+

WO1997037027A1 - Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines - Google Patents

Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines Download PDF

Info

Publication number
WO1997037027A1
WO1997037027A1 PCT/US1997/005296 US9705296W WO9737027A1 WO 1997037027 A1 WO1997037027 A1 WO 1997037027A1 US 9705296 W US9705296 W US 9705296W WO 9737027 A1 WO9737027 A1 WO 9737027A1
Authority
WO
WIPO (PCT)
Prior art keywords
ubc
seq
cyclin
set forth
amino acid
Prior art date
Application number
PCT/US1997/005296
Other languages
English (en)
Inventor
Joan V. Rudderman
Avram Hershko
Marc W. Kirschner
Fiona Townsley
Alexander Aristarkov
Esther Eytan
Hongtao Yu
Original Assignee
President And Fellows Of Harvard College
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Priority to BR9710415A priority Critical patent/BR9710415A/pt
Priority to NZ332482A priority patent/NZ332482A/xx
Priority to EP97917760A priority patent/EP0900276A1/fr
Priority to AU26006/97A priority patent/AU732547B2/en
Priority to JP53553297A priority patent/JP2002503949A/ja
Publication of WO1997037027A1 publication Critical patent/WO1997037027A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to cell cycle regulation. More specifically, this invention relates to novel ubiquitin carrier polypeptides (Ubc's) involved in the ubiquitination and degradation of cyclins, and to nucleic acid encoding these proteins. This invention also relates to inhibitors of such Ubc's and to kits for and methods of screening for compounds which inhibit the ubiquitination, and hence the destruction, of cyclins.
  • Ubc's novel ubiquitin carrier polypeptides
  • Mitotic entry and exit in most organisms is controlled by the synthesis and destruction of cyclin B, a positive regulatory subunit of the protein kinase Cdc2, the catalytic component of mitosis promoting factor (MPF) (Norbury et al. (1992) Ann.. Rev. Biochem. 61:441-470; Murray (1995) Cell 81:149-152). Cyclins are marked for destruction by the covalent addition of ubiquitin at the end of mitosis (Glotzer et al. (1991) Nature 349:132-138; Hershko et al. (1991) J. Biol. Chem. 266:16376-16379; Hershko et al. (1994) J. Biol. Chem.
  • Cyclosome-associated E3-C catalyzes cyclin ubiquitination using a specialized ubiquitin conjugating enzyme or carrier protein (E2); also called Ubc, originally identified in clam as E2-C (Hershko et al. (1994) ⁇ . Biol. Chem. 269:4940-4946).
  • E2 ubiquitin conjugating enzyme or carrier protein
  • E2's Structurally, all known E2's share a conserved domain of approximately 16 kD. This domain contains the cysteine (Cys) residue required for the formation of ubiquitin-E2 thiol ester. Certain E2 enzymes contain additional typical domains. Based on their structure, the E2 enzymes can be divided into three groups (Jentsch (1992) Ann.. Rev. Genet. 26:179-207)). Class I E2's consist almost exclusively of the conserved domain. Class II proteins have C-terminal extensions that may contribute to substrate recognition or to cellular localization. For example, yeast Ubc2 and Ubc3 have a highly acidic C-terminal domain that promote interaction with basic substrates such as histones (Jentsch (1992) Ann.. Rev. Genet.
  • Ubc's have different cellular functions. Two closely related Ubc's, Ubc4 and Ubc5, appear responsible for ubiquitin-dependent degradation of most short-lived and abnormal proteins (Jentsch (1992) Ann. Rev. Genetics 26:179- 207). Ubc2 (RAD6) is required for several functions, including DNA repair, sporulation (Sung et al. (1988) Genes & Dev. 2:1476-1485) and N-end rule degradation (Dohmen et al (1991) Proc. Natl. Acad. Sci. USA 88:7351-7355).
  • Ubc9 is required for cell cycle progression in late G2 or early M; both CLB5, an S phase cyclin, and CLB2, an M phase cyclin, are stable in Ubc9 mutants, suggesting that Ubc9 may be responsible for cyclin ubiquitination (Seufert et al (1995) Nature 373:78- 81) .
  • E2-C a clam Ubc was determined to be one of the components of the clam oocyte system responsible for the specific ubiquitination of cyclin (Hershko et al. (1994) J. Biol. Chem. 269:4940-
  • the invention provides an isolated and purified, non-xenopal, ubiquitin carrier polypeptide (Ubc) involved in the ubiquitination of cyclin A and/or B.
  • Ubc ubiquitin carrier polypeptide
  • isolated and purified refers to polypeptides which are substantially free of contaminating cellular or other associated components, including, but not limited to proteinaceous, carbohydrate, or lipid impurities. This term is also meant to encompass molecules which are homogeneous by one or more purity or homogeneity characteristics used by those with skill in the art. For example, an isolated and purified Ubc will show constant and reproducible characteristics within standard experimental deviations for parameters such as molecular weight, chromatographic migration, amino acid composition, HPLC profile, biological activity, and other such parameters. The term is not meant to exclude artificial and synthetic mixtures of the Ubc with other compounds.
  • non-xenopal refers to Ubc's which are not derived from frog cells or encoded by frog nucleic acid.
  • the term "involved in” means “which takes part in” and is meant to encompass the role played or function that a Ubc has during ubiquitination of cyclin A and/or B. This role includes an enzymatic activity required for transporting ubiquitin to cyclin A or B.
  • the "Ubc-specific N-terminal extension" referred to in this aspect of the invention is used to describe a unique (outside of the conserved domain) amino acid sequence of at least 5, or preferably, at least 10, more preferably, at least 15, more preferably at least 20, more preferably, at least 25, most preferably between 30-32 amino acid residues having sequence homology to the unique amino acid sequence(s) found in clam E2-C, human UbcHlO, and frog Ubc-x.
  • the Ubc is recombinantly produced.
  • fragments of the Ubc are provided which are enzymatically active and demonstrate the same or substantially similar ubiquitin carrier polypeptide function as the full length Ubc.
  • a "fragment" of a molecule such as E2-C, UbcHlO, or inhibitors thereof, refers to any smaller polypeptide subset of that molecule.
  • the Ubc is a clam or human Ubc.
  • the Ubc has an amino acid sequence with about 61-100%, more preferably, about 75-100%, and most preferably with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:l or 3
  • homology is meant sequence identity or similarity.
  • the Ubc has the amino acid sequence set forth as SEQ ID NO:l or 3.
  • the polypeptide is encoded by a nucleic acid hybridizable with a second nucleic acid set forth as SEQ ID NO:2 or 4.
  • the polypeptide is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid having SEQ ID NO:2 or 4.
  • Stringent hybridization conditions are known by those with skill in the art (see, e.g., Ausebel et al.
  • the N-terminal extension has about 61-100% homology, preferably 75-100%, and more preferably has about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9 or 10.
  • the N- terminal extension has the amino acid sequence set forth as SEQ ID NO:9 or 10.
  • the N-terminal extension is encoded by a nucleic acid hybridizable, preferably under stringent conditions, with a second nucleic acid encoding the amino acid sequence set forth as SEQ ID NO:9 or 10.
  • the invention provides a nucleic acid encoding the Ubc's, and fragments thereof, of the invention as described above.
  • the nucleic acid is a cDNA, and in particular embodiments, the cDNA has the nucleotide sequence set forth as SEQ ID NO:2 or 4.
  • the nucleic acid of the invention encodes a human Ubc having an amino acid sequence with about 61-100% homology, preferably about 74-100%, and more preferably, with about 94- 100% homology with the amino acid sequence set forth as SEQ ID NO:l.
  • nucleic acid of the invention encodes a clam Ubc having an amino acid sequence with about 61-100%, preferably with about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:3.
  • nucleic acid hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:2 or 4.
  • the present invention provides a selective inhibitor of Ubc polypeptide function.
  • Ubc function is meant to encompass the enzymatic transfer of ubiquitin from El to E2 and from E2 to a protein target, e.g., cyclin A or B.
  • Ubc function also refers to the association of E2 and E3.
  • inhibitors of Ubc function is meant to include agents that block the transfer of ubiquitin from El to E2 and agents that block the transfer of ubiquitin from E2 to a protein target, e.g., cyclin A or B.
  • inhibitortors of Ubc function is also meant to include agents that block association between E2 and E3.
  • the agent prevent cyclin ubiquitination. It is preferred that the agent be a selective inhibitor of Ubc function, more preferably wherein the Ubc is selected from the group consisting of clam E2- C, human UbcHlO, and an enzymatically active fragment thereof.
  • Suitable assays for measuring Ubc function according to the present invention include those which allow measurement of the formation of E-2-ubiquitin thiol ester, measurement of the formation of ubiquitin- or multi-ubiquitin-conjugates of a cyclin, or measurement of cyclin degradation. Assays that allow measurement of cell cycle progression may also be used according to the present invention.
  • agents screened in the above-described assay methods can be, but are not limited to peptides, polypeptides, antibodies, carbohydrates, vitamin derivatives, or other pharmaceutical agents. These agents can be selected and screened 1) at random, 2) by a rational selection, or 3) by design using, for example, protein or ligand modeling techniques.
  • agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to or block the activity of the Ubc.
  • agents may be rationally selected or designed.
  • an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration of the above-described Ubc or known ligand.
  • the present invention further relates to selective inhibitors of Ubc function or cyclin ubiquitination identified by the above-described screening and assay methods, which can include peptides, polypeptides, antibodies, carbohydrates, vitamin derivatives, or other pharmaceutical agents.
  • the inhibitor is a dominant negative mutant of a ubiquitin carrier protein, or a fragment thereof capable of inhibiting Ubc function.
  • a mutant of UbcHlO containing a cysteine serine mutation at residue 114 is as a dominant negative mutant.
  • the dominant negative mutant overcomes the activity of wild type UbcHlO and inhibits cyclin ubiquitination and degradation.
  • a "selective inhibitor” is a compound which preferentially interferes with Ubc function. Preferably, the selective inhibitor reduces the enzymatic function of the novel Ubc's of the invention.
  • the inhibitor is a dominant negative mutant.
  • a "dominant negative mutant” is a polypeptide variant of a wild type Ubc with which it competes or interferes for its ubiquitin carrier function. Dominant negative mutants of the novel Ubc's of the invention inhibit cell cycle progression, blocking both the destruction of mitotic cyclins A and B, and the onset of anaphase. In some embodiments, the dominant negative mutant is recombinantly produced.
  • dominant negative mutants of the invention have a serine residue in place of a cysteine residue in a conserved region of the polypeptide.
  • the dominant negative mutant of the invention comprises a serine residue at position 114 substituted for a cysteine residue.
  • the dominant negative mutant inhibits the function of a human or clam Ubc.
  • the dominant negative mutant has an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, about 94-100%, homology to the amino acid sequence set forth as SEQ ID NO:5 or 7 in some embodiments.
  • the dominant negative mutant is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6 or 8.
  • the invention provides a fragment of the dominant negative mutant which inhibits Ubc function.
  • the invention also provides a nucleic acid encoding the dominant negative mutant described herein.
  • the nucleic acid is hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6 or 8.
  • the nucleic acid may be a cDNA which, in some embodiments, has the nucleotide sequence set forth as SEQ ID NO:6 or 8.
  • the nucleic acid of the invention encodes a dominant negative mutant having an amino acid sequence with about 61-100% homology, preferably about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:5 or 7.
  • Kits useful for the ubiquitination and degradation of a cyclin are also provided by the invention. These kits include (a) a ubiquitin- human ubiquitin carrier polypeptide complex, wherein the ubiquitin carrier polypeptide is an isolated and purified, non-xenopal, Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension.
  • the Ubc is clam E2-C, human UbcHlO, or an enzymatically active fragment of clam E2-C or UbcHlO; and (b) a ubiquitin ligase (E3) .
  • the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises human UbcHlO having an amino acid sequence set forth as SEQ ID NO:l.
  • the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier polypeptide complex comprises clam E2-C having an amino acid sequence set forth as SEQ ID NO:3.
  • the ubiquitin-ubiquitin carrier protein complex comprises a Ubc having an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1 or 3.
  • the Ubc in the complex has the amino acid sequence set forth as SEQ ID NO:1 or 3.
  • the Ubc in the complex is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid set forth as SEQ ID NO:2 or 4.
