EP1185652A2 - Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto - Google Patents
Nucleic acid encoding proteins involved in protein degradation, products and methods related theretoInfo
- Publication number
- EP1185652A2 EP1185652A2 EP00942718A EP00942718A EP1185652A2 EP 1185652 A2 EP1185652 A2 EP 1185652A2 EP 00942718 A EP00942718 A EP 00942718A EP 00942718 A EP00942718 A EP 00942718A EP 1185652 A2 EP1185652 A2 EP 1185652A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- smdp
- scp
- nucleic acid
- degradation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 233
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 209
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 126
- 238000000034 method Methods 0.000 title claims abstract description 101
- 102000039446 nucleic acids Human genes 0.000 title claims description 118
- 108020004707 nucleic acids Proteins 0.000 title claims description 118
- 230000017854 proteolysis Effects 0.000 title claims description 56
- 208000034840 pagnamenta type spondylometaphyseal dysplasia Diseases 0.000 claims abstract description 142
- 241000282414 Homo sapiens Species 0.000 claims abstract description 41
- 239000000203 mixture Substances 0.000 claims abstract description 26
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 16
- 230000009261 transgenic effect Effects 0.000 claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims description 121
- 230000027455 binding Effects 0.000 claims description 65
- 102100021838 E3 ubiquitin-protein ligase SIAH1 Human genes 0.000 claims description 62
- 101710128187 E3 ubiquitin-protein ligase Siah1 Proteins 0.000 claims description 61
- 239000012634 fragment Substances 0.000 claims description 54
- 239000002299 complementary DNA Substances 0.000 claims description 49
- 108020004414 DNA Proteins 0.000 claims description 47
- 238000006731 degradation reaction Methods 0.000 claims description 39
- 150000001875 compounds Chemical class 0.000 claims description 38
- 230000000694 effects Effects 0.000 claims description 37
- 230000015556 catabolic process Effects 0.000 claims description 35
- 230000014509 gene expression Effects 0.000 claims description 33
- 239000013598 vector Substances 0.000 claims description 33
- 101100223242 Helianthus annuus SAMDC gene Proteins 0.000 claims description 32
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 31
- 108020001507 fusion proteins Proteins 0.000 claims description 30
- 102000037865 fusion proteins Human genes 0.000 claims description 30
- 230000001404 mediated effect Effects 0.000 claims description 28
- 108020004999 messenger RNA Proteins 0.000 claims description 26
- 239000002773 nucleotide Substances 0.000 claims description 26
- 125000003729 nucleotide group Chemical group 0.000 claims description 26
- 101000957351 Homo sapiens Myc-associated zinc finger protein Proteins 0.000 claims description 25
- 102100038750 Myc-associated zinc finger protein Human genes 0.000 claims description 25
- 239000005557 antagonist Substances 0.000 claims description 20
- 239000000556 agonist Substances 0.000 claims description 18
- 238000009396 hybridization Methods 0.000 claims description 18
- 239000013615 primer Substances 0.000 claims description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 15
- 108090000848 Ubiquitin Proteins 0.000 claims description 15
- 102000044159 Ubiquitin Human genes 0.000 claims description 15
- 108091034117 Oligonucleotide Proteins 0.000 claims description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 12
- 230000007170 pathology Effects 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 11
- 238000004166 bioassay Methods 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 10
- 230000004663 cell proliferation Effects 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- 101000863884 Homo sapiens Sialic acid-binding Ig-like lectin 8 Proteins 0.000 claims description 8
- 241000124008 Mammalia Species 0.000 claims description 8
- 102100029964 Sialic acid-binding Ig-like lectin 8 Human genes 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 8
- 210000000170 cell membrane Anatomy 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 230000002159 abnormal effect Effects 0.000 claims description 5
- 230000001413 cellular effect Effects 0.000 claims description 5
- 239000012051 hydrophobic carrier Substances 0.000 claims description 4
- 108091008819 oncoproteins Proteins 0.000 claims description 4
- 102000027450 oncoproteins Human genes 0.000 claims description 4
- 230000009257 reactivity Effects 0.000 claims description 4
- 230000028327 secretion Effects 0.000 claims description 4
- 230000030833 cell death Effects 0.000 claims description 3
- 230000000593 degrading effect Effects 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 239000003155 DNA primer Substances 0.000 claims description 2
- 230000012292 cell migration Effects 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 claims description 2
- 108020001019 DNA Primers Proteins 0.000 claims 1
- 206010061218 Inflammation Diseases 0.000 claims 1
- 230000003321 amplification Effects 0.000 claims 1
- 230000004054 inflammatory process Effects 0.000 claims 1
- 238000003199 nucleic acid amplification method Methods 0.000 claims 1
- 238000003556 assay Methods 0.000 abstract description 14
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 102000036366 SCF complex Human genes 0.000 abstract description 4
- 108091007047 SCF complex Proteins 0.000 abstract description 4
- 238000007877 drug screening Methods 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 166
- 108090000765 processed proteins & peptides Proteins 0.000 description 106
- 102000004196 processed proteins & peptides Human genes 0.000 description 100
- 229920001184 polypeptide Polymers 0.000 description 89
- 108020004635 Complementary DNA Proteins 0.000 description 62
- 235000001014 amino acid Nutrition 0.000 description 59
- 229940024606 amino acid Drugs 0.000 description 58
- 150000001413 amino acids Chemical class 0.000 description 55
- 238000010804 cDNA synthesis Methods 0.000 description 46
- 102000053602 DNA Human genes 0.000 description 38
- 239000013612 plasmid Substances 0.000 description 31
- 239000000523 sample Substances 0.000 description 26
- 230000003993 interaction Effects 0.000 description 23
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 21
- 230000006870 function Effects 0.000 description 19
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 18
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 description 15
- 101710089792 BAG family molecular chaperone regulator 1 Proteins 0.000 description 15
- -1 carboxyl amino Chemical group 0.000 description 15
- 102100037152 BAG family molecular chaperone regulator 1 Human genes 0.000 description 14
- 206010028980 Neoplasm Diseases 0.000 description 14
- 108010005774 beta-Galactosidase Proteins 0.000 description 14
- 230000002950 deficient Effects 0.000 description 14
- 235000002639 sodium chloride Nutrition 0.000 description 14
- 102000018700 F-Box Proteins Human genes 0.000 description 13
- 108010066805 F-Box Proteins Proteins 0.000 description 13
- 230000037361 pathway Effects 0.000 description 13
- 108060000903 Beta-catenin Proteins 0.000 description 11
- 102000015735 Beta-catenin Human genes 0.000 description 11
- 230000000875 corresponding effect Effects 0.000 description 11
- 230000012010 growth Effects 0.000 description 11
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 10
- 102000003714 TNF receptor-associated factor 6 Human genes 0.000 description 10
- 108090000009 TNF receptor-associated factor 6 Proteins 0.000 description 10
- 102000005936 beta-Galactosidase Human genes 0.000 description 10
- 230000004071 biological effect Effects 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- 230000001177 retroviral effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000006467 substitution reaction Methods 0.000 description 10
- 230000034512 ubiquitination Effects 0.000 description 10
- 238000001727 in vivo Methods 0.000 description 9
- 238000010798 ubiquitination Methods 0.000 description 9
- 238000001086 yeast two-hybrid system Methods 0.000 description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 8
- 229920002477 rna polymer Polymers 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 102100031748 E3 ubiquitin-protein ligase SIAH2 Human genes 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 7
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- 238000013519 translation Methods 0.000 description 7
- 108020004705 Codon Proteins 0.000 description 6
- 101710128185 E3 ubiquitin-protein ligase Siah2 Proteins 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 108010079855 Peptide Aptamers Proteins 0.000 description 6
- 239000004480 active ingredient Substances 0.000 description 6
- 230000000692 anti-sense effect Effects 0.000 description 6
- 239000005022 packaging material Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 238000010396 two-hybrid screening Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 201000009030 Carcinoma Diseases 0.000 description 5
- 108090000538 Caspase-8 Proteins 0.000 description 5
- 102000004091 Caspase-8 Human genes 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 108090000925 TNF receptor-associated factor 2 Proteins 0.000 description 5
- 102000004393 TNF receptor-associated factor 2 Human genes 0.000 description 5
- 102000003431 Ubiquitin-Conjugating Enzyme Human genes 0.000 description 5
- 108060008747 Ubiquitin-Conjugating Enzyme Proteins 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000025084 cell cycle arrest Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 238000003348 filter assay Methods 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 108060008226 thioredoxin Proteins 0.000 description 5
- 239000013603 viral vector Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 4
- 230000004568 DNA-binding Effects 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 101000966793 Homo sapiens 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase Proteins 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 4
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 4
- 108091008324 binding proteins Proteins 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000022131 cell cycle Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 102000057053 human ADI1 Human genes 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000012423 maintenance Methods 0.000 description 4
- 238000013507 mapping Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000013011 mating Effects 0.000 description 4
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 4
- 239000002853 nucleic acid probe Substances 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 101150054147 sina gene Proteins 0.000 description 4
- 229940094937 thioredoxin Drugs 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 3
- 108050006400 Cyclin Proteins 0.000 description 3
- 102000016736 Cyclin Human genes 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 101710116362 E3 ubiquitin-protein ligase sina Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 3
- 241000713869 Moloney murine leukemia virus Species 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 244000097202 Rathbunia alamosensis Species 0.000 description 3
- 235000009776 Rathbunia alamosensis Nutrition 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 102000000341 S-Phase Kinase-Associated Proteins Human genes 0.000 description 3
- 108010055623 S-Phase Kinase-Associated Proteins Proteins 0.000 description 3
- 101150103019 SCP gene Proteins 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102000002933 Thioredoxin Human genes 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 3
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 108700000707 bcl-2-Associated X Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000032823 cell division Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002405 diagnostic procedure Methods 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 238000001378 electrochemiluminescence detection Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000000520 microinjection Methods 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 108020001580 protein domains Proteins 0.000 description 3
- 108010014186 ras Proteins Proteins 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 239000000225 tumor suppressor protein Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 102000005483 Cell Cycle Proteins Human genes 0.000 description 2
- 108010031896 Cell Cycle Proteins Proteins 0.000 description 2
- 102000003903 Cyclin-dependent kinases Human genes 0.000 description 2
- 108090000266 Cyclin-dependent kinases Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- 101100044298 Drosophila melanogaster fand gene Proteins 0.000 description 2
- 108091060211 Expressed sequence tag Proteins 0.000 description 2
- 101150064015 FAS gene Proteins 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 108010054278 Lac Repressors Proteins 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 241000713333 Mouse mammary tumor virus Species 0.000 description 2
- 102000007474 Multiprotein Complexes Human genes 0.000 description 2
- 108010085220 Multiprotein Complexes Proteins 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 101100335198 Pneumocystis carinii fol1 gene Proteins 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108091007354 SIAHs Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 102100036407 Thioredoxin Human genes 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 102000000160 Tumor Necrosis Factor Receptor-Associated Peptides and Proteins Human genes 0.000 description 2
- 108010080432 Tumor Necrosis Factor Receptor-Associated Peptides and Proteins Proteins 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 102000055102 bcl-2-Associated X Human genes 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 108700000015 c-MYC-associated zinc finger Proteins 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 238000002825 functional assay Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000003607 modifier Substances 0.000 description 2
- 230000036963 noncompetitive effect Effects 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000000861 pro-apoptotic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004844 protein turnover Effects 0.000 description 2
- 102000016914 ras Proteins Human genes 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 101150012083 siah-1 gene Proteins 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N thiocyanic acid Chemical compound SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- JVJUWEFOGFCHKR-UHFFFAOYSA-N 2-(diethylamino)ethyl 1-(3,4-dimethylphenyl)cyclopentane-1-carboxylate;hydrochloride Chemical compound Cl.C=1C=C(C)C(C)=CC=1C1(C(=O)OCCN(CC)CC)CCCC1 JVJUWEFOGFCHKR-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- SPBDXSGPUHCETR-JFUDTMANSA-N 8883yp2r6d Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O[C@@H]([C@@H](C)CC4)C(C)C)O3)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1C[C@H](C)[C@@H]([C@@H](C)CC)O[C@@]21O[C@H](C\C=C(C)\[C@@H](O[C@@H]1O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C1)[C@@H](C)\C=C\C=C/1[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\1)O)C[C@H]4C2 SPBDXSGPUHCETR-JFUDTMANSA-N 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 1
- 101710112282 Adenomatous polyposis coli protein Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 206010002961 Aplasia Diseases 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 108091007914 CDKs Proteins 0.000 description 1
- 241000222178 Candida tropicalis Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 108700020475 Cullin Proteins 0.000 description 1
- 102000052581 Cullin Human genes 0.000 description 1
- 108010088874 Cullin 1 Proteins 0.000 description 1
- 102000036364 Cullin Ring E3 Ligases Human genes 0.000 description 1
- 108091007045 Cullin Ring E3 Ligases Proteins 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101710201734 E3 protein Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000002927 Hamartoma Diseases 0.000 description 1
- 108050008339 Heat Shock Transcription Factor Proteins 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000616722 Homo sapiens E3 ubiquitin-protein ligase SIAH1 Proteins 0.000 description 1
- 101000707245 Homo sapiens E3 ubiquitin-protein ligase SIAH2 Proteins 0.000 description 1
- 101000911513 Homo sapiens Uncharacterized protein FAM215A Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 101000668058 Infectious salmon anemia virus (isolate Atlantic salmon/Norway/810/9/99) RNA-directed RNA polymerase catalytic subunit Proteins 0.000 description 1
- 208000002260 Keloid Diseases 0.000 description 1
- 206010023330 Keloid scar Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102100025169 Max-binding protein MNT Human genes 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101000740063 Mus musculus BAG family molecular chaperone regulator 1 Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 102000010803 Netrins Human genes 0.000 description 1
- 108010063605 Netrins Proteins 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical class [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 210000001033 R7 photoreceptor cell Anatomy 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 206010038997 Retroviral infections Diseases 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 102000000369 SOCS box domains Human genes 0.000 description 1
- 108050008939 SOCS box domains Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 1
- 101150014014 Traf6 gene Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 102100026728 Uncharacterized protein FAM215A Human genes 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- GELXFVQAWNTGPQ-UHFFFAOYSA-N [N].C1=CNC=N1 Chemical compound [N].C1=CNC=N1 GELXFVQAWNTGPQ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007818 agglutination assay Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 231100001075 aneuploidy Toxicity 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000003141 anti-fusion Effects 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 101150042295 arfA gene Proteins 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004640 cellular pathway Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 125000002668 chloroacetyl group Chemical group ClCC(=O)* 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000011490 co-immunoprecipitation assay Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 230000002153 concerted effect Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229940043378 cyclin-dependent kinase inhibitor Drugs 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 150000002169 ethanolamines Chemical class 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000001400 expression cloning Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000004524 haematopoietic cell Anatomy 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 101150032598 hisG gene Proteins 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000012750 in vivo screening Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002642 intravenous therapy Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 210000001117 keloid Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 108010025821 lamin C Proteins 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 101150087557 omcB gene Proteins 0.000 description 1
- 101150115693 ompA gene Proteins 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000008196 pharmacological composition Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 108091007054 readthrough proteins Proteins 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 230000008684 selective degradation Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108091006024 signal transducing proteins Proteins 0.000 description 1
- 102000034285 signal transducing proteins Human genes 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091006107 transcriptional repressors Proteins 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011820 transgenic animal model Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000012863 translational readthrough Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 238000003160 two-hybrid assay Methods 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000012130 whole-cell lysate Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 238000003158 yeast two-hybrid assay Methods 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- 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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to nucleic acids and proteins encoded thereby.
- cyclins A, B, C, DI, E cyclin-dependent kinase inhibitory proteins
- cdk cyclin-dependent kinase
- El proteins form thioester bonds in which the sulfhydryl group of internal cysteine residues binds the carboxyl amino acid of ubiquitin, thereby activating ubiquitin for subsequent transfer to E2-family proteins.
- E2 family proteins then transfer activated ubiquitin to the free amino-groups of lysine side chains in target proteins directly. More often, however, E2-family proteins collaborate with E3 proteins which bind particular target proteins and orchestrate their interactions with E2s, coordinating the polyubiquitination of these target proteins in highly regulated manners Ciechanover, A. 1998 supra .
- E3 functions are sometimes embodied in multiprotein complexes rather than mediated by a single protein.
- SFC complexes The ubiquitination and degradation of a variety of cyclins, cyclin-dependent kinases (cdks) and cdk- inhibitors is temporally controlled during the cell cycle by SFC complexes.
- F-box proteins contain a conserved motif, the F- box, which mediates their interactions with Skp-1.
- the F-box proteins also contain other domains which allow them to simultaneously bind specific substrate proteins, which are then targeted for degradation via polyubiquitination.
- One such F-box protein identified in humans is b-Trcp, which forms a SCF complex with Skp-1 and Cul-1, and which interacts with ⁇ -catenin, targeting it for degradation (Latres, et al . , 1999, Oncogene, 18:849-854, and Winston, J.J. et al . , 1999, Genes & Dev. , 13:270-283) .
- Siah-family proteins represent mammalian homologs of the Drosophila Sina protein.
- Sina is required for R7 photoreceptor cell differentiation within the sevenless pathway.
- Sina binds a ubiquitin- conjugating enzyme (E2) via an N-terminal RING domain.
- E2 ubiquitin- conjugating enzyme
- Heteroco plexes of Sina and another protein called Phyllopodia form a E3-complex which interacts with a transcriptional repressor called Tramtrack, targeting it for polyubiqitination and proteosome-mediated degradation in the fly (Tang, A.H. et al., 1997, Cell, 90:459-467 and Li, S. et al., 1997, Cell, 469-478).
- the destruction of Tramtrack is necessary for differentiation of R7 cells.
- novel isolated nucleic acids encoding a variety of Siah-Mediated-Degradation-Proteins (SMDPs) involved in the Siah-mediated protein degradation pathways and/or SCF-Complex-Proteins (SCPs) involved in SCF-mediated protein degradation pathways.
- SMDPs Siah-Mediated-Degradation-Proteins
- SCPs SCF-Complex-Proteins
- vectors containing invention nucleic acids, probes that hybridize thereto, host cells transformed therewith, antisense-nucleic acids thereto and related compositions are provided.
- the nucleic acid molecules described herein can be incorporated into a variety of expression systems known to those of skill in the art.
- nucleic acid molecules of the present invention are useful as probes for assaying for the presence and/or amount of a SMDP and/or SCP gene or mRNA transcript in a given sample.
- the nucleic acid molecules described herein, and oligonucleotide fragments thereof, are also useful as primers and/or templates in a PCR reaction for amplifying genes encoding SMDP and/or SCP proteins.
- isolated mammalian SMDP and/or SCP proteins are also provided. These proteins, or fragments thereof, are useful in bioassays, as immunogens for producing anti- SMDP and/or SCP antibodies, or in therapeutic compositions containing such proteins and/or antibodies. Also provided are transgenic non-human mammals that express, or fail to express (e.g., knock-out), the invention protein.
- Antibodies that are immunoreactive with invention SMDP and/or SCP proteins are also provided. These antibodies are useful in diagnostic assays to determine levels of SMDP and/or SCP proteins present in a given sample, e.g., tissue samples, Western blots, and the like. The antibodies can also be used to purify SMDP and/or SCP proteins from crude cell extracts and the like. Moreover, these antibodies are considered therapeutically useful to modulate the biological effect of SMDP and/or SCP proteins in vivo.
- bioassays for identifying compounds that modulate the activity of invention SMDP and/or SCP proteins.
- Methods and diagnostic systems for determining the levels of SMDP and/or SCP protein in various tissue samples are also provided. These diagnostic methods can be used for monitoring the level of therapeutically administered SMDP and/or SCP or fragments thereof to facilitate the maintenance of therapeutically effective amounts. These diagnostic methods can also be used to diagnose physiological disorders that result from abnormal levels of SMDP and/or SCP.
- SMDPs for targeting any desired protein for ubiquitination and degradation, thus enabling novel gene discovery through functional genomics strategies or providing the basis for ablating target proteins involved in diseases for therapeutic purposes.
- Figure 1 shows an amino acid sequence comparison of SIP-L (SEQ ID NO: 4) and SIP-S (SEQ ID NO:6) .
- FIG. 1 shows the Destruction-box amino acid consensus sequence of SAD.
- Figure 3 shows the Mapping of Siah-APC interaction domains as described in Example 12.
- Figure 4 shows the results of the cell proliferation functional assay of SIP/Siah interaction described in Example 7.
- Figures 5A and 5B show in vitro and in vivo interaction assays of between Siah-1 and SIP-L as described in Examples 8 and 9, respectively.
- Figures 6A and 6B show the mapping of SKP1, SIP-L, SAF-1 and SAD interaction domains as described in Example 13.
- Figure 7 shows the effect of Siah-1 overexpression on stability of ⁇ -catenin.
- Figure 8 shows a diagram of how an invention SIP communicates with the Protein Ubiquitination Machinery.
- Figure 9 shows a general diagram of an invention method for inducing targeted degradation of proteins using SIP, exemplified in Example 15.
- FIG. 10 shows the results of the SIP-mediated degradation of the target TRAF6 protein, set forth in Example 15.
- isolated nucleic acids which encode novel mammalian Siah-Mediated-Degradation-Proteins (SMDPs) and/or SCF-Complex-Proteins (SCPs) , and functional fragments thereof.
- SMDPs are involved in the Siah- mediated protein degradation pathways and SCPs are involved in SCF-mediated protein degradation pathways. In some instances, these two pathways for protein degradation may operate in collaboration, particularly in cases where proteins have been identified that physically link SMDPs to SCPs.
- Invention SMDPs and/or SCPs are contemplated herein to regulate protein degradation, either by activating or inhibiting such protein degradation.
- invention SMDPs are proteins that participate in the Siah-mediated protein degradation pathway.
- the term "Siah” refers to the mammalian family of proteins encoded by at least two genes referred to as SIAH1 and SIAH2 (Hu, G. et al., 1997, Genomics, 46:103- 111).
- SIAH1 and SIAH2 Hu, G. et al., 1997, Genomics, 46:103- 111
- the Siah-1 and Siah-2 proteins bind ubiquitin conjugating enzymes (UBCs) via an N-terminal RING domain and target other proteins for degradation.
- UBCs ubiquitin conjugating enzymes
- invention SCPs are proteins that participate in the Skp-1, Cullin, F-box (SCF) protein degradation pathway. These proteins can be components of SCF complexes or proteins that associate with SCF complexes. In some instances an invention protein may fulfill the requirements of both a SMDP and a SCP.
- Siah-1 interacts indirectly with SCF complexes through associations with an invention SIP protein.
- Siah-1 has also been found to be an important regulator of cell growth, through its effects on ubiquitination and degradation of ⁇ -catenin and possibly other target proteins including an invention protein SAD.
- DCC protein has recently been shown to deliver either pro-apoptotic or anti- apoptotic signals, depending on whether it is complexed with its ligand Netrin.
- Siah-1 gene of mice was found among a group of immediate-early genes induced by p53 using a hemopoietic cell line as a model for p53-induced cell cycle arrest and apoptosis (Amson, R.B. et al., 1996, PNAS, USA, 93:3953-3957).
- Expression of Siah-1 was also indirectly correlated with increased apoptosis in tumor xenograph experiments, suggesting that Siah-1 could function as a tumor suppressor in some contexts (Nemani, M. et al., 1996, Proc. Natl. Sci. USA 93:9039-9042).
- Siah-1 over- expression can induce cell cycle arrest independently of apoptosis in epithelial cancer cells (Matsuzawa, et al., 1998, EMBO J. , 17 ( 10 ): 2736-2747 ) . Moreover, UV- irradiation at subapoptotic doses was shown to induce
- Siah-1 gene expression in MCF7 breast cancer cells and to promote cell cycle arrest have implicated Siah-1 in a p53-inducible pathway for cell cycle arrest which runs parallel to the well-studied p21- Wafl pathway (Matsuzawa, et al. 1998, supra) .
