COMPOSITIONS AND METHODS FOR MODULATING TGF- beta SIGNALING 机翻标题: 暂无翻译,请尝试点击翻译按钮。

公开号/公开日
WO200047102 A2 2000-08-17 [WO200047102]WO200047102 A3 2002-10-10 [WO200047102] / 2000-08-172002-10-10
申请号/申请日
2000WO-US03561 / 2000-02-11
发明人
WANG TONGWEN;
申请人
GENERAL HOSPITAL;
主分类号
IPC分类号
A61K-038/00C07K-014/47
摘要
(WO200047102) The invention provides novel compositions comprising a Smad protein and an isolated protein component of the proteasome-mediated degradation pathway.  The invention also provides novel compositions comprising a Smad1 protein and a substrate for proteasome-mediated degradation.  The invention also provides methods of screening for compounds that modulate the interaction between the proteins comprising these compositions.  The invention also provides methods of screening for compounds that modulate the activity of the proteins comprising these compositions.  The invention also provides methods of detecting proteasome-mediated degradation of novel Smad interacting proteins.  A further aspect of the invention is a kit for detecting proteasome-mediated degradation of novel Smad interacting proteins.  The invention also provides methods of treating diseases which are associated with aberrant levels of activity of a TGF- beta superfamily member.
机翻摘要
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地址
代理人
代理机构
;
优先权号
1999US-60119786 1999-02-11
主权利要求
(WO200047102) CLAIMS 1. A purified polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 10, 12, 14, 16, 18, 20 and 21. 2. An expression vector comprising the polynucleotide of claim 1. 3. A host cell transformed with the expression vector of claim 2. 4. An isolated polypeptide encoded by the expression vector of claim 2. 5. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-3, 5, 7-9, 11, 13, 15, 17, 19 and 22. 6. An antibody which binds to the polypeptide of claim 4 or claim 5. 7. A composition comprising Smadl and an interaction partner protein selected from the group consisting of HsN3, antizyme, PAG, GST, tumor associated gene, AIP4, UI SnRNP, TRIP4, Ran GTP binding protein 5, P0 acidic ribosomal phosphoprotein, β- tubulin, KIAA 00104, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, and SEQ ID No. 19. 8. A composition comprising Smad2 and an interaction partner protein selected from the group consisting of GST, AIP4, TRIP4, KIAA 00104 and SEQ ID No. 3. 9. A composition comprising Smad3 and an interaction partner protein selected from the group consisting of HsN3, KIAA0104, HEFl, FKBP25, AIP4, SnRNP C, RBP2, TRIP4, hnRNP Al, GST, SEQ ID No. 11, SEQ ID No. 15, SEQ ID No. 13, SEQ ID No. 4, SEQ ID No. 17 and SEQ ID No. 22. 10. A screening method to identify a compound which modulates the interaction of a first protein with a known interaction partner of said first protein, said method comprising the steps of: a) contacting a protein comprising said first protein with a protein comprising said interaction partner in both the presence and absence of said compound; and b) detecting the amount of said interaction partner bound to said first protein, wherein an increase in bound interaction partner in the presence of said compound indicates said compound is an agonist of said interaction, and a decrease in bound interaction partner in the presence of said compound indicates said compound is an antagonist of said interaction. 11. The method of claim 10, wherein said first protein is Smadl and said known interaction partner is selected from the group consisting of HsN3, antizyme, PAG, GST, tumor associated gene, AIP4, UI SnRNP, TRIP4, Ran GTP binding ρrotein5, P0 acidic ribosomal phosphoprotein, β-tubulin, KIAA 00104, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, and SEQ ID No. 19. 12. The method of claim 10, wherein said first protein is Smad2 and said interaction partner is selected from the group consisting of GST, AIP4, TRIP4, KIAA 00104 and SEQ ID No. 3. 13. The method of claim 10, wherein said first protein is Smad3 and said interaction partner protein is selected from the group consisting of HsN3, KIAA0104, HEFl, FKBP25, AIP4, SnRNP C, RBP2, TRIP4, hnRNP Al, GST, SEQ ID No. 11, SEQ ID No. 15, SEQ ID No. 13, SEQ ID No. 4, SEQ ID No. 17 and SEQ ID No. 22. 14. The method of claim 10, wherein said first protein is HsN3 and said interaction partner protein is selected from the group consisting of antizyme, GST, PAG, FKBP25, TRIP4, HEFl, AIP4, SnRNP C, hnRNP Al, TGF-β type II receptors, BMP type I receptor ALK3, FNTA, GGTB, KIAA 00104, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13 and SEQ ID No. 22. 