- dependant increase in the BMP-dependant reporter activity was observed (see Fig. 40 A) demonstrating a role for PTPN12 in the response to the BMP pathway.
  • Fas-interacting ⁇ erine/threonine kinase/homeodomain-interacti ⁇ g protein kinase FIST/HIPK3
  • Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas.
  • the protein contains sequences identical to the catalytic core of many serine-protein kinases and is 54% similar to the yeast protein kinase YAK1 , whose normal role is to restrict growth.
  • the authors therefore designated the protein PKY/HIPK3, for homolog of protein kinase YAK1.
  • PKY/HIPK3 may be identical to a 170-kD kinase identified in the same cell lines by Sampson et al. (1993), the difference in molecular mass being due to posttranslational modifications.
  • PKY/HIPK3 was expressed at higher levels in MDR cells than in their nonresistant parental lines; in addition, a 7-kb PKY/HIPK3 transcript was expressed at high levels in heart and skeletal muscle and at lower levels in placenta, pancreas, and brain.
  • Kim et al. identified in mouse 3 members of a family of cofactors, which they designated homeodomain-interacting protein kinases (HIPKs), that interact with homeoproteins and show the greatest similarity to the yeast YAK1 protein (43% identity in the catalytic domain).
  • HIPKs The corepressor activity of HIPKs depends on both its homeodomain interaction domain and a corepressor domain that maps to the N terminus.
  • Kim et al. (1998) presented evidence that HIPKs can act as transcriptional corepressors for NK homeodomain transcriptionfactors.
  • Nupponen and Visakorpi (1999) mapped the HIPK3 gene to chromosome 11p13. However, no link between HIPK3 and the TGF ⁇ /BMP pathway was previously made.
  • TCTACTTCCA TACCCATCAT CAGCCACCCT CAGTAGTGCT GCACCAGTGG CCCACCTGTT AGCCTCTCCG TGTACCTCAA GACCTATGTT ACAGCATCCA
  • SID Nucleic sequence, SEQ ID No.64 and Proteic sequence, SEQ ID No. 102.
  • Snip1-HIPK3 SID Nucleic sequence, SEQ ID No.62, 63 and Proteic sequence, SEQ ID No. 100, 101.
  • yeast-two-hybrid screens showed that amino-acids 799-1127 from HIPK3 (SEQ ID No.129) interact with amino-acids 1-370 from SnoN (SEQ ID No.26) and that amino-acids 833-930 from HIPK3 interact with amino-acids 1-198 from Snipl (SEQ ID No.24) (see Fig. 41).
  • HIPK3 modulates the BMP signaling
  • HIPK3 Since HIPK3 was found interacting with SnoN and SNIP1 , it was investigated whether HIPK3 could be involved in the TGF ⁇ and/or BMP pathways.
  • HIPK3 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC)i 2 -MLP-Luc reporter and HIPK3-targeting siRNA duplex, a specific, dose-dependant and BMP6-dependant increase in the BMP-dependant reporter activity was observed (see Fig.
  • mammalian baculovirus vector consisted in introduction of mammalian Polymerase ll-type transcriptional units such as a promoter active in mammalian cells (for instance CMV, RSV, albumin or inducible promoters).
  • mammalian Polymerase ll-type transcriptional units such as a promoter active in mammalian cells (for instance CMV, RSV, albumin or inducible promoters).
  • Such plasmids can be used as classical expression vectors to transfect mammalian cells. They can also be used to generate baculoviruses that have the capacity to infect mammalian cells with a high efficiency where they drive the expression of the gene which is under the transcriptional control of the promoter active in mammalian cells (Kost and Condreay, 2002).
  • pV3 and pV ⁇ were prepared from pfastbad vectors (Invitrogen).
  • OI ⁇ 3055 5'-cggaattcTTGGGTCTCCCTATAGTGAGT-3' (SEQ ID No.131 )
  • CTGCA-3 (SEQ ID No.134)
  • AACTGCA-3' (SEQ ID No.136) 3'-
  • pV3 and pV5 vectors thus contain a CMV promoter which controls expression of the proteins of interest fused to the FLAG epitope.
  • the MCS present into pV3 and pV5, contained the Smal/Sfil/pvull/Sfil/Pacl sites. Differences between pV3 and pV5 were in the
  • OH2752 cggactagtCATGTCGTCCATCCTGCCTT (SEQ ID No.138)
  • ON2836 gccttaattaaCTAAGACACACTGGAACAGCGG(SEQ ID No.139)
  • OH3778 gatcggccggacgggccATGGGGAACTGCCTCAAATCCCCC (SEQ ID No.140)
  • LAPTm ⁇ OH3776: gatcggccggacgggccATGGACCCCCGCTTGTCCACTGTC (SEQ ID No.142)
  • OII3777 gatcggccccagtggccTCACACCTCTGAGTATGGGGGTGG (SEQ ID No.143)
  • the PCR program was set up as follows:
  • the amplification was checked by agarose gel electrophoresis.
  • the PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol. The purified
  • PCR fragments were digested with Sfil restriction enzyme (Biolabs) for 1 hour at 60°C.
  • Sfil restriction enzyme Biolabs
  • PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol.
  • PP1ca and pP6-FLJ20037 vectors were digested with Sfil restriction enzyme (Biolabs) for 1 hour at 50°C, extracted, precipitated, and resuspended in water.
  • Sfil restriction enzyme Biolabs
  • the PPIca and FLJ20037 fragments were then purified using Qiaex column (Qiagen) according to the manufacturer's protocol.
  • the pV3 and pV5 vectors were digested with Sfil restriction enzyme (Biolabs) for 1 hour at
  • Digested plasmid vector backbones were purified on a separation column (Chromaspin TE 400, Clontech) according to the manufacturer's protocol.
  • HepG2 cells were propagated in Dulbecco's modified Eagle's medium (Life Technologies, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Life technologies, Invitrogen), 100 units ml-1 penicillin, and 100 ⁇ g.ml-1 streptomycin (Life Technologies, Invitrogen) at 37°C, ⁇ %C02 controlled atmosphere. Cells were regularly passaged to maintain exponential growth. Twenty four hours before transfections, cells were trypsinized and diluted with fresh medium at 2x106 cells/well in a 24 well plate in order to get approximately 60-80% confiuency for transfection.
  • Dulbecco's modified Eagle's medium Life Technologies, Invitrogen
  • FBS fetal bovine serum
  • 100 units ml-1 penicillin 100 units ml-1 penicillin
  • 100 ⁇ g.ml-1 streptomycin Life Technologies, Invitrogen
  • the MLP minimal promoter from an adenovirus Major Late gene containing a TATA box and an initiator element, was first inserted into the Bglll and Hindlll sites of the pGL3 basic vector (Promega) to generate the MLP-Luc plasmid using the oligonucleotides: MLP1: 5'- GATCTGAATTCCATATGCTGCAGGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTC ACTCTCTTCCA-3'(SEQ ID No.144) and the complementary oligonucleotide MLP2 : ⁇ '- AGCTTGGAAGAGAGTGAGGACGAACGCGCCCCCACCCCCTTTTATAGCCCCTGCAGCA TATGGAATTCA-3' (SEQ ID N ⁇ .14 ⁇ )
  • GTCT GTCT 8 -MLP-Luc
  • 2 copies of the following annealed oligonucleotides were inserted into the EcoRI site of MLP-Luc. These oligonucleotides contains 4 copies of 'the GTCT box', a TGF ⁇ -responsive sequence (Zawel et al., 1998).
  • GTCT1 5'-AATTCGTCTAGACAAAAGTCTAGACATTTGTCTAGACTAGTGTCTAGACG-3' (SEQ ID No.146)
  • GTCT2 5'-AATTCGTCTAGACACTAGTCTAGACAAATGTCTAGACTTTTGTCTAGACG-3' (SEQ ID No.147)
  • CAGA 6 -MLP-Luc
  • 1 copy of the following annealed oligonucleotides was inserted into the Xhol and Nhel sites of MLP-Luc.
  • These oligonucleotides contains 6 copies of 'the CAGA box', a TGF -responsive sequence (Dennler et al., 1998).
  • CAGA1 5'- CTAGAGCCAGACAAAAAGCCAGACATTTAGCCAGACAAAAAGCCAGACATTTAGCCAGA
  • CAAAAAGCCAGACA-3' (SEQ ID No.148) and the complementary oligonucleotide CAGA2: ⁇ '-
  • oligonucleotides To construct (GC) 12 -MLP-Luc, 3 copies of the following annealed oligonucleotides were inseted into the Xhol site of MLP-Luc. These oligonucleotides contains 4 copies of 'the GC box', a BMP responsive sequence (Kusanagi et al., 2000).
  • GC1 5'- TCGAGCCGCCGCTTTGCCGCCGCTTTGCCGCCGCTTTGCCGCCGC-3' (SEQ ID NO: 1
  • GC2 5'- TCGAGCGGCGGCAAAGCGGCGGCAAAGCGGCGGCAAAGCGGCGGC-3' (SEQ ID No.151)
  • luciferase reporter 400 ng of luciferase reporter and 100 ng of pRL-TK (Promega), encoding the renilla luciferase and used as an internal transfection efficiency control, were transfected per well of a 24 wells-plate. Variable amounts of expression vectors were co-transfectd as indicated in the figures. When increasing amounts of expression vectors were transfected, total DNA was kept constant by the addition of pV3. 24 hours after the transfection, cells were washed and incubated in a medium without serum. 2 hours later, cells were stimulated with 10 ng/mL of human recombinant TGF ⁇ l (R&D) or 50 ng/mL of human recombinant BMP6 or BMP7 (R&D).
  • Luciferase activities were quantified using the Dual Luciferase reporter assay kit from Promega. Values were normalized with the renilla luciferase activity expressed from pRL-TK. 18-4 Baculovirus infection of mammalian cells
  • baculoviral particles were inserted into the baculoviral genome by transposition into E.
  • Quantitative PCR (Q-PCR) experiments To monitor the biological effects of the proteins of interest in the TGF ⁇ /activin or BMP signaling in cells, quantification of mRNA of genes transcriptionally regulated by TGF ⁇ /activin or BMP by Quantitative-PCR were carried-out using an Applied Biosytems 7000 SDS machine. This quantification follows a transfection of an expression vector of the prey of interest, the transfection of a siRNA or an infection using a genetically-modified baculovirus in mammalian cells such as HepG2, HeLa or HEK 293 cell lines seeded in 24 culture-plate.
  • RNA was extracted using the Rneasy Minikit and the Qia Shredder from Qiagen following the recommendations of the manufacturer. 1 ⁇ g of RNA is then used for a reverse transcription reaction to generate the cDNA which will serve as template in the following Q-PCR reaction.
  • the reverse transcription step was realized in 96 wells-plate with the TaqMan reverse transcription kit (Applied biosystems) following the recommendations of the manufacturer.
  • the cDNA of the gene of interest was then quantified in 96 wells-plate by the SyBR green methodology using the SyBR Green PCR master Mix kit (Applied
  • the forward and reverse oligonucleotides probing the gene of interest were designed using the Primer Express software (Applied Biosystems).These oligonucleotides were validated by
  • the human genes used to monitor the effect of TGF ⁇ are the Plasminogen Activator Inhibitor
  • Type 1 gene hPAI-1
  • JunB gene Type 1 gene and the JunB gene.
  • the human genes used to monitor the effect of BMPs are the JunB gene and the Alcaline Phosphatase gene (hALP).
  • the genes used as internal quantification controls are the Glyceraldehyde Phosphate Dehydrogenase gene
  • hGAPDH forward GGAAGATCGAGGTGAACGAGAGT
  • SEQ ID No.152 Reverse GTCCCAGATGAAGGCGTCTTT
  • hJunB forward ACTCATACACAGCTACGGGATACG(SEQ ID No.154) Reverse GGGTCGGCCAGGTTGAC(SEQ ID No.155)
  • hALP forward CGAGCTGAACAGGAACAACGT(SEQ ID No.156) Reverse CTGCTTGGCTTTTCCTTCATG(SEQ ID No.157)
  • hGAPDH forward GGAGTCAACGGATTTGGTCGTA(SEQ ID No.158)
  • sequences of the oligonucleotides probing the cDNA of the gene targeted by siRNA and used to validate the effect of the siRNA (see siRNA section) or the over-production level following baculovirus infection were:
  • ZNF8 forward CCAGTCAGGCCATTCCAATT(SEQ ID No.162)
  • T ⁇ R1 forward GTGACTACAACATATTGCTGCAATCAG(SEQ ID No.164) Reverse AGCACACTGGTCCAGCAATG(SEQ ID No.165)
  • LAPTm ⁇ forward TGGCCATCTACCATGTGATCA(SEQ ID No.176) Reverse CGATCCTGAGGTAGCCCATCT(SEQ ID No.177)
  • HIPK3 forward TTGTTCAACATATCTACAATCTCGGTACT(SEQ ID No.178)
  • siRNA Chemically synthesized siRNA using RNA phosphoramidites were purchased from Genset Oligos /Proligos (Paris, France). siRNA were ordered deprotected, desalted and duplexed.
  • siRNA duplexes used in these studies were all 19 ribonucteotides long and contained two thymidines nucleotides at their 3' termini. All siRNA duplexes were designed according to the rules edicted by Tuschl and coll. (Elbashir et al., 2001). In the following list, all sequences correspond to the sense DNA in the corresponding CDS
  • T ⁇ RI 5'-GTGTTTCTGCCACCTCTGT-3'(SEQ ID No.182) • mT ⁇ RI: ⁇ '-GTGTGTCTGCAACCTCTGT-3'(SEQ ID No.183)
  • KIAA1196 ⁇ '-CGACTGGAAGGATGAGTTC-3'(SEQ ID No.18 ⁇ )
  • HIPK3 5'-GCAGTTGTGTATTCCAGGA-3'(SEQ ID No.186)
  • ZNF8 5'-GCCTGAAGTCATCTCCCAG-3'(SEQ ID No.187) • PTPN12: 5'-GATATATCCCACAGCCACT-3'(SEQ ID No.188)
  • FLJ20037 5'-CAAGATCATTGCCACCAGG-3'(SEQ ID No.190)
  • HYPA 5'-ATCAATGTGGACTGAACAT-3'(SEQ ID No.191 )
  • Reporter and carrier plasmids were amplified in DH ⁇ (Stratagene) and purified using the Qiagen Endofree Maxi plasmid Kit.
  • Oligofectamine (Life Technologies, Invitrogen) and 4 to 40 nM siRNA duplex per well in a 24 well plate.
  • Anti-SARA rabbit polyclonal antibody was purchased from Santa-Cruz (cat # H-300 sc9135) and used at a 1/150 dilution.
  • Peroxidase-conjugated AffiniPure F(ab')2 fragment donkey anti-Rabbit IgG H+L was used as a secondary reagent (1/10000 dilution) and was purchased from Jackson
  • Cell were harvested in lysis buffer (2%SDS, 1X PBS), denatured 5 minutes at 95°C and quantified using Bradford reagent (BIORAD) according to the manufacturer's specifications.
  • Cell lysates (20 ⁇ g/lane) were resolved on a 4-12% NuPAGE gradient gel (Novex, Invitrogen), transfered to 20 ⁇ m nitrocellulose membrane (Schleicher & Schuell) and blocked in 10% fat-free dried milk in 1X PBS, 0,05% Tween20. Revelation was performed using ECL (Amersham Biosciences) chemoluminescent substrat according to the manufacturer's specifications.
  • Azuma T Takei M, Yoshikawa T, Nagasugi Y, Kato M, Otsuka M, Shiraiwa H, Sugano S, itamura K, Sawada S, Masuho Y.Seki N. Identification of candidate genes for Sjogren's syndrome using MRUIpr mouse model of Sjogren's syndrome and cDNA microarray analysis. Immunol Lett. 2002 May 1;81(3):171-6.
  • Bennett D Alphey L. PP1 binds Sara and negatively regulates Dpp signaling in Drosophila melanogaster. Nat Genet. 2002 Aug;31(4):419-23.
  • Mouse transporter protein a membrane protein that regulates cellular multidrug resistance, is localized to lysosomes. Cancer Res. 1999 Oct 1;59(19):4890-7.
  • JunB is involved in the inhibition of myogenic differentiation by bone morphogenetic protein-2. J Biol Chem. 1998 Jan 2;273(1):537-43.
  • RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8.
  • a mammalian lysosomal membrane protein confers multidrug resistance upon expression in Saccharomyces cerevisiae.
  • Keeton MR Curriden SA
  • van Zonneveld AJ Loskutoff DJ.
  • Kenny DA Jurata LW, Saga Y, Gill GN.
  • C6orf37 a novel candidate human retinal disease gene on chromosome 6q14.
  • Huntingtin's WW domain partners in Huntington's disease post-mortem brain fulfill genetic criteria for direct involvement in Huntington's disease pathogenesis.
  • E3 a hematopoietic-specific transcript directly regulated by the retinoic acid receptor alpha.
  • the LIM domain protein LM04 interacts with the cofactor CtlP and the tumor suppressor BRCA1 and inhibits BRCA1 activity.
  • the LIM domain gene LM04 inhibits differentiation of mammary epithelial cells in vitro and is overexpressed in breast cancer.

Abstract

The present invention relates to protein-protein interactions involved in transforming growth factor β disorders and/or diseases. More specifically, the present invention relates to complexes of polypeptides or polynucleotides encoding the polypeptides, fragments of the polypeptides, antibodies to the complexes, Selected Interacting Domains (SID®) which are identified due to the protein-protein interactions, methods for screening drugs for agents which modulate the interaction of proteins and pharmaceutical compositions that are capable of modulating the protein-protein interactions.

Description

PROTEIN-PROTEIN INTERACTIONS INVOLVING TRANSFORMING GROWTH FACTOR β SIGNALING OR INVOLVING TRANSDUCTION SIGNALS OF TRANSFORMING FACTOR β FAMILY MEMBERS
The present application claims priority to US provisional applications No. 60/333,348 filed on November 26, 2001 , No. 60/384,537 filed on May 31 , 2002 and No. 60/422,471 filed on October 30, 2002.
BACKGROUND AND PRIOR ART
Most biological processes involve specific protein-protein interactions. Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition. Thus, protein-protein interactions are involved, for example, in the assembly of enzyme subunits, in antibody-antigen recognition, in the formation of biochemical complexes, in the correct folding of proteins, in the metabolism of proteins, in the transport of proteins, in the localization of proteins, in protein turnover, in first translation modifications, in the core structures of viruses and in signal transduction. General methodologies to identify interacting proteins or to study these interactions have been developed. Among these methods are the two-hybrid system originally developed by Fields and co-workers and described, for example, in U.S. Patent Nos. 5,283,173, 5,468,614 and 5,667,973, which are hereby incorporated by reference.
The earliest and simplest two-hybrid system, which acted as basis for development of other versions, is an in vivo assay between two specifically constructed proteins. The first protein, known in the art as the "bait protein" is a chimeric protein which binds to a site on DNA upstream of a reporter gene by means of a DNA-binding domain or BD. Commonly, the binding domain is the DNA-binding domain from either Gal4 or native E. coli LexA and the sites placed upstream of the reporter are Gal4 binding sites or LexA operators, respectively.
The second protein is also a chimeric protein known as the "prey" in the art. This second chimeric protein carries an activation domain or AD. This activation domain is typically derived from Gal4, from VP16 or from B42.
Besides the two-hybrid systems, other improved systems have been developed to detected protein-protein interactions. For example, a two-hybrid plus one system was developed that allows the use of two proteins as bait to screen available cDNA libraries to detect a third partner. This method permits the detection between proteins that are part of a larger protein complex such as the RNA polymerase II holoenzyme and the TFIIH or TFIID complexes. Therefore, this method, in general, permits the detection of ternary complex formation as well as inhibitors preventing the interaction between the two previously defined fused proteins.
Another advantage of the two-hybrid plus one system is that it allows or prevents the formation of the transcriptional activator since the third partner can be expressed from a conditional promoter such as the methionine-repressed Met25 promoter which is positively regulated in medium lacking methionine. The presence of the methionine-regulated promoter provides an excellent control to evaluate the activation or inhibition properties of the third partner due to its "on" and "off' switch for the formation of the transcriptional activator. The three-hybrid method is described, for example in Tirode ef al., The Journal of Biological Chemistry, 272, No. 37 pp. 22995-22999 (1997) incorporated herein by reference.
Besides the two and two-hybrid plus one systems, yet another variant is that described in Vidal et al, Proc. Natl. Sci. 93 pgs. 10315-10320 called the reverse two- and one-hybrid systems where a collection of molecules can be screened that inhibit a specific protein- protein or protein-DNA interactions, respectively. A summary of the available methodologies for detecting protein-protein interactions is described in Vidal and Legrain, Nucleic Acids Research Vol. 27, No. 4 pgs. 919-929 (1999) and Legrain and Selig, FEBS Letters 480 pgs. 32-36 (2000) which references are incorporated herein by reference.
However, the above conventionally used approaches and especially the commonly used two-hybrid methods have their drawbacks. For example, it is known in the art that, more often than not, false positives and false negatives exist in the screening method. In fact, a doctrine has been developed in this field for interpreting the results and in common practice an additional technique such as co-immunoprecipitation or gradient sedimentation of the putative interactors from the appropriate cell or tissue type are generally performed. The methods used for interpreting the results are described by Brent and Finley, Jr. in Ann. Rev. Genet, 31 pgs. 663-704 (1997). Thus, the data interpretation is very questionable using the conventional systems.
One method to overcome the difficulties encountered with the methods in the prior art is described in WO99/42612, incorporated herein by reference. This method is similar to the two-hybrid system described in the prior art in that it also uses bait and prey polypeptides. However, the difference with this method is that a step of mating at least one first haploid recombinant yeast cell containing the prey polypeptide to be assayed with a second haploid recombinant yeast cell containing the bait polynucleotide is performed. Of course the person skilled in the art would appreciate that either the first recombinant yeast cell or the second recombinant yeast cell also contains at least one detectable reporter gene that is activated by a polypeptide including a transcriptional activation domain. The method described in W099/42612 permits the screening of more prey polynucleotides with a given bait polynucleotide in a single step than in the prior art systems due to the cell to cell mating strategy between haploid yeast cells. Furthermore, this method is more thorough and reproducible, as well as sensitive. Thus, the presence of false negatives and/or false positives is extremely minimal as compared to the conventional prior art methods.
Transforming growth factor β (TGFβ) belongs to a super-family of cytokines, including TGFβl , TGFβ2, TGFβ3, activins and Bone Morphologenetic Proteins (hereinafter BMP), which are synthesized by many cell types and have a variety of cellular and biological effects, including control of proliferation, differentiation, migration, angiogenesis, immunity and regulation of the turnover of the extracellular matrix. A number of disease states are known to be associated with variations in expression of genes which are controlled by TGFβ and related ,cytokines, including fibrotic disorders, abnormal wound healing, abnormal bone formation, cancer and tumor development, neurologic disorders, haematopoiesis and immune and inflammatory disorders.
Signaling by this family of cytokines is transduced by heteromeric complexes of transmembrane Ser/Thr kinase receptors. Upon ligand binding, type II receptor phosphorylates and activates type I receptor which then propagates signals to downstream targets, in particular the Smad proteins. Ten mammalian Smad proteins have been identified and divided into three classes.
The first includes pathway-restricted proteins such as Smadl , Smad5 and Smadδ which are specifically involved in BMP signaling and Smad2 and Smad3 which are restricted to TGFβ/activin pathway. The second class contains the common-mediator Smad4 implicated in both BMP and TGFβ/activin pathways. The third class contains the inhibitory Smads, Smad6 and Smad7. At least Smad2 and Smad3 are retained in the cytoplasm by binding to the SARA protein. After phosphorylation by TGFβ-activated type I receptor on their carboxy- terminal SSXS sequence, pathway-restricted Smads form heteromeric complexes with Smad4 and then translocate to the nucleus where they control expression of diverse genes involved in various biological processes such as control of cellular proliferation and differentiation, regulation of the immune system and regulation of the extracellular matrix formation.
Several proteins such as TGIF, Ski, SnoN, SNIP1 and CBP have been identified as Smad transcriptional co-regulators and shown to modulate the transcriptional ability of Smad proteins by direct interactions. Finally, proteins such Smurfl and Smurf2 are involved in degradation of Smad proteins by the proteasome machinery.
Most biological processes involve specific protein-protein interactions. Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition. Thus, protein-protein interactions are involved, for example, in the assembly of enzyme subunits, in antibody-antigen recognition, in the formation of biochemical complexes, in the correct folding of proteins, in the metabolism of proteins, in the transport of proteins, in the localization of proteins, in protein turnover, in first translation modifications, in the core structures of viruses and in signal transduction.
Several members of the TGFβ/BMP pathways (SARA, Smurfl , Smurf2, Smadl , Smad2/hMAD2, Smad3/hMAD-3, Smad4, Smad5/MADH5, Smad7, Smad9/MADH6, SNIP1 , SnoN) have been used as baits in yeast-two hybrid screening experiments. Several proteins have been identified as interactors with thoses baits (Figure 10). It was showed here functional data in mammalian cells that validate that those interactants are proteins involved in TGFβ/BMP signaling.
Thus, there is the still a need to explore all mechanisms relating to transforming growth factor β protein and to identify drug targets for fibrotic disorders, abnormal wound healing, abnormal bone formation, cancer and tumor development, neurologic disorders, haematopoiesis and immune and inflammatory disorders and/or diseases.
SUMMARY OF THE PRESENT INVENTION
Thus, it is an aspect of the present invention to identify protein-protein interactions involving proteins of the transforming growth factor β super-family of cytokines transduction pathway and to identify drug targets for fibrotic disorders, abnormal wound healing, abnormal bone formation, cancer and tumor development, neurologic disorders, haematopoiesis and immune and inflammatory disorders and/or disease.
It is another aspect of the present invention to identify protein-protein interactions involved in transforming growth factor β-mediated disorders and/or diseases for the development of more effective and better targeted therapeutic treatments.
It is yet another aspect of the present invention to identify complexes of polypeptides or polynucleotides encoding the polypeptides and fragments of the polypeptides of the transforming growth factor β super-family of cytokines transduction pathway.
It is yet another aspect of the present invention to identify antibodies to these complexes of polypeptides or polynucleotides encoding the polypeptides and fragments of the polypeptides involving transforming growth factor β signaling including polyclonal, as well as monoclonal antibodies that are used for detection.
It is still another aspect of the present invention to identify selected interacting domains of the polypeptides, called SID® polypeptides. It is still another aspect of the present invention to identify selected interacting domains of the polynucleotides, called SID® polynucleotides. It is still another aspect of the present invention to provide a diagnostic kit to test for deficiencies in the transforming growth factor β super-family of cytokines transduction pathway.
It is another aspect of the present invention to identify interacting proteins in the transforming growth factor β super-family of cytokines transduction pathway that can be used in pharmaceutical compositions or for diagnostic purposes.
It is another aspect of the present invention to generate protein-protein interactions maps called PIM®s.
It is yet another aspect of the present invention to provide a method for screening drugs for agents which modulate the interaction of proteins and pharmaceutical compositions that are capable of modulating the protein-protein interactions involved in transforming growth factor β disorders and/or diseases.
It is another aspect to administer the nucleic acids of the present invention via gene therapy. It is yet another aspect of the present invention to provide protein chips or protein microarrays.
It is yet another aspect of the present invention to provide a report in, for example paper, electronic and/or digital forms, concerning the protein-protein interactions, the modulating compounds and the like as well as a PIM®. These and other aspects are achieved by the present invention as evidenced by the summary of the invention, description of the preferred embodiments and the claims.
Thus the present invention relates to a complex of interacting proteins of columns 1 and 4 of Table 2.
Furthermore, the present invention provides SID® polynucleotides and SID® polypeptides of Table 3, as well as a PIM® involved in transforming growth factor β-mediated disorders and/or diseases.
The present invention also provides antibodies to the protein-protein complexes involved in transforming growth factor β-mediated disorders and/or diseases.
In another embodiment the present invention provides a method for screening drugs for agents that modulate the protein-protein interactions and pharmaceutical compositions that are capable of modulating protein-protein interactions.
In another embodiment the present invention provides protein chips or protein microarrays.