- SMDP and/or SCP refers to substantially pure native SMDP and/or SCP, or recombinantly produced proteins, including naturally occurring allelic variants thereof encoded by mRNA generated by alternative splicing of a primary transcript, and further including fragments thereof which retain at least one native biological activity, such as immunogenicity, the ability to bind to a SMDP and/or SCP, and the like.
- Exemplary SMDPs and/or SCPs referred to herein include amino acid sequences set forth in SEQ ID Nos:2 (Siah-l ), 4 (SIP-L), 6 (SIP-S), 8 (SAF-l ⁇ ), 10 (SAF-l ⁇ ), 12 (SAF-2) and 14 (SAD).
- Invention isolated SMDPs and/or SCPs are substantially pure and free of cellular components and/or contaminants normally associated with a native in vivo environment.
- Siah-l ⁇ refers to a splice-variant member of the mammalian, preferably human, Siah-family of proteins.
- the invention Siah-l ⁇ protein, or functional fragment thereof, is characterized by having the ability to bind to at least one or more of the proteins selected from APC (Kinzler K.W., et al., 1996, Cell, 87 (2) :159-170) ; BAG-1 (Takayama et al., 1995, Cell, 80 (2) :279-284) ; SIP-L; SIP-S; or other Siah proteins, such as Siah-1.
- APC Korean K.W., et al., 1996, Cell, 87 (2) :159-170
- BAG-1 Takayama et al., 1995, Cell, 80 (2) :279-284
- SIP-L SIP-S
- Siah proteins such as Siah-1.
- Siah-l ⁇ proteins differ from Siah-l ⁇ (set forth as SIAH-1 in Hu et al., 1997, Genomics , 46:103-111) by containing an additional 16 amino acids at the amino-terminus .
- preferred invention Siah-l ⁇ proteins, and fragments thereof comprise at least a portion of the 16 N-terminal amino acids of SEQ ID NO: 2.
- a particularly preferred Siah-l ⁇ protein is set forth in SEQ ID NO:2.
- Siah-1 has been found to interact with an invention protein referred to herein as the "SIP" family.
- SIP refers to any species, preferably mammalian, more preferably human, Siah-1- .
- Interacting Protein SIP
- the invention SIP proteins, or functional fragments thereof, are characterized by having the ability to bind to at least one or more of the proteins selected from Siah-1, Skpl, or other SIP proteins.
- homodimers of invention SIP proteins are contemplated herein.
- SIP-L L, for long; SEQ ID NO:4
- SIP-S S, for short; SEQ ID NO: 6
- a sequence comparison of SIP-L to SIP-S is set forth in Figure 1.
- yeast two-hybrid screens of cDNA libraries were performed using the invention human SIP-L protein (SEQ ID NO:4) as a bait. Such screen resulted in the identification of Skpl (Zhang et al., 1995, Cell, 82 (6) :915-925) .
- an invention SIP protein binds simultaneously to Siah and Skp, proteins known to bind directly or indirectly, respectively, to ubiquitin- conjugating enzymes (E2s). Therefore, in accordance with the present invention, this characteristic of SIP is useful for methods for targeting desired proteins for degradation, via a ubiquitin/proteosome-dependent mechanism (see, e.g., Example 15).
- SAF-1 and SAD two additional invention SMDP and/or SCP proteins were identified as Skpl-interacting proteins, which are referred to herein as SAF-1 and SAD.
- SAF-1 refers to Skpl-Associated F-box protein-1.
- the invention SAF-1 proteins, or functional fragments thereof, are characterized by having the ability to bind to at least one or more of the proteins selected from Skpl, SIP, such as SIP-L, or SAD.
- Invention SAF-1 proteins are further characterized as containing an "F-box" amino acid domain.
- Exemplary SAF-1 proteins include SAF-l ⁇ (SEQ ID NO: 8) and SAF-l ⁇ (SEQ ID NO: 10).
- An exemplary F-box domain is set forth as amino acids 256-296 of SEQ ID NO: 8 and amino acids 335-375 of SEQ ID NO: 10 (see Figure 6).
- SAF-1 beta has the same F-Box as alfa, the location is in amino acids 335-375.
- a homologue of SAF-1 protein has also been identified in the NCBI BLAST data base (Human DNA sequence from clone 341E18 on chromosome 6pll.2-12.3, AL031178) which shares significant homology with F-box domain of SAF-1.
- the invention homolog is referred to herein as SAF-2 and is set forth in SEQ ID Nos: 11 and 12.
- SAD refers to Skpl-
- SAF-1 proteins are characterized by having the ability to bind to at least one or more of the proteins selected from Skpl, SIP, such as SIP-L, or SAF-1. It is also contemplated herein that SAD has the ability to bind to SAF-2.
- invention SAD proteins are further characterized as containing an " D- box" (Destruction-box) amino acid domain.
- An exemplary SAD protein is set forth herein as SEQ ID NO: 14.
- the D-box domain comprises the consensus amino acid sequence -DSGX ⁇ S-, wherein X ⁇ is preferably selected from a hydrophobic amino acid, such as Y, I, L, M, F, W, or V; and X 2 is any amino acid (see Figure 2) .
- a preferred D-box domain comprises the sequence set forth as amino acids 144-149 of SEQ ID NO: 14.
- exemplary functional fragments of an invention SAD protein comprise at least amino acids 144- 149 of SEQ ID NO: 14.
- functional fragments of inventions SAD proteins that bind to a SIP protein and preferably comprise at least amino acids 360-447 of SEQ ID NO: 14.
- Functional fragments of inventions SAD proteins that bind to a Skpl protein preferably comprise at least amino acids 128-359 of SEQ ID NO: 14.
- Functional fragments of inventions SAD proteins that bind to a SAF-1 protein preferably comprise at least amino acids 1-127 of SEQ ID NO: 14.
- nucleic acid molecules described herein are useful for producing invention proteins, when such nucleic acids are incorporated into a variety of protein expression systems known to those of skill in the art.
- nucleic acid molecules or fragments thereof can be labeled with a readily detectable substituent and used as hybridization probes for assaying for the presence and/or amount of an invention SMDP and/or SCP gene or mRNA transcript in a given sample.
- the nucleic acid molecules described herein, and fragments thereof are also useful as primers and/or templates in a PCR reaction for amplifying genes encoding invention proteins described herein.
- nucleic acid encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes, oligonucleotides, and primers.
- DNA can be either complementary DNA (cDNA) or genomic DNA, e.g. a gene encoding a SMDP and/or SCP.
- cDNA complementary DNA
- genomic DNA e.g. a gene encoding a SMDP and/or SCP.
- One means of isolating a nucleic acid encoding an SMDP and/or SCP polypeptide is to probe a mammalian genomic library with a natural or artificially designed DNA probe using methods well known in the art. DNA probes derived from the SMDP and/or SCP gene are particularly useful for this purpose.
- DNA and cDNA molecules that encode SMDP and/or SCP polypeptides can be used to obtain complementary genomic DNA, cDNA or RNA from mammalian (e.g., human, mouse, rat, rabbit, pig, and the like), or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, by methods described in more detail below.
- Examples of nucleic acids are RNA, cDNA, or isolated genomic DNA encoding an SMDP and/or SCP polypeptide.
- Such nucleic acids may include, but are not limited to, nucleic acids comprising substantially the same nucleotide sequence as set forth in SEQ ID Nos:l
- DNA, RNA, polypeptides or proteins are useful in ways described herein that the DNAs, RNAs, polypeptides or proteins as they naturally occur are not.
- Invention proteins can be obtained from any species of organism, such as prokaryotes, eukaryotes, plants, fungi, vertebrates, invertebrates, and the like.
- a particular species can be d mammalian,
- mammalian refers to a subset of species from which an invention SMDP and/or SCP is derived, e.g., human, rat, mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and the like.
- a preferred SMDP and/or SCP herein is human SMDP and/or SCP.
- cDNAs encoding the invention SMDPs and/or SCPs disclosed herein comprise substantially the same nucleotide sequence as the coding region set forth in any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- Preferred cDNA molecules encoding the invention proteins comprise the same nucleotide sequence as as the coding region set forth in any of SEQ ID N0s:l, 3, 5, 7, 9, 11 and 13.
- the term "substantially the same nucleotide sequence” refers to DNA having sufficient identity to the reference polynucleotide, such that it will hybridize to the reference nucleotide under moderately stringent hybridization conditions.
- DNA having substantially the same nucleotide sequence as the reference nucleotide sequence encodes substantially the same amino acid sequence as that set forth in any of SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14.
- DNA having "substantially the same nucleotide sequence" as the reference nucleotide sequence has at least 60% identity with respect to the reference nucleotide sequence. DNA having at least 70%, more preferably at least 90%, yet more preferably at least 95%, identity to the reference nucleotide sequence is preferred.
- nucleic acids which differ from the nucleic acids shown in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, but which have the same phenotype. Phenotypically similar nucleic acids are also referred to as “functionally equivalent nucleic acids”. As used herein, the phrase "functionally equivalent nucleic acids” encompasses nucleic acids characterized by slight and non-consequential sequence variations that will function in substantially the same manner to produce the same protein product (s) as the nucleic acids disclosed herein. In particular, functionally equivalent nucleic acids encode polypeptides that are the same as those encoded by the nucleic acids disclosed herein or that have conservative amino acid variations. For example, conservative variations include substitution of a non-polar residue with another non-polar residue, or substitution of a charged residue with a similarly charged residue. These variations include those recognized by skilled artisans as those that do not substantially alter the tertiary structure of the protein.
- nucleic acids encoding SMDP and/or SCP polypeptides that, by virtue of the degeneracy of the genetic code, do not necessarily hybridize to the invention nucleic acids under specified hybridization conditions.
- Preferred nucleic acids encoding the invention SMDPs and/or SCPs are comprised of nucleotides that encode substantially the same amino acid sequence as set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14.
- an exemplary nucleic acid encoding an invention SMDP and/or SCP may be selected from:
- Hybridization refers to the binding of complementary strands of nucleic acid (i.e., sense : antisense strands or probe : target-DNA) to each other through hydrogen bonds, similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art.
- stringent hybridization is used herein to refer to conditions under which polynucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T n ) of the hybrids. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions.
- moderately stringent hybridization refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA; with greater than about 90% identity to target-DNA being especially preferred.
- moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5X Denhart ' s solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65°C.
- high stringency hybridization refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein) .
- High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart ' s solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0. IX SSPE, and 0.1% SDS at 65°C.
- low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart ' s solution, 6X SSPE, 0.2% SDS at 42°C, followed by washing in IX SSPE, 0.2% SDS, at 50°C.
- Denhart ' s solution and SSPE are well known to those of skill in the art as are other suitable hybridization buffers .
- the term “degenerate” refers to codons that differ in at least one nucleotide from a reference nucleic acid, e.g., SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, but encode the same amino acids as the reference nucleic acid.
- a reference nucleic acid e.g., SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, but encode the same amino acids as the reference nucleic acid.
- codons specified by the triplets "UCU”, “UCC”, “UCA”, and “UCG” are degenerate with respect to each other since all four of these codons encode the amino acid serine.
- Preferred nucleic acids encoding the invention polypeptide hybridize under moderately stringent, preferably high stringency, conditions to substantially the entire sequence, or substantial portions (i.e., typically at least 15-30 nucleotides) of the nucleic acid sequence set forth in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- nucleic acids can be produced by a variety of methods well-known in the art, e.g., the methods described herein, employing PCR amplification using oligonucleotide primers from various regions of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, and the like.
- optionally labeled SMDP and/or SCP- encoding cDNAs, or fragments thereof can be employed to probe library (ies) (e.g., cDNA, genomic, and the like) for additional nucleic acid sequences encoding novel mammalian SMDPs and/or SCPs.
- libraries e.g., cDNA, genomic, and the like
- Construction of suitable mammalian cDNA libraries is well-known in the art. Screening of such a cDNA library is initially carried out under low-stringency conditions, which comprise a temperature of less than about 42°C, a formamide concentration of less than about 50%, and a moderate to low salt concentration.
- Presently preferred probe-based screening conditions comprise a temperature of about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5X standard saline citrate (SSC; 20X SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0).
- SSC standard saline citrate
- Such conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology.
- the phrase "substantial similarity" refers to sequences which share at least 50% homology.
- hybridization conditions will be selected which allow the identification of sequences having at least 70% homology with the probe, while discriminating against sequences which have a lower degree of homology with the probe.
- nucleic acids having substantially the same nucleotide sequence as SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13 are obtained.
- a nucleic acid "probe” is single-stranded DNA or RNA, or analogs thereof, that has a sequence of nucleotides that includes at least 14, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous bases that are the same as (or the complement of) any contiguous bases set forth in any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- Preferred regions from which to construct probes include 5 ' and/or 3 ' coding regions of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- SMDP and/or SCP may be used as a probe.
- Probes may be labeled by methods well-known in the art, as described hereinafter, and used in various diagnostic kits .
- a SMDP and/or SCP-encoding nucleic acid molecule specifically excludes nucleic acid molecules consisting of any of the nucleotide sequences having the Genbank (gb) , EMBL (emb) or DDBJ (dbj) accession numbers described below.
- a SMDP and/or SCP polypeptide fragment specifically excludes the amino acid fragments encoded by the nucleotide sequences having the GenBank accession numbers described below.
- GenBank accession numbers specifically excluded include NCBI ID: AA054272, AA258606, AA923663, AA418482, and AI167464.
- the human sequence referenced as GenBank accession No. AL031178 is also specifically excluded from an invention SMDP and/or SCP-encoding nucleic acid.
- label and "indicating means” in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal. Any label or indicating means can be linked to invention nucleic acid probes, expressed proteins, polypeptide fragments, or antibody molecules. These atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry.
- the labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturation to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
- a fluorochrome a fluorochrome that is a useful immunofluorescent tracer.
- a description of immunofluorescent analytic techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., eds . , John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.
- the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, and the like.
- HRP horseradish peroxidase
- radioactive elements are employed labeling agents.
- the linking of a label to a substrate, i.e., labeling of nucleic acid probes, antibodies, polypeptides, and proteins, is well known in the art.
- an invention antibody can be labeled by metabolic incorporation of radiolabeled amino acids provided in the culture medium. See, for example, Galfre et al., Meth. Enzymol . , 73:3-46 (1981). Conventional means of protein conjugation or coupling by activated functional groups are particularly applicable.
- SMDPs Siah-Mediated-Degradation-Proteins
- SCPs SCF- Complex-Proteins
- SMDP and/or SCP refers to substantially pure native SMDP and/or SCP, or recombinantly produced proteins, including naturally occurring allelic variants thereof encoded by mRNA generated by alternative splicing of a primary transcript, and further including fragments thereof which retain at least one native biological activity, such as immunogenicity, the ability to bind to another member of the SMDP and/or SCP families, or to homodimerize .
- SMDPs and/or SCPs referred to herein are those polypeptides specifically recognized by an antibody that also specifically recognizes a SMDP and/or SCP (preferably human) including an amino acid sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14.
- Invention isolated SMDPs and/or SCPs are substantially pure and free of cellular components and/or contaminants normally associated with a native in vivo environment.
- SMDPs and/or SCPs of the invention include proteins that comprise substantially the same amino acid sequences as the protein sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14, as well as biologically active, functional fragments thereof.
- the term "substantially the same amino acid sequence” refers to amino acid sequences having at least about 70% identity with respect to the reference amino acid sequence, and retaining comparable functional and biological activity characteristic of the protein defined by the reference amino acid sequence.
- proteins having "substantially the same amino acid sequence” will have at least about 80%, more preferably 90% amino acid identity with respect to the reference amino acid sequence; with greater than about 95% amino acid sequence identity being especially preferred. It is recognized, however, that polypeptides (or nucleic acids referred to hereinbefore) containing less than the described levels of sequence identity arising as splice variants or that are modified by conservative amino acid substitutions, or by substitution of degenerate codons are also encompassed within the scope of the present invention.
- biologically active when used herein as a modifier of invention SMDP and/or SCP(s), or polypeptide fragments thereof, refers to a polypeptide that exhibits functional characteristics similar to SMDP and/or SCP.
- one biological activity of SMDP and/or SCP is the ability to bind, preferably in vivo, to at least one other member of the SMDP and/or SCP families of proteins, or to homodimerize, or to mediate protein degradation via an SFC complex as described herein.
- Such SMDP and/or SCP binding activity can be assayed, for example, using the methods described herein.
- Another biological activity of SMDP and/or SCP is the ability to act as an immunogen for the production of polyclonal and monoclonal antibodies that bind specifically to an invention SMDP and/or SCP.
- an invention nucleic acid encoding SMDP and/or SCP will encode a polypeptide specifically recognized by an antibody that also specifically recognizes the SMDP and/or SCP protein (preferably human) including the amino acid set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14.
- Such immunologic activity may be assayed by any method known to those of skill in the art.
- a test- polypeptide encoded by a SMDP and/or SCP cDNA can be used to produce antibodies, which are then assayed for their ability to bind to an invention SMDP and/or SCP protein including the sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14. If the antibody binds to the test- polypeptide and the protein including the sequence encoded by SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14 with substantially the same affinity, then the polypeptide possesses the requisite immunologic biological activity.
- SMDPs and/or SCPs can be isolated by a variety of methods well-known in the art, e.g., recombinant expression systems described herein, precipitation, gel filtration, ion-exchange, reverse- phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al . , Guide to Protein Purification: Methods in Enzvmoloqy
- the isolated polypeptides of the present invention can be obtained using well-known recombinant methods as described, for example, in Sambrook et al., supra., 1989).
- An example of the means for preparing the invention polypeptide (s ) is to express nucleic acids encoding the SMDP and/or SCP in a suitable host cell, such as a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte) , or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods.
- a suitable host cell such as a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte) , or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods.
- Invention polypeptides can be isolated directly from cells that have been transformed with expression vectors as described below herein.
- the invention polypeptide, biologically functional fragments, and functional equivalents thereof can also be produced by chemical synthesis.
- synthetic polypeptides can be produced using Applied Biosystems, Inc. Model 430A or
- SMDP and/or SCP are functional fragments or polypeptide analogs thereof.
- the term "functional fragment” refers to a peptide fragment that is a portion of a full length SMDP and/or SCP protein, provided that the portion has a biological activity, as defined above, that is characteristic of the corresponding full length protein.
- a functional fragment of an invention SMDP and/or SCP protein can have the protein: protein binding activity prevalent in SMDPs and/or SCPs.
- the characteristic of a functional fragment of invention SMDP and/or SCP proteins to elicit an immune response is useful for obtaining an anti-SMDP and/or SCP antibodies.
- the invention also provides functional fragments of invention SMDP and/or SCP proteins, which can be identified using the binding and routine methods, such as bioassays described herein.
- polypeptide analog includes any polypeptide having an amino acid residue sequence substantially the same as a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to functionally mimic an SMDP and/or SCP as described herein.
- conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
- amino acid length of functional fragments or polypeptide anlogs of the present invention can range from about 5 amino acids up to the full-length protein sequence of an invention SMDP and/or SCP.
- the amino acid lengths include, for example, at least about 10 amino acids, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250 or more amino acids in length up to the full-length SMDP and/or SCP protein sequence.
- the phrase "conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the required binding activity.
- chemical derivative refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
- derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides , p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
- Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine . Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
- Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions of residues, relative to the sequence of a polypeptide whose sequence is shown herein, so long as the required activity is maintained.
- the present invention also provides compositions containing an acceptable carrier and any of an isolated, purified SMDP and/or SCP mature protein or functional polypeptide fragments thereof, alone or in combination with each other.
- acceptable carrier encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
- SMDP and/or SCP compositions described herein can be used, for example, in methods described hereinafter.
- antisense-nucleic acids having a sequence capable of binding specifically with full-length or any portion of an mRNA that encodes SMDP and/or SCP polypeptides so as to prevent translation of the mRNA.
- the antisense-nucleic acid may have a sequence capable of binding specifically with any portion of the sequence of the cDNA encoding SMDP and/or SCP polypeptides.
- binding specifically encompasses the ability of a nucleic acid sequence to recognize a complementary nucleic acid sequence and to form double-helical segments therewith via the formation of hydrogen bonds between the complementary base pairs.
- An example of an antisense- nucleic acid is an antisense-nucleic acid comprising chemical analogs of nucleotides.
- compositions comprising an amount of the antisense-nucleic acid, described above, effective to reduce expression of SMDP and/or SCP polypeptides by passing through a cell membrane and binding specifically with mRNA encoding SMDP and/or SCP polypeptides so as to prevent translation and an acceptable hydrophobic carrier capable of passing through a cell membrane are also provided herein.
- Suitable hydrophobic carriers are described, for example, in U.S. Patent Nos. 5,334,761; 4,889,953; 4,897,355, and the like.
- the acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type.
- the structure may be part of a protein known to bind to a cell-type specific receptor.
- Antisense-nucleic acid compositions are useful to inhibit translation of mRNA encoding invention polypeptides.
- Synthetic oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding SMDP and/or SCP polypeptides and inhibit translation of mRNA and are useful as compositions to inhibit expression of SMDP and/or SCP associated genes in a tissue sample or in a subject.
- kits for detecting mutations, duplications, deletions, rearrangements and aneuploidies in SMDP and/or SCP genes comprising at least one invention probe or antisense nucleotide.
- the present invention provides means to modulate levels of expression of SMDP and/or SCP polypeptides by employing synthetic antisense-nucleic acid compositions (hereinafter SANC) which inhibit translation of mRNA encoding these polypeptides.
- SANC synthetic antisense-nucleic acid compositions
- Synthetic oligonucleotides, or other antisense-nucleic acid chemical structures designed to recognize and selectively bind to mRNA are constructed to be complementary to full-length or portions of an SMDP and/or SCP coding strand, including nucleotide sequences set forth in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- the SANC is designed to be stable in the blood stream for administration to a subject by injection, or in laboratory cell culture conditions.
- the SANC is designed to be capable of passing through the cell membrane in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SANC which render it capable of passing through cell membranes, for example, by designing small, hydrophobic SANC chemical structures, or by virtue of specific transport systems in the cell which recognize and transport the SANC into the cell.
- the SANC can be designed for administration only to certain selected cell populations by targeting the SANC to be recognized by specific cellular uptake mechanisms which bind and take up the SANC only within select cell populations.
- the SANC is an antisense oligonucleotide .
- the SANC may be designed to bind to a receptor found only in a certain cell type, as discussed supra .
- the SANC is also designed to recognize and selectively bind to target mRNA sequence, which may correspond to a sequence contained within the sequences shown in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- the SANC is designed to inactivate target mRNA sequence by either binding thereto and inducing degradation of the mRNA by, for example, RNase I digestion, or inhibiting translation of mRNA target sequence by interfering with the binding of translation-regulating factors or ribosomes, or inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups which either degrade or chemically modify the target mRNA.
- SMDPs and/or SCPs by expressing the above-described nucleic acid sequences in suitable host cells.
- Recombinant DNA expression systems that are suitable to produce SMDPs and/or SCPs described herein are well-known in the art.
- the above-described nucleotide sequences can be incorporated into vectors for further manipulation.
- vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.
- Suitable expression vectors are well-known in the art, and include vectors capable of expressing DNA operatively linked to a regulatory sequence, such as a promoter region that is capable of regulating expression of such DNA.
- an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA.
- Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
- a promoter region refers to a segment of DNA that controls transcription of DNA to which it is operatively linked. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, and the like.
- CMV cytomegalovirus
- MMTV mouse mammary tumor virus
- MMLV Moloney murine leukemia virus
- expression refers to the process by which polynucleic acids are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
- Prokaryotic transformation vectors are well-known in the art and include pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, CA) , and the like. Other suitable vectors and promoters are disclosed in detail in U.S. Patent No. 4,798,885, issued January 17, 1989, the disclosure of which is incorporated herein by reference in its entirety.
- suitable vectors for transformation of E. coli cells include the pET expression vectors (Novagen, see U.S patent 4,952,496), e.g., pETlla, which contains the T7 promoter, T7 terminator, the inducible E . coli lac operator, and the lac repressor gene; and pET 12a-c, which contain the T7 promoter, T7 terminator, and the E . coli ompT secretion signal.