  • the Ubc has an N-terminal extension which has about 61-100%, preferably about 75-100%, and more preferably about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9 or 10.
  • the Ubc in the complex has an N-terminal extension with an amino acid sequence set forth as SEQ ID NO:9 or 10.
  • the invention provides other kits useful for the ubiquitination and degradation of a cyclin including ubiquitin, a ubiquitin activating enzyme (El) , ATP, a ubiquitin carrier protein selected from the group consisting of clam E2-C, human UbcHlO, and an enzymatically active fragment thereof, and a ubiquitin ligase (E3) .
  • the cyclin to be degraded is cyclin A or cyclin B and the ubiquitin-ubiquitin carrier protein complex comprises human UbcHlO having an amino acid sequence set forth as SEQ ID NO:l.
  • the cyclin to be degraded is cyclin A and/or cyclin B and the ubiquitin-ubiquitin carrier protein complex comprises clam E2-C having an amino acid sequence set forth as SEQ ID NO:3.
  • the invention also provides a method of ubiquitinating a cyclin and/or targeting a cyclin for degradation, comprising the step of contacting the cyclin with a ubiquitin-ubiquitin carrier protein complex, the ubiquitin carrier polypeptide being an isolated and purified non-xenopal Ubc involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension; and a ubiquitin ligase (E3) .
  • the Ubc is selected from the group consisting of clam E2-C, human UbcHlO, and an enzymatically active fragment thereof.
  • the ubiquitin-ubiquitin carrier protein complex comprises a Ubc having an amino acid sequence with about 61-100%, preferably about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:1 or 3.
  • the Ubc in the complex has the amino acid sequence set forth as SEQ ID NO:1 or 3.
  • the Ubc in the complex is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid set forth as SEQ ID NO:2 or 4.
  • the Ubc has an N-terminal extension which has about 61- 100% and more preferably, about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9 or 10.
  • the Ubc in the complex has an N-terminal extension with an amino acid sequence set forth as SEQ ID NO:9 or 10.
  • a method of inhibiting Ubc function is also provided by the invention.
  • an inhibitor of a Ubc is administered to the cell in an amount sufficient to inhibit the Ubc function, e.g., by inhibiting the ubiquitination of a cyclin.
  • the inhibitor is a dominant negative mutant according to the invention and as described above.
  • the Ubc is a mutant clam E2-C.
  • the Ubc is a mutant human UbcHlO.
  • the dominant negative mutant is recombinantly produced.
  • the dominant negative mutant of the invention comprises a serine residue at position 114 substituted for a cysteine residue.
  • the dominant negative mutant inhibits the function of a human or clam Ubc.
  • the dominant negative mutant has an amino acid sequence with about 61-100%, more preferably, about 75-100%, and most preferably, about 94-100%, homology to the amino acid sequence set forth as SEQ ID NO:5 or 7 in some embodiments.
  • the dominant negative mutant is encoded by a nucleic acid hybridizable under stringent conditions with a second nucleic acid having the nucleotide sequence set forth as SEQ ID NO:6 or 8.
  • the invention provides a fragment of the dominant negative mutant which inhibits Ubc function.
  • the method of inhibiting Ubc function results in the inhibition of cell proliferation.
  • the present invention further relates to a method of screening for compounds which inhibit
  • an assay for measuring Ubc function, wherein the assay comprises a ubiquitin carrier polypeptide selected from the group consisting of a non-xenopal ubiquitin carrier polypeptide involved in the ubiquitination of cyclin a and/or B and having a Ubc-specific N-terminal extension and an enzymatically active fragment thereof.
  • the assay is performed in the presence and absence of a compound to-be-tested.
  • the amount of change in Ubc function measured in the presence of the compound as compared to Ubc function measured in the absence of the compound is then determined, a reduction of Ubc function measured in the presence of the compound indicating that the compound is an inhibitor of Ubc function.
  • the ubiquitin carrier polypeptide is selected from the group consisting of clam E2-C, human UbcHlO, and an enzymatically active fragment thereof. More preferably, the ubiquitin carrier polypeptide is isolated and purified.
  • the invention provides a method of screening for compounds which inhibit the ubiquitination of cyclins.
  • ubiquitin a ubiquitin activating enzyme (El)
  • ATP an isolated and purified, non-xenopal
  • Ubc involved in the ubiquitination of cyclin A and/or B
  • Ubc-specific N-terminal extension a ubiquitin ligase (E3)
  • Cdc2 Cdc2
  • a cyclin are incubated in the presence and in the absence of a compound to be tested.
  • cyclin-ubiquitin-Cdc2 complex refers to ubiquitin covalently bound to cyclin B complexed to Cdc2.
  • the Ubc is selected from the group consisting of clam E2-C, human UbcHlO, or an enzymatically active portion thereof.
  • the ubiquitin carrier polypeptide is isolated and purified.
  • the human UbcHlO or clam E2-C has an amino acid sequence with about 61- 100%, preferably about 75-100%, and more preferably, with about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:l or 3, respectively.
  • UbcHlO and E2-C have the amino acid sequences set forth as SEQ ID NO:l and 3, respectively.
  • UbcHlO and E2-C are encoded by a nucleic acid hybridizable under stringent conditions, with a second nucleic acid set forth as SEQ ID NO:2 and 4, respectively.
  • UbcHlO has an N-terminal extension which has about 61-100%, preferably about 75-100%, and more preferably about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:9
  • E2-C has an N-terminal extension which has about 61-100%, preferably about 75-100%, and more preferably, about 94-100% homology with the amino acid sequence set forth as SEQ ID NO:10.
  • the N-terminal extension of UbcHlO and E2-C has the amino acid sequence set forth as SEQ ID NO:9 and 10, respectively.
  • antibodies specific for E2-C and for UbcHlO are also provided by the invention.
  • antisense oligonucleotides specific for E2-C or UbcHlO nucleic acids are also provided by the invention.
  • the invention provides therapeutic formulations comprising a selective inhibitor of ubiquitin carrier protein function in an amount sufficient to inhibit the ubiquitination of a cyclin, and a pharmaceutically acceptable carrier.
  • the inhibitor comprises a dominant negative mutant of a ubiquitin carrier protein, or a fragment thereof capable of inhibiting Ubc function.
  • the dominant negative mutant has a serine residue at position 114 substituted for a cysteine residue.
  • the dominant negative mutant has an amino acid sequence which is at least about 90-95% homologous with the amino acid sequence set forth as SEQ ID NO:5 or 7.
  • the dominant negative mutant is encoded by a nucleic acid which is hybridizable under stringent conditions with the nucleic acid having a nucleotide sequence set forth as SEQ ID NO:6 or 8.
  • FIG. 1 is a diagrammatic representation of the ubiquitin-proteasome pathway for protein degradation
  • FIG. 2 is a diagrammatic representation of the ubiquitin-proteasome pathway for cyclin B degradation
  • FIG. 3 is a diagrammatic representation of the involvement of various cyclins during the cell cycle
  • FIG. 4 is a schematic representation of the nucleotide sequence of clam E2-C cDNA (SEQ ID NO: ) and its deduced amino acid sequence (SEQ ID NO:3), wherein the four peptides obtained by microsequencing are underlined;
  • FIG. 5A is a nucleotide sequence of human UbcHlO cDNA (SEQ ID NO:2) and its deduced amino acid sequence (SEQ ID NO:l);
  • FIG. 5B is a schematic representation of the comparison of clam E2-C protein with human UbcHlO protein
  • FIG. 6 is a representation of a polyacrylamide gel illustrating the covalent affinity purification of clam oocyte E2-C, wherein lane 1 contains the peak of E2-C from the Mono S column El, and MgATP; lane 2 contains the peak of E2-C and MgATP; lane 3 contains El and MgATP; and the E2-C activity in these fractions are expressed as the percentage of total E2-C activity applied to the ubiquitin-Sepharose beads;
  • FIG. 7A is a representation of a polyacrylamide gel of filtration fractions of affinity purified E2-C, wherein “Cont.” refers to contamination in the preparation of 125 I-cyclin, “Cyc” refers to free 125 I-cyclin, and molecular mass markers are indicated on the right;
  • FIG. 7B is a representation of a polyacrylamide gel of gel filtration fractions of affinity purified E2-C, wherein “Cont.” refers to contamination in the preparation of 12S I-ubiquitin; "El-Ub, " “E2-C-Ub, “ and “E2-A-Ub” indicate the positions of the corresponding adducts, and molecular mass markers are indicated on the right;
  • FIG. 8 is a representation of a polyacrylamide gel illustrating the thiolester formation between ubiquitin and bacterially expressed E2-C, wherein the samples were either boiled with 5% mercaptoethanol for 5 minutes ("+ME") or were not treated ("-ME") prior to electrophoresis, the numbers on the left indicate the position of molecular mass marker proteins, "El-Ub, " "E2-C-Ub, " “E2A-Ub” indicate the position of the corresponding 125 I-ubiquitin-enzyme adducts, and "*" indicates the position of the fast migrating adduct of E2-C with 125 I-ubiquitin;
  • FIG. 9A is a representation of a polyacrylamide gel showing the activity of different Ubc's in the ligation of 125 I-cyclin to ubiquitin, wherein fraction 1 is a preparation of activated E3-C purified by gel filtration on Superose-6, numbers on the left indicate the position of molecular mass markers, and "Cyc.” indicates the position of free 12S I-cyclin;
  • FIG. 9B is a representation of a polyacrylamide gel showing the ability of different E2-C to ligate 125 I-ubiquitin to proteins, wherein "E2-C-Ub" denotes the position of the autoubiquitination product of E2-C, and the numbers on the right indicate the position of molecular mass marker proteins;
  • FIG. 10 is a graphic representation of the hydrophilicity of clam E2-C
  • FIG. 11 is a diagrammatic representation of various E2-C mutants and their enzymatic activity in cyclin-ubiquitination assays in vitro; including the dominant negative E2-C;
  • FIG. 12A is a graphic representation of the ability of different concentrations of mutant E2-C C(114)S to inhibit 125 I-cyclin ligation to ubiquitin in the presence of wild type E2-C;
  • FIG. 12B is a graphic representation illustrating the ability of mutant E2-C C(114)S to be a competitive inhibitor of cyclin ubiquitination;
  • FIG. 12C is a graphic representation illustrating that the competition between wild type E2-C and dominant negative inhibitor E2-C- C(114)S does not involve the N-terminal region 1- 21 amino acids of E2-C;
  • FIG. 13A is a graphic representation of the ability of human UbcHlO and clam E2-C to stimulate cyclin-ubiquitin ligation,-
  • FIG. 13B is a graphic representation of the ability of recombinant human mutant UbcH10-C(114)S to act as a dominant negative inhibitor of cyclin- dependent ubiquitination;
  • FIG. 13C is a graphic representation of the inhibition of cyclin-ubiquitin ligation by C(114)S mutants of human UbcHlO, wherein recombinant UbcHlO was added at the concentrations indicated in the absence ( o , control) or presence ( o ) of the C(114)S mutant (1 ⁇ M) ;
  • FIG. 13D is a representation of an autoradiogram demonstrating the effects of human and clam Ubc C(114)S mutants on the degradation of clam cyclin B
  • FIG. 13E is a representation of an autoradiogram showing the reversal of the effects of human and clam Ubc C(114)S mutants (shown in FIG. 13D) by wild-type human Ubc, wherein the polypeptides were added at the concentrations indicated;
  • FIG. 14 is a diagrammatic representation of recombinantly expressed clam E2-C and human UbcHlO constructs and their enzymatic activity in cyclin- ubiquitin assays in vitro;
  • FIG. 15A is a diagrammatic representation of the plasmid pUHDl5-l neo used to express UbcHlO wild type and mutant genes in mammalian cells in vivo;
  • FIG. 15B is a diagrammatic representation of the plasmid pUHDlO-3 used for tTA-dependent expression of the UbcHlO wild type and mutant genes in mammalian cells in vivo;
  • FIG. 16A is a schematic representation of the nucleotide sequence of human dominant negative mutant UbcHlO C(114)S cDNA (SEQ ID NO:5) and its corresponding amino acid sequence (SEQ ID NO:6);
  • FIG. 16B is a schematic representation of the nucleotide sequence of clam dominant negative mutant E2-C C(114)S cDNA (SEQ ID NO:7) and its corresponding amino acid sequence (SEQ ID NO:8); and FIG. 17 is a representation of an autoradiogram showing enhancement of the destruction of human cyclin A and B by the addition of UbcHlO and showing blockage of that destruction by UbcHlO C(114)S.
  • the addition of ubiquitin to mitotic cyclins occurs only during a brief period near the end of mitosis.
  • complexes of mitotic cyclins with the protein kinase Cdc2 become activated.
  • Mitotic cyclin/Cdc2 complexes then catalyze entry into mitosis.
  • the cyclosome/anaphase promoting complex (APC) becomes activated for a brief period.
  • Active cyclosome/APC catalyses the transfer of ubiquitin from E2-C or UbcHlO to the target cyclin protein.
  • Ubiquitinated cyclin is then recognized and proteolyzed by the proteasome.