In yet another embodiment the present invention provides a report in, for example, paper, electronic and/or digital forms.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic representation of the pB6 plasmid. Fig. 2 is a schematic representation of the pB20 plasmid.
Fig. 3 is a schematic representation of the pP6 plasmid.
Fig. 4 is a schematic representation of vectors expressing the T25 fragment.
Fig. 5 is a schematic representation of vectors expressing the T18 fragment. Fig. 6 is a schematic representation of various vectors of pCmAHLI , pT25 and pT18.
Fig. 7 is a schematic representation identifying the SID®'s of proteins of the present invention. In this figure the "Full-length prey protein" is the Open Reading Frame (ORF) or coding sequence (CDS) where the identified prey polypeptides are included. The Selected
Interaction Domain (SID®) is determined by the commonly shared polypeptide domain of every selected prey fragment.
Fig. 8 is a protein map (PIM®).
Fig. 9 is a schematic representation of the pB27 plasmid.
Fig. 10 is a schematic representation of the pB28 plasmid.
Fig. 11 is a schematic representation of a protein interaction map around the newly functionally characterized proteins described in the present invention. These 10 proteins are highlighted by the symbol "*". The Predicted Biological Score (PBS) is represented by a code on each line and classified from A to E (Rain et al., 2001 ). PP1ca is also named PPP1CA. MADH5 and MADH6 correspond to Smadδ and Smad9, respectively. hMAD-2 and h-MAD-3 correspond to Smad2 and Smad3, respectively. MAN1 is the orthologous of SANE, a protein recently identified as involved in the BMP pathway (Raju et al., 2002)
Fig. 12 is a schematic representation of a protein interaction map between ZNF8 and Smad proteins. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, 2NF8 was shown to interact with Smadl (A), Smad4 (B), Smad5 (C) and Smad9 (D). Amino-acid position are indicated. Fig. 13 A, B and C are graphs showing that ZNF8 siRNA represses TGFβ- and BMP- dependent luciferase reporter activities. HepG2 cells were transiently transfected in 24 well- plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC)12-MLP-Luc (A & B) or the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc (C). All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGFβ pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co- transfection of ZNF8-targeting siRNA duplex was tested in cells treated or not with 50ng/ml BMP7 (A), 50ng/ml BMP6 (B) or 5 ng/ml TGFβl (C) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
Fig. 14A, B and C are graphs showing that ZNF8 siRNA specifically represses BMP- dependent markers. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or ZNF8-targeting siRNA duplex. Cells were treated or not with 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed either at 0.5nM and 2.5nM (A & B) or at 4 and 40nM (C) of duplex. Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the BMP pathway markers junB (A) and alkaline phosphatase (B& C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using either GAPDH (A & B) or hGUS (C). Fig. 15 A and B are graphs showing that ZNF8 siRNA does not repress BMP- independent markers. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or ZNF8- targeting siRNA duplex. Cells were treated or not with 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed either at 0.5nM and 2.5nM (A) or at 4 and 40nM (B) of duplex. Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGFβ pathway marker PAI-1 (PAI-1 hereinafter Plasminogen Activator inhibitor I) (A) and an unrelated marker, hGUS (B). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using either GAPDH (A) or relative levels (B).
Fig. 16 is a schematic representation of an Interaction between LAPTmδ and Smurf2. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, interaction between Smurf2 and LAPTmδ was found in both directions. Smurf2 was shown to interact with the C-terminal domain of LAPTmδ.
Fig. 17 A and B are graphs showing that LAPTmδ specifically inhibits the TGFβ pathway. The effect of LAPTmδ over-expression was studied using the following Luciferase reporter vectors: a TGFβ responsive element (TGF-RE = p(GTCT)8-MLP-Luc), a BMP- responsive element (BMP-RE = p(GC)12-MLP-Luc) and an unrelated reporter (pGL3 control) (see Materials & Methods). The effect was studied in the presence or absence of TGFβ (10 ng/ml) or BMP7 (50 ng/ml), as described. This study was performed with 0, 2 or 10 ng of pV3-LAPTm5 in HepG2 cells (A) or with 0, 0.5, 2, 10 or 50 ng of pV3-LAPTm5 in HEK293 cells (B). The specific Luciferase activity was normalized using the pRL-TK vector. Experiments were performed in triplicate.
Fig. 18 A and B are graphs showing that LAPTmδ expression is up-regulated by TGFβ The endogenous level of LAPTmδ mRNA was determined in several cell lines by Q-PCR experiments using the LAPTmδ probe (see Materials & Methods). Ct levels of LAPTmδ mRNA is given for each cell lines (A). The endogenous level of mRNA was determined in HepG2 cells in the presence or absence of TGFβ (10 ng/ml) with or without a TβRI-targeting siRNA duplex (B) (TβRI hereinafter Transforming Growth Factor β Receptor I. Fig. 19 A and B are graphs showing that LAPTmδ siRNA up-regulates BMP and TGFβ- dependent reporter activities. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the TGFβ reponsive luciferase reporter, p(GTCT)B-MLP-Luc (A) or the BMP responsive luciferase reporter, p(GC)ι2-MLP-Luc (B). All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGFβ pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co-transfection of LAPTmδ-targeting siRNA duplex was tested in cells treated or not with δng/ml recombinant human TGFβ (A), δOng/ml recombinant human BMP7 (B) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
Fig. 20 A, B, C and D are graphs showing that LAPTmδ siRNA up-regulates BMP and TGFβ-dependent markers. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or LAPTmδ-targeting siRNA duplex. Cells were treated or not with δ ng/ml of recombinant human TGFβl or 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed at 40nM of duplex (A, B, C & D). Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGFβ pathway markers PAI-1 and junB (A & B, respectively) and a BMP pathway marker, alkaline phosphatase (C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A, B & C). Relative levels of hGUS in the same experiment are also shown (D). Fig. 21 is a schematic representation of an Interaction between RNF11 Smurfl, Smurf2 and SARA.The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, RNF11 was shown to interact with Smurfl (A), Smurf2 (B), and SARA (C). Amino-acid positions are indicated. Fig. 22 is a gel showing that RNF11 is involved in regulating SARA protein levels.
Transfection experiments with pV3-SARA (200 ng) and/or pV3-RNF11 (300 ng) in the presence or absence of TGFβ (10 ng/ml) were performed. After TGFβ induction for 18H, cells' lysates were resolved on a 4-12% NuPAGE gradient gel, transfered and revealed using anti-SARA antibody (see Materials & Methods). Fig. 23 is a schematic diagram showing the Interaction between KIAA1196 and Smadl .
The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, KIAA1196 was shown to interact with Smadl .
Fig. 24 A and B are graphs showing that KIAA1196 siRNA specifically represses TGFβ-dependent markers in HepG2 cells.HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc (A) or the BMP reponsive luciferase reporter, p(GC)ι2-MLP-Luc (B). All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGFβ pathway. A mutated version of the T Rl-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co- transfection of KIAA1196-targeting siRNA duplex was tested in cells treated or not with 5ng/ml recombinant human TGFβ (A) and 50ng/ml recombinant human BMP6 (B) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
Fig. 25 is a graph showing that KIAA1196 siRNA specifically represses TGFβ- dependent reporter activity in HEK293 cells. HEK 293 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc. All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGFβ pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 30nM. Co-transfection of K1AA1196-targeting siRNA duplex was tested in cells treated or not with 5ng/ml recombinant human TGFβ for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
Fig. 26 A, B, C and D are graphs showing that KIAA1196 siRNA specifically represses TGFβ-dependent markers. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or KIAA1196-targeting siRNA duplex. Cells were treated or not with 5 ng/ml of recombinant human TGFβl or 50ng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed at 40nM of duplex (A, B, C & D). Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGFβ pathway markers PAI-1 and junB (A & B, respectively) and a BMP pathway marker, alkaline phosphatase (C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A, B & C). Relative levels of hGUS in the same experiment are also shown (D).
Fig. 27 is a schematic representation showing the Interaction between LM04 and Smad9. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, LM04 was shown to interact with Smad9. Fig. 28 A, B and C are graphs showing that LM04 siRNA specifically repress a BMP- dependent luciferase reporter. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC)12- MLP-Luc (A) or the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc (B). All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGF pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co-transfection of LM04-targeting siRNA duplex was tested in cells treated or not with 50ng/ml recombinant human BMP7 or BMP6 (A & B, respectively) and 5ng/ml recombinant human TGFβ (C) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are. representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
Fig. 29 A and B are graphs showing that LM04 siRNA specifically represses BMP- induced markers in BMP7-treated HepG2 cells. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or LM04-targeting siRNA duplex. Cells were treated or not with 50ng/ml of recombinant human BMP7 for 18 hours in ceils pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed at O.δ or 2.δnM of duplex (A) and 4 or 40nM of duplex (B). Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the BMP pathway marker alkaline phosphatase (A & B). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A, B).
Fig. 30 A, B and C are graphs showing that LM04 siRNA does not repress BMP- independent markers in BMP7-treated HepG2 cells.HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3- targeting siRNA) or LM04-targeting siRNA duplex. Cells were treated or not with δOng/ml of recombinant human BMP7 for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed at 4 or40nM of duplex (A, B) and 0.5 or 2.5nMof duplex (C). Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGFβ and BMP pathways marker junB (A) and a TGFβ pathway marker, PAI-1 (C). Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using hGUS (A) or using GAPDH (C). Relative levels of hGUS in the same experiment are also shown (B).
Fig. 31 is a schematic diagram showing the interaction between PPIca and SARA. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, PPIca was shown to interact with SARA. Fig. 32 A and B are graphs showing that PPIca stimulates the TGFβ pathway. The effect of PPIca over-expression was studied using the following luciferase reporter vectors: a TGFβ responsive element (TGF-RE = p(GTCT)8-MLP-Luc), a BMP-responsive element (BMP-RE = p(GC)12-MLP-Luc) and an unrelated reporter (pGL3 control) (see Materials & Methods). The effect was studied in the presence or absence of TGFβ (10 ng/ml) or BMP7 (60 ng/ml), as described. This study was performed with 0, 10, 60 or 200 ng of pV3- PPIca in HepG2 cells (A) or in HEK293 cells (B). The specific Luciferase activity was normalized using the pRL-TK vector. Experiments were performed in triplicate.
Fig. 33 A, B and C are graphs showing that PPIca stimulates PAI-1 mRNA expression. Baculoviruses containing the Smad3 or PPIca genes under the control of the CMV promoter were generated and used to infect HepG2 cells (see Materials & Methods). The over- expression level was checked and quantified by Q-PCR (A). The endogenous PAI-1 mRNA levels were measured by Q-PCR 24 hours post infection with Smad3 or PP1ca-containing baculoviruses in the presence or absence of TGFβ (10 ng/ml). The value 1 is attributed to the mRNA amount of PAI-1 in the absence of TGFβ and in the absence of infection (B).
Fig. 34 is a schematic diagram showing the Interaction between HYPA and Smad4. The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, HYPA was shown to interact with Smad4. Fig. 3δ A, B and C are graphs showing that HYPA siRNA specifically represses BMP- dependent reporter activity. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC)12- MLP-Luc (A & B) or the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc (C). All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGFβ pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co-transfection of HYPA-targeting siRNA duplex was tested in cells treated or not with δOng/ml recombinant human BMP7 or BMP6 (A & B, respectively) and δng/ml recombinant human TGFβ (C) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases. Fig. 36 is a graph showing that HYPA siRNA represses BMP-dependent markers.
HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or HYPA-targeting siRNA duplex. Cells were treated or not with δOng/ml of recombinant human BMP7 for 18 hours in cells pre- starved for 2 hours in serum-free culture medium. SiRNA transfections were performed at 0.5 or 2.5nM of duplex. Cells were harvested and lysed 48 hours after transfection. Total RNA were extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the BMP pathway marker alkaline phosphatase. Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using GAPDH. Fig. 37 is a schematic diagram showing the Interaction between FLJ20037 and SARA.
The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, FLJ20037 was shown to interact with SARA.
Fig. 38 A, B and C are graphs showing that FLJ20037 stimulates PAI-1 mRNA expression. Baculoviruses containing the Smad3 or FLJ20037 genes under the control of the CMV promoter were generated and used to infect HepG2 cells (see Materials & Methods). The over-expression level was checked and quantified by Q-PCR (A). The endogenous PAI- 1 mRNA levels were measured by Q-PCR 24 hours post
Figure imgf000014_0001
containing baculoviruses in the presence or absence of TGFβ (10 ng/mL). The value 1 is attributed to the mRNA amount of PAI-1 in the absence of TGFβ and in the absence of infection (B). Fig. 39 is a graph showing that FLJ20037 siRNA down-regulates TGFβ-dependent markers.HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with a control siRNA (pGL3-targeting siRNA) or FLJ20037-targeting siRNA duplex. Cells were treated or not with δng/ml of recombinant human TGFβ for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. SiRNA transfections were performed at O.δ or 2.δnM of duplex. Cells were harvested and lysed 48 hours after transfection. Total RNA was extracted as described under Materials & Methods and quantitative PCR analysis were performed in order to quantitate the endogenous levels of the TGFβ pathway marker PAI-1. Data are representative of two or three independant duplicated experiments and are presented as normalized RNA levels using GAPDH. Fig. 40 is a schematic diagram showing the Interaction between PTPN12 and Smadδ.
The full-length proteins are represented in grey and black boxes correspond to the interaction domains. Using two-hybrid screening, PTPN12 was shown to interact with Smadδ. Amino-acid positions are indicated.
Fig. 41 A and B are graphs showing that PTPN12 siRNA up-regulates BMP and TGFβ- dependent reporter activities. HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC)12- MLP-Luc (A) or the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc (B). All experiments included pRL-TK as an internal transfection control. A TβRI-targeting siRNA duplex was used as a positive control for disruption of the TGF pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co-transfection of PTPN12-targeting siRNA duplex was tested in cells treated or not with 50ng/ml recombinant human BMP6 (A) and 5ng/ml recombinant human TGFβ (B) for 18 hours in cells pre-starved for 2 hours in serum-free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
Fig. 42 A and B are schematic diagrams showing the Interaction between HIPK3, SnoN and SNIP1. The full-length proteins are represented in grey and black boxes correspohd to the interaction domains. Using two-hybrid screening, HIPK3 was shown to interact with the N-terminal domains of SNIP1 (A) and SnoN (B). Amino-acid positions are indicated. Fig. 43 A and B are graphs showing that HIPK3 siRNA specifically up-regulates BMP- dependent reporter activities.
HepG2 cells were transiently transfected in 24 well-plates as described under Materials & Methods with the BMP reponsive luciferase reporter, p(GC)ι2-MLP-Luc (A) or the TGFβ responsive luciferase reporter, p(GTCT)8-MLP-Luc (B). All experiments included pRL-TK as an internal transfection control. A T Rl-targeting siRNA duplex was used as a positive control for disruption of the TGF pathway. A mutated version of the TβRI-targeting siRNA duplex (2 mismatches versus consensus sequence) was used as a negative control. SiRNA transfections were performed at 4 and 40nM. Co-transfection of HIPK3-targeting siRNA duplex was tested in cells treated or not with δOng/ml recombinant human BMP6 (A) and δng/ml recombinant human TGFβ (B) for 18 hours in cells pre-starved for 2 hours in serum- free culture medium. Cells were harvested 48 hours after transfection and 10μl of lysates were used for the Dual Luciferase Assay. Data are representative of two or three independant duplicated experiments and are presented as a ratio between firefly and renilla luciferases.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As used herein the terms "polynucleotides", "nucleic acids" and "oligonucleotides" are used interchangeably and include, but are not limited to RNA, DNA, RNA/DNA sequences of more than one nucleotide in either single chain or duplex form. The polynucleotide sequences of the present invention may be prepared from any known method including, but not limited to, any synthetic method, any recombinant method, any ex vivo generation method and the like, as well as combinations thereof.
The term "polypeptide" means herein a polymer of amino acids having no specific length. Thus, peptides, oligopeptides and proteins are included in the definition of "polypeptide" and these terms are used interchangeably throughout the specification, as well as in the claims. The term "polypeptide" does not exclude post-translational modifications such as polypeptides having covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like. Also encompassed by this definition of "polypeptide" are homologs thereof. By the term "homologs" is meant structurally similar genes contained within a given species, orthologs are functionally equivalent genes from a given species or strain, as determined for example, in a standard complementation assay. Thus, a polypeptide of interest can be used not only as a model for identifying similiar genes in given strains, but also to identify homologs and orthologs of the polypeptide of interest in other species. The orthologs, for example, can also be identified in a conventional complementation assay. In addition or alternatively, such orthologs can be expected to exist in bacteria (or other kind of cells) in the same branch of the phylogenic tree, as set forth, for example, at frτι://ftp.cme.msu.edu/pub/rdp/SSIJ-rR A/SSU Prok.Ωhylo.
As used herein the term "prey polynucleotide" means a chimeric polynucleotide encoding a polypeptide comprising (i) a specific domain; and (ii) a polypeptide that is to be tested for interaction with a bait polypeptide. The specific domain is preferably a transcriptional activating domain.
As used herein, a "bait polynucleotide" is a chimeric polynucleotide encoding a chimeric polypeptide comprising (i) a complementary domain; and (ii) a polypeptide that is to be tested for interaction with at least one prey polypeptide. The complementary domain is preferably a DNA-binding domain that recognizes a binding site that is further detected and is contained in the host organism.
As used herein "complementary domain" is meant a functional constitution of the activity when bait and prey are interacting; for example, enzymatic activity.
As used herein "specific domain" is meant a functional interacting activation domain that may work through different mechanisms by interacting directly or indirectly through intermediary proteins with RNA polymerase II or Ill-associated proteins in the vicinity of the transcription start site.
As used herein the term "complementary" means that, for example, each base of a first polynucleotide is paired with the complementary base of a second polynucleotide whose orientation is reversed. The complementary bases are A and T (or A and U) or C and G.
The term "sequence identity" refers to the identity between two peptides or between two nucleic acids. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of sequence identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acid sequences that are shared between these sequences. Since two polypeptides may each (i) comprise a sequence (i.e., a portion of a complete polynucleotide sequence) that is similar between two polynucleotides, and (ii) may further comprise a sequence that is divergent between two polynucleotides, sequence identity comparisons between two or more polynucleotides over a "comparison window" refers to the conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference nucleotide sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
To determine the percent identity of two amino acids sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino acid sequence or a first nucleic acid sequence for optimal alignment with the second amino acid sequence or second nucleic acid sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Hence % identity = number of identical positions / total number of overlapping positions X 100.
In this comparison the sequences can be the same length or may be different in length. Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 91 (2) pgs. 370-380 (1981 ), by the homology alignment algorithm of Needleman and Wunsch, J. Miol. Biol., 48(3) pgs. 443-463 (1972), by the search for similarity via the method of Pearson and Lipman, PNAS, USA, 85(δ) pgs. 2444-2448 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetic Computer Group, 675, Science Drive, Madison, Wisconsin) or by inspection.
The best alignment (i.e., resulting in the highest percentage of identity over the comparison window) generated by the various methods is selected. The term "sequence identity" means that two polynucleotide sequences are identical
(i.e., on a nucleotide by nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) and multiplying the result by 100 to yield the percentage of sequence identity. The same process can be applied to polypeptide sequences.
The percentage of sequence identity of a nucleic acid sequence or an amino acid sequence can also be calculated using BLAST software (Version 2.06 of September 1998) with the default or user defined parameter. The term "sequence similarity" means that amino acids can be modified while retaining the same function. It is known that amino acids are classified according to the nature of their side groups and some amino acids such as the basic amino acids can be interchanged for one another while their basic function is maintained. The term "isolated" as used herein means that a biological material such as a nucleic acid or protein has been removed from its original environment in which it is naturally present. For example, a polynucleotide present in a plant, mammal or animal is present in its natural state and is not considered to be isolated. The same polynucleotide separated from the adjacent nucleic acid sequences in which it is naturally inserted in the genome of the plant or animal is considered as being "isolated."
The term "isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with the biological activity and which may be present, for example, due to incomplete purification, addition of stabilizers or mixtures with pharmaceutically acceptable excipients and the like. "Isolated polypeptide" or "isolated protein" as used herein means a polypeptide or protein which is substantially free of those compounds that are normally associated with the polypeptide or protein in a naturally state such as other proteins or polypeptides, nucleic acids, carbohydrates, lipids and the like.
The term "purified" as used herein means at least one order of magnitude of purification is achieved, preferably two or three orders of magnitude, most preferably four or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term "purified" as utilized herein does not mean that the material is 100% purified and thus excludes any other material.
The term "variants" when referring to, for example, polynucleotides encoding a polypeptide variant of a given reference polypeptide are polynucleotides that differ from the reference polypeptide but generally maintain their functional characteristics of the reference polypeptide. A variant of a polynucleotide may be a naturally occurring alleiic variant or it may be a variant that is known naturally not to occur. Such non-naturally occurring variants of the reference polynucleotide can be made by, for example, mutagenesis techniques, including those mutagenesis techniques that are applied to polynucleotides, cells or organisms.
Generally, differences are limited so that the nucleotide sequences of the reference and variant are closely similar overall and, in many regions identical.
Variants of polynucleotides according to the present invention include, but are not limited to, nucleotide sequences which are at least 95% identical after alignment to the reference polynucleotide encoding the reference polypeptide. These variants can also have 96%, 97%, 98% and 99.999% sequence identity to the reference polynucleotide. Nucleotide changes present in a variant polynucleotide may be silent, which means that these changes do not alter the amino acid sequences encoded by the reference polynucleotide.
Substitutions, additions and/or deletions can involve one or more nucleic acids. Alterations can produce conservative or non-conservative amino acid substitutions, deletions and/or additions.
Variants of a prey or a SID® polypeptide encoded by a variant polynucleotide can possess a higher affinity of binding and/or a higher specificity of binding to its protein or polypeptide counterpart, against which it has been initially selected. In another context, variants can also loose their ability to bind to their protein or polypeptide counterpart.
By "fragment of a polynucleotide" or "fragment of a SID® polynucleotide" is meant that fragments of these sequences have at least 12 consecutive nucleotides, or between 12 and 5,000 consecutive nucleotides, or between 12 and 10,000 consecutive nucleotides, or between 12 and 20,000 consecutive nucleotides. By "fragment of a polypeptide" or "fragment of a SID® polypeptide" is meant that fragments of these sequences have at least 4 consecutive amino acids, or between 4 and 1 ,700 consecutive amino acids, or between 4 and 3,300 consecutive amino acids, or between 4 and 6,600 consecutive amino acids.
By "anabolic pathway" is meant a reaction or series of reactions in a metabolic pathway that synthesize complex molecules from simpler ones, usually requiring the input of energy. An anabolic pathway is the opposite of a catabolic pathway.
As used herein, a "catabolic pathway" is a series of reactions in a metabolic pathway that break down complex compounds into simpler ones, usually releasing energy in the process. A catabolic pathway is the opposite of an anabolic pathway. As used herein, "drug metabolism" is meant the study of how drugs are processed and broken down by the body. Drug metabolism can involve the study of enzymes that break down drugs, the study of how different drugs interact within the body and how diet and other ingested compounds affect the way the body processes drugs.
As used herein, "metabolism" means the sum of all of the enzyme-catalyzed reactions in living cells that transform organic molecules.
By "secondary metabolism" is meant pathways producing specialized metabolic products that are not found in every cell.
As used herein, "SID®" means a Selected Interacting Domain and is identified as follows: for each bait polypeptide screened, selected prey polypeptides are compared. Overlapping fragments in the same ORF or CDS define the selected interacting domain. As used herein the term "PIM®" means a protein-protein interaction map. This map is obtained from data acquired from a number of separate screens using different bait polypeptides and is designed to map out all of the interactions between the polypeptides.
The term "affinity of binding", as used herein, can be defined as the affinity constant Ka when a given SID® polypeptide of the present invention which binds to a polypeptide and is the following mathematical relationship:
[SID®/polypeptide complex]
Ka =
[free SID®] [free polypeptide] wherein [free SID®], [free polypeptide] and [SID®/polypeptide complex] consist of the concentrations at equilibrium respectively of the free SID® polypeptide, of the free polypeptide onto which the SID® polypeptide binds and of the complex formed between SID® polypeptide and the polypeptide onto which said SID® polypeptide specifically binds. The affinity of a SID® polypeptide of the present invention or a variant thereof for its polypeptide counterpart can be assessed, for example, on a Biacore™ apparatus marketed by Amersham Pharmacia Biotech Company such as described by Szabo et al. (Curr Opin Struct Biol 5 pgs. 699-705 (1995)) and by Edwards and Leartherbarrow (Anal. Biochem 246 pgs. 1-6 (1997)).
As used herein the phrase "at least the same affinity" with respect to the binding affinity between a SID® polypeptide of the present invention to another polypeptide means that the Ka is identical or can be at least two-fold, at least three-fold or at least five fold greater than the Ka value of reference.
As used herein, the term "modulating compound" means a compound that inhibits or stimulates or can act on another protein which can inhibit or stimulate the protein-protein interaction of a complex of two polypeptides or the protein-protein interaction of two polypeptides.
More specifically, the present invention comprises complexes of polypeptides or polynucleotides encoding the polypeptides composed of a bait polypeptide, or a bait polynucleotide encoding a bait polypeptide and a prey polypeptide or a prey polynucleotide encoding a prey polypeptide. The prey polypeptide or prey polynucleotide encoding the prey polypeptide is capable of interacting with a bait polypeptide of interest in various hybrid systems.
As described in the background of the present invention, there are various methods known in the art to identify prey polypeptides that interact with bait polypeptides of interest. These methods include, but are not limited to, generic two-hybrid systems as described by
Fields et al. (Nature, 340:245-246 (1989)) and more specifically in U.S. Patent Nos.
5,283,173, 5,468,614 and 5,667,973, which are hereby incorporated by reference; the reverse two-hybrid system described by Vidal ef al. (supra); the two plus one hybrid method described, for example, in Tirode et al. (supra); the yeast forward and reverse 'n'-hybrid systems as described in Vidal and Legrain (supra); the method described in WO 99/42612; those methods described in Legrain et al. (FEBS Letters 480 pgs. 32-36 (2000)) and the like. The present invention is not limited to the type of method utilized to detect protein- protein interactions and therefore any method known in the art and variants thereof can be used. It is however better to use the method described in W099/42612 or WO00/66722, both references incorporated herein by reference due to the methods' sensitivity, reproducibility and reliability. Protein-protein interactions can also be detected using complementation assays such as those described by Pelietier et al. at http://www.abrf.org/JBT/ATticles/JBT0012/ibtO012.htmI. WO 00/07038 and WO98/34120.
Although the above methods are described for applications in the yeast system, the present invention is not limited to detecting protein-protein interactions using yeast, but also includes similar methods that can be used in detecting protein-protein interactions in, for example, mammalian systems as described, for example in Takacs et al. (Proc. Natl. Acad. Sci., USA, 90 (21 ): 10375-79 (1993)) and Vasavada et al. (Proc. Natl. Acad. Sci., USA, 88 (23): 10686-90 (1991)), as well as a bacterial two-hybrid system as described in Karimova et al. (1998), W099/28746, WO00/66722 and Legrain et al. (FEBS Letters, 480 pgs. 32-36 (2000)).
The above-described methods are limited to the use of yeast, mammalian cells and Escherichia coli cells, the present invention is not limited in this manner. Consequently, mammalian and typically human cells, as well as bacterial, yeast, fungus, insect, nematode and plant cells are encompassed by the present invention and may be transfected by the nucleic acid or recombinant vector as defined herein.
Examples of suitable cells include, but are not limited to, VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines such as ATCC No. CCL61 , COS cells such as COS-7 cells and ATCC No. CRL 1650 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361 , A549, PC12, K662 cells, 293 cells, Sf9 cells such as ATCC No. CRL1711 and Cv1 cells such as ATCC No. CCL70.
Other suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5-α), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus. Further suitable cells that can be used in the present invention include yeast cells such as those of Saccharomyces such as Saccharomyces cerevisiae. The bait polynucleotide, as well as the prey polynucleotide can be prepared according to the methods known in the art such as those described above in the publications and patents reciting the known method perse.