- Another suitable vector is the pIN-IIIompA2 (see Duffaud et al., Meth. in Enzvmolo ⁇ v, 153:492-507, 1987), which contains the lpp promoter, the lacUV5 promoter operator, the ompA secretion signal, and the lac repressor gene.
- Exemplary, eukaryotic transformation vectors include the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system [described by Mulligan and Berg, Nature Vol. 277:108-114 (1979)] the Okayama-Berg cloning system [Mol. Cell Biol. Vol. 2:161- 170 (1982)], and the expression cloning vector described by Genetics Institute [Science Vol. 228:810-815 (1985)], are available which provide substantial assurance of at least some expression of the protein of interest in the transformed eukaryotic cell line.
- pSV-2 gpt system described by Mulligan and Berg, Nature Vol. 277:108-114 (1979)
- the Okayama-Berg cloning system Mol. Cell Biol. Vol. 2:161- 170 (1982)
- Genetics Institute Genetics Institute
- nucleic acid molecules i.e., DNA or mRNA
- suitable host cells preferably bacterial cells, and more preferably E . coli cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art.
- Exemplary methods of transformation include, e.g., transformation employing plasmids, viral, or bacterial phage vectors, transfection, electroporation, Hpofection, and the like.
- the heterologous DNA can optionally include sequences which allow for its extrachromosomal maintenance, or said heterologous DNA can be caused to integrate into the genome of the host
- Host organisms contemplated for use in the practice of the present invention include those organisms in which recombinant production of heterologous proteins has been carried out.
- Examples of such host organisms include bacteria (e.g., E . coli ) , yeast (e.g., Saccharomyces cerevisiae, Candida tropicalis , Hansenula polymorpha and P. pastoris ; see, e.g., U.S. Patent Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk " cells), insect cells, and the like.
- Presently preferred host organisms are bacteria. The most preferred bacteria is E . coli .
- nucleic acids encoding the invention SMDPs and/or SCPs can be delivered into mammalian cells, either in vivo or in vitro using suitable viral vectors well-known in the art.
- suitable viral vectors well-known in the art.
- Suitable retroviral vectors, designed specifically for "gene therapy" methods are described, for example, in WIPO publications WO 9205266 and WO 9214829, which provide a description of methods for efficiently introducing nucleic acids into human cells.
- the introduction of the antisense strand of the invention nucleic acid is contemplated.
- Viral based systems provide the advantage of being able to introduce relatively high levels of the heterologous nucleic acid into a variety of cells.
- Suitable viral vectors for introducing invention nucleic acid encoding an SMDP and/or SCP protein into mammalian cells are well known in the art.
- These viral vectors include, for example, Herpes simplex virus vectors (e.g., Geller et al., Science, 241:1667-1669 (1988)), Vaccinia virus vectors (e.g., Piccini et al., Meth.
- Moloney murine leukemia virus vectors (Danos et_al., PNAS , USA, 85:6469 (1980)), adenovirus vectors (e.g., Logan et al., PNAS, USA, 81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner, Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098 (1992); Graham et al . , Meth. Mol . Biol .
- adenovirus vectors e.g., Logan et al., PNAS, USA, 81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner, Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098 (1992); Graham et al .
- adeno-associated virus vectors adeno-associated virus vectors
- retrovirus vectors see, e.g., U.S. Patent 4,405,712 and 4,650,764
- Especially preferred viral vectors are the adenovirus and retroviral vectors.
- adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes (Wagner et al., PNAS, USA, 89:6099-6103 (1992); Curiel et al., Hum. Gene Ther. ,
- Retroviral vector refers to the well-known gene transfer plasmids that have an expression cassette encoding an heterologous gene residing between two retroviral LTRs .
- Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Patent 4,650,764).
- retroviral vectors for use herein are described, for example, in U.S. Patent 5,252,479, and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated herein by reference, which provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors.
- retroviral vectors include, for example, the mouse mammary tumor virus vectors (e.g., Shackleford et al., PNAS, USA, 85:9655-9659 (1988)), and the like.
- anti-SMDP and/or SCP antibodies having specific reactivity with an SMDP and/or SCP polypeptides of the present invention.
- Active fragments of antibodies are encompassed within the definition of "antibody”.
- Invention antibodies can be produced by methods known in the art using invention polypeptides, proteins or portions thereof as antigens.
- polyclonal and monoclonal antibodies can be produced by methods well known in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory (1988)), which is incorporated herein by reference.
- Invention polypeptides can be used as immunogens in generating such antibodies.
- synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens.
- Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide.
- Altered antibodies such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods described, for example, in Sambrook et al., supra., and Harlow and Lane, supra . Both anti-peptide and anti-fusion protein antibodies can be used, (see, for example, Bahouth et al., Trends Pharmacol . Sci .
- Antibody so produced can be used, inter alia, in diagnostic methods and systems to detect the level of SMDP and/or SCP present in a mammalian, preferably human, body sample, such as tissue or vascular fluid. Such antibodies can also be used for the immunoaffinity or affinity chromatography purification of the invention SMDP and/or SCP.
- methods are contemplated herein for detecting the presence of an invention SMDP and/or SCP protein in a tissue or cell, comprising contacting the cell with an antibody that specifically binds to SMDP and/or SCP polypeptides, under conditions permitting binding of the antibody to the SMDP and/or SCP polypeptides, detecting the presence of the antibody bound to the SMDP and/or SCP polypeptide, and thereby detecting the presence of invention polypeptides.
- the antibodies can be used for in vi tro diagnostic or in vivo imaging methods .
- Immunological procedures useful for in vi tro detection of target SMDP and/or SCP polypeptides in a sample include immunoassays that employ a detectable antibody.
- Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art.
- An antibody can be made detectable by various means well known in the art.
- a detectable marker can be directly or indirectly attached to the antibody.
- Useful markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.
- invention anti-SMDP and/or SCP antibodies are contemplated for use herein to modulate the activity of the SMDP and/or SCP polypeptide in living animals, in humans, or in biological tissues or fluids isolated therefrom.
- modulate refers to a compound's ability to increase (e.g., via an agonist) or inhibit (e.g., via an antagonist) the biological activity of an invention SMDP and/or SCP protein, such as the participation in Siah-Mediated-Degradation via an SFC complex and the 26S proteosome.
- compositions comprising a carrier and an amount of an antibody having specificity for SMDP and/or SCP polypeptides effective to inhibit naturally occurring ligands or other SMDP and/or SCP-binding proteins from binding to invention SMDP and/or SCP polypeptides are contemplated herein.
- an antibody having specificity for SMDP and/or SCP polypeptides effective to inhibit naturally occurring ligands or other SMDP and/or SCP-binding proteins from binding to invention SMDP and/or SCP polypeptides are contemplated herein.
- a monoclonal antibody directed to an epitope of an invention SMDP and/or SCP polypeptide including an amino acid sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 or 14, can be useful for this purpose.
- the present invention further provides transgenic non-human mammals that are capable of expressing exogenous nucleic acids encoding SMDP and/or SCP polypeptides.
- exogenous nucleic acid refers to nucleic acid sequence which is not native to the host, or which is present in the host in other than its native environment (e.g., as part of a genetically engineered DNA construct) .
- invention SMDPs and/or SCPs can either be overexpressed or underexpressed (such as in the well- known knock-out transgenics) in transgenic mammals.
- transgenic non-human mammals capable of expressing nucleic acids encoding SMDP and/or SCP polypeptides so mutated as to be incapable of normal activity, i.e., do not express native SMDP and/or SCP.
- the present invention also provides transgenic non-human mammals having a genome comprising antisense nucleic acids complementary to nucleic acids encoding SMDP and/or SCP polypeptides, placed so as to be transcribed into antisense mRNA complementary to mRNA encoding SMDP and/or SCP polypeptides, which hybridizes to the mRNA and, thereby, reduces the translation thereof.
- the nucleic acid may additionally comprise an inducible promoter and/or tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types.
- tissue specific regulatory elements are DNA or cDNA having a coding sequence substantially the same as the coding sequence shown in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- An example of a non-human transgenic mammal is a transgenic mouse.
- tissue specificity- determining elements are the metallothionein promoter and the L7 promoter.
- Animal model systems which elucidate the physiological and behavioral roles of SMDP and/or SCP polypeptides are also provided, and are produced by creating transgenic animals in which the expression of the SMDP and/or SCP polypeptide is altered using a variety of techniques. Examples of such techniques include the insertion of normal or mutant versions of nucleic acids encoding an SMDP and/or SCP polypeptide by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos to produce a transgenic animal. (See, for example, Hogan et al . , Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, (1986) ) .
- homologous recombination of mutant or normal versions of SMDP and/or SCP genes with the native gene locus in transgenic animals is also contemplated herein, to alter the regulation of expression or the structure of SMDP and/or SCP polypeptides (see, Capecchi et al . , Science 244:1288 (1989); Zi mer et al., Nature 338:150 (1989); which are incorporated herein by reference) .
- Homologous recombination techniques are well known in the art.
- Homologous recombination replaces the native (endogenous) gene with a recombinant or mutated gene to produce an animal that cannot express native (endogenous) protein but can express, for example, a mutated protein which results in altered expression of SMDP and/or SCP polypeptides.
- microinjection adds genes to the host genome, without removing host genes.
- Microinjection can produce a transgenic animal that is capable of expressing both endogenous and exogenous SMDP and/or SCP.
- Inducible promoters can be linked to the coding region of nucleic acids to provide a means to regulate expression of the transgene.
- Tissue specific regulatory elements can be linked to the coding region to permit tissue-specific expression of the transgene.
- Transgenic animal model systems are useful for in vivo screening of compounds for identification of specific ligands, i.e., agonists and antagonists, which activate or inhibit SMDP and/or SCP protein responses.
- SMDP and/or SCP proteins such as Siah-1, are contemplated herein to be a tumor suppressor proteins.
- Tumor suppressor proteins generally are thought to have a function in signal transduction . Mutation results in loss of function whereupon a signal pathway that the suppressor protein regulates is left in the "on" position, which results in unregulated cell proliferation resulting in cancerous tumor formation. Nearly all tumor suppressors regulate cell division, and proliferation, and may have involvement in biochemical pathways of development and the cell cycle.
- invention SMDP and/or SCP proteins support the role of both Siah and the invention SMDPs and/or SCPs in cellular pathways that affect protein degradation, such as by activating or inhibiting protein degradation, cell division and proliferation. Accordingly, invention SMDP and/or SCP proteins provide targets for treating a broad variety of pathologies, such as proliferative diseases, cancer pathologies, and the like.
- Siah-1 has been found to bind to the protein APC (Kinzler, et al., 1996, supra) .
- the APC protein is known to bind to ⁇ -catenin and target it for ubiquitination and degradation (Korinek, V. et al., 1997, Science, 275:1784-1786, Rubinfeld, B. et al . , 1997, Science, 275:1790-1792, and Morin, P.J. et al., 1997, Science, 275:1787-1790).
- Siah-1 and antagonist or agonists thereof, are contemplated for use in methods for treating a variety of cancers, such as epithelial cancer and the like, preferably by modulating ⁇ -catenin degradation.
- fragments comprising the carboxy terminus of Siah-1, preferably comprising at least amino acids 252-298 of SEQ ID NO:2, are employed (See Figure 3) .
- Invention nucleic acids, oligonucleotides (including antisense), vectors containing same, transformed host cells, polypeptides and combinations thereof, as well as antibodies of the present invention can be used to screen compounds in vi tro to determine whether a compound functions as a potential agonist or antagonist to invention polypeptides.
- vi tro screening assays provide information regarding the function and activity of invention polypeptides, which can lead to the identification and design of compounds that are capable of specific interaction with one or more types of invention proteins or fragments thereof.
- invention proteins may be employed in a competitive binding assay.
- Such an assay can accommodate the rapid screening of a large number of compounds to determine which compounds, if any, are capable of binding to SMDPs and/or SCPs. Subsequently, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as modulators, agonists or antagonists of invention SMDP and/or SCP proteins.
- Compounds that bind to and/or modulate invention SMDPs and/or SCPs can be used to treat a variety of pathologies mediated by invention SMDPs and/or SCPs, as described herein.
- transformed host cells that recombinantly express invention polypeptides can be contacted with a test compound, and the modulating effect (s) thereof can then be evaluated by comparing the SMDP-mediated response (e.g., the degradation of a known Siah-mediated target, such as DCC or ⁇ -catenin) in the presence and absence of test compound, or by comparing the response of test cells or control cells (i.e., cells that do not express SMDP and/or SCP polypeptides) , to the presence of the compound.
- SMDP-mediated response e.g., the degradation of a known Siah-mediated target, such as DCC or ⁇ -catenin
- test cells or control cells i.e., cells that do not express SMDP and/or SCP polypeptides
- modulates the activity of invention polypeptides refers to a compound or a signal that alters the activity of SMDP and/or SCP polypeptides so that the activity of the invention polypeptide is different in the presence of the compound or signal than in the absence of the compound or signal.
- compounds or signals include agonists and antagonists.
- An agonist encompasses a compound or a signal that activates SMDP and/or SCP protein expression.
- an antagonist includes a compound or signal that interferes with SMDP and/or SCP expression.
- the effect of an antagonist is observed as a blocking of agonist-induced protein activation.
- Antagonists include competitive and non-competitive antagonists.
- a competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding.
- a non-competitive antagonist or blocker inactivates the function of the polypeptide by interacting with a site other than the agonist interaction site.
- control is a cell or culture that is treated substantially the same as the test cell or test culture exposed to the compound, with the distinction that the "control" cell or culture is not exposed to the compound.
- control cell or culture may be a cell or culture that is identical to the transfected cells, with the exception that the "control” cell or culture do not express native proteins. Accordingly, the response of the transfected cell to compound is compared to the response (or lack thereof) of the "control" cell or culture to the same compound under the same reaction conditions.
- bioassay for evaluating whether test compounds are capable of acting as agonists or antagonists for SMDP and/or SCP proteins, wherein said bioassay comprises:
- DNA which expresses an SMDP and/or SCP or functional fragments thereof wherein said culturing is carried out in the presence of at least one compound whose ability to modulate an activity of an SMDP and/or SCP is sought to be determined, wherein said activity is selected from a protein : protein binding activity or a protein degradation activity and thereafter (b) monitoring said cells for either an increase or decrease in the level of protein: protein binding or protein degradation.
- Methods well-known in the art for measuring protein:protein binding or protein degradation can be employed in bioassays described herein to identify agonists and antagonists of SMDP and/or SCP proteins.
- the Siah-1 over-expression assay described in Example 14 can be used to evaluate the cell degradation activity of recombinant SMDP and/or SCP proteins or mutants and/or analogs thereof, expressed in mammalian host cells.
- ability to modulate protein degradation activity of an SMDP and/or SCP refers to a compound that has the ability to either induce (agonist) or inhibit (antagonist) the protein degradation activity of SMDP and/or SCP proteins within a cell.
- Host cells contemplated for use in the bioassay(s) of the present invention include human and other mammalian cells (readily avaialable from American Type Culture Collection) , as well as genitically engineered yeast or bacteria that express human SMDPs and/or SCPs, and the like.
- SMDP and/or SCP protein comprising: contacting an SMDP and/or SCP protein with an effective, modulating amount of an agonist or antagonist identified by the above-described bioassays.
- kits for treating pathologies comprising administering an effective amount of an invention therapeutic composition.
- Such compositions are typically administered in a physiologically compatible composition.
- Exemplary diseases related to abnormal cell proliferation contemplated herein for treatment according to the present invention include cancer pathologies, keratin hyperplasia, neoplasia, keloid, benign prothetic hypertrophy, inflammatory hyperplasia, and the like.
- Exemplary cancer pathologies contemplated herein for treatment include, gliomas, carcinomas, sarcomas, melanomas, hamartomas, leukemias, lymphomas, and the like.
- invention pharmaceutical compositions for the treatment of pathological disorders in which there is too little cell division, such as, for example, bone marrow aplasias, immunodeficiencies due to a decreased number of lymphocytes, and the like.
- Methods of treating a variety of inflammatory diseases with invention therapeutic compositions are also contemplated herein, such as treatment of sepsis, fibrosis (e.g., scarring), arthritis, graft versus host disease, and the like.
- compositions of the present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein.
- Therapeutic compositions of the present invention contain a physiologically compatible carrier together with an invention SMDP and/or SCP (or functional fragment thereof) , a SMDP and/or SCP modulating agent, such as a compound (agonist or antagonist) identified by the methods described herein, or an anti-SMDP and/or SCP antibody, as described herein, dissolved or dispersed therein as an active ingredient.
- the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.
- pharmaceutically acceptable “physiologically compatible” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, and the like.
- compositions that contains active ingredients dissolved or dispersed therein are well known in the art.
- compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension, in liquid prior to use can also be prepared.
- the preparation can also be emulsified.
- the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein.
- Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations of any two or more thereof.
- the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.
- the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
- Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.
- Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and organic bases such as mono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like) .
- inorganic bases such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like
- organic bases such as mono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine,
- Physiologically tolerable carriers are well known in the art.
- Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH, physiological saline or both, such as phosphate-buffered saline.
- aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
- Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.
- additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions .
- an "effective amount" is a predetermined amount calculated to achieve the desired therapeutic effect, e.g., to modulate the protein degradation activity of an invention SMDP and/or SCP protein.
- the required dosage will vary with the particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. It may be particularly advantageous to administer such compounds in depot or long-lasting form as discussed hereinafter.
- a therapeutically effective amount is typically an amount of an SMDP and/or SCP-modulating agent or compound identified herein that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of from about 0.1 ⁇ g/ml to about 100 ⁇ g/ml, preferably from about 1.0 ⁇ g/ml to about 50 ⁇ g/ml, more preferably at least about 2 ⁇ g/ml and usually 5 to 10 ⁇ g/ml.
- Therapeutic invention anti-SMDP and/or SCP antibodies can be administered in proportionately appropriate amounts in accordance with known practices in this art.
- invention SMDPs and/or SCPs, or functional fragments thereof, for targeting any desired protein for ubiquitination and degradation, thus enabling novel gene discovery through functional genomics strategies or providing the basis for ablating target proteins involved in diseases for therapeutic purposes.
- the phrase "inducing the degradation of the function” refers to deleting, altering, modifying and/or degrading a target protein so that it no longer has the ability to perform its native physiological function. This method is useful to determine the physiological/cellular function of the degraded protein. Thus, this invention method is useful in alleviating one of the rate limiting steps in functional genomics.
- the invention methods take advantage of the invention SMDP and/or SCP proteins provided herein to create a system that targets specific proteins for degradation by recruiting them to a SCF complex for ubiquitination and subsequent degradation by an appropriate proteosome, such as 26S proteosome.
- a protein or peptide fragment is selected that binds the protein targeted for degradation in a cell, which is referred to herein as the "target-protein binding domain.”
- Such protein or peptide fragment could be, for example: a domain of any known protein that interacts with the target protein; the Fab region of an anti-target-protein antibody (e.g., sFv, and the like); or a peptide aptamer obtained by screening (using, for example, yeast two-hybridization, phage display, or other screening methods) a random library of peptide aptamers.
- the target-protein binding domain is then fused (by engineering cDNAs in expression plasmids to form a chimera) with an appropriate protein-degradation binding domain selected from an invention SMDP and/or SCP, such as Siah, SIP, SAF-1, ⁇ AF-2 or SAD, and the like; or other known proteins involved in protein degradation, such as F-box containing proteins, (e.g., Skpl, and the like), SOCS-box containing proteins (see, e.g., Kamura et al., 1998, Genes Dev, 12:3872; and Starr and Hilton, 1999, Bioessays.21 : 47 ) , HECT family proteins (see, e.g., Huibregtse et al.
- protein-degradation binding domain refers to a protein region that functions to recruit the target protein into a member of the superfamily of E3 ubiquitin ligase complexes, such as the SCF complex, or to Siah-family proteins which may target some proteins for degradation independently of SCF complexes, where the protein-degradation binding domain and/or the target protein become ubiquitinated and then degraded, such as by the 26S proteosome, lysosomes and/or vacuoles (see, e.g., Tyers and Williams, 1999, Science, 284:601-604; and Ciechanover, 1998, supra) .
- Exemplary protein-degradation domains can be obtained from a protein member of the ubiquitin-mediated protein- degradation family.
- a protein member of the ubiquitin-mediated protein- degradation family refers to one of the numerous proteins that are known to interact, via protein: protein binding, in the ubiquitin system of intracellular protein degradation (see, e.g., Ciechanover et al . , 1998, supra; and Tyers and Williams, supra, and the like) .
- methods are provided of identifying a nucleic acid molecules encoding a chimeric protein that modulates a cellular phenotype, said method comprising: (a) expressing, in a cell, a chimeric nucleic acid comprising a member of a nucleic acid library fused to nucleic acid encoding a protein degradation binding domain of a protein member of the ubiquitin-mediated protein degradation family; and
- unbiased nucleic acid libraries can be constructed wherein, each member of the nucleic acid library is expressed as an encoded protein fused to a particular protein-degradation binding domain, such as invention SMDPs and/or SCPs, e.g., Siah-1, SIP (see, e.g., Example 15), SAF-1, SAF-2, or SAD; or other known proteins involved in protein degradation, such as Skpl, F-box containing proteins, HECT family proteins, or any other subunit of an E3 ubiquitin ligase complex, and the like.
- SMDPs and/or SCPs e.g., Siah-1, SIP (see, e.g., Example 15), SAF-1, SAF-2, or SAD
- Skpl F-box containing proteins
- HECT family proteins or any other subunit of an E3 ubiquitin ligase complex, and the like.
- nucleic acid library comprises cDNA libraries, YAC libraries, BAC libraries, cosmid libraries, or any other source of nucleic acid encoding polypeptides.
- the chimeric nucleic acid encoding these fusion proteins is then introduced into cells possessing a particular phenotype to be assayed.
- the cells are then subjected to a "screening" step which comprises selecting one or more cells in which the desired phenotype has been modulated (e.g., suppressed or enhanced) .
- the phenotypes to be screened may be any chemical or physical representation of a cellular process, including but not limited to: cell proliferation in either an attached or detached (i.e., anchorage-independent) state, cell survival, cell death, cell secretion, cell migration, abnormal cell morphology, chemical reactivity (e.g., heavy metals, antibiotics, etc.), physical reactivity (e.g., heat, light, radiation, etc.), and the like.
- cDNAs are identified and isolated whose expression products function to modulate the desired phenotype within cells.
- the cDNAs identified by the invention method encode an invention chimeric protein that interacts, preferably by direct binding, with another protein in the cells that is targeted for degradation, thereby eliminating its physiological function. Accordingly, any target-protein that has one or more protein-binding or protein-interacting partners, or which homodimerizes/homo-oligomerizes is contemplated for degradation in the invention methods.
- the cDNA identified by the above-described method can be used to perform a two-hybrid screen, as described herein, to identify the protein-binding region of the partner to the target protein, or directly sequenced to determine the identity of the target protein if homo-dimerization or homo-oligomerization situation occurs.
- chimeric proteins and encoding nucleic acids, comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein-degradation family.
- Exemplary proteins whose function can be targeted for degradation according to the invention methods include any protein encoded by a known gene or cDNA whose function is desired.
- Exemplary targets include, for example, apoptosis-related proteins, cell- cycle regulatory proteins, heat shock proteins, transcription factors, or any other target protein which, when degraded, will modulate the phenotype of a cell.
- methods for treating a disease by degrading the function of a target protein comprising introducing, into a cell, a chimeric protein comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein- degradation family.
- the chimeric protein of the invention may be used to therapeutically treat the disease, by way of reducing or completely eliminating, via protein degradation, the pathology causing protein.
- This treatment comprises fusion of a protein domain which binds the target pathology causing protein (i.e., the protein which causes the illness) with a particular protein-degradation binding domain as described herein.
- This chimeric protein may then be delivered to the location of the protein which causes the illness by intravenous therapy or gene therapy employing the methods described herein, or any other method well-known to one skilled in the art for delivering a protein to its binding target.
- treatment of a disease refers to a reduction in the effects of the disease, including reducing the symptoms of the disease.