  • E2/Ubc and E3 enzyme activities are responsible for recognizing the specific target proteins which are to be ubiquitinated. Genetic and biochemical studies in yeast, humans, and other organisms have identified several different E2/Ubc family members, but none were known to be the E2/Ubc responsible for the ubiquitination of the mitotic cyclins A or B.
  • the present invention is directed to the
  • E2/Ubc's responsible for the ubiquitination of the mitotic cyclins A or B are non- xenopal, ubiquitin carrier polypeptides involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension. They may be be isolated and purified, for example, from natural sources, or they may be biochemically or recombinantly synthesized.
  • the E2-C or UbcHlO polypeptides of this invention may be purified from biological material.
  • clam E2-C can be purified from clam oocytes as described in the Examples below.
  • these proteins may be obtained by expression from recombinant DNA, as described below.
  • DNA sequences coding for E2-C and UbcHlO are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
  • human UbcHlO genomic DNA can be extracted and purified from any human cell or tissue, and clam E2-C DNA can be extracted from clam oocytes or any clam cell or tissue, by means well known in the art (for example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, 1989) .
  • such genomic DNA may be obtained in association with the 5 ' promoter region of the UbcHlO gene sequences and/or with the 3 ' translational termination region.
  • genomic DNA may be obtained in association with DNA sequences which encode the 5 ' nontranslated region of the UbcHlO mRNA and/or with the genetic sequences which encode the 3 ' nontranslated region.
  • a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then the 5 ' and/or 3 ' nontranscribed regions of the native gene, and/or, the 5' and/or 3' nontranslated regions of the mRNA, may be retained and employed for transcriptional and translational regulation.
  • UbcHlO or E2-C mRNA can be isolated from any cell which expresses UbcHlO or E2-C, and used to produce cDNA by means v/ell known in the art (for example, see Sambrook et al., supra) .
  • the mRNA preparation used will be enriched in mRNA coding for Ubc, either naturally, by isolation from cells which produce large amounts of Ubc, or in vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both.
  • Ubc mRNA may be obtained from mammalian tissue and cells, or cell lines derived therefrom.
  • suitable DNA preparations are randomly sheared or enzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) library.
  • a DNA sequence encoding Ubc may be inserted into a vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra, and are well known in the art.
  • Ubc clones may be screened and the Ubc clones identified by any means which specifically selects the Ubc DNA such as, for example: 1) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein; or 2) by hybridization-selected translational analysis in which native mRNA hybridizes to the clone in question, is translated in vitro, and the translation products are further characterized; or, 3) if the cloned DNA sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated Ubc product produced by the host containing the clone.
  • any means which specifically selects the Ubc DNA such as, for example: 1) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein; or 2) by hybridization-selected translational analysis in which native mRNA hybridizes to the clone in question, is translated in vitro, and the translation products are further characterized; or,
  • a cDNA library can be prepared in Mgtll vector and screened using Ubc-specific antibodies (Huynh et al., "Constructing and Screening cDNA Libraries in MgtlO and Mgtll, " in DNA Cloning: A Practical Approach, Vol. I, Glover, D. M. (Ed.), IRL Press, Washington, D.C. pp. 49-78 (1985)) .
  • Oligonucleotide probes specific for Ubc which can be used to identify clones to this protein can be designed from knowledge of the amino acid sequence of the corresponding Ubc. For example, the sequence of such oligonucleotide probes can be based upon the amino acid sequence of peptide fragment.
  • oligonucleotides can be identified, each of which would be capable of encoding Ubc polypeptides.
  • the oligonucleotide, or set of oligonucleotides, containing a sequence most likely capable of identifying the Ubc gene sequence fragments is used to identify the sequence of a complementary set of oligonucleotides which is capable of hybridizing to the sequence, or set of sequences.
  • An oligonucleotide sequence containing such a complementary sequence can be employed as a probe to identify and isolate Ubc gene sequence (for example, see Sambrook et al. , supra) .
  • the suitable oligonucleotide, or set of oligonucleotides may be synthesized by means well known in the art (for example, see Sambrook et al., supra) . Techniques of nucleic acid hybridization and clone identification are disclosed by Sambrook et al. , supr . Those members of the above-described gene library which are found to be capable of such hybridization are then analyzed to determine the extent and nature of the Ubc encoding sequences which they contain. In order to further characterize the Ubc- encoding DNA sequences, and in order to produce the recombinant protein, the DNA sequences are expressed. These sequences are capable of expressing a polypeptide if they contain expression control sequences "operably linked" to the nucleotide sequence which encodes the protein. The control sequences contain transcriptional regulatory information and such sequences.
  • Recombinant prokaryotic host cells can express the Ubc polypeptide. Alternatively, recombinant Ubc can be expressed by such cells as a fusion protein.
  • Useful prokaryotic host cells are is E. coli and B. subtillus .
  • the present invention also encompasses the expression of Ubc in eucaryotic cells, and especially mammalian, insect, and yeast cells.
  • Preferred eucaryotic hosts are mammalian cells which provide post- translational modifications to recombinant Ubc including folding and/or phosphorylation.
  • Useful mammalian host cells include Chinese hamster ovary cells, rat pituitary cells, HeLa cells, and rat hepatoma cells.
  • the Ubc protein-encoding sequence and an operably linked promotor may be introduced into eucaryotic cells either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule.
  • the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • clam E2-C was first partially purified by cation exchange chromatography and then subjected to covalent affinity chromatography on ubiquitin- Sepharose.
  • E2's bind to immobilized ubiquitin by thiolester linkage; ubiquitin-bound enzymes can then be eluted with high concentrations of DTT or by raising the pH (Hershko et al. (1983) J. Biol. Chem. 258:8206-8214) .
  • ubiquitin-Sepharose beads were mixed with three kinds of mixtures. The complete mixture contained the peak of E2-C from the Mono S column, El purified from human erythrocytes and MgATP; the two others were controls, lacking either El or the source of E2-C.
  • the protein composition of the pH 9 eluates of these treatments was examined by SDS- polyacrylamide gel electrophoresis and silver staining. As shown in FIG. 6 (upper panel), the pH 9 eluate of the complete reaction mix (lane 1) contained several protein bands. These include an approximately 105 kD protein identified as El (which also binds to the ubiquitin column and is eluted at pH 9 (Ciechanover et al. (1982) J. Biol.
  • E2-C and E2-A are present in fractions 21-23 of the Mono S column used for affinity purification, so both are expected to bind to the ubiquitin beads under the conditions employed.
  • both proteins are absent from the pH 9 eluate of the control lacking El (FIG. 6, lane 2), indicating that both are E2's.
  • they were also absent in the control containing El, but lacking the source of E2-C (FIG.
  • the pH 9 eluate of the preparation purified on ubiquitin Sepharose was subjected to gel filtration on Superose-12.
  • the activity of E2-C (determined by the cyclin-ubiquitin ligation assay) eluted mainly in fractions 33-34 (FIG. 7A) , coincident with the 27 kD ubiquitin-thiolester band (FIG. 7B) . It was partially separated from the 18 kD E2-A-ubiquitin thiolester that eluted at a lower size during gel filtration (FIG. 7B) .
  • the anomalously migrating 27 kD adduct is the ubiquitin thiolester of the 21 kD E2-C protein.
  • the 21 kD E2-C was chosen for microsequencing.
  • Material originating from 100 ml of clam oocyte extract was processed by the Mono S and ubiquitin-Sepharose steps described above and the 21 kD band was digested with trypsin. Sequences of four tryptic peptides were obtained, as shown in FIG. 4 (underlined sequences) .
  • a degenerate oligonucleotide primer corresponding to the second peptide was designed, and then with a Mgt22 primer to screen a clam ovary cDNA library using PCR, as described in the Examples below.
  • a partial length cDNA clone containing sequences corresponding to three of the four peptides was obtained and used to select several candidate clones encoding full length E2-C.
  • the first peptide sequence was identified in the N-terminal region (FIG. 4) .
  • the same coding sequence was found in other independently isolated cDNA clones .
  • the sequence obtained contains only one long open reading frame which initiates at the first methionine codon (FIG. 4) .
  • the size of the presumed translation product is 20 kD, in good agreement with the size of purified E2-C observed by SDS polyacrylamide gel electrophoresis.
  • the encoded protein is clearly an E2, as demonstrated by its extensive alignment with other cloned Ubc's.
  • Clam E2-C does not appear to be a Ubc2 homolog, since Ubc2 's from several different species show much higher conserved sequence similarities within the family (-70%) .
  • the clam sequence contains a novel 30-32 amino acid N-terminal extension not found in any other Ubc besides the frog and human.
  • Other unique regions include the adjacent sequence beginning at position 42 (TLLMSGD) , and a short C- terminal extension (KYKTAQSDK) .
  • PT7-7 the protein was induced, and a crude lysate was assayed in two different ways.
  • ubiquitin- thiolesters of a mix of natural E2-C and E2-A were separated on the same gel.
  • the recombinant protein formed an adduct with ubiquitin.
  • the electrophoretic mobility of the ubiquitin thiolester of the recombinant E2 was identical to that of the 27 kD adduct with native E2-C (FIG. 8, lanes 2 and 3) .
  • FIG. 8, lane 3 a minor species of a more rapidly migrating ubiquitin adduct of the recombinant protein (labelled *) was observed (FIG. 8, lane 3) .
  • This may be a cleavage product or, more likely, an incompletely denatured conformer of a E2-C/ubiquitin thiolester.
  • Multiple bands of thiolesters have been observed previously with some other E2's, and have been attributed to the incomplete denaturing conditions necessary for the preservation of the labile thiolester linkage during electrophoresis (Haas et al. (1988) J. Biol. Chem. 263:13258-13267; Sullivan et al (1991) ⁇ . Biol. Chem.
  • the ability of the recombinant E2 to promote cyclin-ubiquitin ligation was tested in the presence of activated, partially purified E3- C/cyclosome complexes. As shown in FIGS. 9A and 9B, the recombinant E2 efficiently promoted this process, as compared to the action of natural E2- C.
  • the recombinant E2 stimulated cyclin ubiquitination at remarkable low concentrations: half-maximal activation was obtained with 0.05 ⁇ M recombinant E2. Since it has been reported that Ubc4 can support cyclin B ubiquitination in a Xenopus egg extract (King et al.
  • the recombinant UbcH5 protein had to be added at a 20-fold higher molar concentration than the recombinant clam E2-C.
  • Ubc4 supports cyclin ubiquitination much less efficiently than the new Ubc protein cloned here.
  • the protein substrates for ubiquitin ligation are presumably clam oocyte proteins present in the partially purified preparation of the non-specific E3.
  • UbcH5 strongly stimulated the ligation of 15 I-ubiquitin to high molecular weight conjugates in the presence of non-specific E3 from clam oocytes.
  • This finding indicates that the human Ubc4 homolog can act with an appropriate clam E3.
  • the formation of the high molecular weight conjugates required the addition of both UbcH5 and the non-specific E3.
  • the recombinant clam E2 had no significant influence on the formation of ubiquitin-protein conjugates by the non-specific E3 (FIG. 9B, lane 3) .
  • the only stable adduct formed in the presence of the recombinant clam E2-C is a 30 kD auto- ubiquitination product.
  • the formation of this product does not require the presence of the non ⁇ specific E3.
  • the amount of the product is higher in FIGS. 9A and 9B than in FIGS. 4A and 4B due to the longer incubation time. Its apparent 30 kD size in the denaturing conditions of gel electrophoresis is close to that expected for recombinant E2-ubiquitin adduct (29.5 kD) .
  • a similar auto-ubiquitination product with native E2-C is seen with a mix of natural E2-C and E2-A (FIGS. 9A and 9B, lane 2) .
  • the cDNA clone described here encodes the cyclin- selective E2-C that is responsible for the cell cycle stage-selective ubiquitination and destruction of the mitotic cyclins A and B.
  • E2 E2
  • M phase mitosis
  • E2-C is a novel Ubc and reveals the presence of several unique sequence domains, including an N-terminal 32 amino acid extension not seen in any other Ubc family, a 7 amino acid region immediately downstream of this extension, and a short C-terminal extension.