The bait and the prey polynucleotide of the present invention is obtained from transforming growth factor β cDNA, or variants of cDNA fragment from a library of transforming growth factor β, and fragments from the genome or transcriptome of transforming growth factor β cDNA ranging from about 12 to about 5,000, or about 12 to about 10,000 or from about 12 to about 20,000. The prey polynucleotide is then selected, sequenced and identified. A transforming growth factor β super-family of cytokines prey library is prepared from the transforming growth factor β cDNA and constructed in the specially designed prey vector pP6 as shown in Figure 3 after ligation of suitable linkers such that every cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcription activation domain of a reporter gene. Any transcription activation domain can be used in the present invention. Examples include, but are not limited to, Gal4,YP16, B42, His and the like. Toxic reporter genes, such as CATR, CYH2, CYH1 , URA3, bacterial and fungi toxins and the like can be used in reverse two-hybrid systems.
The polypeptides encoded by the nucleotide inserts of the transforming growth factor β prey library thus prepared are termed "prey polypeptides" in the context of the presently described selection method of the prey polynucleotides.
The bait polynucleotides can be inserted in bait plasmid pB27 or pB28 as illustrated in Figure 8 and Figure 9. The bait polynucleotide insert is fused to a polynucleotide encoding the binding domain of, for example, the Gal4 DNA binding domain and the shuttle expression vector is used to transform cells. The bait polynucleotides used in the present invention are described in Table 1.
As stated above, any cells can be utilized in transforming the bait and prey polynucleotides of the present invention including mammalian cells, bacterial cells, yeast cells, insect cells and the like.
In an embodiment, the present invention identifies protein-protein interactions in yeast. In using known methods a prey positive clone is identified containing a vector which comprises a nucleic acid insert encoding a prey polypeptide which binds to a bait polypeptide of interest. The method in which protein-protein interactions are identified comprises the following steps: i) mating at least one first haploid recombinant yeast cell clone from a recombinant yeast cell clone library that has been transformed with a plasmid containing the prey polynucleotide to be assayed with a second haploid recombinant yeast cell clone transformed with a plasmid containing a bait polynucleotide encoding for the bait polypeptide; ii) cultivating diploid cell clones obtained in step i) on a selective medium; and iii) selecting recombinant cell clones which grow on the selective medium. This method may further comprise the step of: iv) characterizing the prey polynucleotide contained in each recombinant cell clone which is selected in step iii).
In yet another embodiment of the present invention, in lieu of yeast, Escherichia coli is used in a bacterial two-hybrid system, which encompasses a similar principle to that described above for yeast, but does not involve mating for characterizing the prey polynucleotide. In yet another embodiment of the present invention, mammalian cells and a method similar to that described above for yeast for characterizing the prey polynucleotide are used. By performing the yeast, bacterial or mammalian two-hybrid system, it is possible to identify for one particular bait an interacting prey polypeptide. The prey polynucleotide that has been selected by testing the library of preys in a screen using the two-hybrid, two plus one hybrid methods and the like, encodes the polypeptide interacting with the protein of interest.
The present invention is also directed, in a general aspect, to a complex of polypeptides, polynucleotides encoding the polypeptides composed of a bait polypeptide or bait polynucleotide encoding the bait polypeptide and a prey polypeptide or prey polynucleotide encoding the prey polypeptide capable of interacting with the bait polypeptide of interest. These complexes are identified in Table 2.
In another aspect, the present invention relates to a complex of polynucleotides consisting of a first polynucleotide, or a fragment thereof, encoding a prey polypeptide that interacts with a bait polypeptide and a second polynucleotide or a fragment thereof. This fragment has at least 12 consecutive nucleotides, but can have between 12 and 5,000 consecutive nucleotides, or between 12 and 10,000 consecutive nucleotides or between 12 and 20,000 consecutive nucleotides.
The complexes of the two interacting polypeptides listed in Table 2 and the sets of two polynucleotides encoding these polypeptides also form part of the present invention. In yet another embodiment, the present invention relates to an isolated complex of at least two polypeptides encoded by two polynucleotides wherein said two polypeptides are associated in the complex by affinity binding and are depicted in columns 1 and 4 of Table 2.
In yet another embodiment, the present invention relates to an isolated complex comprising at least a polypeptide as described in column 1 of Table 2 and a polypeptide as described in column 4 of Table 2. The present invention is not limited to these polypeptide complexes alone but also includes the isolated complex of the two polypeptides in which fragments and/or homologous polypeptides exhibit at least 95% sequence identity, as well as from 96% sequence identity to 99.999% sequence identity.
Also encompassed in another embodiment of the present invention is an isolated complex in which the SID® of the prey polypeptides encoded by SEQ ID N°27 to 64 in Table 3 form the isolated complex.
Besides the isolated complexes described above, nucleic acids coding for a Selected Interacting Domain (SID®) polypeptide or a variant thereof or any of the nucleic acids set forth in Table 3 can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such transcription elements include a regulatory region and a promoter. Thus, the nucleic acid which may encode a marker compound of the present invention is operably linked to a promoter in the expression vector. The expression vector may also include a replication origin.
A wide variety of host/expression vector combinations are employed in expressing the nucleic acids of the present invention. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col El, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith et al (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM989, as well as other phage DNA such as M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2m plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or the expression control sequences; and the like.
For example in a baculovirus expression system, both non-fusion transfer vectors, such as, but not limited to pVL941 (BamHI cloning site Summers), pVL1393 (BamHI, Smal, Xba\,
EcoRI, Notl, XmaUl, Bg\\\ and Psfl cloning sites; Invitrogen), pVL1392 (Sg/lll, Psfl, Notl,
Xmalll, EcoRI, Xbaft, Sma\ and Sa HI cloning site; Summers and Invitrogen) and pBlueSaclll (BamHI, BglW, Pst\, Λ/col and Hindltt cloning site, with blue/white recombinant screening, Invitrogen), and fusion transfer vectors such as, but not limited to, pAc700 (BamHI and pnl cloning sites, in which the SamHI recognition site begins with the initiation codon; Summers), pAc701 and pAc70-2 (same as pAc700, with different reading frames), pAc360 (BamHI cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen (1995)) and pBlueBacHisA, B, C (three different reading frames with BamHI, BglW, Pst\, Λcol and Hind\l\ cloning site, an N-terminal peptide for ProBond purification and blue/white recombinant screening of plaques; Invitrogen (220) can be used. Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase promoters, any expression vector with a DHFR expression cassette or a DHFR/methotrexate co-amplification vector such as pED (Psfl, Sail, Sbal, Smal and EcoRI cloning sites, with the vector expressing both the cloned gene and DHFR; Kaufman, 1991). Alternatively a glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (Hindlll, Xball, Smal, Sbal, EcoRI and Bell cloning sites in which the vector expresses glutamine synthetase and the cloned gene; Celltech). A vector that directs episomal expression under the control of the Epstein Barr Virus (EBV) or nuclear antigen (EBNA) can be used such as pREP4 (BamHI, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull and Kpnl cloning sites, constitutive RSV- LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHI, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull and Kpnl cloning sites, constitutive hCMV immediate early gene promoter, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, Hindlll, Notl, Xhol, Sfil, BamHI cloning sites, inducible methallothionein Ha gene promoter, hygromycin selectable marker, Invitrogen), pREP8 (BamHI, Xnol, Notl, HindlM, Nhel and Kpnl cloning sites, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (Kpnl, Nhel, Hindlll, Notl, Xhol, Sfil, BamHI cloning sites, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). Selectable mammalian expression vectors for use in the invention include, but are not limited to, pRc/CMV (Hindlll, BstXl, Notl, Sbal and Apal cloning sites, G418 selection, Invitrogen), pRc/RSV (Hindll, Spel, BstXl, Notl, Xbal cloning sites, G418 selection, Invitrogen) and the like. Vaccinia virus mammalian expression vectors (see, for example Kaufman 1991 that can be used in the present invention include, but are not limited to, pSC11 (Smal cloning site, TK- and β-gal selection), pMJ601 (Sail, Smal, Afll, Naή, SspMII, BamHI, Apal, Nhel, Sac l, Kpnl and Hindlll cloning sites; TK- and β-gal selection), pTKgptFIS (EcoRI, Psfl, Sa/ll, Accl, Hindll, Sbal, BamHI and Hpa cloning sites, TK or XPRT selection) and the like.
Yeast expression systems that can also be used in the present include, but are not limited to, the non-fusion pYES2 vector (Xόal, Spήl, Shol, Notl, GstXl, EcoRI, BsfXI, BamHI, Sacl, Kpnl and Hindlll cloning sites, Invitrogen), the fusion pYESHisA, B, C (Xball, Sphl, Shol, Notl, BstXl, EcoRI, BamHI, Sacl, Kpnl and Hindlll cloning sites, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), pRS vectors and the like. Consequently, mammalian and typically human cells, as well as bacterial, yeast, fungi, insect, nematode and plant cells an used in the present invention and may be transfected by the nucleic acid or recombinant vector as defined herein.
Examples of suitable cells include, but are not limited to, VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines such as ATCC No. CCL61, COS cells such as COS-7 cells and ATCC No. CRL 1660 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361,
A549, PC12, K562 cells, 293 ceils, Sf9 cells such as ATCC No. CRL1711 and Cv1 cells such as ATCC No. CCL70.
Other suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5-α), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus.
Further suitable cells that can be used in the present invention include yeast cells such as those of Saccharomyces such as Saccharomyces cerevisiae. Besides the specific isolated complexes, as described above, the present invention relates to and also encompasses SID® polynucleotides. As explained above, for each bait polypeptide, several prey polypeptides may be identified by comparing and selecting the intersection of every isolated fragment that are included in the same polypeptide. Thus the SID® polynucleotides of the present invention are represented by the shared nucleic acid sequences of SEQ ID N° 27 to 64 encoding the SID® polypeptides of SEQ ID N° 6δ to 102 in columns δ and 7 of Table 3, respectively.
The present invention is not limited to the SID® sequences as described in the above paragraph, but also includes fragments of these sequences having at least 12 consecutive nucleic acids, between 12 and δ,000 consecutive nucleic acids and between 12 and 10,000 consecutive nucleic acids and between 12 and 20,000 consecutive nucleic acids, as well as variants thereof. The fragments or variants of the SID® sequences possess at least the same affinity of binding to its protein or polypeptide counterpart, against which it has been initially selected. Moreover this variant and/or fragments of the SID® sequences alternatively can have between 95% and 99.999% sequence identity to its protein or polypeptide counterpart.
According to the present invention variants of polynucleotide or polypeptides can be created by known mutagenesis techniques either in vitro or in vivo. Such a variant can be created such that it has altered binding characteristics with respect to the target protein and more specifically that the variant binds the target sequence with either higher or lower affinity. Polynucleotides that are complementary to the above sequences which include the polynucleotides of the SID®'s, their fragments, variants and those that have specific sequence identity are also included in the present invention.
The polynucleotide encoding the SID® polypeptide, fragment or variant thereof can also be inserted into recombinant vectors which are described in detail above.
The present invention also relates to a composition comprising the above-mentioned recombinant vectors containing the SID® polynucleotides in Table 3, fragments or variants thereof, as well as recombinant host cells transformed by the vectors. The recombinant host cells that can be used in the present invention were discussed in greater detail above. The compositions comprising the recombinant vectors can contain physiological acceptable carriers such as diluents, adjuvants, excipients and any vehicle in which this composition can be delivered therapeutically and can include, but is are not limited to sterile liquids such as water and oils.
In yet another embodiment, the present invention relates to a method of selecting modulating compounds, as well as the modulating molecules or compounds themselves which may be used in a pharmaceutical composition. These modulating compounds may act as a cofactor, as an inhibitor, as antibodies, as tags, as a competitive inhibitor, as an activator or alternatively have agonistic or antagonistic activity on the protein-protein interactions. The activity of the modulating compound does not necessarily, for example, have to be
100% activation or inhibition. Indeed, even partial activation or inhibition can be achieved that is of pharmaceutical interest.
The modulating compound can be selected according to a method which comprises:
(a) cultivating a recombinant host cell with a modulating compound on a selective medium and a reporter gene the expression of which is toxic for said recombinant host cell wherein said recombinant host cell is transformed with two vectors:
(i) wherein said first vector comprises a polynucleotide encoding a first hybrid polypeptide having a DNA binding domain; (ii)wherein said second vector comprises a polynucleotide encoding a second hybrid polypeptide having a transcriptional activating domain that activates said toxic reporter gene when the first and second hybrid polypeptides interact;
(b) selecting said modulating compound which inhibits or permits the growth of said recombinant host cell. Thus, the present invention relates to a modulating compound that inhibits the protein- protein interactions of a complex of two polypeptides of columns 1 and 4 of Table 2. The present invention also relates to a modulating compound that activates the protein-protein interactions of a complex of two polypeptides of columns 1 and 4 of Table 2.
In yet another embodiment, the present invention relates to a method of selecting a modulating compound, which modulating compound inhibits the interactions of two polypeptides of columns 1 and 4 of Table 2. This method comprises:
(a) cultivating a recombinant host cell with a modulating compound on a selective medium and a reporter gene the expression of which is toxic for said recombinant host cell wherein said recombinant host cell is transformed with two vectors:
(i) wherein said first vector comprises a polynucleotide encoding a first hybrid polypeptide having a first domain of an enzyme;
(ii) wherein said second vector comprises a polynucleotide encoding a second hybrid polypeptide having an enzymatic transcriptional activating domain that activates said toxic reporter gene when the first and second hybrid polypeptides interact; (b) selecting said modulating compound which inhibits or permits the growth of said recombinant host cell.
In the two methods described above any toxic reporter gene can be utilized including those reporter genes that can be used for negative selection including the URA3 gene, the CYH1 gene, the CYH2 gene and the like. In yet another embodiment, the present invention provides a kit for screening a modulating compound. This kit comprises a recombinant host cell which comprises a reporter gene the expression of which is toxic for the recombinant host cell. The host cell is transformed with two vectors. The first vector comprises a polynucleotide encoding a first hybrid polypeptide having a DNA binding domain; and the second vector comprises a polynucleotide encoding a second hybrid polypeptide having a transcriptional activating domain that activates said toxic reporter gene when the first and second hybrid polypeptides interact.
In yet another embodiment, a kit is provided for screening a modulating compound by providing a recombinant host cell, as described in the paragraph above, but instead of a DNA binding domain, the first vector encodes a first hybrid polypeptide containing a first domain of a protein. The second vector encodes a second polypeptide containing a second part of a complementary domain of a protein that activates the toxic reporter gene when the first and second hybrid polypeptides interact. in the selection methods described above, the activating domain can be p42 Gal 4, YP16 (HSV) and the DNA-binding domain can be derived from Gal4 or Lex A. The protein or enzyme can be adenylate cyclase, guanylate cyclase, DHFR and the like. Examples of modulating compounds are set forth in Table 3. In yet another embodiment, the present invention relates to a pharmaceutical composition comprising the modulating compounds for preventing or treating disorders and/or diseases involving members of the TGFβ family of cytokines in a human or animal, most preferably in a mammal. This pharmaceutical composition comprises a pharmaceutically acceptable amount of the modulating compound. The pharmaceutically acceptable amount can be estimated from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range having the desired effect in an in vitro system. This information can thus be used to accurately determine the doses in other mammals, including humans and animals.
The therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or in experimental animals. For example, the LD50 (the dose lethal to 50% of the population) as well as the ED50 (the dose therapeutically effective in 50% of the population) can be determined using methods known in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index which can be expressed as the ratio between LD 50 and ED50 compounds that exhibit high therapeutic indexes.
The data obtained from the cell culture and animal studies can be used in formulating a range of dosage of such compounds which lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
The pharmaceutical composition can be administered via any route such as locally, orally, systemically, intravenously, intramuscularly, mucosally, using a patch and can be encapsulated in liposomes, microparticles, microcapsules, and the like. The pharmaceutical composition can be embedded in liposomes or even encapsulated.
Any pharmaceutically acceptable carrier or adjuvant can be used in the pharmaceutical composition. The modulating compound will be preferably in a soluble form combined with a pharmaceutically acceptable carrier. The techniques for formulating and administering these compounds can be found in "Remington's Pharmaceutical Sciences" Mack Publication Co., Easton, PA, latest edition.
The mode of administration optimum dosages and galenic forms can be determined by the criteria known in the art taken into account the seriousness of the general condition of the mammal, the tolerance of the treatment and the side effects.
The present invention also relates to a method of treating or preventing diseases involving the trasduction pathways of members of the transforming growth factor β super- family of cytokines in a human or mammal in need of such treatment. This method comprises administering to a mammal in need of such treatment a pharmaceutically effective amount of a modulating compound which binds to a targeted mammalian or human or inner ear cell protein. In a preferred embodiment, the modulating compound is a polynucleotide which may be placed under the control of a regulatory sequence which is functional in the mammal or human. In yet another embodiment, the present invention relates to a pharmaceutical composition comprising a SID® polypeptide, a fragment or variant thereof. The SID® polypeptide, fragment or variant thereof can be used in a pharmaceutical composition provided that it is endowed with highly specific binding properties to a bait polypeptide of interest. The original properties of the SID® polypeptide or variants thereof interfere with the naturally occurring interaction between a first protein and a second protein within the cells of the organism. Thus, the SID® polypeptide binds specifically to either the first polypeptide or the second polypeptide.
Therefore, the SID® polypeptides of the present invention or variants thereof interfere with protein-protein interactions between mammalian and especially human protein.
Thus, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable amount of a SID® polypeptide or variant thereof, provided that the variant has the above-mentioned two characteristics; i.e., that it is endowed with highly specific binding properties to a bait polypeptide of interest and is devoid of biological activity of the naturally occurring protein.
In yet another embodiment, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a polynucleotide encoding a SID® polypeptide or a variant thereof wherein the polynucleotide is placed under the control of an appropriate regulatory sequence. Appropriate regulatory sequences that are used are polynucleotide sequences derived from promoter elements and the like.
Polynucleotides that can be used in the pharmaceutical composition of the present invention include the nucleotide sequences of SEQ ID N° 27 to 64.
Besides the SID® polypeptides and polynucleotides, the pharmaceutical composition of the present invention can also include a recombinant expression vector comprising the polynucleotide encoding the SID® polypeptide, fragment or variant thereof.
The above described pharmaceutical compositions can be administered by any route such as orally, systemically, intravenously, intramuscularly, intradermally, mucosally, encapsulated, using a patch and the like. Any pharmaceutically acceptable carrier or adjuvant can be used in this pharmaceutical composition. The SID® polypeptides as active ingredients will be preferably in a soluble form combined with a pharmaceutically acceptable carrier. The techniques for formulating and administering these compounds can be found in "Remington's Pharmaceutical Sciences" supra.
The amount of pharmaceutically acceptable SID® polypeptides can be determined as described above for the modulating compounds using cell culture and animal models. Such compounds can be used in a pharmaceutical composition to treat or prevent transforming growth factor β-mediated disorders and/or diseases.
Thus, the present invention also relates to a method of preventing or treating transforming growth factor β-mediated disorders and/or diseases in a mammal said method comprising the steps of administering to a mammal in need of such treatment a pharmaceutically effective amount of:
(1) a SID® polypeptide of SEQ ID N°6δ to 10δ or a variant thereof which binds to a targeted mammalian or typically human protein; or
(2) or SID® polynucleotide encoding a SID® polypeptide of SEQ ID N° 6δ to 102 or a variant or a fragment thereof wherein said polynucleotide is placed under the control of a regulatory sequence which is functional in said mammal.
In another embodiment the present invention nucleic acids comprising a sequence of SEQ ID N° 27 to 64 which encodes the protein of sequence SEQ ID N° 66 to 102 and/or functional derivatives thereof are administered to modulate complex (from Table 2) function by way of gene therapy. Any of the methodologies relating to gene therapy available within the art may be used in the practice of the present invention such as those described by Goldspiel et al Clin. Pharm. 12 pgs. 488-δOδ (1993).
Delivery of the therapeutic nucleic acid into a patient may be direct in vivo gene therapy (i.e., the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or indirect ex vivo gene therapy (i.e., cells are first transformed with the nucleic acid in vitro and then transplanted into the patient).
For example for in vivo gene therapy, an expression vector containing the nucleic acid is administered in such a manner that it becomes intracellular; i.e., by infection using a defective or attenuated retroviral or other viral vectors as described, for example in U.S. Patent 4,980,286 or by Robbins et al, Pharmacol. Ther. , 80 No. 1 pgs. 3δ-47 (1998). The various retroviral vectors that are known in the art are such as those described in
Miller et al. (Meth. Enzymol. 217 pgs. 681-699 (1993)) which have been modified to delete those retroviral sequences which are not required for packaging of the viral genome and subsequent integration into host cell DNA. Also adenoviral vectors can be used which are advantageous due to their ability to infect non-dividing cells and such high-capacity adenoviral vectors are described in Kochanek (Human Gene Therapy, 10, pgs. 2461-2459 (1999)). Chimeric viral vectors that can be used are those described by Reynolds et al. (Molecular Medecine Today, pgs. 25 -31 (1999)). Hybrid vectors can also be used and are described by Jacoby et al. (Gene Therapy, 4, pgs. 1282-1283 (1997)).
Direct injection of naked DNA or through the use of microparticle bombardment (e.g., Gene Gun®; Biolistic, Dupont) or by coating it with lipids can also be used in gene therapy. Cell-surface receptors/transfecting agents or through encapsulation in liposomes, microparticles or microcapsules or by administering the nucleic acid in linkage to a peptide which is known to enter the nucleus or by administering it in linkage to a ligand predisposed to receptor-mediated endocytosis (See Wu & Wu, J. Biol. Chem., 262 pgs. 4429-4432 (1987)) can be used to target cell types which specifically express the receptors of interest. In another embodiment a nucleic acid ligand compound may be produced in which the ligand comprises a fusogenic viral peptide designed so as to disrupt endosomes, thus allowing the nucleic acid to avoid subsequent lysosomal degradation. The nucleic acid may be targeted in vivo for cell specific endocytosis and expression by targeting a specific receptor such as that described in WO92/06180, W093/14188 and WO 93/20221. Alternatively the nucleic acid may be introduced intracellularly and incorporated within the host cell genome for expression by homologous recombination (See Zijlstra et al, Nature, 342, pgs. 435-428 (1989)).
In ex vivo gene therapy, a gene is transferred into cells in vitro using tissue culture and the cells are delivered to the patient by various methods such as injecting subcutaneously, application of the cells into a skin graft and the intravenous injection of recombinant blood cells such as hematopoietic stem or progenitor cells.
Cells into which a nucleic acid can be introduced for the purposes of gene therapy include, for example, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes and blood cells. The blood cells that can be used include, for example, T- lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryotcytes, granulocytes, hematopoietic cells or progenitor cells and the like.
In yet another embodiment the present invention relates to protein chips or protein microarrays. It is well known in the art that microarrays can contain more than 10,000 spots of a protein that can be robotically deposited on a surface of a glass slide or nylon filter. The proteins attach covalently to the slide surface, yet retain their ability to interact with other proteins or small molecules in solution. In some instances the protein samples can be made to adhere to glass slides by coating the slides with an aldehyde-containing reagent that attaches to primary amines. A process for creating microarrays is described, for example by MacBeath and Schreiber (Science, Volume 289, Number 5486, pgs, 1760-1763 (2000)) or (Service, Science, Vol, 289, Number 5485 pg. 1673 (2000)). An apparatus for controlling, dispensing and measuring small quantities of fluid is described, for example, in U.S. Patent No. 6,112,605. The present invention also provides a record of protein-protein interactions, PIM®'s and any data encompassed in the following Tables. It will be appreciated that this record can be provided in paper or electronic or digital form.
The present invention also relates to the use of a SID® or an interaction or a prey to screen molecules that inhibit TGFβ or a TGFβ super-family of cytokines pathway, as well as molecules that inhibit TGFβ or a TGFβ super-family of cytokines pathway obtained by this screening method. The screening can occur in mammalian or yeast cells. Furthermore, the inhibition can be detected by fluorescence polarization, FRET, BRET, filter binding assays or radioactive techniques. In order to fully illustrate the present invention and advantages thereof, the following specific examples are given, it being understood that the same are intended only as illustrative and in nowise limitative.
EXAMPLES
EXAMPLE 1 : Preparation of a collection of random-primed cDNA fragments
1.A. Collection preparation and transformation in Escherichia coli
1.A.1. Random-primed cDNA fragment preparation
For mRNA sample from transforming growth factor β, random-primed cDNA was prepared from 5 μg of polyA+ mRNA using a TimeSaver cDNA Synthesis Kit (Amersham Pharmacia Biotech) and with 5 μg of random N9-mers according to the manufacturer's instructions. Following phenolic extraction, the cDNA was precipitated and resuspended in water. The resuspended cDNA was phosphorylated by incubating in the presence of T4
DNA Kinase (Biolabs) and ATP for 30 minutes at 37°C. The resulting phosphorylated cDNA was then purified over a separation column (Chromaspin TE 400, Cloπtech), according to the manufacturer's protocol.
1.A.2. Liqation of linkers to blunt-ended cDNA
Oligonucleotide HGX931 (δ' end phosphorylated) 1 μg/μl and HGX932 1 μg/μl were used.
Sequence of the oligo HGX931: δ'-GGGCCACGAA-3' (SEQ ID No.103) Sequence of the oligo HGX932: 5'-TTCGTGGCCCCTG-3' (SEQ ID No.104)
Linkers were preincubated (5 minutes at 9δ°C, 10 minutes at 68°C, 1δ minutes at 42°C) then cooled down at room temperature and ligated with cDNA fragments at 16°C overnight.
Linkers were removed on a separation column (Chromaspin TE 400, Clontech), according to the manufacturer's protocol. 1 A3. Vector preparation
Plasmid pP6 (see Figure 3) was prepared by replacing the SpellXhol fragment of pGAD3S2X with the double-stranded oligonucleotide: δ'CTAGCCATGGCCGCAGGGGCCGCGGCCGCACTAGTGGGGATCCTTAATTAAGGGCC ACTGGGGCCCCC3' (SEQ ID No.105)
5 CGAGGGGGCCCCAGTGGCCCTTAATTAAGGATCCCCACTAGTGCGGCCGCGGCCC
CTGCGGCCATGG3' (SEQ ID No.106)
The pP6 vector was successively digested with Sf1 and BamHI restriction enzymes (Biolabs) for 1 hour at 37°C, extracted, precipitated and resuspended in water. Digested plasmid vector backbones were purified on a separation column (Chromaspin TE 400, Clontech), according to the manufacturer's protocol. 1.A.4. Liαation between vector and insert of cDNA
The prepared vector was ligated overnight at 15°C with the blunt-ended cDNA described in section 2 using T4 DNA ligase (Biolabs). The DNA was then precipitated and resuspended in water.
1.A.5. Library transformation in Escherichia coli
The DNA from section 1.A.4 was transformed into Electromax DH10B electrocompetent cells (Gibco BRL) with a Cell Porator apparatus (Gibco BRL). 1 ml SOC medium was added and the transformed cells were incubated at 37°C for 1 hour. 9 mis of
SOC medium per tube was added and the cells were plated on LB+ampicillin medium. The colonies were scraped with liquid LB medium, aliquoted and frozen at -80°C.
1.B. Collection transformation in Saccharomyces cerevisiae
The Saccharomyces cerevisiae strain (YHGX13 (MATα Gal4Δ GalδOΔ ade2- 101::KANR, his3, leu2-3, -112, trp1-901, ura3-62 URA3::UASGAL1-LacZ, Met)) was transformed with the cDNA library.
The plasmid DNA contained in E. coli wer extracted (Qiagen) from aliquoted E. coli frozen cells (1.A.6.). Saccharomyces cerevisiae yeast YHGX13 in YPGIu were grown.
Yeast transformation was performed according to standard protocol (Giest et al. Yeast, 11, 356-360, 1995) using yeast carrier DNA (Clontech). This experiment leads to 104 to δ x 104 cells/μg DNA. 2 x 104 cells were spread on DO-Leu medium per plate. The cells were aliquoted into vials containing 1 ml of cells and frozen at -80°C. 1.C. Construction of bait plasmids
For fusions of the bait protein to the DNA-binding domain of the GAL4 protein of S. cerevisiae, bait fragments were cloned into plasmid pB27and pB28.