- methods for diagnosing cancer comprising: detecting, in said subject, a defective sequence or mutant of SEQ ID NOs:l, 3, 5, 7, 9,
- diagnostic systems preferably in kit form, comprising at least one invention nucleic acid in a suitable packaging material.
- the diagnostic nucleic acids are derived from the
- the diagnostic nucleic acids are derived from any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
- Invention diagnostic systems are useful for assaying for the presence or absence of nucleic acid encoding SMDP and/or SCP in either genomic DNA or in transcribed nucleic acid (such as mRNA or cDNA) encoding SMDP and/or SCP.
- a suitable diagnostic system includes at least one invention nucleic acid, preferably two or more invention nucleic acids, as a separately packaged chemical reagent (s) in an amount sufficient for at least one assay. Instructions for use of the packaged reagent are also typically included. Those of skill in the art can readily incorporate invention nucleic probes and/or primers into kit form in combination with appropriate buffers and solutions for the practice of the invention methods as described herein.
- the phrase "packaging material” refers to one or more physical structures used to house the contents of the kit, such as invention nucleic acid probes or primers, and the like.
- the packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment.
- the packaging material has a label which indicates that the invention nucleic acids can be used for detecting a particular sequence encoding SMDP and/or SCP including the nucleotide sequences set forth in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13 or mutations or deletions therein, thereby diagnosing the presence of, or a predisposition for, cancer.
- the packaging material contains instructions indicating how the materials within the kit are employed both to detect a particular sequence and diagnose the presence of, or a predisposition for, cancer.
- packaging materials employed herein in relation to diagnostic systems are those customarily utilized in nucleic acid-based diagnostic systems.
- the term "package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits an isolated nucleic acid, oligonucleotide, or primer of the present invention.
- a package can be a glass vial used to contain milligram quantities of a contemplated nucleic acid, oligonucleotide or primer, or it can be a microtiter plate well to which microgram quantities of a contemplated nucleic acid probe have been operatively affixed.
- Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
- the mouse BAG-1 amino acid sequence was cloned into the pGilda plasmid and used as bait to screen a human Jurkat T-cell cDNA library. From an initial screen of -1.6 X 10' transformants, 298 clones were identified that trans-activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 30 colonies were also positive for ⁇ -galactosidase.
- cDNA fragments containing the 5' end of human Siah 1 were PCR-amplified from Jurkat randomly primer cDNAs by using aforward primer 5' GGGAATTCGGACTTATGGCATGTAAACA-3' (SEQ ID NO: 42) containing an EcoRI site and a reverse primer 5' TAGCCAAGTTGCGAATGGA-3' (SEQ ID NO:43), based on sequences of EST database clones (NCBI ID: AA054272, AA258606, AA923663, AA418482, and AI167464).
- the PCR products were digested with EcoRI and BamHI, then directly subcloned into the EcoRI and Sail sites of pel plasmid into which the cDNA derived from pJG4-5-Siah (22-298) had previously been cloned, as a BamHI - Xhol fragment.
- the complete human Siah-l ⁇ cDNA and amino acid sequence is set forth in SEQ ID Nos : 1 and 2, respectively.
- the human Siah-l ⁇ sequence contains 16 N-terminal amino acids that are not present in the human Siah-l ⁇ protein.
- Human Siah-l ⁇ cDNA encoding amino acids 22-298 of SEQ ID NO:l (corresponding to amino acids 6-282 set forth in Nemani et al., supra) was cloned into the pGilda plasmid and used as a bait to screen a human embryonic brain cDNA library (Invitrogen) in EGY48 strain S . cerevisiae . From an initial screen of -2.0 X 10 7 transformants, 322 clones were identified that trans- activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 32 colonies were also positive for ⁇ -galactosidase.
- DNA sequencing analysis revealed 5 cDNAs encoding portions of SIP-L, 1 cDNA encoding portions of SIP-S, 3 cDNAs encoding portions of of APC (2681-2843) , and 2 cDNAs encoding portions of Siah- 1.
- the SIP-L and SIP-S clones were sequenced and the resulting nucleotide sequences are set forth in SEQ ID Nos:3 and 5, respectively.
- Human Skpl cDNA encoding amino acids 91-163 of (Zhang et al., 1995, Cell, 82:915-925) was cloned into the pGilda plasmid as a bait to screen a human embryonic brain cDNA library (Invtrogen) in EGY48 strain S . cerevisiae . From an initial screen of ⁇ 1.2 X 10 ft transformants, 130 clones were identified that trans- activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 36 colonies were also positive for ⁇ -galactosidase.
- DNA sequencing analysis revealed 12 cDNAs encoding portions of SAF-1 and 9 cDNAs encoding portions of SAD.
- the SAF-1 and SAD clones were sequenced and the resulting nucleotide sequences are set forth in SEQ ID Nos:7 (SAF-l ⁇ ), 9 (SAF-l ⁇ ), and 13 (SAD). 5. Isolation of full-length SAF-2 cDNAs .
- cDNA encoding a human SAF-2 protein was PCR-amplified from ZAPII Jurkat cDNA labrary (Stratagene) by using a forward primer 5'- GTGAATTCATGCAACTTGTACCTGATATAGAGTTC-3' (SEQ ID NO: 44) containing an EcoRI site and a reverse primer 5'- GGACTCGAGGCTCTACAGAGGCC-3 ' (SEQ ID NO: 45), based on human DNA sequence from clone 341E18 on chromosome 6pll.2-12.3 (AL031178) .
- the PCR products were digested with EcoRI and Xhol, then directly subcloned into the EcoRI and Xhol sites of the plasmid pCDNA3. The corresponding plasmid was sequenced and the results are set forth in SEQ ID Nos: 11 and 12.
- the human SIP-L cDNA encoding full-length SIP-L was cloned into the pGilda plasmid as a bait to screen a human embryonic brain cDNA library (Invtrogen) in EGY48 strain S . cerevisiae . From an initial screen of -1.5 X 10 7 transformants, 410 clones were identified that trans- activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 68 colonies were also positive for ⁇ -galactosidase.
- DNA sequencing analysis revealed 3 cDNAs encoding portions of Skpl, 1 cDNA encoding portions of Siah-1, and 11 cDNAs encoding portions of SIP-L. These results indicate that SIP-L binds to Skpl and Siah-1 proteins, and is able to homodimerize with SIP isoforms.
- Siah-1 As revealed in Figure 4, over-expression of Siah-1 resulted in decreased numbers of viable cells after 24 hours, without an increase in cell death. Thus, Siah-1 suppresses proliferation of 293 cells. Co- transfection of SIP-L with Siah-1 did not substantially alter Siah-1-mediated growth suppression. In contrast, the SIP-S protein abrogated the growth suppressive effects of Siah-1, which indicates that the invention SIP-S protein affects Siah-1 intracellularly in a different manner than SIP-L. 8. In vitro SIP: Siah-1 protein interaction assays.
- the eluted proteins were analyzed by SDS-PAGE (12%) and detected by fluorography. Use of equivalent amounts of intact GST-fusion proteins and successful IVT of each protein was confirmed by SDS- PAGE analysis using Coomassie staining or autoradiography, respectively.
- Two x 10 6 293T cells in 100 mm plates were transiently transfected with 10 ⁇ g of pCDNA3-myc-SIP-L and 10 mg of pcDNA3-HA-Siah-l (amino acids 97-298 of SEQ ID NO:2). Twenty-four hours later, cells were disrupted by sonication in 1 ml of HKMEN solution containing 0.2% NP-40, 0.1 ⁇ M PMSF, 5 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, and 1 ⁇ g/ml pepstatin.
- plasmids encoding fusion proteins of the LexA DNA-binding domain fused to Siah-1, Siah-2, BAG-1, Bax, Ras, Fas, FLICE, and SIP-L were co-tansformed into yeast strain EGY48 with 1 ⁇ g of pJG4-5 plasmid encoding fusion proteins of the B42 trans-activation domain fused to SIP-L, SIP-S, Siah-1, Siah-2, BAG-1, Bax, and Ras.
- Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control which resulted in equivalent amounts of growth for all transformants .
- Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) .
- ⁇ -galactosidase activity of each colony was tested by filter assay and scored as blue (+) versus white (-) after 60 minutes.
- SIP-S was also found to interact with Siah-1.
- Table G also reveals that the SIP-L homodimerization domain is within amino acids 73-228 of SIP-L (SEQ ID NO:4)
- Siah-l ⁇ fragments corresponding to: SEQ ID NO: 2 amino acids 22-298; 22-251; 22-193; 97-298; and 46-102, fused to the B-42 trans-activation domain were co-transformed into yeast EGY48 cells with a plasmid encoding a chimeric fusion protein of the Lex A DNA-binding domain fused to amino acids 2681-2843 of APC "APC (2681-2843) .”
- Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control. Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) .
- ⁇ - galactosidase activity for each colony was tested by filter assay and scored as blue (+) versus white (-) ( ⁇ - gal) based on a 1 hour of color development.
- SAF-l-fragment- and SAD-fragment-B-42 fusion proteins were co-transformed into yeast EGY48 cells with a plasmid encoding a chimeric fusion protein of the Lex A DNA-binding domain fused to either SKP1, SIP-L, SAF-1, or SAD.
- Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control. Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) .
- ⁇ -galactosidase activity for each colony was tested by filter assay and scored as blue (+) versus white (-) ( ⁇ -gal) based on a 1 hour of color development.
- Figure 6A indicates that SAF-1 interacts by direct binding to Skpl, SIP-L and SAD, but does not interact with Siah-1.
- a region within the SAF-1 fragment corresponding to amino acids 80-257 of SEQ ID NO: 8 is required for SIP-L interaction, whereas a region within amino acids 258-443 of SAF-1 is required for Skpl and SAD interaction.
- Figure 6B indicates that SAD interacts by direct binding to Skpl, SIP-L and SAF-1, but does not interact with Siah-1.
- a region within the SAD fragment corresponding to amino acids 1-127 of SEQ ID NO: 14 is required for SAF-1 interaction; a region within amino acids 128-359 of SAD is required for Skpl interaction; and a region within amino acids 360-447 of SEQ ID NO: 14 is required for SIP-L interaction.
- 293T cells were transiently transfected with a plasmid encoding myc-tagged ⁇ -catenin and either pcDNA3, pcDNA3-Siah-l, or pcDNA3-Siah-l ( 97-298 ; amino acids 97- 298 of SEQ ID NO:2).
- Whole cell lysates were prepared, normalized for total protein content (25 ⁇ g per lane) and analyzed by SDS-PAGE/immunoblotting using an anti-Myc tag antibody.
- Figure 7 indicates that expression of full- length Siah-1 abolishes, by degradation, the presence of ⁇ -catenin within cells, whereas expression of amino acids 97-298 of Siah-1 (SEQ ID NO:2) does not result in ⁇ -catenin degradation.
- a region within amino acids 1-96 of SEQ ID N0:2 (Siah-l ⁇ ), which contains the N- terminal "Ring" domain, is required for protein degradation. 15.
- FIG. 9 shows the strategy employed for targeted degradation of specific TRAF-family proteins.
- a chimeric protein is expressed from the plasmid pcDNA3 in which SIP-L (SEQ ID NO: 3) is fused with bacterial thioredoxin containing various TRAF- binding peptides displayed on the surface of thioredoxin, as described by Brent and colleagues (Colas, et al . Nature, 380: 548, 1996; Cohen, et al . Proc. Natl. Acad. Sci., 95: 14272, 1998; Geyer, et al. Proc. Natl. Acad.
- the TRAF-binding peptide binds to a member of the TRAF-family, and targets the TRAF-protein for ubiquitination and subsequent proteosome-dependent degradation because the SIP-region of the chimeric protein recruits ubiquitin-conjugating enzymes (E2s) to the protein complex.
- E2s ubiquitin-conjugating enzymes
- TRAFs are a family of signal transducing proteins involved in cytokine receptor signaling inside cells.
- the sequences of the resulting TRAF-binding peptides are set forth in (Tables 3 and 4) .
- An invention SIP-fusion chimeric construct is generated by combining the open reading frame (ORF) of SIP L , followed immediately by restriction enzyme sites allowing for subcloning of desired target-protein-binding domains (e.g. peptides or protein domains). These SIP- fusions are then transfected into mammalian cells to eliminate by protein degradation specific target proteins which bind the subcloned peptides/protein domains by recruiting them into the ubiquitin conjugating complex.
- ORF open reading frame
- the parent SIP-vector (SIPpcDNA3.1 ) cassette was engineered as follows:
- Oligonucleotides corresponding to the 5' and 3' end of SIP L were used in PCR to amplify the entire ORF of SIP L (SEQ ID NO: 3) .
- the forward primer contains a Hind III restriction site linker (5'-
- SIPpcDNA3.1 SIP-thioredoxin (Trx) peptide-aptamer fusions
- Trx SIP-thioredoxin
- clones from a peptide-aptamer library screened against Traf6 were amplified by PCR with the following primers: Forward: 5' -CCTCTGAATTCCATATGAGCGATAAAATTATTCACC (SEQ ID NO: 48) EcoRI underlined; Reverse: 5'-
- the resulting PCR products ( ⁇ 350-370bp) contain the ORF of thioredoxin (Trx) with the selected peptide aptamers inserted into its active-loop.
- the products were then digested with EcoRI and Xho I before ligating into the Eco-RI/X oI-digested SIPpcDNA3.1 cassette using T4-DNA ligase.
- Final clone constructs were numbered and were confirmed by sequencing before using in transfection studies .
- HEK293T cells were transiently transfected by a lipofectamine method with various amounts (1 vs 4 ⁇ g) of pcDNA3 plasmids encoding either SIP-TR fusion protein lacking a TRAF6-binding peptide ("SIP") or SIP-TR fusion protein displaying one of the peptides shown in Table 4 above (set forth in Figure 10 as S603, S604, S606) .
- SIP TRAF6-binding peptide
- SIP TRAF6-binding peptide
- SIP proteosome inhbitor MG132 (10 ⁇ M) was added to cultures to prevent protein turnover.
- SIP* in Figure 10 corresponds to the control expression product of parental construct SIP pcDNA3.1
- TRAF6 protein levels were then measured two days later by immunoblotting using a anti-TRAF6-specific antiserum (Santa Cruz Biotech, Inc.) in experiments where HEK293T cell lysates were normalized for total protein content (25 ⁇ g per lane) .
- the cell lysates were analyzed by SDS- PAGE/immunoblotting using an enhanced chemiluminescence detection method, as described previously (Leo, et al. J Biol Chem, 274: 22414, 1999).
- SEQ ID NO:l is a cDNA (and the deduced amino acid sequence) encoding a Siah l ⁇ of the present invention.
- SEQ ID NO: 2 is the deduced amino acid sequence of a Siah l ⁇ protein of the present invention encoded by SEQ ID NO:l.
- SEQ ID NO: 3 is a cDNA (and the deduced amino acid sequence) encoding a human SIP-L polypeptide of the present invention.
- SEQ ID NO: 4 is the deduced amino acid sequence of a human SIP-L protein of the present invention encoded by SEQ ID NO:3.
- SEQ ID NO: 5 is a cDNA (and the deduced amino acid sequence) encoding a human SIP-S polypeptide of the present invention.
- SEQ ID NO: 6 is the deduced amino acid sequence of a human SIP-S protein of the present invention encoded by SEQ ID NO:5.
- SEQ ID NO: 7 is a cDNA (and the deduced amino acid sequence) encoding a human SAF-l ⁇ polypeptide of the present invention.
- SEQ ID NO: 8 is the deduced amino acid sequence of a SAF-l ⁇ protein of the present invention encoded by SEQ ID NO:7.
- SEQ ID NO: 9 is a cDNA (and the deduced amino acid sequence) encoding a human SAF-l ⁇ polypeptide of the present invention.
- SEQ ID NO: 10 is the deduced amino acid sequence of a SAF-l ⁇ protein encoded by SEQ ID NO: 9.
- SEQ ID NO: 11 is a cDNA (and the deduced amino acid sequence) encoding a human SAF-2 polypeptide of the present invention.
- SEQ ID NO: 12 is the deduced amino acid sequence of a SAF-2 protein encoded by SEQ ID NO: 11.
- SEQ ID NO: 13 is a cDNA (and the deduced amino acid sequence) encoding a human SAD polypeptide of the present invention.
- SEQ ID NO: 14 is the deduced amino acid sequence of a SAD protein encoded by SEQ ID NO: 13.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pain & Pain Management (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
In accordance with the present invention, there are provided novel Siah-Mediated-Degradation-Proteins (SMDPs) and/or SCF-Complex Proteins (SCPs). Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention SMDPs and/or SCPs can be employed in a variety of ways, for example, for the production of anti-SMDP and/or SCP antibodies thereto, in therapeutic compositions, and methods employing such proteins and/or antibodies for drug screening, functional genomics and other applications. Also provided are transgenic non-human mammals that express the invention protein.
Description
NUCLEIC ACID ENCODING PROTEINS INVOLVED IN PROTEIN DEGRADATION, PRODUCTS AND METHODS RELATED THERETO
Portions of the invention described herein were made in the course of research supported in part by NIH grant CA67329. The Government may have certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates to nucleic acids and proteins encoded thereby.
BACKGROUND OF THE INVENTION
The temporal coordination of sequential steps within the eukaryotic cell cycle is governed in large part by protein degradation, involving targeted ubiquitination of specific cell cycle regulatory proteins followed by their destruction by the 26S proteasome
(reviewed in Ciechanover, A. 1998, EMBO J.. 17(24) :7151- 7160) . Among the cell cycle regulators whose levels are controlled by ubiquitination and subsequent proteosome- dependent degradation are the cyclins (cyclins A, B, C, DI, E) and several of the cyclin-dependent kinase (cdk) inhibitory proteins including p21-Wafl and p27 Kip. Defects in this highly regulated process of protein turnover have been documented in many types of cancer.
The steps involved in polyubiquitination of specific proteins in cells involve the concerted actions of El, E2, and E3-type enzymes. El proteins form thioester bonds in which the sulfhydryl group of internal cysteine residues binds the carboxyl amino acid of ubiquitin, thereby activating ubiquitin for subsequent
transfer to E2-family proteins. E2 family proteins then transfer activated ubiquitin to the free amino-groups of lysine side chains in target proteins directly. More often, however, E2-family proteins collaborate with E3 proteins which bind particular target proteins and orchestrate their interactions with E2s, coordinating the polyubiquitination of these target proteins in highly regulated manners Ciechanover, A. 1998 supra . E3 functions are sometimes embodied in multiprotein complexes rather than mediated by a single protein.
The ubiquitination and degradation of a variety of cyclins, cyclin-dependent kinases (cdks) and cdk- inhibitors is temporally controlled during the cell cycle by SFC complexes. Theses multiprotein complexes function as E3-like entities, and contain the Skp-1 protein, at least one Cullin-family protein, and at least one F-box protein, thus the acronym SCF: S=Skpl; C=Cullin; F=F-box) (reviewed in Patton, E.E. et al., 1998, TIG 14(6):236- 243) . F-box proteins contain a conserved motif, the F- box, which mediates their interactions with Skp-1. The F-box proteins also contain other domains which allow them to simultaneously bind specific substrate proteins, which are then targeted for degradation via polyubiquitination. One such F-box protein identified in humans is b-Trcp, which forms a SCF complex with Skp-1 and Cul-1, and which interacts with β-catenin, targeting it for degradation (Latres, et al . , 1999, Oncogene, 18:849-854, and Winston, J.J. et al . , 1999, Genes & Dev. , 13:270-283) .
Siah-family proteins represent mammalian homologs of the Drosophila Sina protein. Sina is required for R7 photoreceptor cell differentiation within the sevenless pathway. Sina binds a ubiquitin-
conjugating enzyme (E2) via an N-terminal RING domain. Heteroco plexes of Sina and another protein called Phyllopodia form a E3-complex which interacts with a transcriptional repressor called Tramtrack, targeting it for polyubiqitination and proteosome-mediated degradation in the fly (Tang, A.H. et al., 1997, Cell, 90:459-467 and Li, S. et al., 1997, Cell, 469-478). The destruction of Tramtrack is necessary for differentiation of R7 cells.
At present, little is known about the expression of mammalian genes related to the Siah- mediated-protein-degradation family of proteins in normal cells and cancers. Moreover, the diversity of functions of the Siah-mediated-protein-degradation family proteins remain unclear. Therefore, there continues to be a need in the art for the discovery of additional proteins that interact with the Siah-mediated-protein-degradation pathway, such as proteins that bind Siah in vivo, and especially a need for information serving to specifically identify and characterize such proteins in terms of their amino acid sequence. Moreover, to the extent that such molecules might form the basis for the development of therapeutic and diagnostic agents, it is essential that the DNA encoding them be elucidated. Similarly, a need exists to identify additional components of SCF complexes which may operate in concert with or independently of Siah. The present invention satisfies these needs and provides related advantages as well.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, there are provided novel isolated nucleic acids encoding a variety of Siah-Mediated-Degradation-Proteins (SMDPs) involved in the Siah-mediated protein degradation
pathways and/or SCF-Complex-Proteins (SCPs) involved in SCF-mediated protein degradation pathways. Further provided are vectors containing invention nucleic acids, probes that hybridize thereto, host cells transformed therewith, antisense-nucleic acids thereto and related compositions. The nucleic acid molecules described herein can be incorporated into a variety of expression systems known to those of skill in the art. In addition, the nucleic acid molecules of the present invention are useful as probes for assaying for the presence and/or amount of a SMDP and/or SCP gene or mRNA transcript in a given sample. The nucleic acid molecules described herein, and oligonucleotide fragments thereof, are also useful as primers and/or templates in a PCR reaction for amplifying genes encoding SMDP and/or SCP proteins.
In accordance with the present invention, there are also provided isolated mammalian SMDP and/or SCP proteins. These proteins, or fragments thereof, are useful in bioassays, as immunogens for producing anti- SMDP and/or SCP antibodies, or in therapeutic compositions containing such proteins and/or antibodies. Also provided are transgenic non-human mammals that express, or fail to express (e.g., knock-out), the invention protein.
Antibodies that are immunoreactive with invention SMDP and/or SCP proteins are also provided. These antibodies are useful in diagnostic assays to determine levels of SMDP and/or SCP proteins present in a given sample, e.g., tissue samples, Western blots, and the like. The antibodies can also be used to purify SMDP and/or SCP proteins from crude cell extracts and the like. Moreover, these antibodies are considered
therapeutically useful to modulate the biological effect of SMDP and/or SCP proteins in vivo.
Also provided are bioassays for identifying compounds that modulate the activity of invention SMDP and/or SCP proteins. Methods and diagnostic systems for determining the levels of SMDP and/or SCP protein in various tissue samples are also provided. These diagnostic methods can be used for monitoring the level of therapeutically administered SMDP and/or SCP or fragments thereof to facilitate the maintenance of therapeutically effective amounts. These diagnostic methods can also be used to diagnose physiological disorders that result from abnormal levels of SMDP and/or SCP.
Also provided are systems using invention
SMDPs, SCPs, or functional fragments thereof, for targeting any desired protein for ubiquitination and degradation, thus enabling novel gene discovery through functional genomics strategies or providing the basis for ablating target proteins involved in diseases for therapeutic purposes.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an amino acid sequence comparison of SIP-L (SEQ ID NO: 4) and SIP-S (SEQ ID NO:6) .
Figure 2 shows the Destruction-box amino acid consensus sequence of SAD.
Figure 3 shows the Mapping of Siah-APC interaction domains as described in Example 12.
Figure 4 shows the results of the cell proliferation functional assay of SIP/Siah interaction described in Example 7.
Figures 5A and 5B show in vitro and in vivo interaction assays of between Siah-1 and SIP-L as described in Examples 8 and 9, respectively.
Figures 6A and 6B show the mapping of SKP1, SIP-L, SAF-1 and SAD interaction domains as described in Example 13.
Figure 7 shows the effect of Siah-1 overexpression on stability of β-catenin.
Figure 8 shows a diagram of how an invention SIP communicates with the Protein Ubiquitination Machinery.
Figure 9 shows a general diagram of an invention method for inducing targeted degradation of proteins using SIP, exemplified in Example 15.