  • Clam E2-C has 65% sequence homology with the corresponding frog Ubc-x.
  • E2-C protein exhibits specificities similar to those seen with natural E2-C.
  • the recombinant protein was shown to be responsible for the highly selective ubiquitination of mitotic cyclins during the cell cycle.
  • recombinant Ubc4 protein does not function well in cyclin ubiquitination assays, even when provided at 20-fold higher levels than E2-C.
  • a clam E2-C protein-containing a "PKA site" insertion between Ser2 and Gly3 in the N-terminus has been constructed, confirmed by sequencing and expressed as protein (see FIG. 14) .
  • the PKA site is a 5 amino acid region (arg-arg-ala-ser-val) which, when present in a recombinant protein, can be phosphorylated in vitro by protein kinase A (PKA), yielding a 32 P-labelled protein that can be used as a reagent to detect proteins that interact with E2-C.
  • PKA site is a 5 amino acid region (arg-arg-ala-ser-val) which, when present in a recombinant protein, can be phosphorylated in vitro by protein kinase A (PKA), yielding a 32 P-labelled protein that can be used as a reagent to detect proteins that interact with E2-C.
  • PKA protein kinase A
  • This Ubc has 80% sequence similarity with frog Ubc-x and 61% sequence homology with clam E2-C.
  • UbcHlO and HsRad ⁇ A the most closely related human Ubc family member, have 41% sequence homology.
  • HsRad6A has an active sequence variant with 94% sequence homology with WT.
  • variants of clam and human Ubc's having from about 61-100%, preferably about 75-100%, and most preferably, about 94-100% sequence homology with their wild-type counterparts are expected to have ubiquitmating function.
  • Clam E2-C and human UbcHlO also share an N- terminal 32 amino acid extension which is also conserved in frog Ubc-x.
  • the amino acid sequences of these N-terminal extensions derived from their respective cDNAs are set forth below in Table 3.
  • the present invention is also directed to enzymatically active fragments of the novel Ubc's of the invention which can be obtained, for example, by chemical synthesis, or by proteolytic cleavage of purified Ubc protein.
  • Such enzymatically active fragments retain their Ubc function.
  • the methodology described in U.S. Patent No. 5,384,255 can be performed to prepare such fragments.
  • Representative proteases useful in the preparation of fragments include trypsin, chymotrypsin, papain, and Staphylococcus aureaus V8 protease. Conditions for proteolytic cleavage of a protein are well known to those of skill in the art.
  • tryptic digestion may be performed by: 1) dissolving the Ubc at a concentration between 2 and 10 mg/ml in 0.2 M ammonium bicarbonate; 2) adding a freshly prepared solution of trypsin (DCC-treated bovine trypsin) at a concentration of 1 mg/ml in water, giving a final trypsin/Ubc enzyme ratio of 1:50; and 3) mixing the sample and incubating at 37°C for 48 hours (Gooderham, in Methods in Molecular Biology, Vol.
  • a proteolytic digest of Ubc can be fractionated by a variety of techniques.
  • a proteolytic digest of Ubc can be fractionated by SDS-PAGE, and the fragments can be recovered from the gel by electroelution ⁇ Current
  • E2-C, UbcHlO, and fragments thereof can be tested for the ability to promote the formation of ubiquitin-protein conjugates in the presence of El and E3 (Example 2A) , and for the ability to form 125 I-ubiquitin- thiol esters (Example 2B) .
  • isolated and purified Ubc can be used to generate Ubc-specific antibodies, which in turn, can be used to detect Ubc in a biological sample, and to inhibit Ubc enzyme activity in both commercial and clinical settings.
  • Such purified Ubc can be isolated from tissues, or can be obtained using recombinant DNA technology, as described below.
  • Purified Ubc can also be used to identify an E3 protein ligase in a biological sample. For example, E3 can be identified by determining whether the biological sample promotes the formation of ubiquitin-protein conjugates in the presence of El and purified Ubc (see Example 2A) .
  • purified Ubc may be used to construct an Ubc affinity column, using well known techniques (see Affinity Chromatography: A Practical Approach, Dean et al. (Eds.) IRL Press, Washington, D.C. (1985)).
  • Ubc affinity column may be used, for example, to bind E3 enzyme from a biological sample, as described in U.S. Patent No. 5,384,255.
  • Enzymatically active fragments of E2-C or UbcHlO can also be used to generate antibodies which are specific for particular domains of Ubc enzyme.
  • Ubc fragments can be used to inhibit Ubc-dependent ubiquitination of proteins.
  • the techniques described above can be used to prepare Ubc fragments which contain the domain required for forming ubiquitin- Ubc thiol ester, but lack the domain that recognizes E3 enzyme. The introduction of such a Ubc fragment into a cell would inhibit ubiquitination by decreasing the transfer of ubiquitin to E3.
  • Such Ubc fragments can be introduced into cultured cells, or can be administered therapeutically, as described for the commercial and therapeutic uses of Ubc antibodies, respectively.
  • purified Ubc can be used to screen for inhibitors of the Ubc enzyme activity in vitro .
  • the ability of a substance to inhibit the ubiquitin carrier activity of a Ubc can be determined by observing the inhibition of Ubc-dependent formation of 125 1-ubiquitin thiol esters in the presence of the test substance, by observing the inhibition of Ubc-dependent formation of ubiquitin-protein conjugates in the presence of El, E3 , and the test substance, as described in the exemplification, below.
  • cultured cells can be used for the rapid screening of an inhibitor of Ubc.
  • rapid screening may be performed by introducing the test substance into cultured cells, wherein the cultured cells are known to degrade at least one identified protein via the Ubc-dependent pathway.
  • An inhibition of Ubc dependent degradation is shown by the accumulation of the identified protein within the cultured cells.
  • FIGS. 16A and 16B show the cDNA and corresponding amino acid sequences of two dominant negative mutants in human and clam, respectively.
  • C(114)S changing the catalytic cysteine to serine at position 114
  • Ubc an inhibitor of wild-type E2-C or UbcHlO function, as judged by the in vitro cyclin-ubiquitination assay described herein and shown in FIGS. 12A-12B.
  • 125 I-cyclin B was incubated with native E2-C and different concentrations of E2-C C(114)S mutant protein and E3C/cyclosome preparation and assayed for cyclin ubiquitination as described below in the Examples.
  • FIG. 13A The representative results shown in FIG. 13A demonstrate that wild-type UbcHlO catalyses cyclin ubiquitination in vitro, while UbcHlO C(114)S acts as a dominant negative in vitro (FIG. 13B) .
  • FIG. 12C Representative results are shown in FIG. 12C. These results demonstrate that UbcHlO C(114)S blocks the ubiquitin-mediated destruction of cyclin B. These dominant negative mutant proteins are valuable reagents for interfering with the destruction of mitotic cyclins, other cyclins, and other cell cycle proteins whose level is regulated by ubiquitin-mediated proteolysis.
  • C(114)S mutant The ability of the C(114)S mutant to affect cell cycle progression in living cells was tested in two different system : the somatic cell cycle of mammalian tissue culture cells and the rapid embryonic cell cycle of frog eggs.
  • COS cells were transfected with AUl-tagged wild type or mutant UbcHlO and 48 hours later the distribution of transfectants in interphase versus mitosis was monitored by microscopy.
  • Injected embryos were collected at mid-late blastula stages, fixed, stained with Hoechst 33342 and squashed to examine chromosome spreads.
  • chromosomes in M phase showed the following distribution: 40% in pre-metaphase, 45% in metaphase and 15% in anaphase.
  • Embryos injected with the mutant E2-C showed a striking reduction in the % of pre-metaphase arrays coupled with a corresponding accumulation of metaphase figures (data not shown) .
  • UbcHlO C(114)S was also found to block the ubiquitin-mediated destruction of human cyclins A and B. Using the method of Brandeis and Hunt (EMBO J. (1996) 15:5280-5289) to prepare a human cell free system, it was determined that extra, recombinant wild type UbcHlO accelerates the proteolysis of 35S-methionine-labelled cyclin A and B, while the addition of the dominant negative mutant UbcHlO C(114)S was found to block the proteolysis of cyclin A and B (FIG. 17) .
  • E2-C- ⁇ 1-30 a deletion mutant missing its first N-terminal 30 amino acids has low activity in vitro, indicating that these 30 amino acids in the N-terminal extension are important for enzymatic activity.
  • E2-C- ⁇ 169-177 a deletion mutant missing residues 169-177, has medium-low activity in vitro, indicating that the novel, short C-terminal extension is important.
  • Inhibitors of Ubc function preferably selective inhibitors, such as dominant negative mutants, can be used commercially, e.g., to block cell cycle progression, both in vi tro and in vivo .
  • inhibitors of Ubc function are useful to synchronize or provide non-proliferating cultured cells. These inhibitors are also useful for 7 P
  • Ubc's of the invention can be used in therapeutic formulations, e.g., for the treatment of disorders resulting in the reduction of Ubc's.
  • Ubc inhibitors of the invention, as well as enzymatically active fragments thereof can also be used in therapeutic formulations.
  • the inhibitors have utility as anti-proliferative agents for use in treating diseases, such as psoriasis, autoimmune diseases, and cancer, in which cell proliferation contributes to the pathology of the disease.
  • Anti-proliferative agents can be used to block clonal expansion of B- and T-cells that specifically recognize autoantigens, a hallmark of autoimmune disease.
  • Autoimmune diseases that are treatable with inhibitors of Ubc function or cyclin ubiquitination include, without limitation, arthritis, multiple sclerosis, lupus, and inflammatory bowel disease.
  • the inhibitors of the present invention block tumor cell proliferation and have broad utility for the treatment of cancer. Examples of cancers treatable with these agents include, without limitation, cancers of the breast, prostate, colon or lung.
  • Therapeutic formulations of the invention comprise a selective inhibitor of Ubc function, or an active fragment thereof, in an amount sufficient to inhibit the ubiquitination of a cyclin, and a pharmaceutically acceptable carrier.
  • such formulations may contain a Ubc, or an active fragment thereof, in an amount sufficient to ubiquitinate a cyclin, and a pharmaceutically acceptable carrier.
  • a "pharmaceutically or physiologically acceptable carrier” includes any and all solvents (including but limited to lactose) , dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • solvents including but limited to lactose
  • dispersion media including but limited to lactose
  • coatings including but limited to lactose
  • antibacterial and antifungal agents include antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • isotonic and absorption delaying agents and the like The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical formulation or method that is sufficient to show a meaningful subject or patient benefit, i.e., a reduction in cell proliferation or tumor growth, or in the expression of proteins which cause or characterize the disease.
  • a meaningful subject or patient benefit i.e., a reduction in cell proliferation or tumor growth, or in the expression of proteins which cause or characterize the disease.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • compositions of the invention can be carried out in a variety of conventional ways, such as by oral ingestion, enteral, rectal, or transdermal administration, inhalation, sublingual administration, or cutaneous, subcutaneous, intramuscular, intraocular, intraperitoneal, or intravenous injection, or any other route of administration known in the art for administrating therapeutic agents.
  • the therapeutic formulation will preferably include a physiologically acceptable carrier, such as an inert diluent or an assimilable edible carrier with which the composition is administered.
  • a physiologically acceptable carrier such as an inert diluent or an assimilable edible carrier with which the composition is administered.
  • suitable formulations that include pharmaceutically acceptable excipients for introducing compounds to the bloodstream by other than injection routes can be found in Remington's Pharmaceutical Sciences (18th ed. ) (Genarro, ed. (1990) Mack Publishing Co., Easton, PA).
  • the Ubc, Ubc inhibitor, or fragments thereof, and other ingredients may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet.
  • the therapeutic compositions may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the therapeutic composition When the therapeutic composition is administered orally, it may be mixed with other food forms and pharmaceutically acceptable flavor enhancers.
  • Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated such as those described in U.S. Patent Nos. 4,704,295, 4,556,552, 4,309,404, and 4,309,406.
  • Ubc, Ubc inhibitor, or fragments thereof, of the invention When a therapeutically effective amount of a Ubc, Ubc inhibitor, or fragments thereof, of the invention is administered by injection, the Ubc, Ubc inhibitor, or fragments thereof will preferably be in the form of a pyrogen-free, parenterally-acceptable, aqueous solution.