15. The method of claim 10, wherein said first protein is antizyme and said interaction partner protein is selected from the group consisting of enolase, PAG, tumor associated gene, hnRNP Al, TRIP4, AIP4, HEFl, SnRNP C, KIAA 00104, HnRNPAl, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 11 and SEQ ID No. 22. 16. The method of claim 10, wherein said first protein is SEQ ID No. 3 and said interaction partner protein is selected from the group consisting of HsN3, antizyme, SEQ ID No. 7, AIP4, the cytoplasmic domain of ALK2 and the cytoplasmic domain of ALK5. 17. The method of any one of claims 10-16 performed in vitro. 18. The method of any one of claims 10-16 performed in yeast. 19. The method of any one of claims 10-16 performed in mammalian cells. 20. A method to identify a candidate compound which modulates the interaction between a first protein and a known interaction partner protein of said first protein in yeast cells comprising the steps of: a) transforming yeast cells with expression constmcts comprising: i) a reporter gene functionally linked to a DNA sequence bound by a second protein; ii) a gene comprising a first protein fused to a DNA binding domain of said second protein; and iii) a gene comprising said known interaction partner protein of said first protein and a transactivation domain; b) culturing the transformed yeast cells of a) in the presence and absence of said candidate compound; and c) detecting expression of said reporter gene, wherein an increase in reporter gene expression in the presence of said candidate compound indicates said candidate compound is an agonist of said interaction, and a decrease in reporter gene expression in the presence of said candidate compound indicates said candidate modulator is an antagonist of said interaction. 21. A method to identify a candidate compound which modulates the interaction between a first protein and a known interaction partner protein of said first protein in mammalian cells comprising the steps of: a) transfecting a mammalian cell line with expression constmcts comprising: i) a reporter gene functionally linked to a DNA sequence bound by a second protein; ii) a gene comprising a first protein fused to a DNA binding domain of said second protein; and iii) a gene comprising said known interaction partner protein of said first protein and a transactivation domain; b) culturing the transfected mammalian cell line of a) in the presence and absence of said candidate compound; and c) detecting expression of said reporter gene, wherein an increase in reporter gene expression in the presence of said candidate compound indicates said candidate compound is an agonist of said interaction, and a decrease in reporter gene expression in the presence of said candidate compound indicates said candidate compound is an antagonist of said interaction. 22. The method of claim 21 wherein said first protein and said known interaction partner protein of said first protein participate in cell signaling by TGF-β family member ligands. 23. The method of claim 21 wherein said first protein is a Smad and said known interaction partner of said first protein is a Smad interacting protein. 24. A method to identify a candidate compound which modulates the activity of an enzyme comprising the steps of: a) expressing said enzyme from a recombinant expression constmct; and b) measuring the activity of said enzyme in the presence and absence of said candidate compound, wherein an increase in the activity of said enzyme in the presence of said candidate compound indicates said candidate compound is an agonist of the activity of said enzyme, and a decrease in the activity of said enzyme in the presence of said candidate compound indicates said candidate compound is an antagonist of the activity of said enzyme. 25. The method of claim 24 wherein said enzyme is expressed in a cell free system. 26. The method of claim 24 wherein said enzyme is expressed in cultured cells. 27. The method of any of claims 24-26 wherein said enzyme is GST. 28. The method of any of claims 24-26 wherein said enzyme is a phosphatase. 29. The method of any of claims 24-26 wherein said enzyme participates in cell signaling by TGF-β family member ligands. 30. A method for monitoring the proteasome-mediated proteolysis of a protein comprising the steps of: a) contacting an isolated polypeptide comprising a protein of interest with isolated proteasomes and a mammalian cell extract in the presence and absence of a specific proteasome inhibitor; and b) detecting the amount of said protein of interest, wherein a decrease in said protein of interest occurring in the absence, but not the presence of said proteasome inhibitor indicates proteasome-mediated degradation of said protein. 