Figure 10 shows the results of the SIP-mediated degradation of the target TRAF6 protein, set forth in Example 15.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there are provided isolated nucleic acids, which encode novel mammalian Siah-Mediated-Degradation-Proteins (SMDPs) and/or SCF-Complex-Proteins (SCPs) , and functional fragments thereof. SMDPs are involved in the Siah-
mediated protein degradation pathways and SCPs are involved in SCF-mediated protein degradation pathways. In some instances, these two pathways for protein degradation may operate in collaboration, particularly in cases where proteins have been identified that physically link SMDPs to SCPs. Invention SMDPs and/or SCPs are contemplated herein to regulate protein degradation, either by activating or inhibiting such protein degradation.
As used herein, invention SMDPs are proteins that participate in the Siah-mediated protein degradation pathway. The term "Siah" refers to the mammalian family of proteins encoded by at least two genes referred to as SIAH1 and SIAH2 (Hu, G. et al., 1997, Genomics, 46:103- 111). Like their Drosophila counterpart protein Sina, the Siah-1 and Siah-2 proteins bind ubiquitin conjugating enzymes (UBCs) via an N-terminal RING domain and target other proteins for degradation.
As used herein, invention SCPs are proteins that participate in the Skp-1, Cullin, F-box (SCF) protein degradation pathway. These proteins can be components of SCF complexes or proteins that associate with SCF complexes. In some instances an invention protein may fulfill the requirements of both a SMDP and a SCP.
Using yeast two-hybrid screening methods, targets of Siah-mediated protein degradation have been identified demonstrating the involvement of Siah and other inventions SMDPs and/or SCPs in pathways involved in cell growth regulation in cancers. For example, evidence is provided herein demonstrating that Siah-1 interacts indirectly with SCF complexes through
associations with an invention SIP protein. Siah-1 has also been found to be an important regulator of cell growth, through its effects on ubiquitination and degradation of β-catenin and possibly other target proteins including an invention protein SAD.
Thus far, the only reported target of Siah- mediated degradation is DCC (Hu, G. et al., 1997, Genes & Dev. , 11:2701-2714), a putative tumor suppressor protein encoded by a gene which is commonly disrupted in colon cancers (Fearon, E.R. et al., 1990, Science, 247:49-56). The involvement of DCC in colon cancers however has recently been questioned, and it appears that a different gene located near DCC on 18q21 is the primary target of deletions in this chromosomal region (Fearon, E.R. et al., 1990, supra) . However, the DCC protein has recently been shown to deliver either pro-apoptotic or anti- apoptotic signals, depending on whether it is complexed with its ligand Netrin. These observations suggest that deletion or inactivation of DCC could potentially contribute to tumorigensis by removing a pro-apoptotic influence from cells.
Another connection between the Siah-family of proteins and tumor suppressor genes has been found for p53. For example, the Siah-1 gene of mice was found among a group of immediate-early genes induced by p53 using a hemopoietic cell line as a model for p53-induced cell cycle arrest and apoptosis (Amson, R.B. et al., 1996, PNAS, USA, 93:3953-3957). Expression of Siah-1 was also indirectly correlated with increased apoptosis in tumor xenograph experiments, suggesting that Siah-1 could function as a tumor suppressor in some contexts (Nemani, M. et al., 1996, Proc. Natl. Sci. USA 93:9039-9042).
It has also been found that Siah-1 over- expression can induce cell cycle arrest independently of apoptosis in epithelial cancer cells (Matsuzawa, et al., 1998, EMBO J. , 17 ( 10 ): 2736-2747 ) . Moreover, UV- irradiation at subapoptotic doses was shown to induce
Siah-1 gene expression in MCF7 breast cancer cells and to promote cell cycle arrest. These and other data have implicated Siah-1 in a p53-inducible pathway for cell cycle arrest which runs parallel to the well-studied p21- Wafl pathway (Matsuzawa, et al. 1998, supra) .
The phrase "SMDP and/or SCP" refers to substantially pure native SMDP and/or SCP, or recombinantly produced proteins, including naturally occurring allelic variants thereof encoded by mRNA generated by alternative splicing of a primary transcript, and further including fragments thereof which retain at least one native biological activity, such as immunogenicity, the ability to bind to a SMDP and/or SCP, and the like. Exemplary SMDPs and/or SCPs referred to herein include amino acid sequences set forth in SEQ ID Nos:2 (Siah-l ), 4 (SIP-L), 6 (SIP-S), 8 (SAF-lα), 10 (SAF-lβ), 12 (SAF-2) and 14 (SAD). Invention isolated SMDPs and/or SCPs are substantially pure and free of cellular components and/or contaminants normally associated with a native in vivo environment.
As used herein, the term "Siah-lα" refers to a splice-variant member of the mammalian, preferably human, Siah-family of proteins. The invention Siah-lα protein, or functional fragment thereof, is characterized by having the ability to bind to at least one or more of the proteins selected from APC (Kinzler K.W., et al., 1996, Cell, 87 (2) :159-170) ; BAG-1 (Takayama et al., 1995, Cell, 80 (2) :279-284) ; SIP-L; SIP-S; or other Siah proteins,
such as Siah-1. Thus, homodimers of Siah-lα are contemplated herin. Invention Siah-lα proteins differ from Siah-lβ (set forth as SIAH-1 in Hu et al., 1997, Genomics , 46:103-111) by containing an additional 16 amino acids at the amino-terminus . Thus, preferred invention Siah-lα proteins, and fragments thereof, comprise at least a portion of the 16 N-terminal amino acids of SEQ ID NO: 2. A particularly preferred Siah-lα protein is set forth in SEQ ID NO:2.
In accordance with another embodiment of the invention, Siah-1 has been found to interact with an invention protein referred to herein as the "SIP" family. As used herein, the term "SIP" refers to any species, preferably mammalian, more preferably human, Siah-1- .Interacting Protein (SIP). The invention SIP proteins, or functional fragments thereof, are characterized by having the ability to bind to at least one or more of the proteins selected from Siah-1, Skpl, or other SIP proteins. Thus, homodimers of invention SIP proteins are contemplated herein. The SIP gene has been found to encode at least two proteins through alternative mRNA splicing: SIP-L (L, for long; SEQ ID NO:4), and SIP-S (S, for short; SEQ ID NO: 6). A sequence comparison of SIP-L to SIP-S is set forth in Figure 1. To further identify potential targets of Siah-1-mediated ubiquitin/proteasome protein degradation, yeast two-hybrid screens of cDNA libraries were performed using the invention human SIP-L protein (SEQ ID NO:4) as a bait. Such screen resulted in the identification of Skpl (Zhang et al., 1995, Cell, 82 (6) :915-925) .
As shown in Figure 8, an invention SIP protein binds simultaneously to Siah and Skp, proteins known to bind directly or indirectly, respectively, to ubiquitin-
conjugating enzymes (E2s). Therefore, in accordance with the present invention, this characteristic of SIP is useful for methods for targeting desired proteins for degradation, via a ubiquitin/proteosome-dependent mechanism (see, e.g., Example 15).
In accordance with another embodiment of the invention, using a yeast two-hybrid screen with Skpl as bait (set forth in the Examples), two additional invention SMDP and/or SCP proteins were identified as Skpl-interacting proteins, which are referred to herein as SAF-1 and SAD. As used herein the term "SAF-1" refers to Skpl-Associated F-box protein-1. The invention SAF-1 proteins, or functional fragments thereof, are characterized by having the ability to bind to at least one or more of the proteins selected from Skpl, SIP, such as SIP-L, or SAD. Invention SAF-1 proteins are further characterized as containing an "F-box" amino acid domain. Exemplary SAF-1 proteins include SAF-lα (SEQ ID NO: 8) and SAF-lβ (SEQ ID NO: 10). An exemplary F-box domain is set forth as amino acids 256-296 of SEQ ID NO: 8 and amino acids 335-375 of SEQ ID NO: 10 (see Figure 6). SAF-1 beta has the same F-Box as alfa, the location is in amino acids 335-375. In accordance with the present invention, a homologue of SAF-1 protein has also been identified in the NCBI BLAST data base (Human DNA sequence from clone 341E18 on chromosome 6pll.2-12.3, AL031178) which shares significant homology with F-box domain of SAF-1. The invention homolog is referred to herein as SAF-2 and is set forth in SEQ ID Nos: 11 and 12.
As used herein the term "SAD" refers to Skpl-
Associated Destruction-box protein. The invention SAF-1 proteins, or functional fragments thereof, are characterized by having the ability to bind to at least
one or more of the proteins selected from Skpl, SIP, such as SIP-L, or SAF-1. It is also contemplated herein that SAD has the ability to bind to SAF-2. Invention SAD proteins are further characterized as containing an " D- box" (Destruction-box) amino acid domain. An exemplary SAD protein is set forth herein as SEQ ID NO: 14. As used herein, the D-box domain comprises the consensus amino acid sequence -DSGX^S-, wherein Xλ is preferably selected from a hydrophobic amino acid, such as Y, I, L, M, F, W, or V; and X2 is any amino acid (see Figure 2) . A preferred D-box domain comprises the sequence set forth as amino acids 144-149 of SEQ ID NO: 14.
Thus, exemplary functional fragments of an invention SAD protein comprise at least amino acids 144- 149 of SEQ ID NO: 14. Also contemplated herein are functional fragments of inventions SAD proteins that bind to a SIP protein, and preferably comprise at least amino acids 360-447 of SEQ ID NO: 14. Functional fragments of inventions SAD proteins that bind to a Skpl protein preferably comprise at least amino acids 128-359 of SEQ ID NO: 14. Functional fragments of inventions SAD proteins that bind to a SAF-1 protein preferably comprise at least amino acids 1-127 of SEQ ID NO: 14.
The nucleic acid molecules described herein are useful for producing invention proteins, when such nucleic acids are incorporated into a variety of protein expression systems known to those of skill in the art. In addition, such nucleic acid molecules or fragments thereof can be labeled with a readily detectable substituent and used as hybridization probes for assaying for the presence and/or amount of an invention SMDP and/or SCP gene or mRNA transcript in a given sample. The nucleic acid molecules described herein, and
fragments thereof, are also useful as primers and/or templates in a PCR reaction for amplifying genes encoding invention proteins described herein.
The term "nucleic acid" (also referred to as polynucleotides) encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes, oligonucleotides, and primers. DNA can be either complementary DNA (cDNA) or genomic DNA, e.g. a gene encoding a SMDP and/or SCP. One means of isolating a nucleic acid encoding an SMDP and/or SCP polypeptide is to probe a mammalian genomic library with a natural or artificially designed DNA probe using methods well known in the art. DNA probes derived from the SMDP and/or SCP gene are particularly useful for this purpose. DNA and cDNA molecules that encode SMDP and/or SCP polypeptides can be used to obtain complementary genomic DNA, cDNA or RNA from mammalian (e.g., human, mouse, rat, rabbit, pig, and the like), or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, by methods described in more detail below. Examples of nucleic acids are RNA, cDNA, or isolated genomic DNA encoding an SMDP and/or SCP polypeptide. Such nucleic acids may include, but are not limited to, nucleic acids comprising substantially the same nucleotide sequence as set forth in SEQ ID Nos:l
(Siah-lα), 3 (SIP-L), 5 (SIP-S), 7 (SAF-lα), 9 (SAF-lβ) , 11 (SAF-2) and 13 (SAD) .
Use of the terms "isolated" and/or "purified" in the present specification and claims as a modifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or proteins so designated have been produced in such form by the hand of man, and thus are separated from their native in vivo cellular environment,
and are substantially free of any other species of nucleic acid or protein. As a result of this human intervention, the recombinant DNAs, RNAs, polypeptides and proteins of the invention are useful in ways described herein that the DNAs, RNAs, polypeptides or proteins as they naturally occur are not.
Invention proteins can be obtained from any species of organism, such as prokaryotes, eukaryotes, plants, fungi, vertebrates, invertebrates, and the like. A particular species can be d mammalian, As used herein, "mammalian" refers to a subset of species from which an invention SMDP and/or SCP is derived, e.g., human, rat, mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and the like. A preferred SMDP and/or SCP herein, is human SMDP and/or SCP.
In one embodiment of the present invention, cDNAs encoding the invention SMDPs and/or SCPs disclosed herein comprise substantially the same nucleotide sequence as the coding region set forth in any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. Preferred cDNA molecules encoding the invention proteins comprise the same nucleotide sequence as as the coding region set forth in any of SEQ ID N0s:l, 3, 5, 7, 9, 11 and 13.
As employed herein, the term "substantially the same nucleotide sequence" refers to DNA having sufficient identity to the reference polynucleotide, such that it will hybridize to the reference nucleotide under moderately stringent hybridization conditions. In one embodiment, DNA having substantially the same nucleotide sequence as the reference nucleotide sequence encodes substantially the same amino acid sequence as that set forth in any of SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14. In
another embodiment, DNA having "substantially the same nucleotide sequence" as the reference nucleotide sequence has at least 60% identity with respect to the reference nucleotide sequence. DNA having at least 70%, more preferably at least 90%, yet more preferably at least 95%, identity to the reference nucleotide sequence is preferred.
This invention also encompasses nucleic acids which differ from the nucleic acids shown in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, but which have the same phenotype. Phenotypically similar nucleic acids are also referred to as "functionally equivalent nucleic acids". As used herein, the phrase "functionally equivalent nucleic acids" encompasses nucleic acids characterized by slight and non-consequential sequence variations that will function in substantially the same manner to produce the same protein product (s) as the nucleic acids disclosed herein. In particular, functionally equivalent nucleic acids encode polypeptides that are the same as those encoded by the nucleic acids disclosed herein or that have conservative amino acid variations. For example, conservative variations include substitution of a non-polar residue with another non-polar residue, or substitution of a charged residue with a similarly charged residue. These variations include those recognized by skilled artisans as those that do not substantially alter the tertiary structure of the protein.
Further provided are nucleic acids encoding SMDP and/or SCP polypeptides that, by virtue of the degeneracy of the genetic code, do not necessarily hybridize to the invention nucleic acids under specified hybridization conditions. Preferred nucleic acids
encoding the invention SMDPs and/or SCPs are comprised of nucleotides that encode substantially the same amino acid sequence as set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14.
Thus, an exemplary nucleic acid encoding an invention SMDP and/or SCP may be selected from:
(a) DNA encoding the amino acid sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14, (b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active SMDP and/or SCP, or
(c) DNA degenerate with respect to either (a) or (b) above, wherein said DNA encodes biologically active SMDP and/or SCP.
Hybridization refers to the binding of complementary strands of nucleic acid (i.e., sense : antisense strands or probe : target-DNA) to each other through hydrogen bonds, similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art.
The phrase "stringent hybridization" is used herein to refer to conditions under which polynucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tn) of the hybrids. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but
higher, stringency. Reference to hybridization stringency relates to such washing conditions.
As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA; with greater than about 90% identity to target-DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5X Denhart ' s solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65°C.
The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein) . High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart ' s solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0. IX SSPE, and 0.1% SDS at 65°C.
The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5X Denhart ' s solution, 6X SSPE, 0.2% SDS at 42°C, followed by washing in IX SSPE, 0.2% SDS, at 50°C. Denhart ' s solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers .
As used herein, the term "degenerate" refers to codons that differ in at least one nucleotide from a reference nucleic acid, e.g., SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, but encode the same amino acids as the reference nucleic acid. For example, codons specified by the triplets "UCU", "UCC", "UCA", and "UCG" are degenerate with respect to each other since all four of these codons encode the amino acid serine.
Preferred nucleic acids encoding the invention polypeptide (s) hybridize under moderately stringent, preferably high stringency, conditions to substantially the entire sequence, or substantial portions (i.e., typically at least 15-30 nucleotides) of the nucleic acid sequence set forth in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
The invention nucleic acids can be produced by a variety of methods well-known in the art, e.g., the methods described herein, employing PCR amplification using oligonucleotide primers from various regions of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, and the like.
In accordance with a further embodiment of the present invention, optionally labeled SMDP and/or SCP- encoding cDNAs, or fragments thereof, can be employed to probe library (ies) (e.g., cDNA, genomic, and the like) for additional nucleic acid sequences encoding novel mammalian SMDPs and/or SCPs. Construction of suitable mammalian cDNA libraries is well-known in the art. Screening of such a cDNA library is initially carried out under low-stringency conditions, which comprise a temperature of less than about 42°C, a formamide
concentration of less than about 50%, and a moderate to low salt concentration.
Presently preferred probe-based screening conditions comprise a temperature of about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5X standard saline citrate (SSC; 20X SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Such conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology. The phrase "substantial similarity" refers to sequences which share at least 50% homology. Preferably, hybridization conditions will be selected which allow the identification of sequences having at least 70% homology with the probe, while discriminating against sequences which have a lower degree of homology with the probe. As a result, nucleic acids having substantially the same nucleotide sequence as SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13 are obtained.
As used herein, a nucleic acid "probe" is single-stranded DNA or RNA, or analogs thereof, that has a sequence of nucleotides that includes at least 14, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous bases that are the same as (or the complement of) any contiguous bases set forth in any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. Preferred regions from which to construct probes include 5 ' and/or 3 ' coding regions of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. In addition, the entire cDNA encoding region of an invention SMDP and/or SCP, or the entire sequence corresponding to SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13, may be used as a probe. Probes may be labeled by methods well-known in the art,
as described hereinafter, and used in various diagnostic kits .
It is understood that a SMDP and/or SCP- encoding nucleic acid molecule of the invention, as used herein, specifically excludes previously known nucleic acid molecules consisting of nucleotide sequences having identity with the SMDP and/or SCP-encoding nucleotide sequence (e.g., SEQ ID NO:NOs:l, 3, 5, 7, 9, 11, 13), such as Expressed Sequence Tags (ESTs), Sequence Tagged Sites (STSs) and genomic fragments, deposited in public databases such as the nr, dbest, dbsts, gss and htgs databases, which are available for searching at http: //www. nebi.nlm.nih.gov/blast/blast. cgi?Jform=0, using the program BLASTN 2.0.9 described by Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).
In particular, a SMDP and/or SCP-encoding nucleic acid molecule specifically excludes nucleic acid molecules consisting of any of the nucleotide sequences having the Genbank (gb) , EMBL (emb) or DDBJ (dbj) accession numbers described below. Similarly, a SMDP and/or SCP polypeptide fragment specifically excludes the amino acid fragments encoded by the nucleotide sequences having the GenBank accession numbers described below. GenBank accession numbers specifically excluded include NCBI ID: AA054272, AA258606, AA923663, AA418482, and AI167464. The human sequence referenced as GenBank accession No. AL031178 is also specifically excluded from an invention SMDP and/or SCP-encoding nucleic acid.
As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a
detectable signal. Any label or indicating means can be linked to invention nucleic acid probes, expressed proteins, polypeptide fragments, or antibody molecules. These atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry.
The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturation to form a fluorochrome (dye) that is a useful immunofluorescent tracer. A description of immunofluorescent analytic techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., eds . , John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.
In one embodiment, the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, and the like. In another embodiment, radioactive elements are employed labeling agents. The linking of a label to a substrate, i.e., labeling of nucleic acid probes, antibodies, polypeptides, and proteins, is well known in the art. For instance, an invention antibody can be labeled by metabolic incorporation of radiolabeled amino acids provided in the culture medium. See, for example, Galfre et al., Meth. Enzymol . , 73:3-46 (1981). Conventional means of protein conjugation or coupling by activated functional groups are particularly applicable. See, for example, Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech. , 3:889-894 (1984), and U.S. Patent No. 4,493,795.
In accordance with another embodiment of the present invention, there are provided isolated mammalian Siah-Mediated-Degradation-Proteins (SMDPs) and/or SCF- Complex-Proteins (SCPs) , and fragments thereof encoded by invention nucleic acid. The phrase "SMDP and/or SCP" refers to substantially pure native SMDP and/or SCP, or recombinantly produced proteins, including naturally occurring allelic variants thereof encoded by mRNA generated by alternative splicing of a primary transcript, and further including fragments thereof which retain at least one native biological activity, such as immunogenicity, the ability to bind to another member of the SMDP and/or SCP families, or to homodimerize . In another embodiment, SMDPs and/or SCPs referred to herein, are those polypeptides specifically recognized by an antibody that also specifically recognizes a SMDP and/or SCP (preferably human) including an amino acid sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14. Invention isolated SMDPs and/or SCPs are substantially pure and free of cellular components and/or contaminants normally associated with a native in vivo environment.
Presently preferred SMDPs and/or SCPs of the invention include proteins that comprise substantially the same amino acid sequences as the protein sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14, as well as biologically active, functional fragments thereof.
Those of skill in the art will recognize that numerous residues of the above-described sequences can be substituted with other, chemically, sterically and/or electronically similar residues without substantially altering the biological activity of the resulting receptor species. In addition, larger polypeptide sequences containing substantially the same sequence as
amino acids set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14 therein (e.g., splice variants) are contemplated.
As employed herein, the term "substantially the same amino acid sequence" refers to amino acid sequences having at least about 70% identity with respect to the reference amino acid sequence, and retaining comparable functional and biological activity characteristic of the protein defined by the reference amino acid sequence. Preferably, proteins having "substantially the same amino acid sequence" will have at least about 80%, more preferably 90% amino acid identity with respect to the reference amino acid sequence; with greater than about 95% amino acid sequence identity being especially preferred. It is recognized, however, that polypeptides (or nucleic acids referred to hereinbefore) containing less than the described levels of sequence identity arising as splice variants or that are modified by conservative amino acid substitutions, or by substitution of degenerate codons are also encompassed within the scope of the present invention.
The term "biologically active" or "functional", when used herein as a modifier of invention SMDP and/or SCP(s), or polypeptide fragments thereof, refers to a polypeptide that exhibits functional characteristics similar to SMDP and/or SCP. For example, one biological activity of SMDP and/or SCP is the ability to bind, preferably in vivo, to at least one other member of the SMDP and/or SCP families of proteins, or to homodimerize, or to mediate protein degradation via an SFC complex as described herein. Such SMDP and/or SCP binding activity can be assayed, for example, using the methods described herein. Another biological activity of SMDP and/or SCP is the ability to act as an immunogen for the production
of polyclonal and monoclonal antibodies that bind specifically to an invention SMDP and/or SCP. Thus, an invention nucleic acid encoding SMDP and/or SCP will encode a polypeptide specifically recognized by an antibody that also specifically recognizes the SMDP and/or SCP protein (preferably human) including the amino acid set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14. Such immunologic activity may be assayed by any method known to those of skill in the art. For example, a test- polypeptide encoded by a SMDP and/or SCP cDNA can be used to produce antibodies, which are then assayed for their ability to bind to an invention SMDP and/or SCP protein including the sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14. If the antibody binds to the test- polypeptide and the protein including the sequence encoded by SEQ ID NOs:2, 4, 6, 8, 10, 12 or 14 with substantially the same affinity, then the polypeptide possesses the requisite immunologic biological activity.
The invention SMDPs and/or SCPs can be isolated by a variety of methods well-known in the art, e.g., recombinant expression systems described herein, precipitation, gel filtration, ion-exchange, reverse- phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al . , Guide to Protein Purification: Methods in Enzvmoloqy
Vol. 182, (Academic Press, (1990) ) , which is incorporated herein by reference. Alternatively, the isolated polypeptides of the present invention can be obtained using well-known recombinant methods as described, for example, in Sambrook et al., supra., 1989).
An example of the means for preparing the invention polypeptide (s ) is to express nucleic acids encoding the SMDP and/or SCP in a suitable host cell,
such as a bacterial cell, a yeast cell, an amphibian cell (i.e., oocyte) , or a mammalian cell, using methods well known in the art, and recovering the expressed polypeptide, again using well-known methods. Invention polypeptides can be isolated directly from cells that have been transformed with expression vectors as described below herein. The invention polypeptide, biologically functional fragments, and functional equivalents thereof can also be produced by chemical synthesis. For example, synthetic polypeptides can be produced using Applied Biosystems, Inc. Model 430A or 431A automatic peptide synthesizer (Foster City, CA) employing the chemistry provided by the manufacturer.