  • a preferred pharmaceutical composition for injection should also contain an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and fungi.
  • the carrier can be a solvent or dispersion medium.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents.
  • Prolonged absorption of the injectable therapeutic agents can be brought about by the use of the compositions of agents delaying absorption.
  • Sterile injectable solutions are prepared by incorporating the Ubc, Ubc inhibitor, or fragments of the Ubc or Ubc inhibitor in the required amount in the appropriate solvent, followed by filtered sterilization.
  • the pharmaceutical formulation can be administered in bolus, continuous, or intermittent dosages, or in a combination of continuous and intermittent dosages, as determined by the physician and the degree and/or stage of illness of the patient.
  • the duration of therapy using the pharmaceutical composition of the present invention will vary, depending on the unique characteristics of the Ubc, Ubc inhibitor, or fragments thereof, and the particular therapeutic effect to be achieved, the limitations inherent in the art of preparing such a therapeutic formulation for the treatment of humans, the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. Ultimately the attending physician will decide on the appropriate duration of therapy using the pharmaceutical composition of the present invention.
  • compositions of the invention also include nucleic acids encoding Ubc 's and Ubc inhibitors of the invention in the form of vectors for administration to animal, and more preferably, to mammals such as humans. These vectors may be administered via gene therapy techniques such as those known in the art (see, e.g., Miller (1992) Nature 357:455) .
  • the present invention also is directed to the production and use of Ubc-specific antibodies.
  • antibodies refers to both polyclonal antibodies which are heterogeneous populations, and to monoclonal antibodies which are substantially homogeneous populations. Polyclonal antibodies are derived from the sera of animals immunized with an antigen preparation.
  • Monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler and Milstein (1975) Nature 256:495-497; and Harlow et al., supra) .
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fv, Fab, and F(ab') 2 , which are capable of binding antigen.
  • fragments such as, for example, Fv, Fab, and F(ab') 2 , which are capable of binding antigen.
  • Fab, F(ab') 2 , FV, and other fragment of the antibodies useful in the present invention may be used for the detection and quantitation of Ubc in a biological sample.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments) .
  • enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments) .
  • such fragments can be produced by recombinant means.
  • Antibodies directed against a Ubc can be used to screen biological samples for the presence of Ubc.
  • the antibodies (or fragments thereof) useful in the present invention are particularly suited for use in in vitro immunoassays to detect the presence of Ubc in a biological sample.
  • the antibodies (or antibody fragments) may be utilized in liquid phase or, bound to a solid-phase carrier, as described below.
  • One screening method for determining whether a biological sample contains Ubc utilizes immunoassays employing radioimmunoassay (RIA) or enzyme-linked immunosorbant assay (ELISA) methodologies .
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbant assay
  • antibodies specific for Ubc, or a functional derivative may be detectably labelled with any appropriate marker, for example, a radioisotope, an enzyme, a fluorescent label, a paramagnetic label, or a free radical.
  • the presence of Ubc, such as the novel Ubc's of the invention in a biological sample can be detected by treating the biological sample with nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labelled Ubc- specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support may then be detected by conventional means.
  • solid phase support is intended any support capable of binding antigen or antibodies.
  • Well-known supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labelling the Ubc-specific antibodies or antibody fragments, it is possible to detect Ubc through the use of radioimmune assays.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter, or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: 3 H, 125 I, 131 I, 35 S, "C, and preferably 125 I.
  • fluorescent labelling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.
  • the Ubc-specific antibody also can be detectably labelled by coupling it to a chemiluminescent compound.
  • chemiluminescent labelling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the Ubc-specific antibody of the present invention.
  • Ubiquitin-dependent proteolysis mediates the degradation of abnormal proteins (for example, see Ciechanover et al. (1984) Cell 37:57-66; Seufert et al. (1990) EMBO ⁇ . 9:543-550). Therefore, inhibition of ubiquitin-dependent proteolysis should enhance the yield of recombinant proteins which are "abnormal" to eucaryotic recombinant host cells.
  • the Ubc antibodies of the present invention can be introduced into cultured recombinant host cells which produce recombinant proteins in order to inhibit Ubc-mediated protein degradation.
  • liposomes can be used to administer Ubc antibodies to the cultured cells.
  • cationic lipids can be used to facilitate the transport of Ubc antibodies to the cultured recombinant host cells (for example, see W091/17424; WO91/16024).
  • the antibodies to Ubc's of the present invention can be used to decrease the inappropriately enhanced degradation of "normal" proteins, which occurs in certain pathological conditions.
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, and previous medical history. Generally it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient) , although a lower or higher dosage may also be administered.
  • Ubc antibodies, or fragments thereof may be administered to patients in a pharmaceutically acceptable form intravenously, intramuscularly, subcutaneously, enterally, or parenterally.
  • the administration may be by continuous infusion, or by single or multiple boluses.
  • the antibody of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby Ubc antibodies, or fragments thereof, are combined in a mixture with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences (16th Edition, Osol,
  • Control release preparations can be achieved through the use of polymers to complex or adsorb Ubc antibody, or Ubc antibody fragment.
  • Controlled delivery can be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) , by the concentration of such macromolecules, as well as by methods of incorporation.
  • appropriate macromolecules for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate
  • Ubc antibody or fragment thereof, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • Ubc antibodies or fragments can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl- cellulose or gelatine microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems (for example, liposomes, cationic lipids, albumin microspheres, microemul ⁇ ions, nanoparticles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, cationic lipids, albumin microspheres, microemul ⁇ ions, nanoparticles, and nanocapsules
  • E2-C- or UbcHlO-specific nucleic acid sequences can be used to generate antisense oligonucleotides specific for E2-C and UbcHlO.
  • the synthesis of such oligonucleotides is well known in the art (see, e.g., Protocols for Oligonucleotides and Analogs (Agrawal, ed. ) Meth.
  • Extracts of M-phase oocytes of the clam Spisula s ⁇ lidissima were prepared and fractionated on DEAE- cellulose, as described by Hershko et al. (J. Biol. Chem. (1994) 269:4940-4946) .
  • Fraction 1 the fraction not adsorbed to the resin
  • the resulting high-speed supernatant contains E2-C
  • Fraction IA a subtraction containing active E3-C, was prepared by salt extraction and ammonium sulfate fractionation, as described by Sudakin et al. (Mol. Biol. Cell. (1995) 6:185-198) .
  • Samples of 1 ml were collected at a flow rate of 1 ml/minute into tubes containing 0.5 mg of carrier ovalbumin. Column fractions were concentrated by centrifuge ultrafiltration with Centricon-10 concentrators (Amicon, Beverly, MA) . Salt was removed with a 20-fold dilution of Buffer B, followed by another ultrafiltration to a final volume of 100 ⁇ l.
  • E2-C and E2-A are tentatively identified as E2-C and E2-A by comparison with our previous results (Hershko et al. (1994) ⁇ . Biol. Chem. 269:4940-4946) .
  • E2-A is a low molecular weight E2 coinciding with non ⁇ specific ubiquitination activity in clam oocytes . Also as observed previously, the amount of E2-C was much less than that of E2-A. Other E2 activities eluted at higher salt concentrations, well separated from the region of E2-C activity. This separation was important for the subsequent purification of E2-C, since a major E2 eluting at fraction 28 had size similar to that of E2-C.
  • ubiquitin-Sepharose beads (approximately 20 mg of ubiquitin/ml of swollen gel) were prepared as described by Hershko et al. ⁇ . Biol. Chem. (1983) 258:8206-8214) .
  • One ml of ubiquitin-Sepharose beads were washed twice with 10 volumes of a solution consisting of Buffer A (20 mM Tris-HCI, pH 7.2, 5 mM MgCl 2 , 2 mM ATP, 0.1 mM DTT and 0.2 mg/ml of ovalbumin) .
  • the beads were mixed with an equal volume of Buffer A containing 3 nmol El, and were rotated at room temperature for 10 min. Subsequently, 300 ⁇ l of partially purified E2-C preparation following the MonoS step were added, and rotation was continued at 18°C for another 20 minutes. The beads were spun down (500 rpm, 3 min.) and the supernatant fraction ("flowthrough") was collected for the estimation of the enzyme not bound to Ub-Sepharose.
  • the beads were washed twice with 10 ml of a solution consisting of 20 mM Tris-HCI, pH 7.2, 1 M KCl and 0.2 mg/ml ovalbumin, and then three times with 10 ml portions of a solution consisting of 20 mM Tris-HCI, pH 7.2, and 0.3% (w/v) octyl glucoside (Boehringer-Mannheim, Indianapolis, IN) .
  • Enzymes bound to ubiquitin- Sepharose were eluted by mixing the beads with 2 ml of a solution consisting of 50 mM Tris-HCI, pH 9.0, 5 mM DTT and 0.3% octyl glucoside, at room temperature for 5 minutes.
  • the pH 9 eluate was neutralized by the addition of 0.1 M Tris-HCl at pH 7.2.
  • the preparation was concentrated with
  • Centricon-10 micro-concentrators (Amicon, Beverly, MA) .
  • the solution was then changed by a 20-fold dilution in a buffer consisting of 20 mM Tris-HCl, pH 7.2 and 0.1% octyl glucoside, followed by ultrafiltration to a final volume of 300 ⁇ l.
  • Proteins were resolved by SDS-polyacrylamide gel electrophoresis, stained with Coomassie blue, the 21 kD band was excised and subject to trypsin (Promega) by the in-gel digestion procedure (Rosenfeld et al (1992) Anal. Biochem. 203:173-179).
  • E2-C and UbcHlO activity was determined by the cyclin-Ub ligation assay (Hershko et al. (1991) J. Biol. Chem. 269:4940-4946), under conditions where El and E3-C were in excess while E2-C was limiting.
  • the reaction mixture contained in a volume of 10 ⁇ l: 40 mM Tris-HCl, pH 7.6, 5 mM MgCl 2 , 0.5 mM ATP, 10 mM phosphocreatine, 50 ⁇ g/ml creatine phosphokinase, 1 mg/ml rcm-BSA, 50 ⁇ M ubiquitin (Sigma, St. Louis, MO), 1 ⁇ M ubiquitin aldehyde
  • E2-C activity was tested as described by Sudakin et al. (ibid.). Briefly, 10 ⁇ l reactions contained 40 mM Tris-HCl, pH 7.6, 5 mM MgCl 2 , 1 mM DTT, 0.5 mM ATP, 10 mM creatine phosphate, 50 ⁇ g/ml creatine phosphokinase, 1 mg/ml reduced-carboxymethylated bovine serum albumin, 20 ⁇ M ubiquitin, 3 ⁇ M ubiquitin- aldehyde, 1 ⁇ M ubiquitin-aldehyde, 1 ⁇ M okadaic acid, 1 pmol El, 1-2 pmol 125 I-cyclin B(13-91) ( ⁇ 1- 2x10 s cpm) , 10 ⁇ g protein of Fraction IA from extracts of clam oocytes and E2-C as specified. Following incubation at 18° C for 60 minutes, samples were electrophoresed on 12.5% poly
  • Reaction mixtures contained in a volume of 10 ⁇ l:20 mM Hepes-KOH, pH 7.2, 5 mM MgCl 2 , 0.5 mM ATP, 10 mM phosphocreatine, 50 ⁇ g/ml creatine phosphokinase, 0.1 mM DTT, 1 mg/ml rcm- BSA, 5 ⁇ M X25 I-ubiquitin (-5,000 cpm/pmol) (chloramine T procedure, 0.1 ⁇ M El and E2 's as specified.
  • human cyclin A and B Using an adaptation of the method of Brandeis and Hunt (EMBO J. (1996) 155280-5289), human cyclin A and B, a mutant of human cyclin B lacking the destruction box, and human cyclin F were in vi tro transcribed and translated in a rabbit reticulocyte lysate system. 3 ⁇ l of the translation products were mixed with 5 ⁇ l of a
  • a polyA* clam ovary cDNA library cloned in the phage vector Mgt22 (Stratagene, La Jolla, CA) was screened by PCR. In this library, cDNA inserts were tailed at the 5 ' end with Sail and the 3 ' end with Notl.
  • the successful PCR primer pair consisted of a degenerate oligonucleotide primer encoding E2-C peptide 1 (primer PI)
  • a 900 bp reaction product was purified by agarose gel electrophoresis (Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edition , Cold Spring Harbour, New York: Cold Spring Harbour Laboratory, NY) and cloned into the plasmid vector pCRII vector (TA Cloning Kit, InVitrogen, San Diego, CA) using the manufacturer's protocols.
  • the insert DNA was sequenced using pCRII vector primers (T3 and T7), and, subsequently, internal unique sequence primers CE24