Plasmid pB27 was prepared by replacing the ampicillin resistance of pB20 with the tetracyclin resistance. MCS sequence EcoRI/Pstl:
5' AATTCGGGGCCGGACGGGCCGCGGCCGCACTAGTGGGGATCCTTAATTAAGGGCCAC
TGGGGCCCCTCGACCTGCA 3' (SEQ ID No 107) 5'
GGTCGAGGGGCCCCAGTGGCCCTTAATTAAGGATCCCCACTAGTGCGGCCGCGGCCC
GTCCGGCCCCG 3' (SEQ ID No 108)
Plasmid pB28 was prepared by replacing the EcoRI/Pstl polylinker fragment of pB27 with the double stranded DNA fragment : δ'GAATTCGGGGCCGCAGGGGCCGCGGCCGCACTAGTGGGGATCCTTAATTAAGGGCC ACTGGGGCCCCTCGACCTGCAG 3' (SEQ ID No 109) δ'CTGCAGGTCGAGGGGCCCCAGTGGCCCTTAATTAAGGATCCCCACTAGTGCGGCCG CGGCCCCTGCGGCCCCGAATTC 3'(SEQ ID No 110)
The amplification of the bait ORF was obtained by PCR using the Pfu proof-reading Tag polymerase (Stratagene), 10 pmol of each specific amplification primer and 200 ng of plasmid DNA as template. The PCR program was set up as follows :
94° 45"
x 30 cycles
Figure imgf000035_0001
72° 10'
15° oo
The amplification was checked by agarose gel electrophoresis. The PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol.
Purified PCR fragments were digested with adequate restriction enzymes. The PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol. The digested PCR fragments were ligated into an adequately digested and dephosphorylated bait vector (pB27 or pB28) according to standard protocol (Sambrook et al.) and were transformed into competent bacterial cells. The cells were grown, the DNA extracted and the plasmid was sequenced.
Example 2 : Screening the collection with the two-hybrid in yeast system 2. A. The mating protocol
The mating two-hybrid in yeast system (as described by Legrain et al, Nature Genetics, vol. 16, 277-282 (1997), Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens) was used for its advantages but one could also screen the cDNA collection in classical two-hybrid system as described in Fields et al. or in a yeast reverse two-hybrid system.
The mating procedure allows a direct selection on selective plates because the two fusion proteins are already produced in the parental cells. No replica plating is required.
This protocol was written for the use of the library transformed into the YHGX13 strain.
For bait proteins fused to the DNA-binding domain of GAL4, bait-encoding plasmids were first transformed into S. cerevisiae (CG1945 strain (MATa Gal4-δ42 Gal180-δ38 ade2- 101 his3Δ200, Ieu2-3,112, trpl-901 , ura3-δ2, Iys2-801 , URA3::GAL4 17mers (X3)-
CyCITATA-LacZ, LYS2::GAL1 UAS-GAL1TATA-HIS3 CYHR)) according to step 1.B. and spread on DO-Trp medium.
For bait proteins fused to the DNA-binding domain of LexA, bait-encoding plasmids were first transformed into S. cerevisiae (L40Δgal4 strain (MATa ade2, trpl-901, Ieu2 3,112, Iys2-801 , his3Δ200, LYS2::(lexAop)4-HIS3, ura3-62::URA3 (lexAop)8-LacZ, GAL4::KanR)) according to step 1.B. and spread on DO-Trp medium. Day 1, morning : preculture
The cells carrying the bait plasmid obtained at step 1.C. were precultured in 20 ml DO-Trp medium and grown at 30°C with vigorous agitation. Day 1, late afternoon : culture
The OD600nm of the DO-Trp pre-culture of cells carrying the bait plasmid was measured. The OD6oon must lie between 0.1 and O.δ in order to correspond to a linear measurement.
60 ml DO-Trp at OD600nm 0.006/ml was inoculated and grown overnight at 30°C with vigorous agitation. Day 2 : mating medium and plates 2 YPGIu 1δcm plates 60 ml tube with 13 ml DO-Leu-Trp-His 100 ml flask with δ ml of YPGIu 8 DO-Leu-Trp-His plates 2 DO-Leu-Trp plates
The ODβoonm of the DO-Trp culture was measured. It should be around 1. For the mating, twice as many bait cells as library cells were used. To get a good mating efficiency, one must collect the cells at 108 cells per cm2.
The amount of bait culture (in ml) that makes up 60 OD600nm units for the mating with the prey library was estimated. A vial containing the library of step 1 B was thawed slowly on ice. 1.0ml of the vial was added to 20 ml YPGIu. Those cells were recovered at 30°C, under gentle agitation for 10 minutes. Mating The 50 OD600nm units of bait culture was placed into a 60 ml falcon tube.
The library of step 1B culture was added to the bait culture, then centrifuged, the supernatant discarded and resuspended in 1.6ml YPGIu medium.
The cells were distributed onto two 15cm YPGIu plates with glass beads. The cells were spread by shaking the plates. The plate cells-up at 30°C for 4h30min were incubated. Collection of mated cells
The plates were washed and rinsed with 6ml and 7ml respectively of DO-Leu-Trp-His. Two parallel serial ten-fold dilutions were performed in 500μl DO-Leu-Trp-His up to 1/10,000. δOμl of each 1/1 ,000 dilution was spread onto DO-Leu-Trp plates. 22.4ml of collected cells were spread in 400μl aliquots on DO-Leu-Trp-His+Tet plates. Day 4
Clones that were able to grow on DO-Leu-Trp-His+Tetracyclin were then selected. This medium allows one to isolate diploid clones presenting an interaction. The His+ colonies were counted on control plates.
The number of His+ cell clones will define which protocol is to be processed : Upon 60.106 Trp+Leu+ colonies :
- if the number His+ cell clones <28δ: then use the process stamp overlay protocol on all colonies
- if the number of His+ cell clones >28δ and <5000: then process via overlay and then stamp overlay protocols on blue colonies (2.B and 2.C). - if number of His+ cell clones >6000: repeat screen using DO-Leu-Trp-His+Tetracyclin plates containing 3-aminotriazol. 2.B. The X-Gal overlay assay
The X-Gal overlay assay was performed directly on the selective medium plates after scoring the number of His* colonies. Materials
A waterbath was set up. The water temperature should be 60°C.
• O.δ M Na2HP04 pH 7.6.
• 1.2% Bacto-agar.
• 2% X-Gal in DMF. • Overlay mixture : 0.2δ M Na2HP04 pH7.δ, 0.6% agar, 0.1% SDS, 7% DMF (LABOSI), 0.04% X-Gal (ICN). For each plate, 10 ml overlay mixture are needed.
• DO-Leu-Trp-His plates. • Sterile toothpicks. Experiment
The temperature of the overlay mix should be between 45°C and 50°C. The overlay- mix was poured over the plates in portions of 10 ml. When the top layer was settled, they were collected. The plates were incubated overlay-up at 30°C and the time was noted. Blue colonies were checked for regularly. If no blue colony appeared, overnight incubation was performed. Using a pen the number of positives was marked. The positives colonies were streaked on fresh DO-Leu-Trp-His plates with a sterile toothpick.
2.C. The stamp overlay assay
His+ colonies were grown overnight at 30°C in microtiter plates containing DO-Leu-Trp- His+Tetracyclin medium with shaking. The day after the overnight culture, the 96 colonies were stamped on a 1δcm plate of DO-Leu-Trp-His. 4 control yeast colonies were spotted on the same plate. After 2 days of growing at 30°C, an overlay assay was performed on this plate with 80ml of overlay mixture (see step 2.B.). After 2 hours of incubation, the plate was photographed with a CCD camera. The blue intensity was quantified by Genetools® software (SYNGENE) and normalized to the control spots. Example 3 : Identification of positive clones 3.A. PCR on yeast colonies Introduction
PCR amplification of fragments of plasmid DNA directly on yeast colonies is a quick and efficient procedure to identify sequences cloned into this plasmid. It is directly derived from a published protocol (Wang H. et al., Analytical Biochemistry, 237, 146-146, (1996)). However, it is not a standardized protocol and it varies from strain to strain and it is dependent of experimental conditions (number of cells, Taςf polymerase source, etc). This protocol should be optimized to specific local conditions. Materials
- For 1 well, PCR mix composition was:
32.6 μl water, 5 μl 10X PCR buffer (Pharmacia),
1 μl dNTP IO mM,
0.5 μl Taq polymerase (δu/μl) (Pharmacia),
0.5 μl oligonucleotide ABS1 10 pmole/μl: 5'-GCGTTTGGAATCACTACAGG-3' (SEQ ID
No.111) 0.5 μl oligonucleotide ABS2 10 pmole/μl: 5'-CACGATGCACGTTGAAGTG-3' (SEQ ID
No.112)
- 1 N NaOH. Experiment
The positive colonies were grown overnight at 30°C on a 96 well cell culture cluster
(Costar), containing 150 μl DO-Leu-Trp-His+Tetracyclin with shaking. The culture was resuspended and 100 μl was transferred immediately on a Thermowell 96 (Costar) and centrifuged for 5 minutes at 4,000 rpm at room temperature. The supernatant was removed.
5 μl NaOH was added to each well and shaken for 1 minute.
The Thermowell was placed in the thermocycler (GeneAmp 9700, Perkin Eimer) for 5 minutes at 99.9DC and then 10 minutes at 4°C. In each well, the PCR mix was added and shaken well. The PCR program was set up as followed:
94°C 3 minutes
94°C 30 seconds δ3°C 1 minute 30 36 cycles
72°C 3 minutes
Figure imgf000039_0001
72°C 5 minutes
1δ°C oo
The quality, the quantity and the length of the PCR fragment was checked on an agarose gel. The length of the cloned fragment was the estimated length of the PCR fragment minus 300 base pairs that corresponded to the amplified flanking plasmid sequences.
3.B. Plasmids rescue from veast bv electroporation Introduction
The previous protocol of PCR on yeast cell may not be successful, in such a case, plasmids from yeast by electroporation can be rescued. This experiment allows the recovery of prey plasmids from yeast cells by transformation of E. coli with a yeast cellular extract. The prey plasmid can then be amplified and the cloned fragment can be sequenced. Materials
Plasmid rescue Glass beads 426-600 μm (Sigma) Phenol/chloroform (1/1) premixed with isoamyl alcohol (Amresco)
Extraction buffer : 2% Triton X100, 1% SDS, 100 mM NaCl, 10 mM TrisHCI pH 8.0, 1 mM EDTA pH 8.0.
Mix ethanol/NH4Ac : 6 volumes ethanol with 7.6 M NH4 Acetate, 70% Ethanol and yeast cells in patches on plates. Electroporation
SOC medium M9 medium Selective plates : M9-Leu+Ampicillin
2 mm electroporation cuvettes (Eurogentech)
Experiment
Plasmid rescue The cell patch on DO-Leu-Trp-His was prepared with the cell culture of section 2.C.
The cell of each patch was scraped into an Eppendorf tube, 300 μl of glass beads was added in each tube, then, 200 μl extraction buffer and 200 μl phenol:chloroform:isoamyl alcohol (25:24:1) was added.
The tubes were centrifuged for 10 minutes at 16,000 rpm. 180 μl supernatant was transferred to a sterile Eppendorf tube and 600 μl each of ethanol/NH4Ac was added and the tubes were vortexed. The tubes were centrifuged for 15 minutes at 16,000 rpm at 4°C. The pellet was washed with 200 μl 70% ethanol and the ethanol was removed and the pellet was dried. The pellet was resuspended in 10 μl water. Extracts were stored at -20°C. Electroporation
Materials: Electrocompetent MC1066 cells prepared according to standard protocols (Sambrook et al. supra).
1 μl of yeast plasmid DNA-extract was added to a pre-chilled Eppendorf tube, and kept on ice. 1 μl plasmid yeast DNA-extract sample was mixed and 20 μl electrocompetent cells was added and transferred in a cold electroporation cuvette.
The Biorad electroporator was set on 200 ohms resistance, 26 μF capacity; 2.6 kV. The cuvette was placed in the cuvette holder and electroporation was performed.
1 ml of SOC was added into the cuvette and the cell-mix was transferred into a sterile Eppendorf tube. The cells were recovered for 30 minutes at 37°C, then spun down for
1 minute at 4,000 x g and the supernatant was poured off. About 100 μl medium was kept and used to resuspend the cells and spread them on selective plates (e.g., M9-Leu plates).
The plates were then incubated for 36 hours at 37°C.
One colony was grown and the plasmids were extracted. The presence and the size of the insert were checked for through enzymatic digestion and agarose gel electrophoresis. The insert was then sequenced. Example 4 : Protein-protein interaction
For each bait, the previous protocol leads to the identification of prey polynucleotide sequences. Using a suitable software program (e.g., Blastwun, available on the Internet site of the University of Washington: http://bioweb.pasteur.fr/seganal/interfaces/blastwu.html). the mRNA transcript that is encoded by the prey fragment may be identified and whether the fusion protein encoded is in the same open reading frame of translation as the predicted protein or not can be determined.
Alternatively, prey nucleotide sequences can be compared with one another and those which share identity over a significant region (60nt) can be grouped together to form a contiguous sequence (Contig) whose identity can be ascertained in the same manner as for individual prey fragments described above.
Example 5 : Identification of SID®
By comparing and selecting the intersection of all isolated fragments that are included in the same polypeptide, one can define the Selected Interacting Domain (SID®) is determined as illustrated in Figure 6. The SID® is illustrated in Table 3.
Example 6: Making of polyclonal and monoclonal antibodies
The protein-protein complex of columns 1 and 4 of Table 2 is injected into mice and polyclonal and monoclonal antibodies are made following the procedure set forth in
Sambrook et al supra. More specifically, mice are immunized with an immunogen comprising the above mentionned complexes conjugated to keyhole limpet hemocyanin using glutaraldehyde or
EDC as is well known in the art. The complexes can also be stabilized by crosslinking as described in WO 00/37483. The immunogen is then mixed with an adjuvant. Each mouse receives four injections of 10 μg to 100 μg of immunogen, and after the fourth injection, blood samples are taken from the mice to determine if the serum contains antibodies to the immunogen. Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.
Spleens are removed from immune mice and single-cell suspension is prepared
(Harlow et al 1988). Cell fusions are performed essentially as described by Kohler et al., Briefly, P366.3 myeloma cells (ATTC Rockville, Md) or NS-1 myeloma cells are fused with spleen cells using polyethylene glycol as described by Harlow et al (1989). Cells are plated at a density of 2 x 105 cells/well in 96-well tissue culture plates. Individual wells are examined for growth and the supernatants of wells with growth are tested for the presence of complex-specific antibodies by ELISA or RIA using the protein-protein complex of columns 1 and 4 of Table 2 as a target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.
Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibodies for characterization and assay development. Antibodies are tested for binding to bait polypeptide of column 1 of Table 2 alone or to prey polypeptide of column 4 of Table 2 alone, to determine which are specific for the protein-protein complex of columns 1 and 4 of Table 2 as opposed to those that bind to the individual proteins. Monoclonal antibodies against each of the complexes set forth in columns 1 and 4 of Table 2 are prepared in a similar manner by mixing specified proteins together, immunizing an animal, fusing spleen cells with myeloma cells and isolating clones which produce antibodies specific for the protein complex, but not for individual proteins. Example 7: Modulating compounds identification
Each specific protein-protein complex of columns 1 and 4 of Table 2 may be used to screen for modulating compounds.
One appropriate construction for this modulating compound screening may be:
- bait polynucleotide inserted in pB27 or pB28;
- prey polynucleotide inserted in pP6;
- transformation of these two vectors in a permeable yeast cell;
- growth of the transformed yeast cell on a medium containing compound to be tested,
- and observation of the growth of the yeast cells. Example 8 : ZNF8 (hgx554)
Gl:17482320
The predicted ZNF8 protein (576 aa) contains 7 zinc finger domains (Lania et al., 1990).
A recent paper has shown mouse ZNF8 (mZNF8) as interacting with the smadl protein. In addition, mZNFδ was shown to be involved in the TGFβ-BMP pathway (Jiao et al., 2002).
Nucleic acid sequence :
ATGTGT GTGATGTTTC AGGAACCAGT GACCTTCCGG GATGTGGCTG TGGACTTTAC
CCAGGAGGAA TGGGGGCAGC TGGACCCTAC CCAGAGGATC CTCTACCGTG
ACGTGATGCT GGAGACCTTT GGTCACCTGC TCTCCATAGG TCCTGAGCTT
CCGAAGCCTG AAGTCATCTC CCAGCTGGAG CAAGGGACCG AGCTATGGGT
GGCTGAGAGA GGAACCACCC AGGGCTGCCA TCCAGCCTGG GAGCCTCGAT
CTGAAAGCCA AGCATCACGC AAGGAAGAGG GCCTGCCTGA AGAGGAGCCA
TCCCATGTCA CGGGAAGGGA AGGATTCCCG ACAGATGCTC CTTATCCCAC
CACGTTAGGG AAAGACAGGG AGTGTCAGAG CCAGAGTCTG GCACTCAAGG
AGCAGAATAA CTTGAAGCAG TTGGAATTTG GCCTCAAGGA AGCACCAGTT
CAAGATCAAG GCTACAAAAC TCTCAGACTC AGGGAAAACT GCGTCCTGAG
TTCAAGCCCA AATCCATTCC CAGAGATCTC TAGAGGGGAG TATTTGTATA
CTTACGACTC ACAGATTACA GACTCAGAAC ATAACTCCAG CTTAGTCAGT
CAGCAGACAG GCTCCCCAGG AAAACAGCCC GGTGAAAACA GTGACTGTCA
CAGAGATTCC AGTCAGGCCA TTCCAATTAC GGAACTCACA AAAAGCCAGG
TGCAGGACAA ACCCTACAAA TGTACTGACT GTGGGAAGTC GTTTAACCAT
AACGCACACC TCACCGTGCA CAAGAGGATT CATACGGGAG AAAGACCTTA
TATGTGCAAG GAGTGTGGGA AAGCCTTCAG CCAGAACTCC TCCCTCGTCC AGCATGAGCG CATCCACACT GGAGACAAGC CCTACAAGTG TGCCGAATGT
GGGAAGTCTT TCTGCCATAG TACACACCTT ACCGTCCATC GGAGGATTCA
CACTGGGGAG AAGCCCTATG AGTGTCAGGA CTGTGGGAGG GCCTTCAACC
AGAACTCCTC CCTGGGGCGG CACAAGAGGA CACACACTGG GGAGAAGCCA
TACACCTGCA GTGTGTGTGG GAAATCCTTC TCTCGGACCA CTTGCCTTTT
CCTGCACCTG AGAACTCACA CCGAGGAGAG GCCCTACGAG TGTAACCACT
GCGGGAAGGG CTTCAGGCAC AGCTCATCCC TGGCCCAGCA CCAGCGGAAG
CACGCGGGGG AGAAGCCCTT TGAGTGCCGC CAGAGGCTGA TCI "GAGCA
GACGCCAGCT CTCACAAAGC ATGAATGGAC AGAAGCCCTG GGCTGTGACC
CACCTTTGAG TCAAGATGAG AGGACTCACC GAAGCGACAG ACCCTTCAAA
TGTAATCAGT GTGGGAAGTG TTTCATTCAG AGCTCTCACC TCATCCGGCA
CCAGATAACT CACACCAGAG AGGAGCAGCC CCATGGGCGA AGCCGGCGGC
GTGAACAATC CTCGAGCAGG AACTCACACC TGGTTCAGCA TCAACACCCG
AACTCCAGAA AGAGCTCTGC AGGCGGAGCA AAGGCAGGGC AGCCGGAAAG
CAGAGCCCTG GCTTTGTTTG ACATCCAAAA AATCATGCAA GAGAAAAACC
CTGTGCACGT TATTGGGGTG GAAGAGCCTT CTGTGGGTGC TTCCATGTTA TTTGACATCA GAGAATCCAC ATAG (SEQ ID N0.113)
Protein sequence
MDPEDEGVAGVMSVGPPAARLQEPVTFRDVAVDFTQEEWGQLDPTQRILYRDVMLETFGH
LLSIGPELPKPEVISQLEQGTELWVAERGTTQGCHPAWEPRSESQASRKEEGLPEEEPSHV
TGREGFPTDAPYPTTLGKDRECQSQSLALKEQNNLKQLEFGLKEAPVQDQGYKTLRLREN
CVLSSSPNPFPEISRGEYLYTYDSQITDSEHNSSLVSQQTGSPGKQPGENSDCHRDSSQAI .
PITELTKSQVQDKPYKCTDCGKSFNHNAHLTVHKRIHTGERPYMCKECGKAFSQNSSLVQH
ERIHTGDKPYKCAECGKSFCHSTHLTVHRRIHTGEKPYECQDCGRAFNQNSSLGRHKRTHT
GEKPYTCSVCGKSFSRTTCLFLHLRTHTEERPYECNHCGKGFRHSSSLAQHQRKHAGEKP
FECRQRLIFEQTPALTKHEWTEALGCDPPLSQDERTHRSDRPFKCNQCGKCFIQSSHLIRH
QITHTREEQPHGRSRRREQSSSRNSHLVQHQHPNSRKSSAGGAKAGQPESRALALFDIQKI
MQEKNPVHVIGVEEPSVGASMLFDIREST (SEQ ID No.114)
I. ZNF8 interacts with several members of the BMP and TGFβ pathways
By two-hybrid screening in yeast (Placenta library) it was shown that ZNF8 interacts with several members of the BMP pathway:
Smadl -ZNF8
SID : Nucleic sequence, SEQ ID No.27 and Proteic sequence, SEQ ID No. 65 SID : Nucleic sequence, SEQ ID No.31 and Proteic sequence, SEQ ID No. 69
Smad5-ZNF8
SID : Nucleic sequence, SEQ ID Nό.42 and Proteic sequence, SEQ ID No. 80 Smad9-ZNF8
SID : Nucleic sequence, SEQ ID No.45 and Proteic sequence, SEQ ID No. 83
In addition, ZNF8 was also found interacting with smad proteins using other libraries
Smad1-ZNF8
SID : Nucleic sequence, SEQ ID No.28, 29, 30 and Proteic sequence, SEQ ID
No. 66, 67, 68 SID : Nucleic sequence, SEQ ID No.32, 33, 34 and Proteic sequence, SEQ ID No. 70, 71, 72
Smad4-ZNF8
SID : Nucleic sequence, SEQ ID No.38 and Proteic sequence, SEQ ID No. 76 Rebound screening experiments using ZNF8 as bait (nt 732-1301 ) on Placenta library allowed us to confirm the Smadl -ZNF8 and Smad9-ZNF8 interactions ZNF8-Smad1
ZNF8-Smad9
In summary, Yeast-two-hybrid screens show that amino-acids 22-268 from Smadl (SEQ ID No.14) interact with amino-acids 364-433 from ZNF8 (SEQ ID No.114) (see . 11 A). Amino-acids 1-162 from Smad4 (SEQ ID No.17) interact with amino-acids 172-441 from ZNF8 (see fig. 11B). Amino-acids 1-268 from Smadδ (SEQ ID No.19) interact with amino- acids 276-437 from ZNF8 (see fig. 11C). Finally, amino-acids 1-233 from Smad9 (SEQ ID No.20) interact with amino-acids 208-1209 from ZNF8 (see fig. 11D).
Interestingly, the full-length ZNF8 protein used as bait behaved as autoactivator. This finding as well as the presence of 7 zinc binding domains led us to hypothesise that ZNF8 could be a transcription factor.
II. ZNF8 is an essential player in the TGFβ and BMP pathways
In order to validate ZNFδ's involvement in the TGFβ/BMP pathways, ZNF8 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and ZNF8-targeting siRNA duplex, a specific dose-dependant repression of the TGFβ-dependant reporter activity was observed (see Fig. 12A) demonstrating a function for ZNF8 in the response to the TGFβ pathway. The repressive effect of ZNF8-targeting siRNA duplex was observed at low concentration of siRNA duplex (4nM) and was enhanced at higher concentrations (40nM). While transiently co-transfecting HepG2 cells using the p(GC)ι2-MLP-Luc reporter and ZNF8-targeting siRNA duplex, a specific dose-dependant repression of the BMP-dependant reporter activity was observed (see Fig. 12B) demonstrating a function for ZNF8 on the response to the BMP pathway on a minimal BMP responsive element. Similar results were obtained using either BMP6 instead of BMP7 (see Fig. 12C). Modulation of these TGFβ/BMP luciferase reporter activities using ZNF8 cellular knock-down suggest an implication of this putative transcription factor in the regulation of these two pathways. In order to further elucidate its role on the expression of genes naturally controlled by
TGFβ and/or BMPs in cells, a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGFβ or BMP-dependant markers was performed. PAI-1 was a well-known target of TGFβ and was strongly induced by TGFβ in many cell types (Keeton et al., 1991). Osteoblastic differentiation was characterized by expression of alkaline phosphatase as an early pre-osteoblastic marker and alkaline phosphatase transcription is directly controled by BMP signals (Wagner EF and Karsenty G, 2001). Modulation of AP1/jun expression by TGFβ is a cell-type specific phenomenon as TGFβ activates c-jun expression only in epithelial cells, whereas it induces junB in mesenchymal cells. JunB is also an immediate early gene induced by BMP-2 (Mauviel ef al., 1996; Chalaux er a/., 1998).
Endogenous levels of alkaline phosphatase and junB mRNA were specifically and dose-dependently decreased following transient transfection of ZNF8-targeting siRNA duplex in HepG2 cells treated with BMP7 (see Fig. 13A, 13B & 13C respectively). As expected, endogenous PAI-1 mRNA levels were not affected following the same transfection experiments induced by BMP7 (see Fig. 14A). Expression levels of various controls were not affected at all following the same ZNF8-targeting siRNA duplex transfection: hGUS (human beta-glucuronidase, Oshima etal., 1987, see Fig. 14B) HPRT (hypoxanthine-guanine phosphoribosyltransferase, Patel et al., 1986, data not shown), GAPDH (giyceraldehyde-3- phosphate dehydrogenase, Allen etal., 1987, data not shown) and 18S ribosomal RNA (Schmittgen ef al., 2000, data not shown). Example 9 : LAPTmS (hgx596) GI: 1255239
Using subtractive hybridization, Adra ef al. (1996) cloned the cDNA coding for a gene preferentially expressed in adult hematopoietic tissues. The predicted protein (262 amino- acids) contained δ highly hydrophobic transmembrane domains. Immuno-cytological and cell fractionation studies with a specific antibody revealed a protein localizing in lysosomes. In addition, the gene, named LAPTmδ, was found to interact with ubiquitin. Recently, a new rat gene which exhibits 80% of identity with LAPTmδ, called GCD-10, was identified as activated in response to neuronal apoptosis (Origasa ef al., 2001). LAPTmδ has also been found to be an immediate-early gene induced by retinoic acid during granulocytic differentiation in murine retinoic acid-inducible MPRO promyelocyte cell line (Scott et al., 1996). Finally, LAPTmδ was shown to be up-regulated in the Sjogren's syndrome which is a chronic autoimmune disease (Azuma ef al., 2002) and to be co-expressed with activated macrophage genes in rheumatoid arthritis (Walker et al., 2002). Despite being structurally highly related to a family of lysosomal transporter proteins shown to regulate cellular multidrug resistance ( Cabrita et al., 1999; Hogue ef al., 1999), no function has been attributed to this gene, yet. Nucleic acid sequence
ATGGACCCCC GCTTGTCCAC TGTCCGCCAG ACCTGCTGCT GCTTCAATGT CCGCATCGCA ACCACCGCCC TGGCCATCTA CCATGTGATC ATGAGCGTCT TGTTGTTCAT CGAGCACTCA GTAGAGGTGG CCCATGGCAA GGCGTCCTGC AAGCTCTCCC AGATGGGCTA CCTCAGGATC GCTGACCTGA TCTCCAGCTT CCTGCTCATC ACCATGCTCT TCATCATCAG CCTGAGCCTA CTGATCGGCG TAGTCAAGAA CCGGGAGAAG TACCTGCTGC CCTTCCTGTC CCTGCAAATC ATGGACTATC TCCTGTGCCT GCTCACCCTG CTGGGCTCCT ACATTGAGCT GCCCGCCTAC CTCAAGTTGG CCTCCCGGAG CCGTGCTAGC TCCTCCAGTT CCCCCTGATG ACGCTGCAGC TGCTGGACTT CTGCCTGAGC ATCCTGACCC TCTGCAGCTC CTACATGGAA GTGCCCACCT ATCTCAACTT CAAGTCCATG AACCACATGA ATTACCTCCC CAGCCAGGAG GATATGCCTC ATAACCAGTT CATCAAGATG ATGATCATCT TTTCCATCGC CTTCATCACT GTCCTTATCT TCAAGGTCTA CATGTTCAAG TGCGTGTGGC GGTGCTACAG ATTGATCAAG TGCATGAACT CGGTGGAGGA GAAGAGAAAC TCCAAGATGC TCCAGAAGGT GGTCCTGCCG TCCTACGAGG AAGCCCTGTC TTTGCCATCG AAGACCCCAG AGGGGGGCCC AGCACCACCC CCATACTCAG AGGTGTGA (SEQ ID No.1 16) Protein sequence MDPRLSTVRQTCCCFNVRIATTALAIYHVIMSVLLFIEHSVEVAHGKASCKLSQMGYLRIADLI SSFLLITMLFIISLSLLIGWKNREKYLLPFLSLQIMDYLLCLLTLLGSYIELPAYLKLASRSRASS SKFPLMTLQLLDFCLSILTLCSSYMEVPTYLNFKSMNHMNYLPSQEDMPHNQFIKMMIIFSIA FITVLIFKVYMFKCVWRCYRLIKCMNSVEEKRNSKMLQKWLPSYEEALSLPSKTPEGGPAP PPYSEV (SEQ ID No.116) I. LAPTmδ interacts with Smurf2, a protein involved in the TGFβ pathway
We showed by two-hybrid screening in yeast that LAPTmδ interacts with Smurf2, a E3 ubiquitin ligase known to regulate the protein level of Smadl , 2, 7, SnoN and the TGFβ- activated type I receptor (TβRI). Smurf2-LAPTmδ SID : Nucleic sequence, SEQ ID No.47, 48, 49 and Proteic sequence, SEQ ID
No. 85, 86, 87 Rebound screening experiments using LAPTmδ as bait (nt 654-786) on placenta library allowed us to confirm the Smurf2-LAPTm5 interaction:
LAPTm5-Smurf2 Thus, yeast-two-hybrid screens showed that amino-acids 234-335 from Smurf2 (SEQ ID No.22) interact with amino-acids 261-262 from LAPTmδ (SEQ ID No.116) (see Fig.16). II. LAPTmδ modulates the TGFβ pathway
The two-hybrid screening results led the involvement of the LAPTmδ protein in the TGFβ pathway. To demonstrate a functional effect in mammalian cells, the LAPTmδ c-DNA was cloned into the pV3 vector and used in our TGFβ reporter assay (see Materials & Methods). Over-expression of LAPTmδ (2 and 10 ng of pV3-LAPTm5) results in a dose dependant 2-fold decrease of TGFβ signaling in HepG2 (Figure 16A). In addition, a similar LAPTmδ over-expression in HEK293 cells (O.δ, 2, 10 and 50 ng) results also in a 2-fold decrease of TGFβ signaling (Figure 16B). This LAPTmδ effect was not observed when the BMP signaling and the pGL3-control were tested thus showing a reproducible and specific effect of LAPTmδ (Fig. 16A & B, right panel for both figures).