Also encompassed by the term SMDP and/or SCP are functional fragments or polypeptide analogs thereof. The term "functional fragment" refers to a peptide fragment that is a portion of a full length SMDP and/or SCP protein, provided that the portion has a biological activity, as defined above, that is characteristic of the corresponding full length protein. For example, a functional fragment of an invention SMDP and/or SCP protein can have the protein: protein binding activity prevalent in SMDPs and/or SCPs. In addition, the characteristic of a functional fragment of invention SMDP and/or SCP proteins to elicit an immune response is useful for obtaining an anti-SMDP and/or SCP antibodies. Thus, the invention also provides functional fragments of invention SMDP and/or SCP proteins, which can be identified using the binding and routine methods, such as bioassays described herein.
The term "polypeptide analog" includes any polypeptide having an amino acid residue sequence substantially the same as a sequence specifically shown
herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to functionally mimic an SMDP and/or SCP as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
The amino acid length of functional fragments or polypeptide anlogs of the present invention can range from about 5 amino acids up to the full-length protein sequence of an invention SMDP and/or SCP. In certain embodiments, the amino acid lengths include, for example, at least about 10 amino acids, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250 or more amino acids in length up to the full-length SMDP and/or SCP protein sequence.
As used herein the phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the required binding activity. The phrase "chemical derivative" refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules
include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides , p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine . Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions of residues, relative to the sequence of a polypeptide whose sequence is shown herein, so long as the required activity is maintained.
The present invention also provides compositions containing an acceptable carrier and any of an isolated, purified SMDP and/or SCP mature protein or functional polypeptide fragments thereof, alone or in combination with each other. These polypeptides or proteins can be recombinantly derived, chemically synthesized or purified from native sources. As used herein, the term "acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The SMDP and/or SCP compositions
described herein can be used, for example, in methods described hereinafter.
Also provided are antisense-nucleic acids having a sequence capable of binding specifically with full-length or any portion of an mRNA that encodes SMDP and/or SCP polypeptides so as to prevent translation of the mRNA. The antisense-nucleic acid may have a sequence capable of binding specifically with any portion of the sequence of the cDNA encoding SMDP and/or SCP polypeptides. As used herein, the phrase "binding specifically" encompasses the ability of a nucleic acid sequence to recognize a complementary nucleic acid sequence and to form double-helical segments therewith via the formation of hydrogen bonds between the complementary base pairs. An example of an antisense- nucleic acid is an antisense-nucleic acid comprising chemical analogs of nucleotides.
Compositions comprising an amount of the antisense-nucleic acid, described above, effective to reduce expression of SMDP and/or SCP polypeptides by passing through a cell membrane and binding specifically with mRNA encoding SMDP and/or SCP polypeptides so as to prevent translation and an acceptable hydrophobic carrier capable of passing through a cell membrane are also provided herein. Suitable hydrophobic carriers are described, for example, in U.S. Patent Nos. 5,334,761; 4,889,953; 4,897,355, and the like. The acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type. The structure may be part of a protein known to bind to a cell-type specific receptor.
Antisense-nucleic acid compositions are useful to inhibit translation of mRNA encoding invention polypeptides. Synthetic oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding SMDP and/or SCP polypeptides and inhibit translation of mRNA and are useful as compositions to inhibit expression of SMDP and/or SCP associated genes in a tissue sample or in a subject.
In accordance with another embodiment of the invention, kits are provided for detecting mutations, duplications, deletions, rearrangements and aneuploidies in SMDP and/or SCP genes comprising at least one invention probe or antisense nucleotide.
The present invention provides means to modulate levels of expression of SMDP and/or SCP polypeptides by employing synthetic antisense-nucleic acid compositions (hereinafter SANC) which inhibit translation of mRNA encoding these polypeptides. Synthetic oligonucleotides, or other antisense-nucleic acid chemical structures designed to recognize and selectively bind to mRNA, are constructed to be complementary to full-length or portions of an SMDP and/or SCP coding strand, including nucleotide sequences set forth in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. The SANC is designed to be stable in the blood stream for administration to a subject by injection, or in laboratory cell culture conditions. The SANC is designed to be capable of passing through the cell membrane in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SANC which render it capable of passing through cell membranes, for example, by designing small, hydrophobic SANC chemical structures, or by virtue of specific transport systems in
the cell which recognize and transport the SANC into the cell. In addition, the SANC can be designed for administration only to certain selected cell populations by targeting the SANC to be recognized by specific cellular uptake mechanisms which bind and take up the SANC only within select cell populations. In a particular embodiment the SANC is an antisense oligonucleotide .
For example, the SANC may be designed to bind to a receptor found only in a certain cell type, as discussed supra . The SANC is also designed to recognize and selectively bind to target mRNA sequence, which may correspond to a sequence contained within the sequences shown in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. The SANC is designed to inactivate target mRNA sequence by either binding thereto and inducing degradation of the mRNA by, for example, RNase I digestion, or inhibiting translation of mRNA target sequence by interfering with the binding of translation-regulating factors or ribosomes, or inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups which either degrade or chemically modify the target mRNA. SANCs have been shown to be capable of such properties when directed against mRNA targets (see Cohen et al., TIPS, 10:435 (1989) and Weintraub, Sci. American, January (1990), pp.40; both incorporated herein by reference).
In accordance with yet another embodiment of the present invention, there is provided a method for the recombinant production of invention SMDPs and/or SCPs by expressing the above-described nucleic acid sequences in suitable host cells. Recombinant DNA expression systems that are suitable to produce SMDPs and/or SCPs described herein are well-known in the art. For example, the
above-described nucleotide sequences can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.
Suitable expression vectors are well-known in the art, and include vectors capable of expressing DNA operatively linked to a regulatory sequence, such as a promoter region that is capable of regulating expression of such DNA. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
As used herein, a promoter region refers to a segment of DNA that controls transcription of DNA to which it is operatively linked. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, and the like.
As used herein, expression refers to the process by which polynucleic acids are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleic acid is derived from
genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
Prokaryotic transformation vectors are well- known in the art and include pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, CA) , and the like. Other suitable vectors and promoters are disclosed in detail in U.S. Patent No. 4,798,885, issued January 17, 1989, the disclosure of which is incorporated herein by reference in its entirety.
Other suitable vectors for transformation of E. coli cells include the pET expression vectors (Novagen, see U.S patent 4,952,496), e.g., pETlla, which contains the T7 promoter, T7 terminator, the inducible E . coli lac operator, and the lac repressor gene; and pET 12a-c, which contain the T7 promoter, T7 terminator, and the E . coli ompT secretion signal. Another suitable vector is the pIN-IIIompA2 (see Duffaud et al., Meth. in Enzvmoloαv, 153:492-507, 1987), which contains the lpp promoter, the lacUV5 promoter operator, the ompA secretion signal, and the lac repressor gene.
Exemplary, eukaryotic transformation vectors, include the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system [described by Mulligan and Berg, Nature Vol. 277:108-114 (1979)] the Okayama-Berg cloning system [Mol. Cell Biol. Vol. 2:161- 170 (1982)], and the expression cloning vector described by Genetics Institute [Science Vol. 228:810-815 (1985)], are available which provide substantial assurance of at
least some expression of the protein of interest in the transformed eukaryotic cell line.
In accordance with another embodiment of the present invention, there are provided "recombinant cells" containing the nucleic acid molecules (i.e., DNA or mRNA) of the present invention. Methods of transforming suitable host cells, preferably bacterial cells, and more preferably E . coli cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art.
See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989) .
Exemplary methods of transformation include, e.g., transformation employing plasmids, viral, or bacterial phage vectors, transfection, electroporation, Hpofection, and the like. The heterologous DNA can optionally include sequences which allow for its extrachromosomal maintenance, or said heterologous DNA can be caused to integrate into the genome of the host
(as an alternative means to ensure stable maintenance in the host) .
Host organisms contemplated for use in the practice of the present invention include those organisms in which recombinant production of heterologous proteins has been carried out. Examples of such host organisms include bacteria (e.g., E . coli ) , yeast (e.g., Saccharomyces cerevisiae, Candida tropicalis , Hansenula polymorpha and P. pastoris ; see, e.g., U.S. Patent Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk" cells), insect cells,
and the like. Presently preferred host organisms are bacteria. The most preferred bacteria is E . coli .
In one embodiment, nucleic acids encoding the invention SMDPs and/or SCPs can be delivered into mammalian cells, either in vivo or in vitro using suitable viral vectors well-known in the art. Suitable retroviral vectors, designed specifically for "gene therapy" methods, are described, for example, in WIPO publications WO 9205266 and WO 9214829, which provide a description of methods for efficiently introducing nucleic acids into human cells. In addition, where it is desirable to limit or reduce the in vivo expression of the invention SMDP and/or SCP, the introduction of the antisense strand of the invention nucleic acid is contemplated.
Viral based systems provide the advantage of being able to introduce relatively high levels of the heterologous nucleic acid into a variety of cells. Suitable viral vectors for introducing invention nucleic acid encoding an SMDP and/or SCP protein into mammalian cells (e.g., vascular tissue segments) are well known in the art. These viral vectors include, for example, Herpes simplex virus vectors (e.g., Geller et al., Science, 241:1667-1669 (1988)), Vaccinia virus vectors (e.g., Piccini et al., Meth. in Enzymology, 153:545-563 (1987); Cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzman and S.H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84), Moloney murine leukemia virus vectors (Danos et_al., PNAS , USA, 85:6469 (1980)), adenovirus vectors (e.g., Logan et al., PNAS, USA, 81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner,
Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098 (1992); Graham et al . , Meth. Mol . Biol . , 7:109-127 (1991)), adeno-associated virus vectors, retrovirus vectors (see, e.g., U.S. Patent 4,405,712 and 4,650,764), and the like. Especially preferred viral vectors are the adenovirus and retroviral vectors.
For example, in one embodiment of the present invention, adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes (Wagner et al., PNAS, USA, 89:6099-6103 (1992); Curiel et al., Hum. Gene Ther. ,
3:147-154 (1992); Gao et al., Hum. Gene Ther. , 4:14-24 (1993)) are employed to transduce mammalian cells with heterologous SMDP and/or SCP nucleic acid. Any of the plasmid expression vectors described herein may be employed in a TfAdpl-DNA complex.
As used herein, "retroviral vector" refers to the well-known gene transfer plasmids that have an expression cassette encoding an heterologous gene residing between two retroviral LTRs . Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Patent 4,650,764).
Suitable retroviral vectors for use herein are described, for example, in U.S. Patent 5,252,479, and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated herein by reference, which provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Other retroviral vectors
include, for example, the mouse mammary tumor virus vectors (e.g., Shackleford et al., PNAS, USA, 85:9655-9659 (1988)), and the like.
In accordance with yet another embodiment of the present invention, there are provided anti-SMDP and/or SCP antibodies having specific reactivity with an SMDP and/or SCP polypeptides of the present invention. Active fragments of antibodies are encompassed within the definition of "antibody". Invention antibodies can be produced by methods known in the art using invention polypeptides, proteins or portions thereof as antigens. For example, polyclonal and monoclonal antibodies can be produced by methods well known in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory (1988)), which is incorporated herein by reference. Invention polypeptides can be used as immunogens in generating such antibodies. Alternatively, synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens. Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide. Altered antibodies such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods described, for example, in Sambrook et al., supra., and Harlow and Lane, supra . Both anti-peptide and anti-fusion protein antibodies can be used, (see, for example, Bahouth et al., Trends Pharmacol . Sci . 12:338 (1991); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, NY (1989) which are incorporated herein by reference).
Antibody so produced can be used, inter alia, in diagnostic methods and systems to detect the level of SMDP and/or SCP present in a mammalian, preferably human, body sample, such as tissue or vascular fluid. Such antibodies can also be used for the immunoaffinity or affinity chromatography purification of the invention SMDP and/or SCP. In addition, methods are contemplated herein for detecting the presence of an invention SMDP and/or SCP protein in a tissue or cell, comprising contacting the cell with an antibody that specifically binds to SMDP and/or SCP polypeptides, under conditions permitting binding of the antibody to the SMDP and/or SCP polypeptides, detecting the presence of the antibody bound to the SMDP and/or SCP polypeptide, and thereby detecting the presence of invention polypeptides. With respect to the detection of such polypeptides, the antibodies can be used for in vi tro diagnostic or in vivo imaging methods .
Immunological procedures useful for in vi tro detection of target SMDP and/or SCP polypeptides in a sample include immunoassays that employ a detectable antibody. Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art. An antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.
Invention anti-SMDP and/or SCP antibodies are contemplated for use herein to modulate the activity of
the SMDP and/or SCP polypeptide in living animals, in humans, or in biological tissues or fluids isolated therefrom. The term "modulate" refers to a compound's ability to increase (e.g., via an agonist) or inhibit (e.g., via an antagonist) the biological activity of an invention SMDP and/or SCP protein, such as the participation in Siah-Mediated-Degradation via an SFC complex and the 26S proteosome. Accordingly, compositions comprising a carrier and an amount of an antibody having specificity for SMDP and/or SCP polypeptides effective to inhibit naturally occurring ligands or other SMDP and/or SCP-binding proteins from binding to invention SMDP and/or SCP polypeptides are contemplated herein. For example, a monoclonal antibody directed to an epitope of an invention SMDP and/or SCP polypeptide including an amino acid sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 or 14, can be useful for this purpose.
The present invention further provides transgenic non-human mammals that are capable of expressing exogenous nucleic acids encoding SMDP and/or SCP polypeptides. As employed herein, the phrase "exogenous nucleic acid" refers to nucleic acid sequence which is not native to the host, or which is present in the host in other than its native environment (e.g., as part of a genetically engineered DNA construct) . In addition to naturally occurring levels of SMDP and/or SCP, invention SMDPs and/or SCPs can either be overexpressed or underexpressed (such as in the well- known knock-out transgenics) in transgenic mammals.
Also provided are transgenic non-human mammals capable of expressing nucleic acids encoding SMDP and/or SCP polypeptides so mutated as to be incapable of normal
activity, i.e., do not express native SMDP and/or SCP. The present invention also provides transgenic non-human mammals having a genome comprising antisense nucleic acids complementary to nucleic acids encoding SMDP and/or SCP polypeptides, placed so as to be transcribed into antisense mRNA complementary to mRNA encoding SMDP and/or SCP polypeptides, which hybridizes to the mRNA and, thereby, reduces the translation thereof. The nucleic acid may additionally comprise an inducible promoter and/or tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types. Examples of nucleic acids are DNA or cDNA having a coding sequence substantially the same as the coding sequence shown in SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. An example of a non-human transgenic mammal is a transgenic mouse. Examples of tissue specificity- determining elements are the metallothionein promoter and the L7 promoter.
Animal model systems which elucidate the physiological and behavioral roles of SMDP and/or SCP polypeptides are also provided, and are produced by creating transgenic animals in which the expression of the SMDP and/or SCP polypeptide is altered using a variety of techniques. Examples of such techniques include the insertion of normal or mutant versions of nucleic acids encoding an SMDP and/or SCP polypeptide by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos to produce a transgenic animal. (See, for example, Hogan et al . , Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, (1986) ) .
Also contemplated herein, is the use of homologous recombination of mutant or normal versions of SMDP and/or SCP genes with the native gene locus in transgenic animals, to alter the regulation of expression or the structure of SMDP and/or SCP polypeptides (see, Capecchi et al . , Science 244:1288 (1989); Zi mer et al., Nature 338:150 (1989); which are incorporated herein by reference) . Homologous recombination techniques are well known in the art. Homologous recombination replaces the native (endogenous) gene with a recombinant or mutated gene to produce an animal that cannot express native (endogenous) protein but can express, for example, a mutated protein which results in altered expression of SMDP and/or SCP polypeptides.
In contrast to homologous recombination, microinjection adds genes to the host genome, without removing host genes. Microinjection can produce a transgenic animal that is capable of expressing both endogenous and exogenous SMDP and/or SCP. Inducible promoters can be linked to the coding region of nucleic acids to provide a means to regulate expression of the transgene. Tissue specific regulatory elements can be linked to the coding region to permit tissue-specific expression of the transgene. Transgenic animal model systems are useful for in vivo screening of compounds for identification of specific ligands, i.e., agonists and antagonists, which activate or inhibit SMDP and/or SCP protein responses.
SMDP and/or SCP proteins, such as Siah-1, are contemplated herein to be a tumor suppressor proteins.
Tumor suppressor proteins generally are thought to have a function in signal transduction . Mutation results in loss of function whereupon a signal pathway that the
suppressor protein regulates is left in the "on" position, which results in unregulated cell proliferation resulting in cancerous tumor formation. Nearly all tumor suppressors regulate cell division, and proliferation, and may have involvement in biochemical pathways of development and the cell cycle.
The functions of the invention SMDP and/or SCP proteins support the role of both Siah and the invention SMDPs and/or SCPs in cellular pathways that affect protein degradation, such as by activating or inhibiting protein degradation, cell division and proliferation. Accordingly, invention SMDP and/or SCP proteins provide targets for treating a broad variety of pathologies, such as proliferative diseases, cancer pathologies, and the like.
For example, in accordance with yet another embodiment of the present invention, Siah-1 has been found to bind to the protein APC (Kinzler, et al., 1996, supra) . The APC protein is known to bind to β-catenin and target it for ubiquitination and degradation (Korinek, V. et al., 1997, Science, 275:1784-1786, Rubinfeld, B. et al . , 1997, Science, 275:1790-1792, and Morin, P.J. et al., 1997, Science, 275:1787-1790). Defects in the regulation of the APC/β-catenin pathway for cell growth control have been implicated in a variety of cancer pathologies, such as epithelial cancers, and the like. Thus, in accordance with the present invention Siah-1, and antagonist or agonists thereof, are contemplated for use in methods for treating a variety of cancers, such as epithelial cancer and the like, preferably by modulating β-catenin degradation. When used for binding to APC, fragments comprising the carboxy terminus of Siah-1, preferably comprising at least amino
acids 252-298 of SEQ ID NO:2, are employed (See Figure 3) .
Invention nucleic acids, oligonucleotides (including antisense), vectors containing same, transformed host cells, polypeptides and combinations thereof, as well as antibodies of the present invention, can be used to screen compounds in vi tro to determine whether a compound functions as a potential agonist or antagonist to invention polypeptides. These in vi tro screening assays provide information regarding the function and activity of invention polypeptides, which can lead to the identification and design of compounds that are capable of specific interaction with one or more types of invention proteins or fragments thereof.
Thus, in accordance with yet another embodiment of the present invention, there are provided methods for identifying compounds which bind to, and preferably, modulate the activity of SMDP and/or SCP polypeptides. The invention proteins may be employed in a competitive binding assay. Such an assay can accommodate the rapid screening of a large number of compounds to determine which compounds, if any, are capable of binding to SMDPs and/or SCPs. Subsequently, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as modulators, agonists or antagonists of invention SMDP and/or SCP proteins. Compounds that bind to and/or modulate invention SMDPs and/or SCPs can be used to treat a variety of pathologies mediated by invention SMDPs and/or SCPs, as described herein.
In accordance with another embodiment of the present invention, transformed host cells that
recombinantly express invention polypeptides can be contacted with a test compound, and the modulating effect (s) thereof can then be evaluated by comparing the SMDP-mediated response (e.g., the degradation of a known Siah-mediated target, such as DCC or β-catenin) in the presence and absence of test compound, or by comparing the response of test cells or control cells (i.e., cells that do not express SMDP and/or SCP polypeptides) , to the presence of the compound.
As used herein, a compound or a signal that
"modulates the activity" of invention polypeptides refers to a compound or a signal that alters the activity of SMDP and/or SCP polypeptides so that the activity of the invention polypeptide is different in the presence of the compound or signal than in the absence of the compound or signal. In particular, such compounds or signals include agonists and antagonists. An agonist encompasses a compound or a signal that activates SMDP and/or SCP protein expression. Alternatively, an antagonist includes a compound or signal that interferes with SMDP and/or SCP expression. Typically, the effect of an antagonist is observed as a blocking of agonist-induced protein activation. Antagonists include competitive and non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding. A non-competitive antagonist or blocker inactivates the function of the polypeptide by interacting with a site other than the agonist interaction site.
As understood by those of skill in the art, assay methods for identifying compounds that modulate SMDP and/or SCP activity generally require comparison to
a control. For example, one type of a "control" is a cell or culture that is treated substantially the same as the test cell or test culture exposed to the compound, with the distinction that the "control" cell or culture is not exposed to the compound. Another type of
"control" cell or culture may be a cell or culture that is identical to the transfected cells, with the exception that the "control" cell or culture do not express native proteins. Accordingly, the response of the transfected cell to compound is compared to the response (or lack thereof) of the "control" cell or culture to the same compound under the same reaction conditions.
Accordingly, in accordance with another embodiment of the present invention, there is provided a bioassay for evaluating whether test compounds are capable of acting as agonists or antagonists for SMDP and/or SCP proteins, wherein said bioassay comprises:
(a) culturing cells containing:
DNA which expresses an SMDP and/or SCP or functional fragments thereof, wherein said culturing is carried out in the presence of at least one compound whose ability to modulate an activity of an SMDP and/or SCP is sought to be determined, wherein said activity is selected from a protein : protein binding activity or a protein degradation activity and thereafter (b) monitoring said cells for either an increase or decrease in the level of protein: protein binding or protein degradation.
Methods well-known in the art for measuring protein:protein binding or protein degradation can be employed in bioassays described herein to identify agonists and antagonists of SMDP and/or SCP proteins. For example, the Siah-1 over-expression assay described in Example 14 can be used to evaluate the cell degradation activity of recombinant SMDP and/or SCP proteins or mutants and/or analogs thereof, expressed in mammalian host cells.
As used herein, "ability to modulate protein degradation activity of an SMDP and/or SCP" protein refers to a compound that has the ability to either induce (agonist) or inhibit (antagonist) the protein degradation activity of SMDP and/or SCP proteins within a cell. Host cells contemplated for use in the bioassay(s) of the present invention include human and other mammalian cells (readily avaialable from American Type Culture Collection) , as well as genitically engineered yeast or bacteria that express human SMDPs and/or SCPs, and the like.
In yet another embodiment of the present invention, there are provided methods for modulating the protein degradation activity mediated by SMDP and/or SCP protein (s), said method comprising: contacting an SMDP and/or SCP protein with an effective, modulating amount of an agonist or antagonist identified by the above-described bioassays.
Also provided herein are methods of treating pathologies, said method comprising administering an effective amount of an invention therapeutic composition. Such compositions are typically administered in a physiologically compatible composition.
Exemplary diseases related to abnormal cell proliferation contemplated herein for treatment according to the present invention include cancer pathologies, keratin hyperplasia, neoplasia, keloid, benign prothetic hypertrophy, inflammatory hyperplasia, and the like. Exemplary cancer pathologies contemplated herein for treatment include, gliomas, carcinomas, sarcomas, melanomas, hamartomas, leukemias, lymphomas, and the like.
Also contemplated herein, are therapeutic methods using invention pharmaceutical compositions for the treatment of pathological disorders in which there is too little cell division, such as, for example, bone marrow aplasias, immunodeficiencies due to a decreased number of lymphocytes, and the like. Methods of treating a variety of inflammatory diseases with invention therapeutic compositions are also contemplated herein, such as treatment of sepsis, fibrosis (e.g., scarring), arthritis, graft versus host disease, and the like.
Accordingly, the present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention, such as pharmaceutical compositions, contain a physiologically compatible carrier together with an invention SMDP and/or SCP (or functional fragment thereof) , a SMDP and/or SCP modulating agent, such as a compound (agonist or antagonist) identified by the methods described herein, or an anti-SMDP and/or SCP antibody, as described herein, dissolved or dispersed therein as an active ingredient.
In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.
As used herein, the terms "pharmaceutically acceptable", "physiologically compatible" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well known in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension, in liquid prior to use can also be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations of any two or more thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.
The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are
formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.
Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and organic bases such as mono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like) .
Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions .
As described herein, an "effective amount" is a predetermined amount calculated to achieve the desired therapeutic effect, e.g., to modulate the protein degradation activity of an invention SMDP and/or SCP protein. The required dosage will vary with the particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. It may be particularly advantageous to administer such compounds in depot or long-lasting form as discussed hereinafter. A therapeutically effective amount is typically an amount of an SMDP and/or SCP-modulating agent or compound identified herein that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1.0 μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml and usually 5 to 10 μg/ml. Therapeutic invention anti-SMDP and/or SCP antibodies can be administered in proportionately appropriate amounts in accordance with known practices in this art.
Also provided are systems using invention SMDPs and/or SCPs, or functional fragments thereof, for targeting any desired protein for ubiquitination and degradation, thus enabling novel gene discovery through functional genomics strategies or providing the basis for ablating target proteins involved in diseases for therapeutic purposes.
In accordance with another embodiment of the invention, there are provided methods for "inducing the degradation of the function" of a desired protein from a particular cell or cell-system. As used herein the
phrase "inducing the degradation of the function" refers to deleting, altering, modifying and/or degrading a target protein so that it no longer has the ability to perform its native physiological function. This method is useful to determine the physiological/cellular function of the degraded protein. Thus, this invention method is useful in alleviating one of the rate limiting steps in functional genomics.
The invention methods take advantage of the invention SMDP and/or SCP proteins provided herein to create a system that targets specific proteins for degradation by recruiting them to a SCF complex for ubiquitination and subsequent degradation by an appropriate proteosome, such as 26S proteosome. First, a protein or peptide fragment is selected that binds the protein targeted for degradation in a cell, which is referred to herein as the "target-protein binding domain." Such protein or peptide fragment could be, for example: a domain of any known protein that interacts with the target protein; the Fab region of an anti-target-protein antibody (e.g., sFv, and the like); or a peptide aptamer obtained by screening (using, for example, yeast two-hybridization, phage display, or other screening methods) a random library of peptide aptamers. The target-protein binding domain is then fused (by engineering cDNAs in expression plasmids to form a chimera) with an appropriate protein-degradation binding domain selected from an invention SMDP and/or SCP, such as Siah, SIP, SAF-1, ΞAF-2 or SAD, and the like; or other known proteins involved in protein degradation, such as F-box containing proteins, (e.g., Skpl, and the like), SOCS-box containing proteins (see, e.g., Kamura et al., 1998, Genes Dev, 12:3872; and Starr and Hilton, 1999, Bioessays.21 : 47 ) , HECT family proteins (see, e.g.,
Huibregtse et al. 1995 Proc. Natl. Acad. Sci. USA 92:2563-2567), or any other subunit of an E3 ubiquitin ligase complex, and the like (see, e.g., Tyers and Williams, 1999, Science, 284:601-604; incorporated herein by reference in its entirety) .
As used herein, the phrase "protein-degradation binding domain" refers to a protein region that functions to recruit the target protein into a member of the superfamily of E3 ubiquitin ligase complexes, such as the SCF complex, or to Siah-family proteins which may target some proteins for degradation independently of SCF complexes, where the protein-degradation binding domain and/or the target protein become ubiquitinated and then degraded, such as by the 26S proteosome, lysosomes and/or vacuoles (see, e.g., Tyers and Williams, 1999, Science, 284:601-604; and Ciechanover, 1998, supra) . Exemplary protein-degradation domains can be obtained from a protein member of the ubiquitin-mediated protein- degradation family. As used herein, the phrase "a protein member of the ubiquitin-mediated protein- degradation family" refers to one of the numerous proteins that are known to interact, via protein: protein binding, in the ubiquitin system of intracellular protein degradation (see, e.g., Ciechanover et al . , 1998, supra; and Tyers and Williams, supra, and the like) .
In yet another embodiment contemplated by the present invention, methods are provided of identifying a nucleic acid molecules encoding a chimeric protein that modulates a cellular phenotype, said method comprising: (a) expressing, in a cell, a chimeric nucleic acid comprising a member of a nucleic acid library fused to nucleic acid encoding a protein degradation binding domain of a protein
member of the ubiquitin-mediated protein degradation family; and
(b) screening said cells for a modulation of said phenotype.
For example, unbiased nucleic acid libraries can be constructed wherein, each member of the nucleic acid library is expressed as an encoded protein fused to a particular protein-degradation binding domain, such as invention SMDPs and/or SCPs, e.g., Siah-1, SIP (see, e.g., Example 15), SAF-1, SAF-2, or SAD; or other known proteins involved in protein degradation, such as Skpl, F-box containing proteins, HECT family proteins, or any other subunit of an E3 ubiquitin ligase complex, and the like. As used herein a "nucleic acid library" comprises cDNA libraries, YAC libraries, BAC libraries, cosmid libraries, or any other source of nucleic acid encoding polypeptides. The chimeric nucleic acid encoding these fusion proteins is then introduced into cells possessing a particular phenotype to be assayed.
The cells are then subjected to a "screening" step which comprises selecting one or more cells in which the desired phenotype has been modulated (e.g., suppressed or enhanced) . The phenotypes to be screened may be any chemical or physical representation of a cellular process, including but not limited to: cell proliferation in either an attached or detached (i.e., anchorage-independent) state, cell survival, cell death, cell secretion, cell migration, abnormal cell morphology, chemical reactivity (e.g., heavy metals, antibiotics, etc.), physical reactivity (e.g., heat, light, radiation, etc.), and the like.
Next, cDNAs are identified and isolated whose expression products function to modulate the desired phenotype within cells. The cDNAs identified by the invention method encode an invention chimeric protein that interacts, preferably by direct binding, with another protein in the cells that is targeted for degradation, thereby eliminating its physiological function. Accordingly, any target-protein that has one or more protein-binding or protein-interacting partners, or which homodimerizes/homo-oligomerizes is contemplated for degradation in the invention methods. The cDNA identified by the above-described method can be used to perform a two-hybrid screen, as described herein, to identify the protein-binding region of the partner to the target protein, or directly sequenced to determine the identity of the target protein if homo-dimerization or homo-oligomerization situation occurs.
Accordingly, also provided in accordance with the present invention are chimeric proteins, and encoding nucleic acids, comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein-degradation family.
Exemplary proteins whose function can be targeted for degradation according to the invention methods, include any protein encoded by a known gene or cDNA whose function is desired. Exemplary targets include, for example, apoptosis-related proteins, cell- cycle regulatory proteins, heat shock proteins, transcription factors, or any other target protein which, when degraded, will modulate the phenotype of a cell.
Also provided are methods for treating a disease by degrading the function of a target protein, comprising introducing, into a cell, a chimeric protein comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein- degradation family. For example, for a variety of proteins which, when expressed in overabundant or mutated form (e.g., an oncoprotein such as ras, or a genetic mutation, such as in the CF gene (cystic fibrosisgene) result in a known pathology, the chimeric protein of the invention may be used to therapeutically treat the disease, by way of reducing or completely eliminating, via protein degradation, the pathology causing protein. This treatment comprises fusion of a protein domain which binds the target pathology causing protein (i.e., the protein which causes the illness) with a particular protein-degradation binding domain as described herein. This chimeric protein may then be delivered to the location of the protein which causes the illness by intravenous therapy or gene therapy employing the methods described herein, or any other method well-known to one skilled in the art for delivering a protein to its binding target. As used herein, "treatment of a disease" refers to a reduction in the effects of the disease, including reducing the symptoms of the disease.
In accordance with another embodiment of the present invention, there are provided methods for diagnosing cancer, said method comprising: detecting, in said subject, a defective sequence or mutant of SEQ ID NOs:l, 3, 5, 7, 9,
11 and 13.
In accordance with another embodiment of the present invention, there are provided diagnostic systems, preferably in kit form, comprising at least one invention nucleic acid in a suitable packaging material. The diagnostic nucleic acids are derived from the
SMDP and/or SCP-encoding nucleic acids described herein. In one embodiment, for example, the diagnostic nucleic acids are derived from any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13. Invention diagnostic systems are useful for assaying for the presence or absence of nucleic acid encoding SMDP and/or SCP in either genomic DNA or in transcribed nucleic acid (such as mRNA or cDNA) encoding SMDP and/or SCP.
A suitable diagnostic system includes at least one invention nucleic acid, preferably two or more invention nucleic acids, as a separately packaged chemical reagent (s) in an amount sufficient for at least one assay. Instructions for use of the packaged reagent are also typically included. Those of skill in the art can readily incorporate invention nucleic probes and/or primers into kit form in combination with appropriate buffers and solutions for the practice of the invention methods as described herein.
As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as invention nucleic acid probes or primers, and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the invention nucleic acids can be used for detecting a particular sequence encoding SMDP and/or SCP including the nucleotide sequences set forth in SEQ
ID NOs:l, 3, 5, 7, 9, 11 and 13 or mutations or deletions therein, thereby diagnosing the presence of, or a predisposition for, cancer. In addition, the packaging material contains instructions indicating how the materials within the kit are employed both to detect a particular sequence and diagnose the presence of, or a predisposition for, cancer.
The packaging materials employed herein in relation to diagnostic systems are those customarily utilized in nucleic acid-based diagnostic systems. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits an isolated nucleic acid, oligonucleotide, or primer of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated nucleic acid, oligonucleotide or primer, or it can be a microtiter plate well to which microgram quantities of a contemplated nucleic acid probe have been operatively affixed.
"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto. The invention will now be described in greater detail by reference to the following non-limiting examples .
EXAMPLES
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al . , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed . ) , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Technigues Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987) ) .
Two-hybrid assays.
Library screening by the yeast two-hybrid method was performed herein as described (Durfee et al., 1993; Sato et al., 1995; Matsuzawa et al. 1998) using the pGilda plasmid encoding the desired amino acid region as bait, an appropriate cDNA library, and the EGY48 strain S. cerevisiae (MATa, trpl, ura3, his, leu2 : :plexApo6- leu2) . Cells were grown in either YPD medium with 1% yeast extract, 2% polypeptone, and 2% glucose, or in Burkholder's minimal medium (BMM) fortified with appropriate amino-acids as described previously (Sato et al., 1994). Transformations were performed by a LiCl method using 0.25 mg of pJG4-5-cDNA library DNA, and 5 mg of denatured salmon sperm carrier DNA. Clones that formed on Leu deficient BMM plates containing 2% galactose/ 1% raffinose were transferred to BMM plates containing leucine and 2% glucose, and filter assays were
performed for β-galactosidase measurements as previously described.
1. Yeast two-hybrid screen of BAG-1 binding proteins to obtain cDNA encoding Siah-lα.
The mouse BAG-1 amino acid sequence was cloned into the pGilda plasmid and used as bait to screen a human Jurkat T-cell cDNA library. From an initial screen of -1.6 X 10' transformants, 298 clones were identified that trans-activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 30 colonies were also positive for β-galactosidase. These 30 candidate transformants were then cured of the LexA/BAG-1 bait plasmid by growth in media containing histidine and then mated with each of 5 different indicator strains of cells containing one of following LexA bait proteins: BAG-1 (1-219), Bax (1-171), v-Ras, Fas (191-335), or Lamin-C. The mating strain was RFY206 (MATa, his3D200, leu2-3, lys2D201, ura3-52, trplD: : hisG) , which had been transformed with pGilda-BAG-1 or various control proteins and selected on histidine-deficient media. This resulted in 23 clones which displayed specific two-hybrid binding interactions with BAG-1. DNA sequencing analysis revealed 4 cDNAs encoding portions of Siah-1.
2. Isolation of full-length human Siah-lα cDNAs .
To obtain the complete sequence of human Siah-1, cDNA fragments containing the 5' end of human Siah 1 were PCR-amplified from Jurkat randomly primer cDNAs by using aforward primer 5' GGGAATTCGGACTTATGGCATGTAAACA-3' (SEQ ID NO: 42) containing an EcoRI site and a reverse primer 5'
TAGCCAAGTTGCGAATGGA-3' (SEQ ID NO:43), based on sequences of EST database clones (NCBI ID: AA054272, AA258606, AA923663, AA418482, and AI167464). The PCR products were digested with EcoRI and BamHI, then directly subcloned into the EcoRI and Sail sites of pel plasmid into which the cDNA derived from pJG4-5-Siah (22-298) had previously been cloned, as a BamHI - Xhol fragment. The complete human Siah-lα cDNA and amino acid sequence is set forth in SEQ ID Nos : 1 and 2, respectively. The human Siah-lα sequence contains 16 N-terminal amino acids that are not present in the human Siah-lβ protein.
3. Yeast two-hybrid screen of Siah-1 binding proteins to obtain cDNA encoding SIP-L and SIP-S .
Human Siah-lα cDNA encoding amino acids 22-298 of SEQ ID NO:l (corresponding to amino acids 6-282 set forth in Nemani et al., supra) was cloned into the pGilda plasmid and used as a bait to screen a human embryonic brain cDNA library (Invitrogen) in EGY48 strain S . cerevisiae . From an initial screen of -2.0 X 107 transformants, 322 clones were identified that trans- activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 32 colonies were also positive for β-galactosidase. These 32 candidate transformants were then cured of the LexA/Siah-1 bait plasmid by growth in media containing histidine and then mated with each of 5 different indicator strains of cells containing one of following LexA bait proteins: Siah- 1(22-298), Bax (1-171), v-Ras, Fas (191-335), or BAG-1. The mating strain was RFY206 which had been transformed with pGilda-Siah-1 or various control proteins and selected on histidine-deficient media. This resulted in 11 clones which displayed specific two-hybrid interactions with Siah-1. DNA sequencing analysis
revealed 5 cDNAs encoding portions of SIP-L, 1 cDNA encoding portions of SIP-S, 3 cDNAs encoding portions of of APC (2681-2843) , and 2 cDNAs encoding portions of Siah- 1. The SIP-L and SIP-S clones were sequenced and the resulting nucleotide sequences are set forth in SEQ ID Nos:3 and 5, respectively.
4. Yeast two-hybrid screen of Skpl binding proteins to obtain cDNA encoding SAF-1 and SAD.
Human Skpl cDNA encoding amino acids 91-163 of (Zhang et al., 1995, Cell, 82:915-925) was cloned into the pGilda plasmid as a bait to screen a human embryonic brain cDNA library (Invtrogen) in EGY48 strain S . cerevisiae . From an initial screen of ~1.2 X 10ft transformants, 130 clones were identified that trans- activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 36 colonies were also positive for β-galactosidase. These 36 candidate transformants were then cured of the LexA/BAG-.l bait plasmid by growth in media containing histidine and then mated with each of 5 different indicator strains of cells containing one of following LexA bait proteins: Skpl (91- 163), SIP-L, Bax (1-171), v-Ras, Fas (191-335), or Siah- 1. The mating strain was RFY206 which had been transformed with pGilda-Skpl or various control proteins and selected on histidine-deficient media. This resulted in 3 clones which displayed specific two-hybrid interactions with Skpl and 18 clones clones which displayed specific two-hybrid interactions with both Skpl and SIP-L. DNA sequencing analysis revealed 12 cDNAs encoding portions of SAF-1 and 9 cDNAs encoding portions of SAD. The SAF-1 and SAD clones were sequenced and the resulting nucleotide sequences are set forth in SEQ ID Nos:7 (SAF-lα), 9 (SAF-lβ), and 13 (SAD).
5. Isolation of full-length SAF-2 cDNAs .
Full-length cDNA encoding a human SAF-2 protein was PCR-amplified from ZAPII Jurkat cDNA labrary (Stratagene) by using a forward primer 5'- GTGAATTCATGCAACTTGTACCTGATATAGAGTTC-3' (SEQ ID NO: 44) containing an EcoRI site and a reverse primer 5'- GGACTCGAGGCTCTACAGAGGCC-3 ' (SEQ ID NO: 45), based on human DNA sequence from clone 341E18 on chromosome 6pll.2-12.3 (AL031178) . The PCR products were digested with EcoRI and Xhol, then directly subcloned into the EcoRI and Xhol sites of the plasmid pCDNA3. The corresponding plasmid was sequenced and the results are set forth in SEQ ID Nos: 11 and 12.
6. Yeast two-hybrid screen of SIP-L binding proteins .
The human SIP-L cDNA encoding full-length SIP-L was cloned into the pGilda plasmid as a bait to screen a human embryonic brain cDNA library (Invtrogen) in EGY48 strain S . cerevisiae . From an initial screen of -1.5 X 107 transformants, 410 clones were identified that trans- activated the LEU2 reporter gene based on ability to grow on leucine-deficient media. Of those, 68 colonies were also positive for β-galactosidase. These 32 candidate transformants were then cured of the LexA/SIP-L bait plasmid by growth in media containing histidine and then mated with each of 32 different indicator strains of cells containing one of following LexA bait proteins: SIP-L, Bax (1-171), v-Ras, Fas (191-335), or BAG-1. The mating strain was RFY206 which had been transformed with pGilda-SIP-L or various control proteins and selected on histidine-deficient media. This resulted in 16 clones which displayed specific two-hybrid interactions with SIP-L. DNA sequencing analysis revealed 3 cDNAs encoding
portions of Skpl, 1 cDNA encoding portions of Siah-1, and 11 cDNAs encoding portions of SIP-L. These results indicate that SIP-L binds to Skpl and Siah-1 proteins, and is able to homodimerize with SIP isoforms.
7. A cell proliferation functional assay of SIP/Siah interaction
The effects of invention SIP-L and SIP-S proteins on Siah-1-induced cell cycle arrest in 293T epithelial cancer cells was examined and the results are shown in Figure 4. Human embryonic kidney 293 cells were maintained in high-glucose DMEM medium containing 10% fetal calf serum, 1 mM L-glutamine, and antibiotics. Cells (-5 x 105) in 60 mm plates were transfected with a total of 3.0 μg of plasmid DNAs encoding Siah-1 alone or together with SIP or SIP-S by a calcium phosphate precipitation technique. After 24 hours, the cells were harvested and the number of viable and dead cells were counted using trypan blue dye exclusion assays. Efficiency of transient transfection was estimated by in situ β-galactosidase assay using a portion of the transfected cells. The transient transfection efficiency of the T293 cells was consistently 90%.
As revealed in Figure 4, over-expression of Siah-1 resulted in decreased numbers of viable cells after 24 hours, without an increase in cell death. Thus, Siah-1 suppresses proliferation of 293 cells. Co- transfection of SIP-L with Siah-1 did not substantially alter Siah-1-mediated growth suppression. In contrast, the SIP-S protein abrogated the growth suppressive effects of Siah-1, which indicates that the invention SIP-S protein affects Siah-1 intracellularly in a different manner than SIP-L.
8. In vitro SIP: Siah-1 protein interaction assays.
Complementary cDNA encoding SIP-L was cloned into pGEX-4T-l and expressed in XL-1-blue cells (Stratagene, Inc.), and affinity-purified using glutathione-Sepharose as is well-known in the art.
Purified GST-fusion proteins (0.5-1.0 μg immobilized on 10-20 μl of glutathione beads) and 2.5 ul of rat reticulocyte lysates (TNT-Lysates; Promega, Inc.) containing 35S-labeled in vitro translated (IVT) Siah-1 proteins were incubated in 0.1 ml of HKMEN (10 mM HEPES [pH7.2], 142 mM KC1, 5 mM MgC12, 2 mM EGTA, 0.1% NP-40) at 4°C for 30 minutes. The beads were washed 3X with 1 ml HKMEN solution, followed by boiling in 25 μl of Laemmli-SDS sample buffer. The eluted proteins were analyzed by SDS-PAGE (12%) and detected by fluorography. Use of equivalent amounts of intact GST-fusion proteins and successful IVT of each protein was confirmed by SDS- PAGE analysis using Coomassie staining or autoradiography, respectively.
The results are shown in Figure 5A and indicate that Siah-1 binds to SIP-L and homodimerizes in vitro.
9. Co-immunoprecipitation Assay of SIP: Siah-1.
Two x 106 293T cells in 100 mm plates were transiently transfected with 10 μg of pCDNA3-myc-SIP-L and 10 mg of pcDNA3-HA-Siah-l (amino acids 97-298 of SEQ ID NO:2). Twenty-four hours later, cells were disrupted by sonication in 1 ml of HKMEN solution containing 0.2% NP-40, 0.1 μM PMSF, 5 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. After preclearing with normal mouse IgG and 10 ml protein A-agarose, immunoprecipitations were performed using 10 ml of anti-
myc antibody-conjugated sepharose (Santa Cruz) to precipitate the myc-SIP-L fusion, or an anti-IgG as a control at 4°C for 4 hours. After extensive washing in HKMEN solution, immune-complexes were analyzed by SDS- PAGE/immunoblotting using anti-HA antibody 12CA5
(Boehringer Mannheim) , followed by HRPase-conjugated goat anti mouse immunoglobulin (Amersham, Inc.), and detected using an enhanced chemiluminescence (ECL) system (Amersham, Inc.) .
The results are shown in Figure 5B and indicate that SIP proteins bind to Siah-1 intracellularly .
10. Yeast two-hybrid assay of Siah-1:APC binding specificity.
One μg of plasmids encoding fusion proteins of the LexA DNA-binding domain fused to Siah-1, APC (2681- 284), BAG-1, Bax, Ras, Fas, FLICE were co-tansformed into yeast strain EGY48 with 1 μg of pJG4-5 plasmid encoding fusion proteins of the B42 trans-activation domain fused to APC (2681-2843) and Siah-1. Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control which resulted in equivalent amounts of growth for all transformants . Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) . β-galactosidase activity of each colony was tested by filter assay and scored as blue (+) versus white (-) after 60 minutes.
The results are shown in Table 1, and indicate that APC interacts specifically by direct binding with Siah-1, and not with BAG-1, Bax, Ras, Fas nor FLICE.
Table 1 : Specific Interaction of Siah with SIP
Lex A B42 Leu+ β-Gal+
Siah-1 APC (2681- + + 2843)
APC (2681- Siah-1 + +
2843)
BAG-1 APC (2681- — — 2843)
Bax APC (2681- — — 2843)
Ras APC (2681- — — 2843)
Fas APC (2681- — — 2843)
FLICE APC (2681- — ~ 2843) empty APC (2681- — _ 2843)
11. Yeast two-hybrid assay of Siah-1: SIP binding specificity.
One μg of plasmids encoding fusion proteins of the LexA DNA-binding domain fused to Siah-1, Siah-2, BAG-1, Bax, Ras, Fas, FLICE, and SIP-L were co-tansformed into yeast strain EGY48 with 1 μg of pJG4-5 plasmid encoding fusion proteins of the B42 trans-activation domain fused to SIP-L, SIP-S, Siah-1, Siah-2, BAG-1, Bax, and Ras. Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control which resulted in equivalent amounts of growth for all transformants . Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) . β-galactosidase activity of each
colony was tested by filter assay and scored as blue (+) versus white (-) after 60 minutes.
The results are shown in Table 2, and indicate that SIP proteins interact specifically by direct binding with Siah proteins. SIP-L was found to interact with
Siah-1 and Siah-2, and not with BAG-1, Bax, Ras, Fas nor FLICE. SIP-S was also found to interact with Siah-1. Table G also reveals that the SIP-L homodimerization domain is within amino acids 73-228 of SIP-L (SEQ ID NO:4)
Specific Interaction of Siah with SIP
Table 2
Lex A B42 Leu+ β-Gal+
Siah-1 SIP-L + +
Siah-1 SIP-S + +
Siah-2 SIP-L + +
BAG-1 SIP-L - -
Bax SIP-L - -
Ras SIP-L - -
FLICE SIP-L - - empty SIP-L - -
SIP-L Siah-1 + +
SIP-L Siah-2 + +
SIP-L BAG-1 - -
SIP-L Bax - -
SIP-L Ras - -
SIP-L SIP-L + +
SIP-L SIP-S - -
12. Mapping of Siah-APC interaction domains
Expression plasmids encoding fusion proteins of
Siah-lα fragments corresponding to: SEQ ID NO: 2 amino acids 22-298; 22-251; 22-193; 97-298; and 46-102, fused to the B-42 trans-activation domain were co-transformed into yeast EGY48 cells with a plasmid encoding a chimeric fusion protein of the Lex A DNA-binding domain fused to amino acids 2681-2843 of APC "APC (2681-2843) ." Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control. Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) . β-
galactosidase activity for each colony was tested by filter assay and scored as blue (+) versus white (-) (β- gal) based on a 1 hour of color development.