  • Membranes were hybridized with the labelled probe in SSC at 65°C, following several high stringency washes, positive plaques were cored and vortexed in SM buffer (100 mM NaCl, 10 mM Mg 2 SO Register .7H 2 0) , 50 mM Tris-HCl, pH 7.5, 0.01% (w/v) gelatin) to release the phage.
  • SM buffer 100 mM NaCl, 10 mM Mg 2 SO Register .7H 2 0
  • Tris-HCl pH 7.5
  • 0.01% gelatin 0.01%
  • PCR reactions were performed using the library vector primers ⁇ gt22al and ⁇ gt22a2. Several plaques yielded inserts of 1.5 kb. This insert was gel purified, cloned into the pCRII vector, and sequenced using primers T7, CE24, and CE24R.
  • PCR product containing the 1.5 kb E2-C insert was diluted 1:1000 and a second PCR was performed with primers CE2Ful
  • Protein expression was monitored by the appearance of a 21 kD protein band in SDS- polyacrylamide gels stained with Coomassie blue. Bacteria were lysed by 3 cycles of freezing (liquid nitrogen) and thawing (25°C) , followed by passage in a syringe fitted with a 20 gauge needle. Insoluble material was removed by centrifugation (20,000 x g for 15 minutes); the supernatant was used as the source of bacterially expressed E2-C. The concentration of recombinant E2-C was estimated by comparison of the intensity of the 21 kD band on Coomassie-stained SDS- polyacrylamide gel with those of known amounts of bovine serum albumin, separated on the same gel .
  • reactions contained 1 x 10 s pfu of the HeLa cDNA library, 2.5 mM MgCl 2 , 0.25 mM dNTP's, 1 X PCR buffer (Perkin Elmer, Norwalk, CT) , 1.25 U AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT) , 200 pmol primer YE2-C4, and 50 pmol primer T7. Reactions were carried out at 94°C for 1 min, 50°C for 1 min and 72°C for 1 min, for 35 cycles with a final 10 min extension at 72°C.
  • reaction produced a ladder of 5 bands from -390-1000 bp.
  • PCR B a second, nested, PCR reaction
  • reactions contained l/200th of PCR A reaction products, 2.5 mM MgCl 2 , 0.25 mM dNTP's, 1 X PCR buffer (Perkin Elmer, Norwalk, CT) , 1.25 U AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT) , 200 pmol primer YE2-C4, and 200 pmol primer YE2-C2. Reactions were carried out at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, for 35 cycles with a final 10 min extension at 72°C.
  • PCR B produced a PCR product of 258 bp which was cloned directly into the plasmid vector pCRTMII using the TA cloning kit (InVitrogen, San Diego, CA) and following the manufacturer's protocols.
  • the insert DNA was sequenced using the Sequenase 7-deaza-dGTP Sequencing Kit (United States Biochemical, Cleveland, OH) with the vector primers T7 and SP6 5 '-ATTTAGGTGACACTATA-3 '
  • Membranes were hybridized with the labelled probe in hybridization buffer (6X SSC, 20 mM NaH 2 P0 4 , 0.4% SDS, 5X Denhardt' s reagent (Maniatis et al. (1982) Molecular Cloning , p. 448 for 14 hours at 65°C.
  • the filters were washed twice in 2X SSC, 0.1% SDS for 10 min at room temperature, then once in IX SSC, 0.1% SDS for 1 hour at 53°C, and once in 0.1X SSC, 0.1% SDS for 1 hour at 53°C.
  • the membranes were then exposed to x-ray film (Kodak, Rochester, NY) for 72 hours with an intensifying screen and labelled plaques were identified by autoradiography.
  • plaques yielded inserts of about 700 bp and 15 plaques yielded inserts of about 1000 bp.
  • Six of the plaques that yielded inserts of about 1000 bp were selected for in vivo excision of the
  • Bluescript phagemid containing the cloned insert, from the Lambda ZAP vector (Stratagene, La Jolla, CA) using the manufacturer's protocols. Each of these plaques were independent isolates from the primary screen.
  • SEQ ID NO:28 antisense direction
  • the sequences were aligned using the DNA Star Multiple Sequence Alignment program (DNASTAR, Inc., Madison, WI) .
  • Non-transformed cells can be synchronized by deprivation of essential growth factors (see below for method) ; this causes them to enter a quiescent state (GO) and when growth factors are restored to the medium they will traverse the cell cycle in partial synchrony (Resnitzky et al. (1994) Mol. Cell Biol. 14: 1669-1679) .
  • HeLa cells can be synchronized at the Gl/S phase boundary by using a double thymidine block. Thymidine is added to cultures of cells in exponential growth phase to a final concentration of 2 mM and the cells are incubated for 24 hours.
  • the cells are then harvested by centrifugation, rinsed in thymidine-free complete media and incubated for a further 12 hours. Thymidine is added again to the culture medium and the cells are incubated for a further 24 hours. At the conclusion of this incubation, typically >90% of the cell population is synchronized at Gl/S (Brown et al. (1994) J. Cell Biol. 125:1303-1312) .
  • HeLa cells can also be synchronized in early Gl by Lovastatin treatment or mitotic shake off. Semi-confluent cells are incubated in medium containing 20 mM Lovastatin (Merck, Sharp and
  • Total RNA is prepared from synchronized cells at various time points after release from starvation, Lovastatin treatment, or thymidine treatment, using guanidine isothiocyanate as described by Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual , 2nd Edition, Cold Spring Harbour, New York: Cold Spring Harbour Laboratory, NY) .
  • the RNA is resolved by electrophoresis in a formaldehyde agarose gel and transferred onto Hybond-N membrane (Amersham, Chicago, IL) .
  • the UbcHlO cDNA is labelled with a 32 P-dCTP using the T7 QuickPrime kit (Pharmacia, Piscataway, NJ) following the manufacturer's protocols.
  • the membrane is incubated with the labelled cDNA probe and washed according to the manufacturer's protocols (Amersham, Chicago, IL) . It is then exposed to x-ray film (Kodak, Rochester, NY) with an intensifying screen to identify any signal(s) by autoradiography. The intensity of staining in each lane is quantitated to determine if there are differences in the levels of UbcHlO mRNA across the cell cycle. A probe derived from the acting gene is used as a loading control to check the total amount of mRNA in each lane. A mouse acting cDNA clone is labelled using the T7 QuickPrime kit as described above.
  • UbcHlO RNA levels are expected to vary across the cell cycle, making potential therapies involving incubation of cells with membrane- permeable antisense oligonucleotides feasible.
  • UbcHlO protein To monitor the cell cycle profile of UbcHlO protein, antibodies against recombinant UbcHlO protein are generated. Polyclonal antibodies are isolated and purified from sera of animals immunized with an antigen preparation which is comprised of purified UbcHlO and an adjuvant such as Freund's adjuvant (Syntex Research, Palo Alto, CA) (Harlow et al. (1988) Antibodies. A Laboratory Manual, Cold Spring Harbor, New York. : Cold Spring Harbor Laboratory) . The cells are synchronized as described above, and total protein extracts are prepared from the cells at various time points after release from starvation, Lovastatin treatment, or thymidine treatment.
  • an adjuvant such as Freund's adjuvant
  • the cells are washed with phosphate-buffered saline (PBS; 170 mM NaCl, 3 mM KCl, 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 ) and scraped off the plates.
  • PBS phosphate-buffered saline
  • the cells are harvested by centrifugation and mixed with twice the pellet volume of a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.2% Nonidet P-40, 1 mg/ml leupeptin (Sigma Chemical Company, St.
  • the suspension is incubated at 4°C for 45 min, and cell debris is removed by centrifugation in a microfuge for 30 min at 4°C.
  • the protein concentration of the cell lysates is measured using a Bio-Rad protein assay system (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard.
  • Cell extracts are adjusted to the same protein concentration in sodium dodecyl sulphate (SDS)-sample buffer (80 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% ⁇ - mercaptoethanol, 0.025 mg/ml Bromophenol blue) and are resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold
  • Proteins besides A- and B-type cyclins which are degraded during progression through mitosis may be ubiquitinated using E2-C/UbcHl0.
  • E2-C/UbcHl0 proteins besides A- and B-type cyclins which are degraded during progression through mitosis
  • proteins include CENP-E, CENP F, NIMA, thymidine kinase, the Drosophila tumor suppressor protein OHO-31, the Drosophila pimples protein, and the hypothetical "glue" protein required for sister chromatid cohesion.
  • UbcHlO may ubiquitinate other cell cycle regulatory proteins at other cell cycle stages.
  • Reasonable candidates involved in Gl progression include the Gl cyclins, cyclin D and cyclin E, the cyclin dependent kinase (CDK) inhibitor p27, other members of the CDK inhibitor family, and the tumor suppressor gene product p53.
  • CDK cyclin dependent kinase
  • Purified, recombinant versions of the proteins to be tested are assayed for ubiquitination in vitro in the presence of purified, recombinant UbcHlO and a rabbit reticulocyte lysate (RRL) system, which is an established source of ubiquitinating enzymes and proteasome complexes (Hershko (1988) J. Biol. Chem. 263:15237-
  • reaction products are analyzed by immunoblotting with antibodies against the protein to be tested.
  • Ubiquitination of the protein is characterized by the appearance of a ladder of higher molecular weight bands in addition to the immunoreactive band that corresponds to the protein itself; the appearance of these bands should be dependent upon the presence of recombinant UbcHlO.
  • Immunoblotting with an anti- ubiquitin antibody will confirm that these higher molecular weight forms of the protein represent ubiquitinated species.
  • in vivo approaches involving the injection or transfection of a presumptive dominant negative UbcHlO are described below.
  • HSEC encodes the last 5 amino acids of the UbcHlO open reading frame followed by two stop codons then a Hindlll restriction site.
  • the resulting PCR product was digested with Ndel and Hindlll, ligated with Ndel/Hindlll-cut pT7-7 and transformed into BL- 21(DE3) pLysS cells (Novagen) .
  • the UbcHlO C(114)S mutant was generated in two steps by PCR.
  • the amino-terminal portion was amplified from the UbcHlO cDNA clone as above, using the primers HSEN and HSECSR (5' GATGTCCAGGCTTATGTTACC 3', antisense; SEQ ID NO:26) .
  • the carboxyl-terminal portion was amplified using primers HSECSF (5' GGTAACATAAGCCTGGACATC 3', sense; SEQ ID NO:27) and HSEC.
  • HSECSR is the antisense sequence of HSECSF and both encode amino acids GNISLDI which alters residue 114 of UbcHlO from cysteine to serine.
  • the corresponding clam E2-C mutant was generated by amplification of the amino-terminal portion of E2-C cDNA (Aristarkhov et al. (1996) Proc. Natl. Acad. Sci. (USA) 93 : 4294-4299 ) using the primers CE2FULL (5 *GGGCATATGTCGGGACAAAATATAGATC 3' sense; SEQ ID NO:28) and CE2MUTR (5' CCAGACTTATATTTCCTGACTG 3', antisense; SEQ ID NO:29) .
  • the carboxyl-terminal portion was amplified using primers CE2MUTF (5'
  • CE2MUTR has the antisense sequence of CE2MUTF and both encode amino acids ESGNISL which alters residue 114 of
  • E2-C from cysteine to serine was generated.
  • the PCR products from the first step were amplified with primers CE2FULL and CE2REV.
  • the second step PCR product was digested with Nde I and Hindlll and cloned into pT7-7.
  • the AU1 epitope (DTYRYI) was added to the C-terminus of wild-type UbcHlO and the C(114)S mutant by PCR using the primers HSEN and HSEAUC (5'
  • pT7-7 vectors containing the wild-type and mutant genes were used as templates.
  • HSEAUC encodes the last 6 amino acids of the UbcHlO open reading frame followed by amino acids DTYRYI, two stop codons then a Hindlll restriction site.
  • the resulting PCR product was digested with EcoRI and Hindlll and ligated with EcoRI/HindHI- cut pJS55, a derivative of pSG5 (Stratagene) with a modified polylinker (Sparkowski et al . (1994) J.
  • Bacteria were pelleted, washed with PBS and resuspended in 6 ml 50 mM Tris-HCl (pH 7.2), 1 mM DTT, 1 mM EDTA, 10 ⁇ g/ml leupeptin and chymostatin, and sonicated 94 x 30 seconds) and centrifuged at 15,000 x g for 10 minutes. All recombinant E2-C's were in the supernatant fraction.
  • bacterial extracts were diluted with 4 volumes 10 mM potassium phosphate (pH 7.0) and 1 mM DTT, and applied to a column of DE-52 (Whatman) at a ratio of 5 mg of protein per ml of resin. Unadsorbed material was collected and concentrated by centrifuge ultrafiltration (Centriprep-10, Amicon) to 10 mg protein/ml. This fraction 20-30 mg of protein) was applied to a 120-ml column of Superdex-75 (Pharmacia) equilibrated with 50 mM Tris-HCl (pH 7.4), 1 mM EDTA and 1 mM DTT. Fractions of 2.5 ml were collected at a flow rate of 1 ml/min. The various E2 ' s eluted in fractions 28-32, well separated from the majority of bacterial proteins. All E2-C preparations were >95% homogenous.
  • the tagged UbcHlO mutants and tagged and untagged versions of wild-type UbcHlO were cloned into the vector pT7-7 (Tabor and Richardson, 1985) to allow expression of these proteins in E. coli .
  • the recombinant proteins were purified as described above, and the wild-type protein tested for its ability to promote cyclin-ubiquitin ligation in vitro .
  • the tagged protein can promote ubiquitination of cyclin as well as the untagged WT protein.
  • tagged UbcHlO can functionally replace WT UbcHlO.
  • the tagged mutant proteins were then tested for their ability to compete with clam E2-C (and UbcHlO) in the in vitro cyclin ubiquitination assay (see FIGS.