31. A method to identify a candidate compound which modulates the proteolysis of a protein comprising the steps of: a) transforming yeast cells with expression constmcts comprising: i) a hybrid protein comprising from the amino terminus to the carboxyl terminus a DNA binding domain, a protein of interest and a transactivation domain; and ii) a reporter gene comprising a DNA sequence bound by said DNA binding domain and transactivated by said transactivation domain; b) culturing said cells in the presence and absence of said candidate compound; and c) detecting the amount of reporter gene expression, wherein a decrease in reporter gene expression in the presence of said candidate compound indicates said candidate compound is an agonist of the proteolysis of said protein of interest, and an increase in reporter gene expression in the presence of said candidate compound indicates said candidate compound is an antagonist of the proteolysis of said protein of interest. 32. A method to identify a candidate compound which modulates the proteolysis of a protein comprising the steps of: a) transforming yeast cells with expression constmcts comprising: i) a hybrid protein comprising from the amino terminus to the carboxyl terminus a DNA binding domain, a protein of interest and a transactivation domain; ii) a reporter gene comprising a DNA sequence bound by said DNA binding domain and transactivated by said transactivation domain; and iii) a constitutively active mutant of a TGF-β family ligand type I receptor; and b) culturing the yeast cells of a) in the presence and absence of said candidate compound; and c) detecting the amount of reporter gene expression, wherein an increase in reporter gene expression indicates said candidate compound is an antagonist of proteolysis of said protein of interest, and a decrease in reporter gene expression indicates said candidate compound is an agonist of the proteolysis of said protein of interest. 33. The method of claim 31 or 32 wherein said protein of interest participates in cell signaling by TGF-β family member ligands. 34. A method for monitoring the proteolysis of a protein of interest in mammalian cells comprising the steps of: a) transfecting a mammalian cell line with expression constmcts comprising: i) a hybrid protein comprising from the amino terminus to the carboxyl terminus a DNA binding domain, a protein of interest and a transactivation domain; ii) a reporter gene, comprising a DNA sequence bound by said DNA binding domain, and transactivated by said transactivation domain; and b) detecting the expression of said reporter gene, wherein expression of said reporter gene indicates that said protein of interest is intact. 35. A method to identify a candidate compound which modulates the proteolysis of a protein of interest in mammalian cells comprising the steps of: a) transfecting a mammalian cell line with expression constmcts comprising: i) a hybrid protein comprising from the amino terminus to the carboxyl terminus a DNA binding domain, a protein of interest and a transactivation domain; ii) a reporter gene, comprising a DNA sequence bound by said DNA binding domain, and transactivated by said transactivation domain; b) culturing said cell line in the presence and absence of said candidate compound; and c) detecting the amount of reporter gene expression, wherein an increase in said reporter gene expression in the presence of said candidate compound indicates said candidate compound is an antagonist of proteolysis, and a decrease in said reporter gene expression in the presence of said candidate compound indicates said candidate compound is an agonist of proteolysis. 36. A method to identify novel, tissue-specific Smad interactors comprising the steps of: a) transforming yeast cells with expression constmcts comprising: i) a hybrid gene comprising the coding sequences for a full length Smad and a DNA binding domain; ii) a cDNA library, derived from a single tissue or cell type, cloned into a vector which fuses the library sequences to a transactivation domain; and iii) a reporter gene, comprising a DNA sequence bound by said DNA binding domain, and transactivated by said transactivation domain; b) selecting yeast cell clones which express said reporter gene; c) probing DNA isolated from said clones with probes specific for all known Smad interactor proteins to identify clones which are novel; and d) using the sequences of clones identified in c) as probes of multi-tissue Northern (RNA) blots to confirm tissue-specific expression of said clones identified in c). 