Next investigated was the endogenous level of LAPTmδ mRNA using Q-PCR. LAPTmδ mRNA was barely detectable in HepG2, HeLa and WI38 cells. In contrast, a strong amount of LAPTmδ mRNA was observed in hematopoietic cells such as CEM, CEMC7, K662 and Jurkat cells (Figure 17A). Next investigated was the effect of TGFβ on the endogenous level of LAPTmδ mRNA in HepG2 cells. After TGFβ induction for 18 H, a 50-fold induction of LAPTmδ mRNA (Figure 17B) was observed. This induction was TGFβ-specific since no effect was observed using several members of BMP, such as BMP2, 4 and 7 (data not shown).
To confirm LAPTmδ induction by TGFβ in HepG2 cells, a TβRI-targeting siRNA duplex previously shown to dramatically reduce T Rl mRNA levels in HepG2 cells (data not shown) and to inhibit the TGFβ pathway (see Fig 13A and Materials & Methods) was transiently transfected into HepG2 cells. Following quantitative PCR analysis of total RNA, the TGFβ induction of LAPTmδ mRNA was totally abolished thus confirming the regulation of LAPTmδ mRNA expression by TGFβ (Figure 17B, right panel). In order to demonstrate LAPTmδ's involvement in the TGFβ/BMP pathways in a functional cellular assay , LAPTmδ cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and LAPTmδ-targeting siRNA duplex, a specific dose-dependant activation of the TGFβ-dependaπt reporter activity was observed (see Fig. 18 A) demonstrating a function for LAPTmδ in the response to the TGFβ pathway. The activating effect of LAPTmδ-targeting siRNA duplex was observed at low concentration of siRNA duplex (4nM) and was enhanced at higher concentrations (40nM). While transiently co-transfecting HepG2 cells using the p(GC)12-MLP-Luc reporter and LAPTmδ-targeting siRNA duplex, a specific, dose-dependant and BMP-dependant activation of the BMP-dependant reporter activity was observed (see Fig. 18 B) demonstrating a function for LAPTmδ in the response to the BMP pathway. In order to further elucidate its role on the expression of genes naturally controlled by
TGFβ and/or BMPs in cells, a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGFβ or BMP-dependant markers were performed. Endogenous levels of PAI-1 , junB and alkaline phosphatase mRNA were specifically and dose-dependently increased following transient transfection of LAPTmδ-targetiπg siRNA duplex in HepG2 cells treated with either TGFβ (PAI-1 and junB, see Fig. 19 A & B) or BMP7 (alkaline phosphatase, see fig 19 C). Expression levels of various controls were not significantly affected following the same LAPTmδ-targeting siRNA duplex transfection: hGUS (see fig 19 D) HPRT, GAPDH and 18S (data not shown).
The inhibition effect of LAPTmδ on the TGFβ pathway as well as the up-regulation of the LAPTmδ mRNA level by TGFβ led us to conclude that LAPTmδ is involved in the negative feedback of the TGFβ signalling. It has been suggested by Kavsak and coll. (Kavsak ef al. 2000) that Smurf2 could address the TGFβ receptors and smad7 to the lysosome for degradation. Thus, by interacting with smurf2, a specific E3 ubiquitin ligase known to be involved in the degradation of the TGFβ receptors, Smadl , Smad2, Smad3, Smad7 and SnoN, LAPTmδ could be a smurf2 receptor in the lysosomal membrane and could address some TGFβ signaling members to the lysosomal compartment to induce their degradation.
Example 10 : RNF11 (hgx555) Gl:7657519 Seki ef al. (1999) identified a new member of the RING finger family, named RNF11 (164 amino acids). Recently, a differential display analysis of gene expression using NIH 3T3 cells expressing the RET-MEN2A or RET-MEN2B mutant proteins was performed. These germ- line point mutations of the RET gene are responsible for multiple endocrine neoplasia (MEN) type 2A and 2B that develop medullary thyroid carcinoma and pheochromocytoma. It has been shown that RNF11 was up-regulated in these mutant cells (Watanabe ef al., 2002). In addition, GNDF was found to up-regulate RNF11 levels (Watanabe ef al., 2002). However, no function for RNF11 has been attributed yet.
Nucleic acid sequence ATGGGGAACT GCCTCAAATC CCCCACCTCG GATGACATCT CCCTGCTTCA CGAGTCTCAG TCCGACCGGG CTAGCTTTGG CGAGGGGACG GAGCCGGATC AGGAGCCGCC GCCGCCATAT CAGGAACAAG TTCCAGTTCC AGTCTACCAC CCAACACCTA GCCAGACTCG GCTAGCAACT CAGCTGACTG AAGAGGAACA AATTAGGATA GCTCAAAGAA TAGGTCTTAT ACAACATCTG CCTAAAGGAG TTTATGACCC TGGAAGAGAT GGATCAGAAA AAAAGATCCG GGAGTGTGTG ATCTGTATGA TGGACTTTGT TTATGGGGAC CCAATTCGAT TTCTGCCGTG CATGCACATC TATCACCTGG ACTGTATAGA TGACTGGTTG ATGAGATCCT TCACGTGCCC CTCCTGCATG GAGCCAGTTG ATGCAGCACT GCTTTCATCC TATGAGACTA ATTGA (SEQ ID No.117)
Protein sequence
MGNCLKSPTSDDISLLHESQSDRASFGEGTEPDQEPPPPYQEQVPVPVYHPTPSQTRLAT
QLTEEEQIRIAQRIGLIQHLPKGVYDPGRDGSEKKIRECVICMMDFVYGDPIRFLPCMHIYHL DCIDDWLMRSFTCPSCMEPVDAALLSSYETN (SEQ ID No.118)
RNF11 interacts with SARA and Smurf2, proteins involved in the TGFβ pathway By two-hybrid screening in yeast it was shown that RNF11 interacts with SARA, the "Smad anchoring for Receptor Activation", and Smurf2, a E3 ubiquitin ligase known to regulate the protein level of Smadl , 2, 7, SnoN and TβRI. Smurf2-RNF11
SID : Nucleic sequence, SEQ ID No.50, 51 , 52, 53 and Proteic sequence, SEQ ID No. 88, 89, 90, 91 SARA-RNF1 1
SID : Nucleic sequence, SEQ ID No.54, 5δ and Proteic sequence, SEQ ID No. 92, 93
Rebound screening experiments using truncated RNF11 as bait (hgxδ55v1: nt 93-462) and Full-length RNF11 as bait (hgx555v2) on placenta library allowed us to confirm the Smurf2- RNF11 interaction and to find a new interaction: Smurfl -RNF11 : RNF11-Smurf2 RNF11 -Smurfl
Thus, yeast-two-hybrid screens showed that amino-acids 239-335 from Smurf2 (SEQ ID No.22) (aa 239-335) interact with amino-acids 31-84 from RNF11 (SEQ ID No.118) (see Fig. 20 A). Amino-acids 665-1323 from SARA (SEQ ID No.23) interact with amino-acids 61-154 from RNF11 (see Fig. 20B) and amino-acids 236-415 from Smurfl interact with amino-acids 31-154 from RNF11 (see Fig. 20 C).
II. RNF11 regulates the SARA protein level
Since E3 ubiquitin-protein ligase activity is likely to be a general function of the RING finger, the association between SARA and RNF11 thus raised the interesting possibility that RNF11 might function to regulate the protein level of SARA. To test this, SARA was over-expressed in HepG2 cells (300 ng of pCDNA-SARA, a gift from Azzeddine ATFI) in the presence and absence of RNF11 (300 ng of pV3-RNF11) and TGFβ (5 ng/ml for 18H) and examined the SARA protein level using anti-SARA antibody (cf Materials & Methods). Figure 21 shows that the SARA protein level was increased in the presence of RNF11. However, no effect of TGFβ was observed in these conditions. In conclusion, this experiment showed that RNF11 was likely involved in regulation of the SARA protein level. Example 11 : KIAA1196 (hgx559) GI: 18591703
Nagase et al. (1999) newly determined the sequences of 100 cDNA clones of unknown human genes, named KIAA1193 to KIAA1292, from two sets of size-fractionated human adult and fetal brain cDNA libraries. Among these unknown human genes, the hypothetical zinc finger protein KIAA1196 was identified. Since this putative protein contains 7 zinc fingers (C2H2 type), it has been suspected that it may function as a transcription factor. Moreover, KIAA1196 contains a leucine zipper motif in the domain that we have discovered interacting with Smadl and is predicted to be a nuclear protein, reinforcing its potential function as a transcription factor. However, no function for this protein has been attributed, yet.
Nucleic acid sequence ATGCCGGTGG TCCGTGGTGG ACAGACAGTG CCCGGCCAGG CCCCTCTCTG
CTTTGACCCG GGAAGTCCAG CCAGTGACAA GACAGAAGGG AAGAAAAAGG
GGCGGCCAAA AGCCGAGAAC CAGGCCCTCC GAGACATTCC TCTCTCCCTG
ATGAACGACT GGAAGGATGA GTTCAAGGCA CACTCGAGGG TGAAGTGTCC
AAACTCAGGG TGCTGGCTGG AGTTCCCCAG CATCTACGGG CTCAAGTACC
ATTACCAGCG GTGCCAAGGG GGTGCCATCT CAGATCGCCT GGCCTTCCCC
TGCCCCTTCT GCGAGGCCGC ATTCACCTCT AAGACCCAGC TGGAGAAACA
CCGGATCTGG AACCACATGG ACCGACCCCT GCCTGCCTCC AAGCCTGGGC
CCATCAGCAG GCCGGTCACC ATCAGCCGGC CTGTTGGGGT CAGCAAGCCC
ATCGGAGTGA GCAAACCTGT CACTATTGGC AAACCTGTGG GTGTCAGCAA
ACCCATTGGC ATCAGCAAGC CAGTCTCGGT CGGCAGACCC ATGCCAGTCA
CCAAGGCCAT CCCGGTCACT AGGCCCGTGC CAGTCACCAA ACCTGTCACA
GTCAGCAGGC CCATGCCCGT CACCAAGGCC ATGCCGGTCA CCAAACCCAT
CACAGTCACC AAGTCTGTGC CGGTCACCAA ACCCGTACCT GTCACCAAAC
CCATTACGGT AACAAAGCTT GTGACAGTTA CGAAACCCGT GCCGGTCACC
AAGCCAGTGA CAGTCAGCAG GCCCATTGTG GTCAGCAAGC CGGTGACAGT
CAGCAGGCCC ATTGCTATCA GCAGACACAC ACCGCCCTGC AAAATGGTGC
TGCTGACCAG GTCGGAGAAC AAAGCACCTC GTGCCACAGG GAGGAACAGT
GGTAAGAAAA GGGCTGCGGA CAGCCTGGAC ACCTGCCCAA TTCCACCCAA
GCAGGCCAGG CCAGAGAATG GGGAGTACGG CCCCTCCTCC ATGGGCCAGA
GCTCGGCCTT CCAGCTGAGT GCAGACACCA GCAGTGGCTC CTTGTCGCCA
GGCAGCAGGC CGTCAGGGGG CATGGAGGCA CTGAAGGCTG CAGGCCCTGC GTCCCCGCCT GAGGAGGACC CGGAGCGCAC AAAGCACAGA AGGAAACAGA
AAACACCCAA AAAGTTTACA GGGGAGCAGC CATCCATCTC AGGGACCTTT
GGGCTCAAAG GCCTGGTCAA AGCTGAGGAC AAGGCCCGAG TTCACCGCTC
CAAGAAGCAG GAGGGGCCAG GCCCTGAGGA CGCCCGGAAG AAGGTGCCAG CTGCCCCCAT CACTGTCAGC AAGGAGGCAC CGGCCCCTGT GGCCCACCCA
GCTCCAGGTG GCCCTGAAGA GCAGTGGCAG AGGGCCATCC ATGAGCGCGG
GGAAGCCGTC TGCCCCACCT GCAACGTGGT CACCCGGAAG ACTCTCGTGG
GGCTTAAGAA GCACATGGAG GTGTGTCAGA AGCTTCAGGA TGCACTCAAG
TGCCAGCACT GCCGGAAGCA GTTCAAGTCC AAAGCCGGCC TCAACTACCA CACTATGGCC GAGCACAGTG CCAAGCCCTC TGACGCCGAG GCCTCCGAAG
GGGGCGAGCA GGAGGAGCGC GAGAGGCTGC GCAAGGTGCT GAAGCAGATG
GGACGGCTGC GCTGCCCCCA GGAGGGTTGC GGGGCTGCCT TCTCCAGCCT
CATGGGCTAC CAGTACCACC AGCGGCGCTG CGGGAAGCCG CCCTGCGAGG
TGGACAGCCC CTCCTTCCCC TGCACCCACT GTGGCAAGAC GTACCGATCC AAGGCTGGCC ACGACTACCA CGTGCGCTCG GAGCACACGG CCCCCCCCCC
TGAGGAGCCC ACAGACAAGT CCCCTGAGGC TGAGGACCCG CTGGGTGTGG
AGCGGACCCC AAGCGGGCGT GTCCGCCGCA CGTCGGCCCA GGTGGCGGTG
TTCCACCTGC AGGAGATAGC GGAGGACGAG CTGGCCCGCG ACTGGACCAA
GCGGCGCATG AAGGATGACC TTGTGCCCGA GACCTCACAG CTCAACTACA CTCGACCAGG GCTCCCCACG CTGAACCCCC AGCTGCTAGA GGCATGGAAG
AATGAAGTGA AGGAGAAAGG CCACGTCAAC TGTCCCAACG ACTGCTGTGA
AGCCATCTAC TCCAGCGTGT CCGGACTCAA GGCTCATCTC GCCAGCTGCA
GTAAGGGGGC CCACCTGGCA GGGAAGTACC GCTGTCTGCT GTGTCCGAAG
GAGTTCAGTT CTGAGAGTGG CGTCAAATAC CACATCCTGA AGACCCACGC AGAGAACTGG TTCCGAACAT CAGCAGACCC ACCTCCCAAA CACAGGAGCC
AGGACTCATT GGTGCCCAAG AAGGAAAAGA AGAAAAATCT GGCAGGTGGA
AAGAAGCGGG GCCGAAAGCC CAAGGAGCGG ACCCCAGAGG AGCCTGTGGC
CAAGCTGCCC CCGCGCCGGG ACGACTGGCC TCCAGGATGC AGAGACAAGG
GGGCCCGGGG CTCCACCGGC CGGAAGGTGG GAGTCAGCAA GGCGCCTGAA AAGTGA (SEQ ID No.118)
Protein sequence
MPWRGGQTVPGQAPLCFDPGSPASDKTEGKKKGRPKAENQALRDIPLSLMNDWKDEFKA HSRVKCPNSGCWLEFPSIYGLKYHYQRCQGGAISDRLAFPCPFCEAAFTSKTQLEKHRIWN HMDRPLPASKPGPISRPVTISRPVGVSKPIGVSKPVTIGKPVGVSKPIGISKPVSVGRPMPVT r AIP\ TRPVPVTKP\ TVSRPMP KAMPVTKPITVTKSVPVTKPVP KPIT\ TKLVrVTKPVP VTKPVTVSRPIWSKPVTVSRPIAISRHTPPCKMVLLTRSENKAPRATGRNSGKKRAADSLD TCPIPPKQARPENGEYGPSSMGQSSAFQLSADTSSGSLSPGSRPSGGMEALKAAGPASPP EEDPERTKHRRKQKTPKKFTGEQPSISGTFGLKGLVKAEDKARVHRSKKQEGPGPEDARK KVPAAPITVSKEAPAPVAHPAPGGPEEQWQRAIHERGEAVCPTCNWTRKTLVGLKKHMEV CQKLQDALKCQHCRKQFKSKAGLNYHTMAEHSAKPSDAEASEGGEQEERERLRKVLKQM GRLRCPQEGCGAAFSSLMGYQYHQRRCGKPPCEVDSPSFPCTHCGKTYRSKAGHDYHVR SEHTAPPPEEPTDKSPEAEDPLGVERTPSGRVRRTSAQVAVFHLQEIAEDELARDWTKRR MKDDLVPETSQLNYTRPGLPTLNPQLLEAWKNEVKEKGHVNCPNDCCEAIYSSVSGLKAHL ASCSKGAHLAGKYRCLLCPKEFSSESGVKYHILKTHAENWFRTSADPPPKHRSQDSLVPKK EKKKNLAGGKKRGRKPKERTPEEPVAKLPPRRDDWPPGCRDKGARGSTGRKVGVSKAPE K (SEQ ID No.119) KIAA1196 interacts with Smadl a protein involved in the BMP TGFβ pathway
By two-hybrid screening in yeast it was shown that KIAA1196 interacts with Smadl , a protein involved in the BMP/TGFβ pathway. Smad 1-KIAA1196
SID : Nucleic sequence, SEQ ID No.35, 36, 37 and Proteic sequence, SEQ ID No. 73, 74, 75
Rebound screening experiments using truncated KIAA1196 as baits (hgx559v1: nt 1455- 2322 and hgx559v2: nt 1929-2499) on placenta library allowed us to confirm the Smadl - KIAA1196 interaction: KIAA1196-Smad1 Thus, yeast-two-hybrid screens showed that amino-acids 242-465 from Smadl (SEQ ID No.14) interact with amino-acids 643-774 from KIAA1196 (SEQ ID No.14) (see Fig. 22). II. KIAA1196 modulates the TGFβ signaling
It has been shown that TGFβ binds ALK1 (which induces phosphorylation of Smadl and 5) and ALKδ (which induces phosphorylation of Smad2 and 3) in transfected COS cells (Ten Dijke et al., 1994). In addition, recent studies have shown that TGFβ regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signaling (Goumans ef al., 2002). Since KIAA1196 was found interacting with Smadl, it was investigated whether KIAA1196 could be involved in the TGFβ and/or BMP pathways. In order to validate KIAA1196's involvement in the TGFβ/BMP pathways, KIAA1196 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and KIAA1196-targeting siRNA duplex, a specific, dose-dependant and TGFβ-dependant repression of the luciferase reporter activity was observed (see Fig. 23 A) demonstrating a function for KIAA1196 in the TGFβ pathway. The repressive effect of KIAA1196-targeting siRNA duplex was already observed at low concentration of siRNA duplex (4nM) and was further enhanced at higher concentrations (40nM). The same transient transfection experiments performed using p(GC)12-MLP-Luc reporter system and KIAA1196-targeting siRNA-mediated cellular knock-down did not show any impact on the BMP-specific reporter system using BMP6 to activate the pathway (see Fig. 23 B).
SiRNA-mediated KIAA1196 cellular knock-down were also performed in another cell type: HEK293 cells. A specific, dose-dependant and TGFβ-dependaπt repression of the p(GTCT)8-MLP-Luc reporter activity was also observed (see Fig. 24). The extend of the repression of the TGFβ-dependant reporter activity observed using KIAA1196-targeting siRNA duplex was almost as efficient as the repression obseved using the positive control (TβRI-targeting siRNA duplex). Modulation of the TGFβ luciferase reporter activity using KIAA1196 cellular knock-down demonstrated an essential implication of this putative transcription factor in the regulation of the TGFβ pathway.
In order to further elucidate KIAA1196's role on the expression of genes naturally controlled by TGFβ in mammalian cells, a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGFβ-dependant markers were performed. Endogenous levels of PAI-1 and junB mRNA were specifically and dose-dependently decreased following transient transfection of KIAA1196-targeting siRNA duplex in HepG2 cells treated with TGFβ (see Fig. 25 A & B). As expected, endogenous alkaline phosphatase mRNA levels were not stimulated following BMP7 treatment and thus were not affected by KIAA1196-targeting siRNA (see Fig. 25 C). Expression levels of various controls were not affected at all following the same KIAA 196-targeting siRNA duplex transfection: hGUS ( see Fig. 25 D), HPRT, GAPDH and 18S ribosomal RNA ( data not shown). Endogenous levels of alkaline phosphatase mRNA were barely affected (only a slight decrease) following transient transfection of KIAA1196-targeting siRNA duplex in BMP7 treated HepG2 cells (see Fig. 25 C). Note however that the endogenous levels of TGFβ- induced alkaline phosphatase mRNA is strongly repressed following transient transfection of KIAA1 96-targeting siRNA duplex in TGFβ treated HepG2 cells (see Fig. 25 C). Example 12 : LM04 (hgx561) GI: 1914876
LlM-only proteins are transcriptional regulators that function by mediating protein- protein interactions and include the T cell oncogenes LM01 and LM02. By screening expression libraries with the LIM interaction domain of NL1/CLIM2/LDB1 , Kenny ef a/. (1998) isolated and characterized LM04, a novel LlM-only gene. The LM04 gene was further characterized in terms of genomic organization and comparative chromosomal mapping (Tse ef al., 1999). LM04 was found to be a candidate gene associated with prostate cancer progression since LM04 was down-regulated in prostate cancer (Mousses ef al., 2001). In addition, the LMO4 mRNA is over-expressed in human breast cancer cell lines (5 out of 10) and in situ hybridization analysis of 177 primary invasive breast carcinomas revealed over- expression of LM04 in 66% of specimens (Visvader et al., 2001). Finally, a recent paper describes an interaction between BRCA1 and LM04. In functional assays, LM04 was shown to repress BRCA1 -mediated transcriptional activation in mammalian cells, suggesting a role for LM04 as a repressor of BRCA1 activity in breast tissue (Sum ef al., 2002). However, no link between LM04 and the TGFβ/BMP pathways was previously made. Nucleic acid sequence
ATGGTGAATC CGGGCAGCAG CTCGCAGCCG CCCCCGGTGA CGGCCGGCTC
CCTCTCCTGG AAGCGGTGCG CAGGCTGCGG GGGCAAGATT GCGGACCGCT
TTCTGCTCTA TGCCATGGAC AGCTATTGGC ACAGCCGGTG CCTCAAGTGC
TCCTGCTGCC AGGCGCAGCT GGGCGACATC GGCACGTCCT GTTACACCAA
AAGTGGCATG ATCCTTTGCA GAAATGACTA CATTAGGTTA TTTGGAAATA
GCGGTGCTTG CAGCGCTTGC GGACAGTCGA TTCCTGCGAG TGAACTCGTC
ATGAGGGCGC AAGGCAATGT GTATCATCTT AAGTGTTTTA CATGCTCTAC
CTGCCGGAAT CGCCTGGTCC CGGGAGATCG GTTTCACTAC ATCAATGGCA
GTTTATTTTG TGAACATGAT AGACCTACAG CTCTCATCAA TGGCCATTTG
AATTCACTTC AGAGCAATCC ACTACTGCCA GACCAGAAGG TCTGCTAA (SEQ ID No.120)
Protein sequence MVNPGSSSQPPPVTAGSLSWKRCAGCGGKIADRFLLYAMDSYWHSRCLKCSCCQAQLGD IGTSCYTKSGMILCRNDYIRLFGNSGACSACGQSIPASELVMRAQGNVYHLKCFTCSTCRN RLVPGDRFHYINGSLFCEHDRPTALINGHLNSLQSNPLLPDQKVC (SEQ ID No.121)
LM04 interacts with Smad9 a protein involved in the BMP pathway By two-hybrid screening in yeast it was shown that LM04 interacts with Smad9, a protein involved in the BMP pathway.
Smad9-LMQ4
SID : Nucleic sequence, SEQ ID No.44 and Proteic sequence, SEQ ID No. 82 SID : Nucleic sequence, SEQ ID No.46 and Proteic sequence, SEQ ID No. 84 Thus, yeast-two-hybrid screens showed that amino-acids 209-430 from Smad9 (SEQ ID No.20) (aa 209-430) interact with amino-acids 7-126 from LM04 (SEQ ID No.121) (see Fig. 26). II. LM04 modulates BMP signaling
In order to assay LM04's functional involvement in the TGFβ/BMP pathways, LM04 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC)ι2-MLP-Luc reporter and LM04-targeting siRNA duplex, a specific, dose-dependant and BMP7-dependant repression of the BMP-dependant reporter activity was observed (see Fig. 27 A) suggesting a general function for LM04 in the response to the BMP7 pathway. Almost similar results were obtained in HepG2 cells using BMP6 instead of BMP7, further reinforcing the BMP-dependant effect of LM04 siRNA in these cells (see Fig. 27 B).The repressive effect of LM04-targeting siRNA duplex was observed at low concentration of siRNA duplex (4nM) and was further enhanced at higher concentrations (40nM) for both BMP7 and BMP6 (Fig. 27 A & B).
This effect was shown to be specific and restricted to the BMP pathway since LM04 did not show any effect on the TGFβ signaling either at 4 or 40 nM of siRNA duplex (see Fig. 27 C). Modulation of the BMP-specific luciferase reporter activity using LM04 cellular knockdown demonstrates the implication of this putative transcription factor in the regulation of the BMP pathway in HepG2 cells.