  • RNA encoding wild type or mutant E2-C was injected into one cell of two cell frog embryos as described (LaBonne et al. (1995) Develop . 121:1472- 1486) . Embryos were collected at mid-late blastula stage, fixed, stained with Hoechst 33342, squashed and visualized by fluorescence microscopy.
  • the wild-type and mutant UbcHlO genes are cloned into the vector pCS2+ to allow the production of in vitro transcripts.
  • Transcripts are generated using the MEGAscript kit (Ambion Inc., Austin, Texas) following the manufacturer's protocols.
  • mRNAs from the wild- type and mutant UbcHlO genes are micro-injected into one cell of the two-cell stage frog embryo as described by Kay et al. (Meth. Cell Biol . (1991) Vol. 36, San Diego, CA, Academic Press) . Injection of the mutant transcripts inhibit or delay cell division in the micro-injected cell relative to the uninjected cell.
  • the wild-type transcript serves as a control and has no inhibitory effect on cell division.
  • the chromosome morphology will be determined in the arrested or delayed cells.
  • the embryos are fixed in 63% ethanol, 30% distilled H 2 0, 7% glacial acetic acid overnight at 4°C.
  • the embryos are washed twice for 1 hour in H 2 0 then stained in 1 ⁇ g/ml Hoechst 33342 (Sigma Chemical Company, St. Louis, MO) overnight.
  • a portion of the stained embryo is then dissected, placed on a slide, immersed in 10% acetic acid then covered with a coverslip and squashed. Samples are then observed using fluorescent optics.
  • Coverslips were mounted in 70% glycerol containing DABCO (1,4,- diazabicyclo[2, 2,2]octaine, Sigma) as an anti- fading agent in PBS, sealed with nail polish and viewed by fluorescence microscopy.
  • DABCO 1,4,- diazabicyclo[2, 2,2]octaine
  • the recombinant epitope tagged mutant and wild-type UbcHlO proteins are expressed in mammalian cells using an inducible expression system which uses the bacterial tetracycline resistance operator/repressor to establish tight regulation of gene expression.
  • the system is based on two plasmids pUHDl5-l neo (FIG. 15A) and pUHDlO-3 (FIG. 15B) , which can be stably integrated into mammalian cells to establish cell lines (Gossen et al. (1992) Proc. Natl. Acad. Sci. USA
  • the plasmid pUHD15-l neo encodes a chimeric protein composed of the tetracycline repressor
  • HSV herpes simplex virus
  • VP16 the C-terminal 130 amino acids
  • hCMV human cytomegalovirus
  • SV40 downstream simian virus 40 polyadenylation
  • the plasmid also encodes a neomycin resistance gene.
  • the plasmid pUHDlO-3 is used for tTA- dependent expression of the gene of interest. Suitable sites in the polylinker are used to clone the genes encoding WT UbcHlO and the UbcHlO mutants into pUHDlO-3. Upstream of the cloning polylinker is a minimal hCMV promoter, hCMV*-l
  • tetracycline operator sequence 02 of TnlO, a 19 bp inverted repeat which is bound by the tetracycline repressor
  • tetO tetracycline operator
  • Downstream of the polylinker is an SV40 poly(A) sequence.
  • tTA can bind to the tetO sequence and promote transcription of the downstream gene.
  • tetracycline 1-2 mg/ml in the culture medium
  • tTA can no longer bind to tetO, and transcription of the downstream gene is switched off: + tetracycline : Gene OFF; - tetracycline : Gene ON.
  • a suitable cell line is selected for these studies.
  • Stable cell lines that express the tTA transactivator have been described, e.g. the rat embryo fibroblast cell line, Rat-1 (Resnitzky et al. (1994) Mol. Cell Biol. 14:1669-1679), and HeLa cells (Gossen et al. (1992) Proc. Natl. Acad.
  • the tTA may also be expressed in a non-transformed human cell line such as IMR-90 or human foreskin fibroblasts, for example, as these cell lines can be synchronized by a serum starvation/stimulation method, as described.
  • Cells are transfected with 10 ⁇ g of linearized pUHD15-l neo using the calcium phosphate precipitation technique (Chen et al. (1988) BioTechniq. 6: 632-38) . Clones are selected in the presence of 400 ⁇ g/ml of active G418
  • clones expressing tTA are transfected with plasmids carrying tagged wild-type and mutant UbcHlO genes. This is done by co-transfection with a plasmid encoding a hygromycin resistance gene. 10 ⁇ g of linearized UbcHlO plasmid and 0.5 ⁇ g of linearized hygromycin plasmid are co-transfected into the tTA-expressing cell line, using the calcium phosphate precipitation technique.
  • the cells are grown in the presence of tetracycline (1 ⁇ g/ml in the culture medium) and clones are selected in the presence of 150 ⁇ g/ml hygromycin (Calbiochem, San Diego, CA) . Resulting clones are screened for their ability to express the UbcHlO genes by immunoblotting with the AUl antibody as described above. Positive clones are then maintained in medium containing 2 ⁇ g/ml tetracycline, 150 ⁇ g/ml hygromycin, and 350 ⁇ g/ml G418 and used for subsequent experiments.
  • DMEM Dulbecco Modified Eagle's medium
  • FBS fetal bovine serum
  • Dextran (1 mg/ml) in TBS (25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl), and 1.5 ml serum-free DMEM. DNA was added to the DMEM first to prevent precipitation. The DNA mixture was removed and the cells were incubated in DMEM containing 10% FBS and 100 ⁇ g/ml chloroquine for 3-4 hours. At the end of this period the cells were incubated in serum-containing DMEM until fixation or harvesting.
  • non-transformed cells are synchronized using the serum starvation/stimulation technique (Resnitzky et al. (1994) Mol. Cell Biol. 14:1669-1679.
  • Cell lines containing stably integrated and inducible UbcHlO genes are seeded at 2 x 10 5 cells per 60 mm diameter tissue culture plate (at least 2 plates per cell line for comparing expression in the presence and absence of tetracycline) in medium containing 10% fetal calf serum (FCS) and 2 ⁇ g/ml tetracycline.
  • FCS fetal calf serum
  • the medium on the cells is replaced with medium containing 0.1% FCS (serum starvation) and 2 ⁇ g/ml tetracycline. 48 hours later the medium is replaced with medium containing 0.1% FCS with or without 2 ⁇ g/ml tetracycline. 24 hours later the cells are induced to re-enter the cell cycle in synchrony by replacing the medium with medium containing 10% FCS (serum stimulation) with or without 2 mg/ml tetracycline. The cells are harvested at various times after release from starvation for protein/mRNA extraction (see above) or cell cycle analysis (see below) .
  • the cells are fixed at room temperature with 50% vol/vol methanol/acetone for 2 min, or with 3% formaldehyde for 5 min followed by permeabilization with 0.5% Triton X-100 for 10 min. They are then incubated with antibodies against b-tubulin (Amersham, Chicago, IL) diluted to the appropriate concentration in 3% BSA in PBS, for one hour at room temperature. After primary antibody incubation the cells are washed 3 times with 0.5% BSA in PBS, then are incubated with a suitable fluorescent-conjugated secondary antibody (Amersham, Chicago, IL) for one hour at room temperature.
  • b-tubulin Amersham, Chicago, IL
  • the cells are washed as before then incubated with 0.1 ⁇ g/ml 4' -6' diamino-2- phenylindole (DAPI, Sigma, St Louis, MO) in PBS for 10 min at room temperature to stain the DNA. This allows the detection of any delays in the cell cycle and/or disruptions in cell morphology that result from expression of the UbcHlO mutants. If the cells expressing the mutant UbcHlO genes fail to enter S phase, this will indicate that the UbcHlO protein is involved in the Gl/S phase transition and thus is involved in ubiquitinating proteins at cell cycle stages other than mitosis .
  • DAPI diamino-2- phenylindole
  • UbcHlO mutants may block the cells prior to anaphase, indicating that the UbcHlO protein is required for cells to exit mitosis and enter Gl of the next cell cycle. Expression of the wild-type protein is used as a control for these experiments. If the mutant UbcHlO proteins do block cell cycle progression at different stages, then proteins that are known to be degraded during these phases (see above) are monitored to see if they are stabilized in the arrested cells. Protein extracts are prepared from the arrested cells and immunoblotted with appropriate antibodies, as described above, to see if these proteins are present at higher levels than normal in the arrested cells.
  • the cells are synchronized and induced to express the DTYRYI-tagged or untagged WT UbcHlO gene as described above.
  • the cells are removed, fixed and stained with the AUl antibody or anti-UbcHlO antibodies to determine the localization of UbcHlO at each particular time point.
  • the cells are co- stained with b-tubulin antibody and DAPI, as described above, to see if UbcHlO associates with known structures such as microtubules, centrosomes or DNA.
  • the cells are also co-stained with antibodies against human cdcl6Hs and Cdc27Hs, (John Hopkins School of Medicine, Baltimore, MD) to determine if there is any co-localization between UbcHlO and known components of the cyclosome/anaphase promoting complex (APC) (King et al. (1995) Cell 81:279-288; Tugendreich et al.
  • APC cyclosome/anaphase promoting complex
  • UbcHlO peptide compatible domains are identified as follows.
  • the UbcHlO sequence is "mapped" onto the existing Ubc crystal structures (Cook et al. (1992) J. Biol. Chem. 267:15116-21; Cook et al. (1993) Biochem. 32:13809-13817) to identify regions on the surface. Peptides corresponding to these regions are then tested for their effect on cyclin-ubiquitination in vitro using the assay described above. Any peptides that block ubiquitination can be used as "lead" compounds for the rational design of therapeutic agents that are cell permeable and can potentially be used to block cyclin ubiquitination, and thus the cell cycle, in vivo .
  • UbcHlO a cAMP-dependent protein kinase (PKA) phosphorylation site is engineered into the UbcHlO gene using PCR (Kaelin Jr. et al . (1992) Cell 70: 351-364); Songyang et al. (1994) Curr. Biol. 4:973- 982.
  • the modified protein is expressed in E. coli and phosphorylated in vitro with PKA and radiolabelled ATP.
  • Labelled UbcHlO is incubated with El enzyme in the presence of ubiquitin and ATP to form the UbcHlO-ubiquitin thiolester. This is used to probe blots of whole cell lysates and/or purified cyclosome complexes to screen for interacting proteins, as described for clam E2-C (see above) .
  • the AUl antibody is used to immunoprecipitate proteins from total cell extracts to look for proteins that interact with UbcHlO.
  • 2.5 x 10 s cells induced to express WT UbcHlO or the UbcHlO mutants are labelled with 1 ⁇ Ci of 35S-TransLabel, washed twice with complete media, then washed with cold PBS.
  • Extracts for immunoprecipitation are prepared by incubating the cells in 100 ⁇ l lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Na- deoxycholate, 1 ⁇ g/ml N-tosyl-L—phenylalanine chloromethyl ketone, 0.1 ⁇ g/ml Pepstatin, 50 ⁇ g/ml N-tosyl-L-lysine chloromethyl ketone, 50 ⁇ g/ml antipain, 40 ⁇ g/ml PMSF, 12 ⁇ g/ml phosphoamidon, 6 ⁇ g/ml leupeptin, 6 ⁇ g/ml aprotinin) .
  • lysis buffer 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Na- deoxycholate, 1 ⁇ g/ml N-tosyl-
  • the extracts are vortexed and centrifuged for 10 min at 14,000 rpm at 4°C to pellet the nuclei and other insoluble material.
  • An appropriate amount of AUl antibody is added to the extract and the reaction is incubated at 4°C for 1 hour.
  • 25 ml of a 50% vol/vol slurry of protein A-Sepharose beads (Pharmacia, Piscataway, NJ) in PBS is added and the tubes are rotated for 1 hour at 4°C.
  • the beads are collected by centrifugation, washed in RIPA buffer (50 mM Tris- HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS , 1 mM EDTA, 100 ⁇ M PMSF) at 4°C, and boiled in 50 ml SDS-sample buffer. The samples are then resolved by SDS-PAGE and fluorography (Brown et al. (1994) J. Cell Biol.
  • the tagged UbcHlO protein is also tested for its ability to co-precipitate known components of the cyclosome/APC.
  • Immunoprecipitation extracts are prepared as described above but the cells are not labelled. Protein samples from the immunoprecipitation are resolved by SDS-PAGE, the samples are transferred to Immobilon (Millipore, Bedford, MA) , and immunoblotted with antibodies against human Cdcl6Hs and Cdc27Hs following the manufacturer's protocols.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne de nouveaux polypeptides porteurs d'ubiquitine, lesquels sont d'origine humaine ou proviennent de myes, et jouent un rôle dans l'ubiquitination de cyclines A et/ou B. Cette invention concerne également des inhibiteurs de ces polypeptides, des acides nucléiques codant ces polypeptides et les inhibiteurs, des anticorps spécifiques à ces polypeptides, ainsi que des procédés d'utilisation de ces derniers.
PCT/US1997/005296 1996-04-01 1997-03-31 Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines WO1997037027A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
BR9710415A BR9710415A (pt) 1996-04-01 1997-03-31 Novos polipeptideos transportadores de ubiquitina ciclina seletiva
NZ332482A NZ332482A (en) 1996-04-01 1997-03-31 Human and clam cyclin-selective ubiquitin carrier polypeptides (E2) that are involved in the ubiquitination of cyclins A and B, inhibitors of these E2 polypeptides are included
EP97917760A EP0900276A1 (fr) 1996-04-01 1997-03-31 Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines
AU26006/97A AU732547B2 (en) 1996-04-01 1997-03-31 Novel cyclin-selective ubiquitin carrier polypeptides
JP53553297A JP2002503949A (ja) 1996-04-01 1997-03-31 新規なサイクリン選択的ユビキチン輸送ポリペプチド