37. A method to identify novel Smad proteins comprising the steps of: a) transforming yeast cells with expression constmcts comprising: i) a hybrid gene comprising the coding sequences for a full length Smad and a DNA binding domain; ii) a cDNA library, cloned into a vector which fuses the library sequences to a transactivation domain; and iii) a reporter gene, comprising a DNA sequence bound by said DNA binding domain, and transactivated by said transactivation domain; b) selecting yeast cell clones which express said reporter gene; c) probing DNA isolated from said clones with nucleic acid probes derived from known Smads under conditions which permit the identification of yeast colonies which contain sequences which hybridize with known Smad sequences; d) isolating and sequencing the plasmid DNA sequences identified in c); and e) comparing the resulting sequences with known Smad sequences, such that clones with sequences which are not identical to the sequence of any known Smad are identified as novel. 38. A composition comprising a ternary complex comprising Smadl, HsN3 and antizyme. 39. A composition comprising a quartemary complex comprising Smadl, Smad4, HsN3 and antizyme. 40. A composition comprising one or more of antizyme and HsN3; antizyme and HsN3; or HEFl and antizyme. 41. A compositions comprising the SNTPl and CBP/p300.
法律状态
(WO200047102) LEGAL DETAILS FOR WO200047102  Actual or expected expiration date=2002-08-11    Legal state=DEAD    Status=LAPSED     Event publication date=2000-02-11  Event code=WO/APP  Event indicator=Pos  Event type=Examination events  Application details  Application country=WO WOUS0003561  Application date=2000-02-11  Standardized application number=2000WO-US03561     Event publication date=2000-08-17  Event code=WO/A2  Event type=Examination events  International application published without international search report  Publication country=WO  Publication number=WO200047102  Publication stage Code=A2  Publication date=2000-08-17  Standardized publication number=WO200047102     Event publication date=2000-08-17  Event code=WO/AL  Event indicator=Pos  Event type=Designated states  Designated countries for regional patents AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE    Event publication date=2000-08-17  Event code=WO/AK  Event indicator=Pos  Event type=Designated states  Designated states CA JP US    Event publication date=2001-04-05  Event code=WO/DFPE  Event type=Examination events  Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)    Event publication date=2002-08-11  Event code=WO/EETL  Event type=Event indicating Not In Force  PCT Application validity period expired.    Event publication date=2002-10-10  Event code=WO/A3  Event indicator=Pos  Event type=Examination events  Later publication of ISR with revised front page  Publication country=WO  Publication number=WO200047102  Publication stage Code=A3  Publication date=2002-10-10  Standardized publication number=WO200047102     Event publication date=2002-10-10  Event code=WO/AL  Event indicator=Pos  Event type=Designated states  Designated countries for regional patents AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE    Event publication date=2002-10-10  Event code=WO/AK  Event indicator=Pos  Event type=Designated states  Designated states CA JP US LEGAL DETAILS FOR DESIGNATED STATE US2002076799  Actual or expected expiration date=2009-07-13    Legal state=DEAD    Status=LAPSED   Corresponding cc:  Designated or member state=US Corresponding appl: US09927738  Application date in the designated or member state=2001-08-10   Application number in the designated or member state=2000US-09927738 Corresponding cc:  Designated or member state=US Corresponding pat: US2002076799  Publication stage code in the designated or member state=A1  Publication date in the designated or member state=2002-06-20   Publication number in the designated or member state=US20020076799    Event publication date=2001-08-10  Event code=WO/WWE  Event indicator=Pos  Event type=Entry into national phase  Wipo information: entry into national phase Corresponding cc:  Designated or member state=US     Event publication date=2016-06-28  Event code=US/STCHG  Patent Status changed by the national office Corresponding cc:  Designated or member state=US
专利类型码
A2A3
国别省市代码
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