In order to further elucidate LM04's role on the expression of genes naturally controlled by TGFβ and/or BMPs in cells, we performed similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGFβ or BMP-dependant markers. Endogenous levels of alkaline phosphatase mRNA (see Fig. 28 A & B) were specifically and dose-dependently decreased following transient transfection of LM04-targeting siRNA duplex in HepG2 cells treated with BMP7 demonstrating the role played by LM04 in the BMP pathway. However, endogenous levels of junB were not affected at all following transient transfection of LM04-targeting siRNA duplex in HepG2 cells treated with BMP7 (Fig. 29 A). As expected, endogenous PAI-1 mRNA levels were not affected following the same transfection experiments induced by BMP7 ( see Fig. 29 C). Expression levels of various controls were not affected at all following the same LM04-targeting siRNA duplex transfection: hGUS see Fig. 29 B) HPRT, GAPDH and 18S ribosomal RNA (data not shown). Example 13 : PPIca (hgx591) GI: 4506002
Protein phosphatase 1 (PP1) is a major eukaryotic protein serine/threoπine phosphatase that regulates an enormous variety of cellular functions through the interaction of its catalytic subunit (PP1c) with over fifty different established or putative regulatory subunits (see for review; Cohen, 2002). Most of these target PP1c to specific sub-cellular locations and interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif - the RVxF motif - which is often preceded by further basic residues. Recently, Bennett and Alphey (2002) showed that PP1 binds SARA and negatively regulates Dpp signaling in Drosophila melanogaster. Using SARA mutant defective for PP1c binding, they demonstrated that the absence of such interaction resulted in increased expression of TGFβ-reporter gene through increased phosphorylation of type I receptor in the absence of TGFβ. Nucleic acid sequence ATGTCCGACA GCGAGAAGCT CAACCTGGAC TCGATCATCG GGCGCCTGCT
GGAAGTGCAG GGCTCGCGGC CTGGCAAGAA TGTACAGCTG ACAGAGAACG
AGATCCGCGG TCTGTGCCTG AAATCCCGGG AGAI I I I TCT GAGCCAGCCC
ATTCTTCTGG AGCTGGAGGC ACCCCTCAAG ATCTGCGGTG ACATACACGG
CCAGTACTAC GACCTTCTGC GACTATTTGA GTATGGCGGT TTCCCTCCCG
AGAGCAACTA CCTCTTTCTG GGGGACTATG TGGACAGGGG CAAGCAGTCC
TTGGAGACCA TCTGCCTGCT GCTGGCCTAT AAGATCAAGT ACCCCGAGAA
CTTCTTCCTG CTCCGTGGGA ACCACGAGTG TGCCAGCATC AACCGCATCT
ATGGTTTCTA CGATGAGTGC AAGAGACGCT ACAACATCAA ACTGTGGAAA
ACCTTCACTG ACTGCTTCAA CTGCCTGCCC ATCGCGGCCA TAGTGGACGA
AAAGATCTTC TGCTGCCACG GAGGCCTGTC CCCGGACCTG CAGTCTATGG
AGCAGATTCG GCGGATCATG CGGCCCACAG ATGTGCCTGA CCAGGGCCTG
CTGTGTGACC TGCTGTGGTC TGACCCTGAC AAGGACGTGC AGGGCTGGGG
CGAGAACGAC CGTGGCGTCT CI ΓΓACCTT TGGAGCCGAG GTGGTGGCCA
AGTTCCTCCA CAAGCACGAC TTGGACCTCA TCTGCCGAGC ACACCAGGTG
GTAGAAGACG GCTACGAGTT CT ΓGCCAAG CGGCAGCTGG TGACACTTTT
CTCAGCTCCC AACTACTGTG GCGAG1 ΓGA CAATGCTGGC GCCATGATGA
GTGTGGACGA GACCCTCATG TGCTCTTTCC AGATCCTCAA GCCCGCCGAC
AAGAACAAGG GGAAGTACGG GCAGTTCAGT GGCCTGAACC CTGGAGGCCG ACCCATCACC CCACCCCGCA ATTCCGCCAA AGCCAAGAAA TAG (SEQ ID N0122)
Protein sequence MSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFLSQPILLELEAPLKICGDI HGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNHEC ASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGGLSPDLQSMEQIRRIMR PTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEWAKFLHKHDLDLICRAHQW EDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPADKNKGKYGQFSG LNPGGRPITPPRNSAKAKK (SEQ ID No.123)
PPIca interacts with SARA, a protein involved in the TGFβ pathway By two-hybrid screening in yeast it was shown that PPIca interacts with SARA, a protein involved in the TGFβ pathway.
SARA-PP1ca SID : Nucleic sequence, SEQ ID No.56, 57,58, 59 and Proteic sequence, SEQ ID No. 94, 95, 96, 97.
In addition, these screens using SARA as bait gave the two additional isoformε of PP1c: PP1cb an PP1cc. Rebound screening experiments using PPIca as bait (hgx591v2: nt 1-972) on a placenta library allowed us to confirm the SARA-PP1ca interaction.
PP1ca-SARA Thus, yeast-two-hybrid screens showed that amino-acids 668-947 from SARA (SEQ ID No.23) interact with amino-acids 29-296 from PPI ca (SEQ ID No.123) (see Fig. 30). PPIca is a regulator of the TGFβ signaling
The two-hybrid screening results led the involvement of the catalytic subunit of the serine/threonine phosphatase (PP1 c) protein in the TGFβ pathway. Sequence analysis of SARA revealed the presence of the RVxF motif in the C-terminal part of SARA found to interact with PP1c (aa 668-947). To show a functional involvement of PPIca in the TGFβ pathway, the PPIca c-DNA (nt 1-972) was cloned into the pV3 vector and used in our TGFβ reporter assay (cf Materials & Methods).
Over-expression of PPIca at several amounts (10, 50 and 200 ng of pV3-PP1 ca) results in a 3.5-fold increase and a 6-fold increase of TGFβ signaling in HepG2 and HEK293 cells, respectively (Figure 31 A & B, respectively). This PPIca effect was not observed when the BMP signaling and the pGL3-control was tested thus showing a reproducible and specific effect of PPIca on the TGFβ pathway (Figure 31 A & B respectively; right panels).
To confirm this finding, a Baculovirus over-expressing the smad3 protein (as positive control) and the PPI ca protein was generated. This baculovirus expression system has been genetically engineered to allow infection and expression in mammalian cells (see material & methods). Both viruses were used to infect the HepG2 cells for 24 hours with or without TGFβ. First, the over-expression level of our proteins of interest by Q-PCR experiments was checked. In these conditions, the Smad3 and PPIca mRNA were shown to be over- expressed by a 350-fold and 50-fold, respectively, as compared to the endogenous mRNA level (Figure 32 A). Next, the endogenous PAI-1 and JunB mRNA levels was looked at, which were previously shown to be up-regulated by TGF . In the case of PAI-1 expression, in the absence of TGFβ, a 5-fold induction by Smad3 and a 2.5-foid induction by PPIca (Figure 32 B, left panel) was observed. In the presence of TGFβ, a 2.δ-fold induction by Smad3 but no effect of PPIca (Figure 32 B, right panel) was observed. Concerning the Jun- B expression, a 7-fold induction by smad3 was observed in the absence of TGFβ (data not shown). However, no effect was observed following PPIca over-expression. This result suggests that PPIca is involved in regulation of PAI-1 expression. Example 14 : HYPA (hgx530) GI: 3341989 Huntington's disease, with its hallmark choreiform movements and graded loss of striatal neurons, is a dominantly inherited disorder caused by expansion of a CAG repeat in one copy of the HD gene. The HD mutation elongates an N-terminal glutamine segment in the huntingtin protein. HYPA, HYPB and HYPC were found to interact with the huntingtin protein (Faber ef al., 1998). HYPA is a protein containing a WW domain, known to bind proiin-rich peptides stretches. This protein is the human homolog of the essential pre-mRNA splicing factor PrP40 and is also called FBP11. Modification of mutant huntingtin in target neurons may promote an abnormal interaction with one, or all, huntingtin's WW domain partners, perhaps altering ribonucleo-protein function with toxic consequences (Passani ef al., 2000). In addition, HYPA contains a FF domain, with a structure recently determined, which is a 60 amino acid residue phosphopeptide-binding module (Allen ef al., 2002). However, no link between HYPA and the TGFβ/BMP pathway was previously made. Nucleic acid sequence
CTGAGCCCGA CGATGAGGCC GGGGACGGGA GCTGAGCGTG GAGGCCTCAT
GGTGAGTGAA ATGGAGAGCC ATCCTCCCTC GCAGGGTCCT GGGGACGGGG
AGCGGAGATT GTCCGGCTCA AGCCTCTGCT CCGGCTCTTG GGTCTCTGCT
GACGGCTTCC TGAGGAGACG GCCCTCGATG GGGCACCCTG GCATGCATTA
TGCCCCAATG GGAATGCACC CTATGGGTCA GAGAGCGAAT ATGCCTCCTG
TACCTCATGG AATGATGCCG CAGATGATGC CCCCTATGGG AGGGCCACCA
ATGGGACAAA TGCCTGGAAT GATGTCGTCA GTAATGCCTG GAATGATGAT
GTCTCATATG TCTCAGGCTT CCATGCAGCC TGCCTTACCG CCAGGAGTAA
ATAGTATGGA TGTAGCAGCA GGTACAGCAT CTGGTGCAAA ATCAATGTGG
ACTGAACATA AATCACCTGA TGGAAGGACT TACTACTACA ACACTGAAAC
CAAACAGTCT ACCTGGGAGA AACCAGATGA TCTTAAAACA CCTGCTGAGC
AACTCTTATC TAAATGCCCC TGGAAGGAAT ACAAATCAGA TTCTGGAAAG
CCTTACTATT ATAATTCTCA AACAAAAGAA TCTCGCTGGG CCAAACCTAA
AGAACTTGAG GATCTTGAAG GATACCAGAA TACCATTGTT GCTGGAAGTC
TTATTACAAA ATCAAACCTG CATGCAATGA TCAAAGCTGA AGAAAGCAGT
AAGCAAGAAG AGTGCACCAC AACATCAACA GCCCCAGTCC CTACAACAGA
AATTCCGACC ACAATGAGCA CCATGGCTGC TGCCGAAGCA GCAGCTGCTG
TTGTTGCAGC AGCAGCAGCG GCAGCAGCAG CAGCAGCTGC AGCCAATGCT
AATGCTTCCA CTTCTGCTTC TAATACTGTC AGTGGAACTG TTCCAGTTGT
TCCTGAGCCT GAAGTTACTT CCATTGTTGC TACTGTTGTA GATAATGAGA
ATACAGTAAC TATTTCAACT GAGGAACAAG CACAACTTAC TAGTACCCCT
GCTATTCAGG ATCAAAGTGT GGAAGTATCC AGTAATACTG GAGAAGAAAC
ATCTAAGCAA GAAACTGTAG CTGATTTTAC TCCCAAAAAA GAAGAGGAGG
AGAGCCAACC AGCAAAGAAA ACATACACTT GGAATACAAA GGAAGAGGCA
AAGCAAGCTT TTAAAGAATT ATTGAAAGAA AAGCGGGTAC CATCGAATGC
TTCATGGGAG CAGGCTATGA AAATGATTAT TAATGATCCA CGATACAGTG CTTTGGCAAA CTTAAGTGAA AAAAAGCAAG CCTTTAATGC CTATAAAGTC CAGACAG
(SEQ ID No.124)
Protein sequence LSPTMRPGTGAERGGLMVSEMESHPPSQGPGDGERRLSGSSLCSGSWVSADGFLRRRP SMGHPGMHYAPMGMHPMGQRANMPPVPHGMMPQMMPPMGGPPMGQMPGMMSSVMP GMMMSHMSQASMQPALPPGVNSMDVAAGTASGAKSMWTEHKSPDGRTYYYNTETKQST WEKPDDLKTPAEQLLSKCPWKEYKSDSGKPYYYNSQTKESRWAKPKELEDLEGYQNTIVA GSLITKSNLHAMIl AEESSKQEECTTTSTAPVPTTEIPTTMSTMAAAEAAAAVVAAAAAAAAA AAAANANASTSASNTVSGTVPWPEPEVTSIVATWDNENTVTISTEEQAQLTSTPAIQDQSV EVSSNTGEETSKQE ADFTPKKEEEESQPAKKTYTWNTKEEAKQAFKELLKEKRVPSNAS WEQAMKMIINDPRYSALANLSEKKQAFNAYKVQT (SEQ ID No.125)
HYPA interacts with Smad4, a protein involved in the TGFβ/ BMP pathway
By two-hybrid screening in yeast it was shown that HYPA interacts with Smad4, a protein involved in the TGFβ/BMP pathway. Smad4-HYPA SID : Nucleic sequence, SEQ ID No.39, 40, 41 and Proteic sequence, SEQ ID No. 77, 78, 79. Thus, yeast-two-hybrid screens showed that amino-acids 251-552 from Smad4 (SEQ ID No.17) interact with amino-acids 276-387 from HYPA (SEQ ID No.123) (see Fig. 33). HYPA is a regulator of the TGFβ signaling
Since HYPA was found interacting with Smad4, it was investigated whether HYPA could be involved in the TGFβ and/or BMP pathways. In order to assay HYPA's functional involvement in the TGFβ/BMP pathways, HYPA cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC)8-MLP-Luc reporter and HYPA-targeting siRNA duplex, a specific dose-dependant repression of the BMP-dependant reporter activity was observed (see Fig. 34 A) demonstrating a function for HYPA in the response to the BMP pathway. Similar results were also obtained using either BMP6 or BMP7 (see Fig. 34 A & B). The repressive effect of HYPA-targeting siRNA duplex was observed at low concentration of siRNA duplex (4nM) and was enhanced at higher concentrations (40nM). While transiently co-transfecting HepG2 cells using the p(GTCT)ι2-MLP-Luc reporter and HYPA-targeting siRNA duplex, no repression of the TGFβ-dependant reporter activity was observed (see Fig. 34 C) demonstrating a restricted function for HYPA in the response to the BMP pathway. In order to further elucidate HYPA's role on the expression of genes naturally controlled by BMPs in mammalian cells, a similar siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of BMP-dependant markers was performed.
Endogenous levels of alkaline phosphatase mRNA were specifically and dose- dependently decreased following transient transfection of HYPA-targeting siRNA duplex in HepG2 cells treated with BMP7 (see Fig. 35) demonstrating HYPA's role in the BMP pathway. Expression levels of various controls were not affected at all following the same HYPA-targeting siRNA duplex transfection: hGUS, HPRT, GAPDH and 18S ribosomal RNA (data not shown).
Example 15 : FLJ20037 (hgx594) GI: 8923041
Lagali ef al. (2002) have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), also named FLJ20037, that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. The N-terminal part of this protein contains some glycine- rich repeats. However, no link between FLJ20037 and the TGFβ/BMP pathway was previously made.
Nucleic acid sequence ATGGCGGAGG GTGAAGGGTA CTTCGCCATG TCTGAGGACG AGCTGGCCTG CAGCCCCTAC ATCCCCCTAG GCGGCGACTT CGGCGGCGGC GACTTCGGCG
GCGGCGACTT CGGCGGCGGC GACTTCGGCG GCGGCGACTT CGGCGGTGGC
GGCAGCTTCG GTGGGCATTG CTTGGACTAT TGCGAAAGCC CTACGGCGCA CTGCAATGTG CTGAACTGGG AGCAAGTGCA GCGGCTGGAC GGCATCCTGA GTGAGACCAT TCCGATTCAC GGGCGCGGCA ACTTCCCCAC GCTCGAGCTG CAGCCGAGCC TGATCGTGAA GGTGGTGCGG CGGCGCCTGG CCGAGAAGCG CATTGGCGTC CGCGACGTGC GCCTCAACGG CTCGGCAGCC AGCCATGTCC TGCACCAGGA CAGCGGCCTG GGCTACAAGG ACCTGGACCT CATCTTCTGC
GCCGACCTGC GCGGGGAAGG GGAGTTTCAG ACTGTGAAGG ACGTCGTGCT
GGACTGCCTG TTGGACTTCT TACCCGAGGG GGTGAACAAA GAGAAGATCA
CACCACTCAC GCTCAAGGAA GCTTATGTGC AGAAAATGGT TAAAGTGTGC
AATGACTCTG ACCGATGGAG TCTTATATCC CTGTCAAACA ACAGTGGCAA
AAATGTGGAA CTGAAATTTG TGGATTCCCT CCGGAGGCAG TTTGAATTCA GTGTAGATTC TTTTCAAATC AAATTAGACT CTCTTCTGCT CTTTTATGAA
TGTTCAGAGA ACCCAATGAC TGAGACATTT CACCCCACAA TAATCGGGGA
GAGCGTCTAT GGCGATTTCC AGGAAGCCTT TGATCACCTT TGTAACAAGA
TCATTGCCAC CAGGAACCCA GAGGAAATCC GAGGGGGAGG CCTGCTTAAG
TACTGCAACC TCTTGGTGAG GGGCTTTAGG CCCGCCTCTG ATGAAATCAA
GGCCCTTCAA AGGTACATGT GTTCCAGGTT TTTCATCGAC TTCTCAGACA
TTGGAGAGCA GCAGAGAAAA CTGGAGTCCT ATTTGCAGAA CCACTTTGTG
GGATTGGAAG ACCGCAAGTA TGAGTATCTC ATGACCCTTC ATGGAGTGGT
AAATGAGAGC ACAGTGTGCC TGATGGGACA TGAAAGAAGA CAGAC1 AA
ACCTTATCAC CATGCTGGCT ATCCGGGTGT TAGCTGACCA AAATGTCATT
CCTAATGTGG CTAATGTCAC TTGCTATTAC CAGCCAGCCC CCTATGTAGC
AGATGCCAAC TTTAGCAATT ACTACATTGC ACAGGTTCAG CCAGTATTCA CGTGCCAGCA ACAGACCTAC TCCACTTGGC TACCCTGCAA TTAA (SEQ ID No.124)
Protein sequence MAEGEGYFAMSEDELACSPYIPLGGDFGGGDFGGGDFGGGDFGGGDFGGGGSFGGHCL DYCESPTAHCNVLNWEQVQRLDGILSETIPIHGRGNFPTLELQPSLIVKWRRRLAEKRIGVR DVRLNGSAASHVLHQDSGLGYKDLDLIFCADLRGEGEFQTVKDWLDCLLDFLPEGVNKEKI TPLTLKEAYVQKMVKVCNDSDRWSLISLSNNSGKNVELKFVDSLRRQFEFSVDSFQIKLDSL LLFYECSENPMTETFHPTIIGESVYGDFQEAFDHLCNKIIATRNPEEIRGGGLLKYCNLLVRGF RPASDEIKALQRYMCSRFFIDFSDIGEQQRKLESYLQNHFVGLEDRKYEYLMTLHGWNEST VCLMGHERRQTLNLITMLAIRVLADQNVIPNVANVTCYYQPAPYVADANFSNYYIAQVQPVF TCQQQTYSTWLPCN (SEQ ID No.125)
FLJ20037 interacts with SARA, a protein involved in the TGFβ pathway By two-hybrid screening in yeast it was shown that FLJ20037 interacts with SARA, a protein involved in the TGFβ pathway.
SARA-FLJ20037 SID : Nucleic sequence, SEQ ID No.60, 61 and Proteic sequence, SEQ ID No. 98, 99.
Thus, yeast-two-hybrid screens showed that amino-acids 665-1323 from SARA (SEQ ID No.23) interact with amino-acids 58-253 from FLJ20037 (SEQ ID No.125) (see Fig. 36). FLJ20037 modulates the TGFβ signaling
Since FLJ20037 was found as interacting with SARA, it was investigated whether
FLJ20037 could be involved in the TGFβ and/or BMP pathways. To test this, baculoviruses over-expressing the smad3 protein (as positive control) and the FLJ20037 protein were generated. Both viruses were used to infect the HepG2 cells during 24 hours, treated or not with TGFβ. First, the over-expression level of our proteins of interest by Q-PCR experiments was checked. Smad3 and FLJ20037 mRNA were shown to be over-expressed 350-fold and 200- fold, respectively, when compared to their respective endogenous mRNA levels (Figure 37 A). Next, endogenous PAI-1 and JunB mRNA levels were looked at, which were previously shown to be up-regulated by TGFβ. In the case of PAI-1 expression, in the absence of TGFβ, a 5-fold induction by smad3 and a 3.5-fold induction by FLJ20037 were observed (Figure 37 B, left panel). In the presence of TGFβ, we observed a 2.5-fold induction by Smad3 and a 2- fold induction by FLJ20037 (Figure 37 B, right panel). Concerning the Jun-B expression, a 7- fold induction by smad3 was observed in the absence of TGFβ (data not shown). However, no effect on junB expression was observed following FLJ20037 over-expression (data not shown). These results suggested that FLJ20037 was involved in the regulation of PAI-1 expression.