The results are shown in Figure 3 and indicate that a region within the 47 carboxy terminal amino acids of Siah-lα (SEQ ID NO:2) is required for binding to APC.
13. Mapping of SKP-1, SIP-L, SAF-1, and SAD interaction domains .
Expression plasmids encoding fusion proteins of SAF-lα and functional fragments thereof corresponding to SEQ ID NO:8 amino acids 68-443; 80-443; and 258-443, were fused to the B-42 trans-activation domain. Likewise, expression plasmids encoding fusion proteins of SAD and functional fragments thereof corresponding to SEQ ID NO: 14 amino acids 128-447; and 360-447, were fused to the B-42 trans-activation domain. These SAF-l-fragment- and SAD-fragment-B-42 fusion proteins were co-transformed into yeast EGY48 cells with a plasmid encoding a chimeric fusion protein of the Lex A DNA-binding domain fused to either SKP1, SIP-L, SAF-1, or SAD. Transformed cells were grown on semi-solid media lacking leucine or containing leucine as a control. Plasmid combinations that resulted in growth on leucine-deficient media within 4 days were scored as positive (+) . β-galactosidase activity for each colony was tested by filter assay and scored as blue (+) versus white (-) (β-gal) based on a 1 hour of color development.
The results are shown in Figure 6A and 6B. Figure 6A indicates that SAF-1 interacts by direct binding to Skpl, SIP-L and SAD, but does not interact with Siah-1. A region within the SAF-1 fragment
corresponding to amino acids 80-257 of SEQ ID NO: 8 is required for SIP-L interaction, whereas a region within amino acids 258-443 of SAF-1 is required for Skpl and SAD interaction.
Figure 6B indicates that SAD interacts by direct binding to Skpl, SIP-L and SAF-1, but does not interact with Siah-1. A region within the SAD fragment corresponding to amino acids 1-127 of SEQ ID NO: 14 is required for SAF-1 interaction; a region within amino acids 128-359 of SAD is required for Skpl interaction; and a region within amino acids 360-447 of SEQ ID NO: 14 is required for SIP-L interaction.
14. Effect of Siah-1 over-expression on stability of β-catenin.
293T cells were transiently transfected with a plasmid encoding myc-tagged β-catenin and either pcDNA3, pcDNA3-Siah-l, or pcDNA3-Siah-l ( 97-298 ; amino acids 97- 298 of SEQ ID NO:2). Whole cell lysates were prepared, normalized for total protein content (25 μg per lane) and analyzed by SDS-PAGE/immunoblotting using an anti-Myc tag antibody.
Figure 7 indicates that expression of full- length Siah-1 abolishes, by degradation, the presence of β-catenin within cells, whereas expression of amino acids 97-298 of Siah-1 (SEQ ID NO:2) does not result in β-catenin degradation. Thus, a region within amino acids 1-96 of SEQ ID N0:2 (Siah-lα), which contains the N- terminal "Ring" domain, is required for protein degradation.
15. Demonstration of SIP-mediated degradation of a target protein , TRAF6.
An invention SIP-based method for targeted degradation of proteins was applied to the degradation TRAF proteins. The schematic in Figure 9 shows the strategy employed for targeted degradation of specific TRAF-family proteins. A chimeric protein is expressed from the plasmid pcDNA3 in which SIP-L (SEQ ID NO: 3) is fused with bacterial thioredoxin containing various TRAF- binding peptides displayed on the surface of thioredoxin, as described by Brent and colleagues (Colas, et al . Nature, 380: 548, 1996; Cohen, et al . Proc. Natl. Acad. Sci., 95: 14272, 1998; Geyer, et al. Proc. Natl. Acad. Sci., 96: 8562, 1999; Fabbrizio, et al. Oncogene, 18: 4357, 1999) . The TRAF-binding peptide binds to a member of the TRAF-family, and targets the TRAF-protein for ubiquitination and subsequent proteosome-dependent degradation because the SIP-region of the chimeric protein recruits ubiquitin-conjugating enzymes (E2s) to the protein complex.
Isolation of target-protein binding domain peptides that selectively bind TRAF2 and TRAF6.
A peptide aptamer library was screened by the yeast two-hybrid method to identify peptides that bind to either TRAF2 or TRAF6 using the methods described in Leo, et al. J Biol Chem, 274:22414, 1999. TRAFs are a family of signal transducing proteins involved in cytokine receptor signaling inside cells. The sequences of the resulting TRAF-binding peptides are set forth in (Tables 3 and 4) .
TABLE 3
Selected Traf 2 Aptamer Clones
Clones (SEO ID NO: ) SLxCIxLR motif
219 (15) SESPGALRSGSLRCISLRIC
230 (16) VCRGRIRSGSLRCISLRICR
221 (17) LLRLGCIRLLMLRRGVVFRL
208 (18) VLFLSLRFWGLNIVVMGRLL
215 (19) CRSLGVIVGGTEAAGAPTFI
LS motif
208 (20) VLFLSLRFWGLNIVVMGRLL
213 (21) WLRRGLVGVFFLLSRVMVGI
218 (22) SLGLSVCIGRRAGGGFRGFG
237 (23) RFALSIGVCVVVRVGICLGM
LV motif
209 (24) SAVLVLVYVSAALRGRGFGI
227 (25) HGGGRGALVSVMYLCGFIRL
Non-Consensus motif
231 (26) RGRVIGMWVGLRCRMFLV
TABLE 4
Selected Traf 6 Aptamer Clones
Clones (SEO ID NO: ) WR motif
625 (27) VDWAVYSVVWRYTTT*
631 (28) KTSVILVWRLSLFFCLYRSL*
606 (29) ANRCWRE*
628 (30) EGTLSKRMWRTHN*
640 (31) SWRDMTQSGM*
604 (32) DVPWQRACARQ*
607 (33) LERVARWVL*
602 (34) VADVLVFWGYVF* DVxVF motif
602 (34) VADVLVFWGYVF*
613 (35) GDVGVFPE* Non-Consensus motif
603 (36) PEMMLEGPKYCLxLxE*
609 (37) LLYGALA*
612 (38) GAIKFAHESCE*
616 (39) PMAMD*
632 (40) QEEEM*
639 (41) ISVVHGIGSDSD*
Termination codon
SIP-fusion Chimeric protein construction:
An invention SIP-fusion chimeric construct is generated by combining the open reading frame (ORF) of SIPL, followed immediately by restriction enzyme sites allowing for subcloning of desired target-protein-binding domains (e.g. peptides or protein domains). These SIP- fusions are then transfected into mammalian cells to eliminate by protein degradation specific target proteins which bind the subcloned peptides/protein domains by recruiting them into the ubiquitin conjugating complex.
The parent SIP-vector (SIPpcDNA3.1 ) cassette was engineered as follows:
Oligonucleotides corresponding to the 5' and 3' end of SIPL were used in PCR to amplify the entire ORF of SIPL (SEQ ID NO: 3) . The forward primer contains a Hind III restriction site linker (5'-
GATCAAGCTTATGGCTTCAGAAGAGCTACAG; (SEQ ID NO: 46) restriction site is underlined) followed immediately by the SIPL (SEQ ID NO: 3) start codon; the reverse primer contains an EcoRI restriction site and mutations in the stop codon allowing for translational readthrough (5'- GATCGAATTCtccAAATTCCGTGTCTCCTTTGGCTTG; (SEQ ID NO: 47) mutated stop codon is in lowercase) . The generated PCR product was then agarose gel-purified and digested with Hind III and EcoRI restriction enzymes (New England
BioLabs; Beverly, MA) . The product was again gel-purified before ligating into Hind III/ EcoRI digested pcDNA3.1 expression vector (Invitrogen; Carlsbad, CA) with T4-DNA ligase (New England BioLabs). This construct was termed SIPpcDNA3.1.
For the construction of SIP-thioredoxin (Trx) peptide-aptamer fusions, clones from a peptide-aptamer library screened against Traf6 (see Table 4) were amplified by PCR with the following primers: Forward: 5' -CCTCTGAATTCCATATGAGCGATAAAATTATTCACC (SEQ ID NO: 48) EcoRI underlined; Reverse: 5'-
GATCCTCGAGTAGATGGCCAGCTAGGCCAGGTTA (SEQ ID NO: 49) Xho I underlined.
The resulting PCR products (~350-370bp) contain the ORF of thioredoxin (Trx) with the selected peptide aptamers inserted into its active-loop. The products were then digested with EcoRI and Xho I before ligating into the Eco-RI/X oI-digested SIPpcDNA3.1 cassette using T4-DNA ligase. Final clone constructs were numbered and were confirmed by sequencing before using in transfection studies .
Tranfection:
HEK293T cells were transiently transfected by a lipofectamine method with various amounts (1 vs 4 μg) of pcDNA3 plasmids encoding either SIP-TR fusion protein lacking a TRAF6-binding peptide ("SIP") or SIP-TR fusion protein displaying one of the peptides shown in Table 4 above (set forth in Figure 10 as S603, S604, S606) . In some cases, the proteosome inhbitor MG132 (10 μM) was added to cultures to prevent protein turnover. SIP* in Figure 10 corresponds to the control expression product of parental construct SIP pcDNA3.1
To determine the efficacy of the SIP:TRAF- binding peptide chimeric proteins, levels of TRAF6 protein were then measured two days later by immunoblotting using a anti-TRAF6-specific antiserum
(Santa Cruz Biotech, Inc.) in experiments where HEK293T cell lysates were normalized for total protein content (25 μg per lane) . The cell lysates were analyzed by SDS- PAGE/immunoblotting using an enhanced chemiluminescence detection method, as described previously (Leo, et al. J Biol Chem, 274: 22414, 1999). The results shown in the left panel of Figure 10 show that SIP-TR fusion proteins displaying TRAF6-binding peptides (S603, S604, and S613) induce a reduction in TRAF6 protein levels, with the S603 peptide representing the most potent of these.
To determine the specificity of the SIP:TRAF- binding peptide chimeric proteins, the same immunoblots were reprobed with an antiserum against SIP to demonstrate equivalent levels of production of SIP-TR fusion proteins, or with antibodies specific for TRAF2 to reveal selective degradation of TRAF6 but not TRAF2. The results shown in the right panel of Figure 10 show that addition of a proteosome inhibitor, MG132, prevents the reductions in TRAF6. Note also that TRAF2 protein is not degraded, demonstrating the specificity of the targeting approach .
While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.
Summary of Sequences
SEQ ID NO:l is a cDNA (and the deduced amino acid sequence) encoding a Siah lα of the present invention.
SEQ ID NO: 2 is the deduced amino acid sequence of a Siah lα protein of the present invention encoded by SEQ ID NO:l.
SEQ ID NO: 3 is a cDNA (and the deduced amino acid sequence) encoding a human SIP-L polypeptide of the present invention.
SEQ ID NO: 4 is the deduced amino acid sequence of a human SIP-L protein of the present invention encoded by SEQ ID NO:3.
SEQ ID NO: 5 is a cDNA (and the deduced amino acid sequence) encoding a human SIP-S polypeptide of the present invention.
SEQ ID NO: 6 is the deduced amino acid sequence of a human SIP-S protein of the present invention encoded by SEQ ID NO:5.
SEQ ID NO: 7 is a cDNA (and the deduced amino acid sequence) encoding a human SAF-lα polypeptide of the present invention.
SEQ ID NO: 8 is the deduced amino acid sequence of a SAF-lα protein of the present invention encoded by SEQ ID NO:7.
SEQ ID NO: 9 is a cDNA (and the deduced amino acid sequence) encoding a human SAF-lβ polypeptide of the present invention.
SEQ ID NO: 10 is the deduced amino acid sequence of a SAF-lβ protein encoded by SEQ ID NO: 9.
SEQ ID NO: 11 is a cDNA (and the deduced amino acid sequence) encoding a human SAF-2 polypeptide of the present invention.
SEQ ID NO: 12 is the deduced amino acid sequence of a SAF-2 protein encoded by SEQ ID NO: 11.
SEQ ID NO: 13 is a cDNA (and the deduced amino acid sequence) encoding a human SAD polypeptide of the present invention.
SEQ ID NO: 14 is the deduced amino acid sequence of a SAD protein encoded by SEQ ID NO: 13.
Claims
1. Isolated nucleic acid encoding a Siah-Mediated- Degradation-Protein (SMDP) and/or SFC-Complex-Protein (SCP), or a functional fragment thereof.
2. Isolated nucleic acid encoding Siah-Mediated- Degradation-Protein (SMDP) and/or SFC-Complex-Protein (SCP) , or functional fragments thereof, selected from: (a) DNA encoding the amino acid sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14, or (b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active SMDP and/or SCP, or (c) DNA degenerate with respect to either (a) or (b) above, wherein said DNA encodes biologically active SMDP and/or SCP.
3. A nucleic acid according to claim 2, wherein said nucleic acid hybridizes under high stringency conditions to the SMDP and/or SCP coding portion of any of SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
4. A nucleic acid according to claim 2, wherein the nucleotide sequence of said nucleic acid is substantially the same as set forth in any of SEQ ID N0:1, 3, 5, 7, 9, 11 and 13.
5. A nucleic acid according to claim 2, wherein the nucleotide sequence of said nucleic acid is the same as that set forth in any of SEQ ID N0s:l, 3, 5, 7, 9, 11 and 13.
6. A nucleic acid according to claim 2, wherein said nucleic acid is cDNA.
7. A vector containing the nucleic acid of claim 2.
8. Recombinant cells containing the nucleic acid of claim 2.
9. An oligonucleotide comprising at least 15 nucleotides capable of specifically hybridizing with a the nucleotide sequence set forth in any of SEQ ID NOs:l,
3, 5, 7, 9, 11 and 13.
10. An oligonucleotide according to claim 9, wherein said oligonucleotide is labeled with a detectable marker .
11. An antisense-nucleic acid capable of specifically binding to mRNA encoded by said nucleic acid according to claim 2.
12. A kit for detecting the presence of the SMDP and/or SCP cDNA sequence comprising at least one oligonucleotide according to claim 10.
13. An isolated Siah-Mediated-Degradation-Protein (SMDP) and/or SFC-Complex-Protein (SCP) characterized by having ability to bind to at least one SMDP and/or SCP.
14. A SMDP and/or SCP according to claim 13, wherein the amino acid sequence of said protein comprises substantially the same sequence as any of SEQ ID Nos: 2,
4, 6, 8, 10, 12 or 14.
15. A SMDP and/or SCP according to claim 14 comprising the same amino acid sequence as set forth in any of SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14.
16. A SMDP and/or SCP according to claim 13, wherein said protein is encoded by a nucleotide sequence comprising substantially the same nucleotide sequence as set forth in SEQ ID Nos:l, 3, 5, 7, 9, 11 or 13.
17. A SMDP and/or SCP according to claim 16, wherein said protein is encoded by a nucleotide sequence comprising the same sequence as set forth in SEQ ID Nos:l, 3, 5, 7, 9, 11 or 13.
18. A method for expression of a SMDP and/or SCP protein, said method comprising culturing cells of claim
8 under conditions suitable for expression of said SMDP and/or SCP.
19. An isolated anti-SMDP and/or SCP antibody having specific reactivity with a SMDP and/or SCP according to claim 13.
20. Antibody according to claim 19, wherein said antibody is a monoclonal antibody.
21. An antibody according to claim 20, wherein said antibody is a polyclonal antibody.
22. A composition comprising an amount of the antisense-nucleic acid according to claim 11 effective to inhibit expression of a human SMDP and/or SCP and an acceptable hydrophobic carrier capable of passing through a cell membrane.
23. A transgenic nonhuman mammal expressing exogenous nucleic acid encoding a SMDP and/or SCP.
24. A transgenic nonhuman mammal according to claim 23, wherein said nucleic acid encoding said SMDP and/or SCP has been mutated, and wherein the SMDP and/or SCP so expressed is not native SMDP and/or SCP.
25. A transgenic nonhuman mammal according to claim 23, wherein the transgenic nonhuman mammal is a mouse.
26. A method for identifying nucleic acids encoding a mammalian SMDP and/or SCP, said method comprising: contacting a sample containing nucleic acids with an oligonucleotide according to claim 9, wherein said contacting is effected under high stringency hybridization conditions, and identifying compounds which hybridize thereto.
27. A method for detecting the presence of a human SMDP and/or SCP in a sample, said method comprising contacting a test sample with an antibody according to claim 19, detecting the presence of an antibody-SMDP and/or SCP complex, and therefor detecting the presence of a human SMDP and/or SCP in said test sample.
28. Single strand DNA primers for amplification of SMDP and/or SCP nucleic acid, wherein said primers comprise a nucleic acid sequence derived from the nucleic acid sequences set forth as SEQ ID NOs:l, 3, 5, 7, 9, 11 and 13.
29. A method for modulating the activity of an oncogenic protein, comprising contacting said oncogenic proteins with a substantially pure SMDP and/or SCP, or a oncogenic protein-binding fragment thereof.
30. A bioassay for evaluating whether test compounds are capable of acting as agonists or antagonists for SMDP and/or SCP proteins, or functional fragments thereof, wherein said bioassay comprises:
(a) culturing cells containing:
DNA which expresses an SMDP and/or SCP or functional fragments thereof, wherein said culturing is carried out in the presence of at least one compound whose ability to modulate an activity of an SMDP and/or SCP is sought to be determined, wherein said activity is selected from a protein : protein binding activity or a protein degradation activity and thereafter
(b) monitoring said cells for either an increase or decrease in the level of protein: protein binding or protein degradation.
31. A method for modulating an activity mediated by a SMDP and/or SCP protein, said method comprising: contacting said SMDP and/or SCP protein with an effective, modulating amount of said agonist or antagonist identified by claim 30.
32. The method of claim 31, wherein said modulated activity is the binding of Siah-1 to APC.
33. A method for modulating the protein degradation activity mediated by an SMDP and/or SCP protein, said method comprising: contacting said SMDP and/or SCP protein with an effective, modulating amount of said agonist or antagonist identified by claim 30.
34. A therapeutic composition comprising a compound selected from an SMDP and/or SCP, or functional fragment thereof, a SMDP and/or SCP modulating compound identified according to claim 30, or an anti-SMDP and/or SCP antibody; and a pharmaceutically acceptable carrier.
35. A method of treating a pathology characterized by abnormal cell proliferation or abnormal inflammation, said method comprising administering an effective amount of the composition according to claim 34.
36. A method of inducing the degradation of the function of a target protein, said method comprising: expressing, in a cell, a chimeric protein comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein-degradation family.
37. A method of determining the function of a target protein, said method comprising: expressing, in a first cell, a chimeric protein comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein- degradation family; and comparing the phenotype of said first cell to the phenotype of a control second cell.
38. A method of identifying a nucleic acid molecule encoding a protein that modulates a cellular phenotype, said method comprising:
(a) expressing, in a cell, a chimeric nucleic acid comprising a member of a nucleic acid library fused to nucleic acid encoding a protein degradation binding domain of a protein member of the ubiquitin- mediated protein degradation family; and (b) screening said cells for a modulation of said phenotype.
39. The method of claim 38, wherein the phenotype is selected from the group consisting of: cell proliferation, cell survival, cell death, cell secretion, and cell migration.
40. A chimeric nucleic acid identified according to claim 38.
41. A nucleic acid library comprising a plurality of chimeric nucleic acids, wherein each chimeric nucleic acid comprises an SMDP and/or SCP or functional fragment thereof .
42. The method of claim 38 wherein said nucleic acid encoding a protein degradation binding domain is selected from the group consisting of Sia-lα, SIP-L, SIP- S, SAF-1, SAF-2, and SAD, or functional fragments thereof .
43. A method for treating a disease by degrading the function of a target protein comprising: introducing, into a cell, a chimeric protein comprising a target-protein binding domain operatively linked to a protein-degradation binding domain of a protein member of the ubiquitin-mediated protein- degradation family.
44. A chimeric protein comprising the SMDP and/or SCP of claim 13.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US33051799A | 1999-06-11 | 1999-06-11 | |
US330517 | 1999-06-11 | ||
PCT/US2000/015873 WO2000077207A2 (en) | 1999-06-11 | 2000-06-09 | Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1185652A2 true EP1185652A2 (en) | 2002-03-13 |
Family
ID=23290119
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00942718A Withdrawn EP1185652A2 (en) | 1999-06-11 | 2000-06-09 | Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1185652A2 (en) |
JP (1) | JP2003502042A (en) |
AU (1) | AU5730300A (en) |
WO (1) | WO2000077207A2 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001066126A1 (en) | 2000-03-07 | 2001-09-13 | Smithkline Beecham Corporation | Sialoadhesin factor-2 antibodies |
WO2002002819A1 (en) * | 2000-06-30 | 2002-01-10 | University Of Utah Research Foundation | Method of screening for chemotherapeutic treatments for cancer |
AUPR290801A0 (en) * | 2001-02-06 | 2001-03-01 | Peter Maccallum Cancer Institute, The | Substrate binding domain |
GB0309116D0 (en) * | 2003-04-22 | 2003-05-28 | Univ London | Target for cancer therapy and drug discovery |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2240449A1 (en) * | 1995-12-20 | 1997-06-26 | Fondation Jean Dausset-Ceph | Nucleotide sequences, proteins, drugs and diagnostic agents for treating cancer |
IL120485A0 (en) * | 1997-03-19 | 1997-07-13 | Yeda Res & Dev | Modulators of the function of receptors of the TNF/NGF receptor family and other proteins |
JP2002512517A (en) * | 1997-03-25 | 2002-04-23 | ジェネティックス・インスチチュート・インコーポレーテッド | Secreted proteins and polynucleotides encoding them |
US6573094B1 (en) * | 1997-10-16 | 2003-06-03 | Baylor College Of Medicine | F-box genes and proteins |
DE19811193A1 (en) * | 1998-03-10 | 1999-09-16 | Metagen Gesellschaft Fuer Genomforschung Mbh | New nucleic acid expressed at high level in prostatic tumor tissue and encoded polypeptides, useful for treating cancer and screening for therapeutic agents |
JP2002506627A (en) * | 1998-03-19 | 2002-03-05 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | 95 human secreted proteins |
-
2000
- 2000-06-09 JP JP2001503651A patent/JP2003502042A/en not_active Withdrawn
- 2000-06-09 EP EP00942718A patent/EP1185652A2/en not_active Withdrawn
- 2000-06-09 WO PCT/US2000/015873 patent/WO2000077207A2/en active Application Filing
- 2000-06-09 AU AU57303/00A patent/AU5730300A/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO0077207A2 * |
Also Published As
Publication number | Publication date |
---|---|
JP2003502042A (en) | 2003-01-21 |
WO2000077207A3 (en) | 2001-05-10 |
WO2000077207A2 (en) | 2000-12-21 |
AU5730300A (en) | 2001-01-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7982002B2 (en) | Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto | |
US20060199195A1 (en) | Nucleic acid encoding Schwannomin-Binding-Proteins and products related thereto | |
US5942490A (en) | Cell death regulators | |
US7927832B2 (en) | Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto | |
US7626001B2 (en) | Card domain containing polypeptides, encoding nucleic acids, and methods of use | |
US20080109917A1 (en) | Card-domain containing polypeptides, encoding nucleic acids, and methods of use | |
US20110318362A1 (en) | Novel card proteins involved in cell death regulation | |
US7666607B1 (en) | TRAF family proteins | |
WO2001044282A2 (en) | Bcl-g polypeptides, encoding nucleic acids and methods of use | |
US6638734B1 (en) | Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto | |
WO2000077207A2 (en) | Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto | |
WO2001032696A2 (en) | Traf family proteins | |
US20110027260A1 (en) | Bcl-g polypeptides, encoding nucleic acids and methods of use | |
US7041783B2 (en) | Survivin-binding proteins, encoding nucleic acids, and methods of use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20020102 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
17Q | First examination report despatched |
Effective date: 20050615 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20090804 |