In order to further elucidate FLJ20037's role on the expression of genes naturally controlled by TGFβ in mammalian cells, siRNA-mediated knock-down experiments followed by quantitative PCR (Q-PCR) analysis of TGFβ-dependant markers were performed (see Materials & Methods for siRNA sequences and protocols). Endogenous levels of PAI-1 mRNA were specifically decreased following transient transfection of FLJ20037-targeting siRNA duplex in HepG2 cells treated with TGFβ (see Fig. 38). Expression levels of various controls were not affected at all following the same HYPA-targeting siRNA duplex transfection: hGUS, HPRT, GAPDH and 18S ribosomal RNA (data not shown). Example 16-PTPN12 GI: 18375651
Protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) are involved in the regulation of tyrosine phosphorylation-mediated signaling. Such signaling is critical for the regulation of cell proliferation, differentiation, and neoplastic transformation. Tyrosine-phosphorylated proteins can be specifically dephosphorylated through the action of PTPs, which therefore are likely to have as important a role as PTKs in the control of cellular growth and differentiation. Given that hyperphosphorylation of protein tyrosine residues can cause cell transformation, it is plausible that lack of dephosphorylation resulting from loss of PTP function may also wreak an oncogenic effect. Intracellular PTPs are candidates for tumor suppressor genes. From an adult cDNA library, Takekawa et al. (1992) isolated a cDNA encoding a predicted 88-kD protein and Yang et al. (1993) isolated a virtually identical gene from HeLa cell extracts. The protein was designated protein tyrosine phosphatase G1 (PTPG1) or PTPN12 or PTP-PEST. Cong et al. (2000) showed that PSTPIP1 bridges ABL to the PEST-type PTPs. Several experiments suggested that the PEST-type PTPs negatively regulate ABL activity: ABL was hyperphosphorylated in PTP-PEST-deficient cells; disruption of the ABL-PSTPIP1 -PEST-type PTP ternary complex by overexpression of mutants increased ABL phosphotyrosine content; and PDGF-induced ABL kinase activation was prolonged in PTP-PEST-deficient cells. The authors concluded that dephosphorytation of ABL by PSTPIP1 -directed PEST-type PTPs represents a novel mechanism by which ABL activity is regulated. Charest et al. (1996) determined that the mouse PTP 12 gene contains 18 exons spanning about 90 kb of DNA. By fluorescence in situ hybridization (FISH), Takekawa et al. (1994) mapped the PTPN12 gene to 7q11.23. Charest et al. (1996) used FISH to map the mouse Ptpn12 gene to chromosome 6A3 to B, a region with homology of synteny to human chromosome 7q11.23. The potential importance of PTPG1 in tumorigenesis was investigated by Takekawa et al. (1994), who sought abnormalities of the PTPG1 transcript in various human cancer cell lines by use of RT-PCR. In a colorectal carcinoma cell line, DLD-1, they found 3 aberrant transcripts (Sequencing in one demonstrated an A-to-G transition at nucleotide 201 , predicting a change of codon 61 from lysine to arginine): a missense point mutation, a 77-bp deletion, and a 173-bp deletion. However, no link between HIPK3 and the TGF /BMP pathway was previously made. Nucleic acid sequence
ATGGAGCAAG TGGAGATCCT GAGGAAATTC ATCCAGAGGG TCCAGGCCAT
GAAGAGTCCT GACCACAATG GGGAGGACAA CTTCGCCCGG GACTTCATGC
GGTTAAGAAG ATTGTCTACC AAATATAGAA CAGAAAAGAT ATATCCCACA
GCCACTGGAG AAAAAGAAGA AAATGTTAAA AAGAACAGAT ACAAGGACAT
ACTGCCATTT GATCACAGCC GAGTTAAATT GACATTAAAG ACTCCTTCAC
AAGATTCAGA CTATATCAAT GCAAAI I I IA TAAAGGGCGT CTATGGGCCA
AAAGCATATG TAGCAACTCA AGGACCTTTA GCAAATACAG TAATAGATTT
TTGGAGGATG ATATGGGAGT ATAATGTTGT GATCATTGTA ATGGCCTGCC
GAGAATTTGA GATGGGAAGG AAAAAATGTG AGCGCTATTG GCC I I I GTAT
GGAGAAGACC CCATAACGTT TGCACCATTT AAAATTTCTT GTGAGGATGA
ACAAGCAAGA ACAGACTACT TCATCAGGAC ACTCTTACTT GAA I I I CAAA
ATGAATCTCG TAGGCTGTAT CAG I I I CATT ATGTGAACTG GCCAGACCAT
GATGTTCCTT CATCATTTGA TTCTATTCTG GACATGATAA GCTTAATGAG
GAAATATCAA GAACATGAAG ATGTTCCTAT TTGTATTCAT TGCAGTGCAG
GCTGTGGAAG AACAGGTGCC ATTTGTGCCA TAGATTATAC GTGGAAI I I A
CTAAAAGCTG GGAAAATACC AGAGGAATTT AATGTATTTA ATTTAATACA
AGAAATGAGA ACACAAAGGC ATTCTGCAGT ACAAACAAAG GAGCAATATG
AACTTGTTCA TAGAGCTATT GCCCAACTGT TTGAAAAACA GCTACAACTA
TATGAAATTC ATGGAGCTCA GAAAATTGCT GATGGAGTGA ATGAAATTAA
CACTGAAAAC ATGATCAGCT CCATAGAGCC TGAAAAACAA GATTCTCCTC
CTCCAAAACC ACCAAGGACC CGCAGTTGCC TTGTTGAAGG GGATGCTAAA
GAAGAAATAC TGCAGCCACC GGAACCTCAT CCAGTGCCAC CCATCTTGAC ACCTTCTCCC CCTTCAGCTT TTCCAACAGT CACTACTGTG TGGCAGGACA
ATGATAGATA CCATCCAAAG CCAGTGTTGC ATATGGTTTC ATCAGAACAA
CATTCAGCAG ACCTCAACAG AAACTATAGT AAATCAACAG AACTTCCAGG
GAAAAATGAA TCAACAATTG AACAGATAGA TAAAAAATTG GAACGAAATT
TAAGTTTTGA GATTAAGAAG GTCCCTCTCC AAGAGGGACC AAAAAGTTTT
GATGGGAACA CACTTTTGAA TAGGGGACAT GCAATTAAAA TTAAATCTGC
TTCACCTTGT ATAGCTGATA AAATCTCTAA GCCACAGGAA TTAAGTTCAG
ATCTAAATGT CGGTGATACT TCCCAGAATT CTTGTGTGGA CTGCAGTGTA
ACACAATCAA ACAAAGTTTC AGTTACTCCA CCAGAAGAAT CCCAGAATTC
AGACACACCT CCAAGGCCAG ACCGCTTGCC TCTTGATGAG AAAGGACATG
TAACGTGGTC ATTTCATGGA CCTGAAAATG CCATACCCAT ACCTGATTTA
TCTGAAGGCA ATTCCTCAGA TATCAACTAT CAAACTAGGA AAACTGTGAG
TTTAACACCA AGTCCTACAA CACAAGTTGA AACACCTGAT CTTGTGGATC
ATGATAACAC TTCACCACTC TTCAGAACAC CCCTCAGTTT TACTAATCCA CTTCACTCTG ATGACTCAGA CTCAGATGAA AGAAACTCTG ATGGTGCTGT GACCCAGAAT AAAACTAATA TTTCAACAGC AAGTGCCACA GTTTCTGCTG CCACTAGTAC TGAAAGCATT TCTACTAGGA AAGTATTGCC AATGTCCATT GCTAGACATA ATATAGCAGG AACAACACAT TCAGGTGCTG AAAAAGATGT TGATGTTAGT GAAGATTCAC CTCCTCCCCT ACCTGAAAGA ACTCCTGAAT CGTTTGTGTT AGCAAGTGAA CATAATACAC CTGTAAGATC GGAATGGAGT GAACTTCAAA GTCAGGAACG ATCTGAACAA AAAAAGTCTG AAGGCTTGAT AACCTCTGAA AATGAGAAAT GTGATCATCC AGCGGGAGGT ATTCACTATG
AAATGTGCAT AGAATGTCCA CCTACTTTCA GTGACAAGAG AGAACAAATA TCAGAAAATC CAACAGAAGC CACAGATATT GGTTTTGGTA ATCGATGTGG AAAACCCAAA GGACCAAGAG ATCCACCTTC AGAATGGACA TGA (SEQ ID No.126)
Protein sequence MEQVEILRKFIQRVQAMKSPDHNGEDNFARDFMRLRRLSTKYRTEKIYPTATGEKEENVKK NRYKDILPFDHSRVKLTLKTPSQDSDYINANFIKGVYGPKAYVATQGPLANTVIDFWRMIWE YNWIIVMACREFEMGRKKCERYWPLYGEDPITFAPFKISCEDEQARTDYFIRTLLLEFQNES RRLYQFHYVNWPDHDVPSSFDSILDMISLMRKYQEHEDVPICIHCSAGCGRTGAICAIDYTW NLLKAGKIPEEFNVFNLIQEMRTQRHSAVQTKEQYELVHRAIAQLFEKQLQLYEIHGAQKIAD GVNEINTENMISSIEPEKQDSPPPKPPRTRSCLVEGDAKEEILQPPEPHPVPPILTPSPPSAF PTVTTVWQDNDRYHPKPVLHMVSSEQHSADLNRNYSKSTELPGKNESTIEQIDKKLERNLS FEIKKVPLQEGPKSFDGNTLLNRGHAIKIKSASPCIADKISKPQELSSDLNVGDTSQNSCVDC SVTQSNKVSVTPPEESQNSDTPPRPDRLPLDEKGHVTWSFHGPENAIPIPDLSEGNSSDINY QTRKTVSLTPSPTTQVETPDLVDHDNTSPLFRTPLSFTNPLHSDDSDSDERNSDGAVTQNK TNISTASATVSAATSTESISTRKVLPMSIARHNIAGTTHSGAEKDVDVSEDSPPPLPERTPES FVLASEHNTPVRSEWSELQSQERSEQKKSEGLITSENEKCDHPAGGIHYEMCIECPPTFSD KREQISENPTEATDIGFGNRCGKPKGPRDPPSEWT (SEQ ID No.127)
PTPN12 interacts with Smad5, a protein involved in the BMP pathway
By two-hybrid screening in yeast it was shown that PTPN12 interacts with Smad5, a protein involved in the BMP pathway. Smad5-PTPN12 SID : Nucleic sequence, SEQ ID No.43 and Proteic sequence, SEQ ID No. 81. Thus, yeast-two-hybrid screens showed that amino-acids 1-268 from Smadδ (SEQ ID No.19) interact with amino-acids 99-337 from PTPN12 (SEQ ID No.127) (see Fig. 39). PTPN12 modulates the TGFβ and BMP signaling
Since PTPN12 was found interacting with Smadδ, it was investigated whether PTPN12 could be involved in the TGFβ and/or BMP pathways. In order to assay PTPN12's functional involvement in the TGFβ/BMP pathways, PTPN12 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC)12-MLP-Luc reporter and PTPN12-targeting siRNA duplex, a specific BMP6- dependant increase in the BMP-dependant reporter activity was observed (see Fig. 40 A) demonstrating a role for PTPN12 in the response to the BMP pathway. The positive effect of PTPN12-targeting siRNA duplex was already observed at low concentration of siRNA duplex (4nM). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and PTPN12-targeting siRNA duplex, a TGFβ-dependant increase in the reporter activity was also observed (see Fig. 40 B) demonstrating a specific function for PTPN12 on the response to both the TGFβ and BMP pathways. Modulation of the TGF and BMP luciferase reporter activities using PTPN12 cellular knock-down shows its an implication in the regulation of both pathway. Example 17-HIPK3 GI: 11386208
Recently was identified a 130-kD kinase designated Fas-interacting εerine/threonine kinase/homeodomain-interactiπg protein kinase (FIST/HIPK3) as a novel Fas-interacting protein (Rochat-Steiner ef al., 2000). These authors demonstrated that these results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas. Using PCR with degenerate primers based on conserved domains of serine-threonine kinases, Begley et al. (1997) isolated an MDR cell cDNA encoding a 1,215-amino acid protein with a calculated molecular mass of 130 kD. The protein contains sequences identical to the catalytic core of many serine-protein kinases and is 54% similar to the yeast protein kinase YAK1 , whose normal role is to restrict growth. The authors therefore designated the protein PKY/HIPK3, for homolog of protein kinase YAK1. The authors stated that PKY/HIPK3 may be identical to a 170-kD kinase identified in the same cell lines by Sampson et al. (1993), the difference in molecular mass being due to posttranslational modifications. By Northern blot analysis, PKY/HIPK3 was expressed at higher levels in MDR cells than in their nonresistant parental lines; in addition, a 7-kb PKY/HIPK3 transcript was expressed at high levels in heart and skeletal muscle and at lower levels in placenta, pancreas, and brain. Using a yeast 2-hybrid screen, Kim et al. (1998) identified in mouse 3 members of a family of cofactors, which they designated homeodomain-interacting protein kinases (HIPKs), that interact with homeoproteins and show the greatest similarity to the yeast YAK1 protein (43% identity in the catalytic domain). The corepressor activity of HIPKs depends on both its homeodomain interaction domain and a corepressor domain that maps to the N terminus. Kim et al. (1998) presented evidence that HIPKs can act as transcriptional corepressors for NK homeodomain transcriptionfactors. By fluorescence in situ hybridization, Nupponen and Visakorpi (1999) mapped the HIPK3 gene to chromosome 11p13. However, no link between HIPK3 and the TGFβ/BMP pathway was previously made. Nucleic acid sequence
ATGGCCTCAC AAGTCTTGGT CTACCCACCA TATGTTTATC AAACTCAGTC
AAGTGCCTTT TGTAGTGTGA AGAAACTCAA AGTAGAGCCA AGCAGTTGTG
TATTCCAGGA AAGAAACTAT CCACGGACCT ATGTGAATGG TAGAAACTTT
GGAAATTCTC ATCCTCCCAC TAAGGGTAGT GCTTTTCAGA CAAAGATACC
ATTTAATAGA CCTCGAGGAC ACAACTTTTC ATTGCAGACA AGTGCTGTTG
TTTTGAAAAA CACTGCAGGT GCTACAAAGG TCATAGCAGC TCAGGCACAG
CAAGCTCACG TGCAGGCACC TCAGATTGGG GCGTGGCGAA ACAGATTGCA
TTTCCTAGAA GGCCCCCAGC GATGTGGATT GAAGCGCAAG AGTGAGGAGT
TGGATAATCA TAGCAGCGCA ATGCAGATTG TCGATGAATT GTCCATACTT
CCTGCAATGT TGCAAACCAA CATGGGAAAT CCAGTGACAG TTGTGACAGC
TACCACAGGA TCAAAACAGA ATTGTACCAC TGGAGAAGGT GACTATCAGT
TAGTACAGCA TGAAGTCTTA TGCTCCATGA AAAATACTTA CGAAGTCCTT
GATTTTCTTG GTCGAGGCAC GTTTGGCCAG GTAGTTAAAT GCTGGAAAAG
AGGGACAAAT GAAATTGTAG CAATCAAAAT TTTGAAGAAT CATCCTTCTT
ATGCCCGTCA AGGTCAAATA GAAGTGAGCA TATTAGCAAG GCTCAGTACT
GAAAATGCTG ATGAATATAA CTTTGTACGA GCTTATGAAT GCTTTCAGCA
CCGTAACCAT ACTTGTTTAG TCTTTGAGAT GCTGGAACAA AACTTGTATG
ACTTTCTGAA ACAAAATAAA TTTAGTCCCC TGCCACTAAA AGTGATTCGG
CCCATTCTTC AACAAGTGGC CACTGCACTG AAAAAATTGA AAAGTCTTGG
TTTAATTCAT GCTGATCTCA AGCCAGAGAA TATTATGTTG GTGGATCCTG
TTCGGCAGCC TTACAGGGTT AAAGTAATAG ACTTTGGGTC GGCCAGTCAT
GTATCAAAGA CTGTTTGTTC AACATATCTA CAATCTCGGT ACTAGAGAGC TCCAGAGATT ATATTGGGGT TGCCATTTTG TGAAGCCATA GACATGTGGT CATTGGGATG TGTGATTGCA GAATTATTTC TTGGATGGCC GCTCTACCCA GGAGCCTTGG AGTATGATCA GATTCGATAC ATTTCTCAGA CTCAAGGTTT GCCAGGAGAA CAGTTGTTAA ATGTGGGTAC TAAATCCACA AGA I I I I I I I GCAAAGAAAC AGATATGTCT CATTCTGGTT GGAGATTAAA GACATTGGAA GAGCATGAGG CAGAGACAGG AATGAAGTCT AAAGAAGCCA GAAAATACAT TTTCAACAGT CTGGATGATG TAGCGCATGT GAACACAGTG ATGGATTTGG AAGGAAGTGA TCTTTTGGCT GAGAAAGCTG ATAGAAGAGA ATTTGTTAGT CTGTTGAAGA AAATGTTGCT GATTGATGCA GATTTAAGAA TTACTCCAGC TGAGACCCTG AACCATCCTT TTGTTAATAT GAAACATCTT CTAGATTTCC CTCATAGCAA CCATGTAAAG TCCTGTTTTC ATATTATGGA TATTTGTAAG TCCCACCTAA ATTCATGTGA CACAAATAAT CACAACAAAA CTTCACTTTT AAGACCAGTT GCTTCAAGCA GTACTGCTAC ACTGACTGCA AATTTTACTA AAATCGGAAC ATTAAGAAGT CAGGCATTGA CCACATCTGC TCATTCAGTT GTGCACCATG GAATACCTCT GCAGGCAGGA ACTGCTCAGT TTGGTTGTGG TGATGCTTTT CAGCAGACAT TGATTATCTG TCCCCCAGCT ATTCAAGGTA TTCCTGCAAC ACATGGTAAA CCCACCAGTT ATTCAATAAG GGTAGATAAT ACAGTTCCAC TTGTAACTCA GGCCCCAGCT GTGCAGCCAC TACAGATCCG ACCAGGAGTT CTTTCTCAGA CGTGGTCTGG TAGAACACAG CAGATGCTGG TGCCTGCCTG GCAACAGGTG ACACCCCTGG CTCCTGCTAC TACTACACTA ACTTCTGAGA GTGTGGCTGG TTCACACAGG CTTGGAGACT GGGGGAAGAT GATTTCATGC AGCAATCATT ATAACTCAGT GATGCCGCAG CCTCTTCTGA CCAATCAGAT AACTTTATCT GCCCCTCAGC CAGTTAGTGT GGGGATTGCA CATGTTGTCT GGCCTCAGCC TGCCACTACC AAGAAAAATA AACAGTGCCA GAACAGAGGT ATTTTGGTAA AACTAATGGA ATGGGAGCCA GGAAGAGAGG AAATAAATGC TTTCAGTTGG AGTAATTCAT TACAGAATAC CAATATCCCA CATTCAGCAT TTATTTCTCC AAAGATAATT AATGGGAAAG ATGTCGAGGA AGTAAGTTGT ATAGAAACAC AGGACAATCA GAACTCAGAA GGAGAGGCAA GAAATTGCTG TGAAACATCT ATCAGACAGG ACTCTGATTC ATCAGTTTCA GACAAACAGC GGCAAACCAT CATTATTGCC GACTCCCCGA GTCCTGCAGT GAGTGTCATC ACTATCAGCA GTGACACTGA TGAGGAAGAG ACTTCCCAGA GACATTCACT CAGAGAATGT AAAGGTAGTC TAGATTGTGA AGCTTGCCAG AGCACTTTGA ATATTGATCG GATGTGTTCA TTAAGTAGTC CTGATAGTAC TCTGAGTACC AGCTCCTCAG GGCAGTCCAG CCCATCCCCC TGCAAGAGAC CGAATAGTAT GTCAGATGAA GAGCAAGAAA GTAGTTGTGA TACGGTGGAT GGCTCTCCGA CATCTGACTC TTCCGGGCAT GACAGTCCAT TTGCAGAGAG CACTTTTGTG GAGGACACTC ATGAAAACAC AGAATTGGTA TCCTCTGCTG ACACAGAAAC CAAGCCAGCT GTCTGTTCTG TTGTGGTGCC ACCAGTGGAA
CTAGAAAATG GCTTAAATGC CGATGAGCAT ATGGCAAACA CAGATTCTAT ATGCCAGCCA TTAATAAAAG GACGATCTGC CCCTGGAAGA TTAAACCAGC
CTTCTGCAGT GGGTACTCGT CAGCAAAAAT TGACATCAGC ATTCCAGCAG CAGCATTTGA ACTTCAGTCA GGTTCAGCAC TTTGGATCTG GGCATCAAGA
GTGGAATGGA AACTTTGGGC ACAGAAGACA GCAAGCTTAT ATTCCTACTA GTGTTACCAG TAATCCATTC ACTCTTTCTC ATGGAAGTCC CAATCACACA GCAGTGCATG CCCACCTGGC TGGAAATACA CACCTCGGAG GACAGCCTAC
TCTACTTCCA TACCCATCAT CAGCCACCCT CAGTAGTGCT GCACCAGTGG CCCACCTGTT AGCCTCTCCG TGTACCTCAA GACCTATGTT ACAGCATCCA
ACTTATAATA TCTCCCATCC CAGTGGCATA GTTCACCAAG TCCCAGTGGG CTTAAATCCC CGTCTGTTAC CATCCCCAAC CATTCATCAG ACTCAGTACA AACCAATCTT CCCACCACAT TCTTACATTG CAGCATCACC TGCATATACT
GGATTTCCAC TGAGTCCAAC AAAACTCAGC CAGTATCCAT ATATGTGA (SEQ ID No.128)
Protein sequence MASQVLVYPPYVYQTQSSAFCSVKKLKVEPSSCVFQERNYPRTYVNGRNFGNSHPPTKGS AFQTKIPFNRPRGHNFSLQTSAWLKNTAGATKVIAAQAQQAHVQAPQIGAWRNRLHFLEG PQRCGLKRKSEELDNHSSAMQIVDELSILPAMLQTNMGNPVTWTATTGSKQNCTTGEGDY QLVQHEVLCSMKNTYEVLDFLGRGTFGQWKCWKRGTNEIVAIKILKNHPSYARQGQIEVSI LARLSTENADEYNFVRAYECFQHRNHTCLVFEMLEQNLYDFLKQNKFSPLPLKVIRPILQQV ATALKKLKSLGLIHADLKPENIMLVDPVRQPYRVKVIDFGSASHVSKTVCSTYLQSRYYRAPE IILGLPFCEAIDMWSLGCVIAELFLGWPLYPGALEYDQIRYISQTQGLPGEQLLNVGTKSTRFF CKETDMSHSGWRLKTLEEHEAETGMKSKEARKYIFNSLDDVAHVNTVMDLEGSDLLAEKA DRREFVSLLKKMLLIDADLRITPAETLNHPFVNMKHLLDFPHSNHVKSCFHIMDICKSHLNSC DTNNHNKTSLLRPVASSSTATLTANFTKIGTLRSQALTTSAHSWHHGIPLQAGTAQFGCGD AFQQTLIICPPAIQGIPATHGKPTSYSIRVDNTVPLVTQAPAVQPLQIRPGVLSQTWSGRTQQ MLVPAWQQVTPLAPATTTLTSESVAGSHRLGDWGKMISCSNHYNSVMPQPLLTNQITLSAP QPVSVGIAHWWPQPATTKKNKQCQNRGILVKLMEWEPGREEINAFSWSNSLQNTNIPHSA FISPKIINGKDVEEVSCIETQDNQNSEGEARNCCETSIRQDSDSSVSDKQRQTIIIADSPSPAV SVITISSDTDEEETSQRHSLRECKGSLDCEACQSTLNIDRMCSLSSPDSTLSTSSSGQSSPS PCKRPNSMSDEEQESSCDTVDGSPTSDSSGHDSPFAESTFVEDTHENTELVSSADTETKP AVCSVWPPVELENGLNADEHMANTDSICQPLIKGRSAPGRLNQPSAVGTRQQKLTSAFQQ QHLNFSQVQHFGSGHQEWNGNFGHRRQQAYIPTSVTSNPFTLSHGSPNHTAVHAHLAGN THLGGQPTLLPYPSSATLSSAAPVAHLLASPCTSRPMLQHPTYNISHPSGIVHQ (SEQ ID No.129) HIPK3 interacts with SnoN and SNIP1, proteins involved in the TGFβ/BMP pathway
By two-hybrid screening in yeast it was shown that PTPN12 interacts with Smadδ, a protein involved in the BMP pathway. SnoN-HIPK3
SID : Nucleic sequence, SEQ ID No.64 and Proteic sequence, SEQ ID No. 102.
Snip1-HIPK3 SID : Nucleic sequence, SEQ ID No.62, 63 and Proteic sequence, SEQ ID No. 100, 101. Thus, yeast-two-hybrid screens showed that amino-acids 799-1127 from HIPK3 (SEQ ID No.129) interact with amino-acids 1-370 from SnoN (SEQ ID No.26) and that amino-acids 833-930 from HIPK3 interact with amino-acids 1-198 from Snipl (SEQ ID No.24) (see Fig. 41).
HIPK3 modulates the BMP signaling
Since HIPK3 was found interacting with SnoN and SNIP1 , it was investigated whether HIPK3 could be involved in the TGFβ and/or BMP pathways. In order to assay HIPK3's functional involvement in the TGFβ/BMP pathways, HIPK3 cellular knock-down experiments were performed using chemically synthesised siRNA duplexes (see Materials & Methods for siRNA sequences and protocols). While transiently co-transfecting HepG2 cells using the p(GC)i2-MLP-Luc reporter and HIPK3-targeting siRNA duplex, a specific, dose-dependant and BMP6-dependant increase in the BMP-dependant reporter activity was observed (see Fig. 42 A) demonstrating a function for HIPK3 in the response to the BMP pathway. The positive effect of HIPK3-targeting siRNA duplex was already observed at low concentration of siRNA duplex (4nM) and further enhanced at higher duplex concentrations (40nM). While transiently co-transfecting HepG2 cells using the p(GTCT)8-MLP-Luc reporter and HIPK3- targeting siRNA duplex, no TGFβ-dependant variation in the reporter activity was observed (see Fig. 42 B) demonstrating a restrictive function for HIPK3 on the response the BMP pathway. Modulation of BMP luciferase reporter activities using HIPK3 cellular knock-down shows its implication in the regulation of the BMP pathway. Examples 18: The following materials and methods were used to obtain the results in examples 8 to 17
18-1.Expression vectors construction
Construction of mammalian baculovirus vector consisted in introduction of mammalian Polymerase ll-type transcriptional units such as a promoter active in mammalian cells (for instance CMV, RSV, albumin or inducible promoters). Such plasmids can be used as classical expression vectors to transfect mammalian cells. They can also be used to generate baculoviruses that have the capacity to infect mammalian cells with a high efficiency where they drive the expression of the gene which is under the transcriptional control of the promoter active in mammalian cells (Kost and Condreay, 2002). pV3 and pVδ (Figure X) were prepared from pfastbad vectors (Invitrogen). First, the
BamH -EcoRl fragment of pfastbad with the PCR-amplified CMV promoter fragment from pcDNA3.1/Zeo(+) (Invitrogen) was replaced using oligonucleotides oli30δ4 and oli305δ (PCR conditions as above) to generate pBacCMV.
ON3064: δ'-cgggatccCGTTGACATTGATTATTGACTAGTT-3' (SEQ ID No.130)
OIΪ3055: 5'-cggaattcTTGGGTCTCCCTATAGTGAGT-3' (SEQ ID No.131 )
Next, the EcoR\-Not\ fragment of pBacCMV was replaced with the double-stranded oligonucleotide corresponding to the FLAG sequence to generate pBacCMVflag: δ'-AATTCACCATGGATTACAAGGATGACGACGATAAGGC-3' (SEQ ID No.132)
3'-GTGGTACCTAATGTTCCTACTGCTGCTATTCCGCCGG-5' (SEQ ID No.133)
Next, the Xoal-Psfl fragment of pBacCMVflag was replaced with the double-stranded oligonucleotide to generate pV3: 5'-
CTAGACCCGGGTTGGCCGGACGGGCCCAGCTGCGGCCACTGGGGCCCTTAATTAAGTAA
CTGCA-3" (SEQ ID No.134)
3'-TGGGCCCAACCGGCCTGCCCGGGTCGACGCCGGTGACCCCGGGAATTAATTCATTG-5'
(SEQ ID No.135) Or the Xbal-Psfl fragment of pBacCMVflag was replaced with the double-stranded oligonucleotide to generate pV5:
5'-
CTAGACCCGGGTTGGCCGCAGGGGCCCAGCTGCGGCCACTGGGGCCCTTAATTAAGT
AACTGCA-3' (SEQ ID No.136) 3'-
TGGGCCCAACCGGCGTCCCCGGGTCGACGCCGGTGACCCCGGGAATTAATTCATTG-5'
(SEQ ID No.137)
These pV3 and pV5 vectors thus contain a CMV promoter which controls expression of the proteins of interest fused to the FLAG epitope. The MCS, present into pV3 and pV5, contained the Smal/Sfil/pvull/Sfil/Pacl sites. Differences between pV3 and pV5 were in the
Sfil sites:
In pV3, the sfil sites were oriented whereas in pV5, the Sfil sites were non-oriented Since the preys identified by the two-hybrid assays are cloned between Sfil sites (W099/42612), the presence of Sfil sites in pV3 and pVδ allowed the direct cloning preys in these mammalian expression vectors.
18-2-1 Gene cloning Cloning of smad3, RNF11 and LAPTmδ in pV3 was performed by PCR amplification from placenta cDNA library with the following oligonucleotides:
Smad3:
OH2752: cggactagtCATGTCGTCCATCCTGCCTT (SEQ ID No.138) ON2836: gccttaattaaCTAAGACACACTGGAACAGCGG(SEQ ID No.139)
RNF11:
OH3778: gatcggccggacgggccATGGGGAACTGCCTCAAATCCCCC (SEQ ID No.140)
ON3779: gatcggccccagtggccTCAATTAGTCTCATAGGATGAAAG (SEQ ID No.141)
LAPTmδ: OH3776: gatcggccggacgggccATGGACCCCCGCTTGTCCACTGTC (SEQ ID No.142)
OII3777: gatcggccccagtggccTCACACCTCTGAGTATGGGGGTGG (SEQ ID No.143)
The PCR program was set up as follows:
94° 46"
94° 46" + 55° 46" j X35
72° 7' +
72° 7'
15°
The amplification was checked by agarose gel electrophoresis. The PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol. The purified
PCR fragments were digested with Sfil restriction enzyme (Biolabs) for 1 hour at 60°C. The
PCR fragments were purified with Qiaquick column (Qiagen) according to the manufacturer's protocol.
Concerning PPIca, by the two-hybrid assay a clone was obtained corresponding to nucleotide 1-972 of the PPIca into the pP6 vector. Concerning FLJ20037, a clone was obtained corresponding to the full-length FLJ20037 cDNA into the pP6 vector. These pP6-
PP1ca and pP6-FLJ20037 vectors were digested with Sfil restriction enzyme (Biolabs) for 1 hour at 50°C, extracted, precipitated, and resuspended in water. The PPIca and FLJ20037 fragments were then purified using Qiaex column (Qiagen) according to the manufacturer's protocol.
18-2-2 Vector preparation pV3 and pVδ were prepared as previously described (see 4.1).
The pV3 and pV5 vectors were digested with Sfil restriction enzyme (Biolabs) for 1 hour at
50°C, extracted, precipitated, and resuspended in water. Digested plasmid vector backbones were purified on a separation column (Chromaspin TE 400, Clontech) according to the manufacturer's protocol.
18-2-3 Ligation between expression vectors and preys followed by transformation The digested insert fragments were ligated into an adequately digested (Sfil) vector (pV3 and pVδ) according to standard protocol (Sambrook ef al.) and were transformed into competent bacterial cells. The cells were grown, the DNA extracted and the plasmid was sequenced. 18-2-4 Cell culture HEK293 cells were propagated in Minimum Essential Medium Eagle (SIGMA) supplemented with 10% fetal bovine serum (FBS, Life technologies, Invitrogen), 100 units ml-1 penicillin, and 100 μg.ml-1 streptomycin (Life Technologies, Invitrogen) at 37°C, δ%C02 controlled atmosphere. HepG2 cells were propagated in Dulbecco's modified Eagle's medium (Life Technologies, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Life technologies, Invitrogen), 100 units ml-1 penicillin, and 100 μg.ml-1 streptomycin (Life Technologies, Invitrogen) at 37°C, δ%C02 controlled atmosphere. Cells were regularly passaged to maintain exponential growth. Twenty four hours before transfections, cells were trypsinized and diluted with fresh medium at 2x106 cells/well in a 24 well plate in order to get approximately 60-80% confiuency for transfection.
18-3 Reporters and luciferase assays 3 different reporter vectors : (GTCT)δ-MLP-Luc, (CAGA)6-MLP-Luc and (GC)12-MLP-Luc, encoding the firefly luciferase, were generated for luciferase reporter assays. These two first reporters are activated by TGFβ and activins whereas the third one responds to BMPs. To construct these reporters, the MLP minimal promoter from an adenovirus Major Late gene, containing a TATA box and an initiator element, was first inserted into the Bglll and Hindlll sites of the pGL3 basic vector (Promega) to generate the MLP-Luc plasmid using the oligonucleotides: MLP1: 5'- GATCTGAATTCCATATGCTGCAGGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTC ACTCTCTTCCA-3'(SEQ ID No.144) and the complementary oligonucleotide MLP2 : δ'- AGCTTGGAAGAGAGTGAGGACGAACGCGCCCCCACCCCCTTTTATAGCCCCTGCAGCA TATGGAATTCA-3' (SEQ ID Nθ.14δ)
To construct (GTCT)8-MLP-Luc, 2 copies of the following annealed oligonucleotides were inserted into the EcoRI site of MLP-Luc. These oligonucleotides contains 4 copies of 'the GTCT box', a TGFβ-responsive sequence (Zawel et al., 1998). GTCT1 : 5'-AATTCGTCTAGACAAAAGTCTAGACATTTGTCTAGACTAGTGTCTAGACG-3' (SEQ ID No.146) and the complementary oligonucleotide GTCT2 : 5'-AATTCGTCTAGACACTAGTCTAGACAAATGTCTAGACTTTTGTCTAGACG-3' (SEQ ID No.147)
To construct (CAGA)6-MLP-Luc, 1 copy of the following annealed oligonucleotides was inserted into the Xhol and Nhel sites of MLP-Luc. These oligonucleotides contains 6 copies of 'the CAGA box', a TGF -responsive sequence (Dennler et al., 1998). CAGA1 : 5'- CTAGAGCCAGACAAAAAGCCAGACATTTAGCCAGACAAAAAGCCAGACATTTAGCCAGA
CAAAAAGCCAGACA-3' (SEQ ID No.148) and the complementary oligonucleotide CAGA2: δ'-
TCGATGTGTGGCTTTTTGTCTGGCTA TGTCTGGCTTTTTGTCTGGCTAAATGTCTGGC TTTTTGTCTGGCT-3' (SEQ ID No.149)
To construct (GC)12-MLP-Luc, 3 copies of the following annealed oligonucleotides were inseted into the Xhol site of MLP-Luc. These oligonucleotides contains 4 copies of 'the GC box', a BMP responsive sequence (Kusanagi et al., 2000).
GC1: 5'- TCGAGCCGCCGCTTTGCCGCCGCTTTGCCGCCGCTTTGCCGCCGC-3' (SEQ ID
No.150) and the complementary oligonucleotide
GC2: 5'- TCGAGCGGCGGCAAAGCGGCGGCAAAGCGGCGGCAAAGCGGCGGC-3' (SEQ ID No.151)
All these constructs were sequence-checked.
These reporters were used to observe the effects of siRNA in transfection experiments r (see the siRNA section). These reporters were also used to determine the effect of over- expression of some proteins on TGFβ and/or BMP signaling in co-transfection experiments with pV3 and pVδ vectors encoding proteins of interest (cf expression vectors construction section). To this end, HepG2 and HEK293 cells were transiently transfected using the Fugene 6 (Roche) or the Lipofectamine 2000 (InVitrogen) reagent, respectively, according to the manufacturer recommendations. 400 ng of luciferase reporter and 100 ng of pRL-TK (Promega), encoding the renilla luciferase and used as an internal transfection efficiency control, were transfected per well of a 24 wells-plate. Variable amounts of expression vectors were co-transfectd as indicated in the figures. When increasing amounts of expression vectors were transfected, total DNA was kept constant by the addition of pV3. 24 hours after the transfection, cells were washed and incubated in a medium without serum. 2 hours later, cells were stimulated with 10 ng/mL of human recombinant TGFβl (R&D) or 50 ng/mL of human recombinant BMP6 or BMP7 (R&D). 18 to 24 hours after stimulation, Luciferase activities were quantified using the Dual Luciferase reporter assay kit from Promega. Values were normalized with the renilla luciferase activity expressed from pRL-TK. 18-4 Baculovirus infection of mammalian cells
Genetically modified baculoviruses were used to infect mammalian cells (Kost and Condreay, 2002). The pV3 and pVδ mammalian expression vectors, derived from pFastbad (see mammalian expression vectors section) and in which the cDNA of genes of interest has been cloned, can be used to produce baculoviruses that can express the protein encoded by this cDNA in mammalian cells. To prepare the baculoviral particles, these vectors were inserted into the baculoviral genome by transposition into E. coli competent cells to obtain a recombinant bacmid using the BAC-TO-BAC Baculovirus Expression System (Invitrogen) according to the manufacturer's procedure. Next steps consist in transfecting Sf9 insect cells with this Bacmid DNA and harvesting the viral particles. 'Control' vectors in which the cDNA encoding the β-galactosidase gene or the GFP gene were constructed and used to produce baculoviruses which were expressing the β-galactosidase and GFP proteins. These 'control' baculoviruses allowed to quantify the efficiency of baculovirus infection in mammalian cells by determining the in situ production of β-galactosidase and GFP proteins in cells. Thus, HepG2, HEK293 or HeLa cells were infected with an efficiency higher than 80 % with the mammalian baculoviruses (data not shown).
Transfection of the Sf9 cells with the Bacmid was made with the GeneShuttle reagent (Quantum). The supernatant of the Sf9 cells which contained the recombinant baculoviral particles was harvested 72 hours post transfection. This supernatant can be used to re-infect other Sf9 cells in order to amplify the viral stock in T7δ or T160 flasks. In order to check if viral particles have been produced in sufficient amount for the following experiments, viral DNA was quantified by Q-PCR. 600 μL of the Sf9 supernatant, containing the baculoviral particles, supplemented by 1.4 mL of classical cell medium are used per well to infect human cells such as HepG2, HEK 293 or HeLa cells seeded in 24 wells-plate. Then, cellular RNA was extracted 24 to 72 hours post infection to perform Q-PCR experiments (see Q-PCR section). All viruses were conserved at +4°C.
18-5 Quantitative PCR (Q-PCR) experiments To monitor the biological effects of the proteins of interest in the TGFβ/activin or BMP signaling in cells, quantification of mRNA of genes transcriptionally regulated by TGFβ/activin or BMP by Quantitative-PCR were carried-out using an Applied Biosytems 7000 SDS machine. This quantification follows a transfection of an expression vector of the prey of interest, the transfection of a siRNA or an infection using a genetically-modified baculovirus in mammalian cells such as HepG2, HeLa or HEK 293 cell lines seeded in 24 culture-plate. Cells are then lysed and RNA was extracted using the Rneasy Minikit and the Qia Shredder from Qiagen following the recommendations of the manufacturer. 1 μg of RNA is then used for a reverse transcription reaction to generate the cDNA which will serve as template in the following Q-PCR reaction. The reverse transcription step was realized in 96 wells-plate with the TaqMan reverse transcription kit (Applied biosystems) following the recommendations of the manufacturer. The cDNA of the gene of interest was then quantified in 96 wells-plate by the SyBR green methodology using the SyBR Green PCR master Mix kit (Applied
Bisosystems) in an ABI 7000 machine following the recommendations of the manufacturer. For each reaction, δ ng of cDNA was used as template and 300 nM of forward and reverse oligonucleotides probing specifically the gene for which the mRNA was quantified are added.
Values are normalized with the value obtained for the mRNA of the hGAPDH or hGUS genes which serve as internal experimental controls.
The forward and reverse oligonucleotides probing the gene of interest were designed using the Primer Express software (Applied Biosystems).These oligonucleotides were validated by
Q-PCR experiments showing that they allow a quantitative measurement (quantification of cDNA diluted in cascade and PCR efficacy determination).
The human genes used to monitor the effect of TGFβ are the Plasminogen Activator Inhibitor
Type 1 gene (hPAI-1 ) and the JunB gene. The human genes used to monitor the effect of BMPs are the JunB gene and the Alcaline Phosphatase gene (hALP). The genes used as internal quantification controls are the Glyceraldehyde Phosphate Dehydrogenase gene
(hGAPDH) and the the β-Glucoronidase gene (hGUS). The sequences of the oligonucleotides probing these mRNA are: hPAI-1 : forward TGAAGATCGAGGTGAACGAGAGT (SEQ ID No.152) Reverse GTCCCAGATGAAGGCGTCTTT (SEQ ID No.153) hJunB: forward ACTCATACACAGCTACGGGATACG(SEQ ID No.154) Reverse GGGTCGGCCAGGTTGAC(SEQ ID No.155) hALP: forward CGAGCTGAACAGGAACAACGT(SEQ ID No.156) Reverse CTGCTTGGCTTTTCCTTCATG(SEQ ID No.157) hGAPDH: forward GGAGTCAACGGATTTGGTCGTA(SEQ ID No.158)
Reverse GTGGAATCATATTGGAACATGTAAACC(SEQ ID No.159) hGUS: forward CCCGCGGTCGTGATGT(SEQ ID No.160)
Reverse TGAGCGATCACCATCTTCAAGT(SEQ ID No.161)
The sequences of the oligonucleotides probing the cDNA of the gene targeted by siRNA and used to validate the effect of the siRNA (see siRNA section) or the over-production level following baculovirus infection were:
ZNF8: forward CCAGTCAGGCCATTCCAATT(SEQ ID No.162)
Reverse GTGTGCGTTATGGTTAAACGACTTC(SEQ ID No.163)
TβR1: forward GTGACTACAACATATTGCTGCAATCAG(SEQ ID No.164) Reverse AGCACACTGGTCCAGCAATG(SEQ ID No.165)
PPIca : forward CTCCACAAGCACGACTTGGA(SEQ ID No.166)
Reverse GTTGGGAGCTGAGAAAAGTGTCA(SEQ ID No.167) KIAA1196: forward GGCCCTCCGAGACATTCC(SEQ ID No.168)
Reverse TAATGGTACTTGAGCCCGTAGATG(SEQ ID No.169) LM04: forward CAGAAGGTCTGCTAAAAGGTCAGAGT(SEQ ID No.170)
Reverse GGGATCCACCTGTGATGAACA(SEQ ID No.171 ) FLJ20037: forward AACAAAGAGAAGATCACACCACTCA(SEQ ID No.172) Reverse TAAGACTCCATCGGTCAGAGTCA(SEQ ID No.173) HYPA : forward TTCCATGCAGCCTGCCTTA(SEQ ID No.174)
Reverse CAGGTGATTTATGTTCAGTCCACAT(SEQ ID No.175) LAPTmδ: forward TGGCCATCTACCATGTGATCA(SEQ ID No.176) Reverse CGATCCTGAGGTAGCCCATCT(SEQ ID No.177)
HIPK3: forward TTGTTCAACATATCTACAATCTCGGTACT(SEQ ID No.178)
Reverse GAGCGGCCATCCAAGAAATA(SEQ ID No.179) PTPN 12: forward TGTGAGCGCTATTGGCCTTT(SEQ ID No.180)
Reverse TTTTGAAATTCAAGTAAGAGTGTCCTGAT(SEQ ID No.181 ) 18-6 siRNA
Chemically synthesized siRNA using RNA phosphoramidites were purchased from Genset Oligos /Proligos (Paris, France). siRNA were ordered deprotected, desalted and duplexed.
The siRNA duplexes used in these studies were all 19 ribonucteotides long and contained two thymidines nucleotides at their 3' termini. All siRNA duplexes were designed according to the rules edicted by Tuschl and coll. (Elbashir et al., 2001). In the following list, all sequences correspond to the sense DNA in the corresponding CDS
TβRI: 5'-GTGTTTCTGCCACCTCTGT-3'(SEQ ID No.182) • mTβRI: δ'-GTGTGTCTGCAACCTCTGT-3'(SEQ ID No.183)
PP1 ca: δ'-AACCTTCACTGACTGCTTC-S SEQ ID No.184)
KIAA1196: δ'-CGACTGGAAGGATGAGTTC-3'(SEQ ID No.18δ)
HIPK3: 5'-GCAGTTGTGTATTCCAGGA-3'(SEQ ID No.186)
ZNF8: 5'-GCCTGAAGTCATCTCCCAG-3'(SEQ ID No.187) • PTPN12: 5'-GATATATCCCACAGCCACT-3'(SEQ ID No.188)
LM04: δ'-GTGGCATGATCCTTTGCAG-S SEQ ID No.189)
FLJ20037: 5'-CAAGATCATTGCCACCAGG-3'(SEQ ID No.190)
HYPA: 5'-ATCAATGTGGACTGAACAT-3'(SEQ ID No.191 )
LAPTmδ: 5'-ATCATGGACTATCTCCTGT-3'(SEQ ID No.192) As a validation experiment, the efficacy of these siRNA was tested on their targeted mRNA by Q-PCR experiments (see Q-PCR section). Their specificity was assayed on unrelated mRNA by Q-PCR. All these siRNA inhibit at least from 6δ to 9δ % the amount of their targeted mRNA and do not show any effect on other unrelated mRNAs. 18-7 SiRNA transfection
Co-transfection of reporter plasmids and siRNAs was carried out with Lipofectamine 2000 (Life Technologies, Invitrogen) as described by the manufacturer for adherent cell lines.
Per well 0,4 μg specific luciferase reporter plasmid, 0,1 μg pRL-TK (Promega), 0,5 μg carrier
DNA (pBluscript) and 4 to 40 nM siRNA duplex, formulated into liposomes, were applied. The final volume per well was δOOμl. Medium were changed 5 hours post-transfection and cells appeared healthy on next day. Cells were serum-starved for 1-2 hours before cytikines treatment. Cells were treated with TGFβl (R&D, 5 ng/ml), BMP6 (R&D, 50ng/ml)or BMP7
(R&D, δOng/ml) for 18 hours before luciferase assay or total RNA extraction.
Luciferase expression was subsequently monitored with the Dual luciferase assay
(Promega).
Reporter and carrier plasmids were amplified in DHδβ (Stratagene) and purified using the Qiagen Endofree Maxi plasmid Kit.
Transfection of siRNAs for targeting endogenous mRNA was carried out using
Oligofectamine (Life Technologies, Invitrogen) and 4 to 40 nM siRNA duplex per well in a 24 well plate.
Specific silencing of targeted genes was confirmed by at least three independent experiments.
18-8 Antibodies
Anti-SARA rabbit polyclonal antibody was purchased from Santa-Cruz (cat # H-300 sc9135) and used at a 1/150 dilution.
Peroxidase-conjugated AffiniPure F(ab')2 fragment donkey anti-Rabbit IgG (H+L) was used as a secondary reagent (1/10000 dilution) and was purchased from Jackson
Immunoresearch laboratories, Inc.
18-9 Cell lysis and Immunoblot
Cell were harvested in lysis buffer (2%SDS, 1X PBS), denatured 5 minutes at 95°C and quantified using Bradford reagent (BIORAD) according to the manufacturer's specifications. Cell lysates (20μg/lane) were resolved on a 4-12% NuPAGE gradient gel (Novex, Invitrogen), transfered to 20μm nitrocellulose membrane (Schleicher & Schuell) and blocked in 10% fat-free dried milk in 1X PBS, 0,05% Tween20. Revelation was performed using ECL (Amersham Biosciences) chemoluminescent substrat according to the manufacturer's specifications.
The following results obtained from these Examples, as well as the teachings in the specification are set forth in the Tables below. While the invention has been described in terms of the various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions and changes may be made without departing from the scope thereof. Accordingly, it is intended that the present invention be limited by the scope of the following claims, including equivalents thereof
Table 1 : Bait name and sequence
1-Bait 2- 3- Nucleic acid sequence 4 5. 6. Amino Acid Seque Name Nucleic Nuclei Amino acid c acid SEQ ID positi SEQ No. on ID
No.
Human ATGAATGTGACAAGTTTATTTTCCTTTACAAGTCCAGCTGTGAAGAGACTTCTTGGGTGGAAACAGG [1 14 MNVTS FSFTSPAVKRLLG Smadl v2 GCGATGAAGAAGAAAAATGGGCAGAGAAAGCTGTTGATGCTTTGGTGAAAAAACTGAAGAAAAAGAA 802] DEEEKWAEKAVDALVKKLK AGGTGCCATGGAGGAACTGGAAAAGGCCTTGAGCTGCCCAGGGCAACCGAGTAACTGTGTCACCATT AMEELEKALSCPGQPSNCV CCCCGCTCTCTGGATGGCAGGCTGCAAGTCTCCCACCGGAAGGGACTGCCTCATGTCATTTACTGCC SLDGR QVSHRKG PHVIY GTGTGTGGCGCTGGCCCGATCTTCAGAGCCACCATGAACTAAAACCACTGGAATGCTGTGAGTTTCC RWPDLQSHHELKPLECCEF TTTTGGTTCCAAGCAGAAGGAGGTCTGCATCAATCCCTACCACTATAAGAGAGTAGAAAGCCCTGTA KQKEVCINPYHYKRVESPV CTTCCTCCTGTGCTGGTTCCAAGACACAGCGAATATAATCCTCAGCACAGCCTCTTAGCTCAGTTCC LVPRHSEYNPQHSLLAQFR GTAACTTAGGACAAAATGAGCCTCACATGCCACTCAACGCCACTTTTCCAGATTCTTTCCAGCAACC NEPHMPLNATFPDSFQQPN CAACAGCCACCCGTTTCCTCACTCTCCCAATAGCAGTTACCCAAACTCTCCTGGGAGCAGCAGCAGC PHSPNSSYPNSPGSSSSTY ACCTACCCTCACTCTCCCACCAGCTCAGACCCAGGAAGCCCTTTCCAGATGCCAGCTGATACGCCCC TSSDPGSPFQMPADTPPPA CACCTGCTTACCTGCCTCCTGAAGACCCCATGACCCAGGATGGCTCTCAGCCGATGGACACAAACAT EDPMTQDGSQPMDTNMMAP GATGGCGCCTCCCCTGCCCTCAGAAATCAACAGAGGAGATGTTCAGGCGGTTGCTTATGAGGAAC EINRGDVQAVAYEE
Human ATGAATGTGACAAGTTTATTTTCCTTTACAAGTCCAGCTGTGAAGAGACTTCTTGGGTGGAAACAGG [1 15 MNVTSLFSFTSPAVKRLLG Smadl vl GCGATGAAGAAGAAAAATGGGCAGAGAAAGCTGTTGATGCTTTGGTGAAAAAACTGAAGAAAAAGAA 1398] DEEEKWAEKAVDALVKKLK AGGTGCCATGGAGGAACTGGAAAAGGCCTTGAGCTGCCCAGGGCAACCGAGTAACTGTGTCACCATT AMEELEKALSCPGQPSNCV CCCCGCTCTCTGGATGGCAGGCTGCAAGTCTCCCACCGGAAGGGACTGCCTCATGTCATTTACTGCC SLDGRLQVSHRKGLPHVIY GTGTGTGGCGCTGGCCCGATCTTCAGAGCCACCATGAACTAAAACCACTGGAATGCTGTGAGTTTCC RWPDLQSHHELKPLECCEF TTTTGGTTCCAAGCAGAAGGAGGTCTGCATCAATCCCTACCACTATAAGAGAGTAGAAAGCCCTGTA KQKEVCINPYHYKRVESPV CTTCCTCCTGTGCTGGTTCCAAGACACAGCGAATATAATCCTCAGCACAGCCTCTTAGCTCAGTTCC LVPRHSEYNPQHSLLAQFR GTAACTTAGGACAAAATGAGCCTCACATGCCACTCAA.CGCCACTTTTCCAGATTCTTTCCAGCAACC NEPHMPLNATFPDSFQQPN CAACAGCCACCCGTTTCCTCACTCTCCCAATAGCAGTTACCCAAACTCTCCTGGGAGCAGCAGCAGC PHSPNSSYPNSPGSSSSTY ACCTACCCTCACTCTCCCACCAGCTCAGACCCAGGAAGCCCTTTCCAGATGCCAGCTGATACGCCCC TSSDPGSPFQMPADTPPP CACCTGCTTACCTGCCTCCTGAAGACCCCATGACCCAGGATGGCTCTCAGCCGATGGACACAAACAT EDPMTQDGSQPMDTNMMAP GATGGCGCCTCCCCTGCCCTCAGAAATCAACAGAGGAGATGTTCAGGCGGTTGCTTATGAGGAACCA EINRGDVQAVAYEEPKHW AAACACTGGTGCTCTATTGTCTACTATGAGCTCAACAATCGTGTGGGTGAAGCGTTCCATGCCTCCT YELNNRVGEAFHASSTSVL CCACAAGTGTGTTGGTGGATGGTTTCACTGATCCTTCCAACAATAAGAACCGTTTCTGCCTTGGGCT TDPSNNKNRFCLGLLSNVN GCTCTCCAATGTTAACCGGAATTCCACTATTGAAAACACCAGGCGGCATATTGGAAAAGGAGTTCAT IENTRRHIGKGVHLYYVGG CTTTATTATGTTGGAGGGGAGGTGTATGCCGAATGCCTTAGTGACAGTAGCATCTTTGTGCAAAGTC ECLSDSSIFVQSRNCNYH GGAACTGCAACTACCATCATGGATTTCATCCTACTACTGTTTGCAAGATCCCTAGTGGGTGTAGTCT TTVCKIPSGCSLKIFNNQ GAAAATTTTTAACAACCAAGAATTTGCTCAGTTATTGGCACAGTCTGTGAACCATGGATTTGAGACA LAQSVNHGFETVYELTKM
GTCTATGAGCTTACAAAAATGTGTACTATACGTATGAGCTTTGTGAAGGGCTGGGGAGCAGAATACC SFVKGWGAEYHRQDVTSTP ACCGCCAGGATGTTACTAGCACCCCCTGCTGGATTGAGATACATCTGCACGGCCCCCTCCAGTGGCT IHLHGPLQWLDKVLTQMGS GGATAAAGTTCTTACTCAAATGGGTTCACCTCATAATCCTATTTCATCTGTATCTTAA ISSVS
Human GATGGACACAAACATGATGGCGCCTCCCCTGCCCTCAGAAATCAACAGAGGAGATGTTCAGGCGGTT [723 16 DGHKHDGASPALRNQQRRC Smadl v3 GCTTATGAGGAACCAAAACACTGGTGCTCTATTGTCTACTATGAGCTCAACAATCGTGTGGGTGAAG 1395] L*GTKTLVLYCLL*AQQSC CGTTCCATGCCTCCTCCACAAGTGTGTTGGTGGATGGTTTCACTGATCCTTCCAACAATAAGAACCG PCL.LHKCVGGWFH*SFQQ* TTTCTGCCTTGGGCTGCTCTCCAATGTTAACCGGAATTCCACTATTGAAAACACCAGGCGGCATATT PWAALQC*PEFHY*KHQAA GGAAAAGGAGTTCATCTTTATTATGTTGGAGGGGAGGTGTATGCCGAATGCCTTAGTGACAGTAGCA SSSLLCWRGGVCRMP**Q* TCTTTGTGCAAAGTCGGAACTGCAACTACCATCATGGATTTCATCCTACTACTGTTTGCAAGATCCC KSELQLPSWISSYYCLQDP TAGTGGGTGTAGTCTGAAAATTTTTAACAACCAAGAATTTGCTCAGTTATTGGCACAGTCTGTGAAC SENF*QPRICΞVIGTVCEP CATGGATTTGAGACAGTCTATGAGCTTACAAAAATGTGTACTATACGTATGAGCTTTGTGAAGGGCT SL*AYKNVYYTYELCEGLG GGGGAGCAGAATACCACCGCCAGGATGTTACTAGCACCCCCTGCTGGATTGAGATACATCTGCACGG PPGCY*HPLLD*DTSARPP CCCCCTCCAGTGGCTGGATAAAGTTCTTACTCAAATGGGTTCACCTCATAATCCTATTTCATCTGTA *SSYSNGFTS*SYFICI TCT
Human ATGGACAATATGTCTATTACGAATACACCAACAAGTAATGATGCCTGTCTGAGCATTGTGCATAGTT [1 17 MDNMSITNTPTSNDACLSI Smad4 vl TGATGTGCCATAGACAAGGTGGAGAGAGTGAAACATTTGCAAAAAGAGCAATTGAAAGTTTGGTAAA 1656] MCHRQGGESETFAKRAIES GAAGCTGAAGGAGAAAAAAGATGAATTGGATTCTTTAATAACAGCTATAACTACAAATGGAGCTCAT LKEKKDELDSLITAITTNG CCTAGTAAATGTGTTACCATACAGAGAACATTGGATGGGAGGCTTCAGGTGGCTGGTCGGAAAGGAT KCVTIQRTLDGRLQVAGRK TTCCTCATGTGATCTATGCCCGTCTCTGGAGGTGGCCTGATCTTCACAAAAATGAACTAAAACATGT VIYARLWRWPDLHKNELKH TAAATATTGTCAGTATGCGTTTGACTTAAAATGTGATAGTGTCTGTGTGAATCCATATCACTACGAA QYAFDLKCDSVCVNPYHYE CGAGTTGTATCACCTGGAATTGATCTCTCAGGATTAACACTGCAGAGTAATGCTCCATCAAGTATGA PGIDLSGLTLQSNAPSSMM TGGTGAAGGATGAATATGTGCATGACTTTGAGGGACAGCCATCGTTGTCCACTGAAGGACATTCAAT YVHDFEGQPSLSTEGHSIQ TCAAACCATCCAGCATCCACCAAGTAATCGTGCATCGACAGAGACATACAGCACCCCAGCTCTGTTA PPSNRASTETYSTPALLAP GCCCCATCTGAGTCTAATGCTACCAGCACTGCCAACTTTCCCAACATTCCTGTGGCTTCCACAAGTC ATSTANFPNIPVASTSQPA AGCCTGCCAGTATACTGGGGGGCAGCCATAGTGAAGGACTGTTGCAGATAGCATCAGGGCCTCAGCC GSHSEGLLQIASGPQPGQQ AGGACAGCAGCAGAATGGATTTACTGGTCAGCCAGCTACTTACCATCATAACAGCACTACCACCTGG TGQPATYHHNSTTTWTGSR ACTGGAAGTAGGACTGCACCATACACACCTAATTTGCCTCACCACCAAAACGGCCATCTTCAGCACC TPNLPHHQNGHLQHHPPMP ACCCGCCTATGCCGCCCCATCCCGGACATTACTGGCCTGTTCACAATGAGCTTGCATTCCAGCCTCC HYWPVHNELAFQPPISNHP CATTTCCAATCATCCTGCTCCTGAGTATTGGTGTTCCATTGCTTACTTTGAAATGGATGTTCAGGTA WCSIAYFEMDVQVGETFKV GGAGAGACATTTAAGGTTCCTTCAAGCTGCCCTATTGTTACTGTTGATGGATACGTGGACCCTTCTG PIVTVDGYVDPΞGGDRFCL GAGGAGATCGCTTTTGTTTGGGTCAACTCTCCAATGTCCACAGGACAGAAGCCATTGAGAGAGCAAG NVHRTEAlERARLHIGKGV GTTGCACATAGGCAAAGGTGTGCAGTTGGAATGTAAAGGTGAAGGTGATGTTTGGGTCAGGTGCCTT KGEGDVWVRCLSDHAVFVQ AGTGACCACGCGGTCTTTGTACAGAGTTACTACTTAGACAGAGAAGCTGGGCGTGCACCTGGAGATG DREAGRAPGDAVHKIYPSA CTGTTCATAAGATCTACCCAAGTGCATATATAAAGGTCTTTGATTTGCGTCAGTGTCATCGACAGAT FDLRQCHRQMQQQAATAQA GCAGCAGCAGGCGGCTACTGCACAAGCTGCAGCAGCTGCCCAGGCAGCAGCCGTGGCAGGAAACATC QAAAVAGNIPGPGSVGGIA CCTGGCCCAGGATCAGTAGGTGGAATAGCTCCAGCTATCAGTCTGTCAGCTGCTGCTGGAATTGGTG LSAAAGIGVDDLRRLCILR TTGATGACCTTCGTCGCTTATGCATACTCAGGATGAGTTTTGTGAAAGGCTGGGGACCGGATTACCC KGWGPDYPRQSIKETPCWI AAGACAGAGCATCAAAGAAACACCTTGCTGGATTGAAATTCACTTACACCGGGCCCTCCAGCTCCTA HRALQLLDEVLHTMPIADP GACGAAGTACTTCATACCATGCCGATTGCAGACCCACAACCTTTAGAC
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Table 2 ; Bait-prey interactions
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000086_0002
Figure imgf000087_0001
Table 3 : SID®
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000091_0002
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
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Claims

CLAIMSWhat is claimed is:
1. A complex between two interacting proteins as defined in columns 1 and 4 in Table 2.
2. A complex between two polynucleotides encoding for the polypeptides of claim 1.
3. A recombinant host cell expressing the interacting polypeptides of said complex of protein-protein interaction of claim 1.
4. Use of a SID®, an interaction or a prey to screen molecules that inhibit TGFβ or inhibit a TGFβ super-family of cytokines pathway.
5. A molecule that inhibits inhibit TGFβ or inhibits a TGFβ super-family of cytokines pathway.
6. Use according to Claim 4, wherein said screening occurs in mammalian cells or yeast cells.
7. A SID® polypeptide comprising the SEQ ID No 63 to 98.
8. A SID® polynucleotide comprising the SEQ ID No 27 to 62.
9. A vector comprising the SID® polynucleotide comprising the SEQ ID No 27 to 62.
10. A fragment of said SID® polypeptide according to Claim 7.
11. A variant of said SID® polypeptide according to Claim 7.
12. A fragment of said SID® polynucleotide according to Claim 8.
13. A variant of said SID® polynucleotide according to Claim 8.
14. A vector comprising the SID® polynucleotide according to any one of Claims 8, 12 or 13.
15. A recombinant host cell containing the vectors according to Claim 14.
16. A pharmaceutical composition comprising the molecule of claim 5 and a pharmaceutically acceptable carrier.
17. A pharmaceutical composition comprising a SID® polypeptide SEQ ID No 63 to 98 and a pharmaceutically acceptable carrier.
18. A pharmaceutical composition comprising the recombinant host cells of Claim 15 and a pharmaceutically acceptable carrier.
19. A protein chip comprising the polypeptides of Table 2.
20. Use of a ZNF8 protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
21. Use of a LAPTmδ protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
22. Use of a RNF11 protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
23. Use of a LM04 protein for the preparation of a medicament for treating prostate cancer.
24. Use of a PPC1 protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
25. Use of an HYPA protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
26. Use of a PTP protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
27. Use of an HYPK3 protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
28. Use of a KIAA1196 protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
29. Use of a FL20037 protein for the preparation of a medicament for treating diseases and /or disorders linked or involving a TGFβ super-family of cytokines.
30. Use of a complex between two interacting proteins as defined in columns 1 and 4 in table 2 to screen I molecules for diagnosis or treating transforming growth factor β disorders and/or diseases.
PCT/EP2002/013866 2001-11-26 2002-11-26 Protein-protein interactions involving transforming growth factor beta signalling WO2003045990A2 (en)

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