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US1449296P 1996-04-01 1996-04-01
US60/014,492 1996-04-01
US82063997A 1997-03-18 1997-03-18
US08/820,639 1997-03-18

Publications (1)

Publication Number Publication Date
WO1997037027A1 true WO1997037027A1 (fr) 1997-10-09

Family

ID=26686162

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/005296 WO1997037027A1 (fr) 1996-04-01 1997-03-31 Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines

Country Status (7)

Country Link
EP (1) EP0900276A1 (fr)
KR (1) KR20000005413A (fr)
AU (1) AU732547B2 (fr)
BR (1) BR9710415A (fr)
CA (1) CA2250849A1 (fr)
NZ (1) NZ332482A (fr)
WO (1) WO1997037027A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2320570A (en) * 1996-12-20 1998-06-24 Zeneca Ltd Assay for the identification of inhibitors of muscle protein degradation involving ubiquitin and human ubiquitin-carrier proteins
WO1998055510A3 (fr) * 1997-06-05 1999-03-18 Incyte Pharma Inc Proteines liees aux cyclines
EP1167539A1 (fr) * 2000-06-29 2002-01-02 Boehringer Ingelheim International GmbH Méthodes pour l'identification des inhibiteurs des complexes promoteurs de l'anaphase

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023200263A1 (fr) * 2022-04-14 2023-10-19 아주대학교산학협력단 Variant u-box de l'e3 ligase à activité ubiquitine ligase renforcée

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5384255A (en) * 1993-06-21 1995-01-24 Rappaport Family Institute For Research In The Medical Sciences Ubiquitin carrier enzyme E2-F1, purification, production, and use
WO1995018974A2 (fr) * 1994-01-04 1995-07-13 Mitotix, Inc. Enzymes conjuguant l'ubiquitine
WO1996033286A1 (fr) * 1995-04-20 1996-10-24 President And Fellows Of Harvard College Methode et reactifs permettant de detecter la degradation par l'ubiquitine de proteines regulatrices du cycle cellulaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5384255A (en) * 1993-06-21 1995-01-24 Rappaport Family Institute For Research In The Medical Sciences Ubiquitin carrier enzyme E2-F1, purification, production, and use
WO1995018974A2 (fr) * 1994-01-04 1995-07-13 Mitotix, Inc. Enzymes conjuguant l'ubiquitine
WO1996033286A1 (fr) * 1995-04-20 1996-10-24 President And Fellows Of Harvard College Methode et reactifs permettant de detecter la degradation par l'ubiquitine de proteines regulatrices du cycle cellulaire

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ARISTARKHOV, A., ET AL .: "E2-C, A CYCLIN-SELECTIVE UBIQUITIN CARRIER PROTEIN REQUIRED FOR THE DESTRUCTION OF MITOTIC CYCLINS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 93, April 1996 (1996-04-01), pages 4294 - 4299, XP002039884 *
HERSHKO, A., ET AL .: "COMPONENTS OF A SYSTEM THAT LIGATES CYCLIN TO UBIQUITIN AND THEIR REGULATION BY THE PROTEIN KINASE cdc2", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 7, February 1994 (1994-02-01), pages 4940 - 4946, XP002039875 *
HILLIER, L., ET AL .: "THE WASHU-MERCK EST PROJECT", EMBL SEQUENCE DATA LIBRARY, 31 March 1995 (1995-03-31), HEIDELBERG, GERMANY, XP002039876 *
JENTSCH, S.: "THE UBIQUITIN-CONJUGATION SYSTEM", ANNUAL REVIEW OF GENETICS, vol. 26, 1992, pages 179 - 207, XP002039882 *
KING, R.W., ETAL.: "A 20S COMPLEX CONTAINING CDC27 AND CDC16 CATALYZES THE MITOSIS-SPECIFIC CONJUGATION OF UBIQUITIN TO CYCLIN B", CELL, vol. 81, April 1995 (1995-04-01), pages 279 - 288, XP002039877 *
MURRAY, A.: "CYCLIN UBIQUITINATION: THE DESTRUCTIVE END OF MITOSIS", CELL, vol. 81, April 1995 (1995-04-01), pages 149 - 152, XP002039881 *
NUBER, U., ET AL .: "CLONING OF HUMAN UBIQUITIN-CONJUGATING ENZYMES UbcH6 AND UbcH7 (E2-F1) AND CHARACTERIZATION OF THEIR INTERACTION WITH E6-AP AND RSP5", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 5, February 1996 (1996-02-01), pages 2795 - 2800, XP002039878 *
ROLFE, M., ET AL .: "RECONSTITUTION OF p53-UBIQUITINYLATION REACTIONS FROM PURIFIED COMPONENTS: THE ROLE OF HUMAN UBIQUITIN-CONJUGATING ENZYME UBC4 AND E6-ASSOCIATED PROTEIN (E6AP)", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 92, April 1995 (1995-04-01), pages 3264 - 3268, XP002039880 *
SCHEFFNER, M., ET AL .: "IDENTIFICATION OF A HUMAN UBIQUITIN-CONJUGATING ENZYME THAT MEDIATES THE E6-AP-DEPENDENT UBIQUITINATION OF p53", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 91, October 1994 (1994-10-01), pages 8797 - 8801, XP002039879 *
TOWNSLEY, F.M., ET AL .: "DOMINANT-NEGATIVE CYCLIN-SELECTIVE UBIQUITIN CARRIER PROTEIN E2-C/UbcH10 BLOCKS CELLS IN METAPHASE", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 94, March 1997 (1997-03-01), pages 2362 - 2367, XP002039885 *
YU,H. ET AL.: "IDENTIFICATION OF A NOVEL UBIQUITIN-CONJUGATING ENZYME INVOLVED IN MITOTIC CYCLIN DEGRADATION", CURRENT BIOLOGY, vol. 6, no. 4, 1 April 1996 (1996-04-01), pages 455 - 466, XP002039883 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2320570A (en) * 1996-12-20 1998-06-24 Zeneca Ltd Assay for the identification of inhibitors of muscle protein degradation involving ubiquitin and human ubiquitin-carrier proteins
WO1998028436A1 (fr) * 1996-12-20 1998-07-02 Zeneca Limited Detection de proteines
GB2320570B (en) * 1996-12-20 1999-01-27 Zeneca Ltd Identification of inhibitors of muscle protein degradation involving ubiquitin and human ubiquitin carrier proteins
WO1998055510A3 (fr) * 1997-06-05 1999-03-18 Incyte Pharma Inc Proteines liees aux cyclines
EP1167539A1 (fr) * 2000-06-29 2002-01-02 Boehringer Ingelheim International GmbH Méthodes pour l'identification des inhibiteurs des complexes promoteurs de l'anaphase
WO2002000923A1 (fr) * 2000-06-29 2002-01-03 Boehringer Ingelheim International Gmbh Procedes servant a identifier des inhibiteurs du complexe de promotion d'anaphase

Also Published As

Publication number Publication date
AU2600697A (en) 1997-10-22
AU732547B2 (en) 2001-04-26
NZ332482A (en) 2000-05-26
CA2250849A1 (fr) 1997-10-09
BR9710415A (pt) 1999-08-17
KR20000005413A (ko) 2000-01-25
EP0900276A1 (fr) 1999-03-10

Similar Documents

Publication Publication Date Title
US5733730A (en) Telomere repeat binding factor and diagnostic and therapeutic use thereof
US20080020462A1 (en) Atm kinase compositions and methods
JP2010063461A (ja) 細胞のＮＦ−κＢ活性化を調節するための組成物および方法
US5621075A (en) Insulin receptor substrate
US5384255A (en) Ubiquitin carrier enzyme E2-F1, purification, production, and use
JP2011097945A (ja) 刺激誘導性Ｉ（κ）Ｂキナーゼ［ＩＫＫ］シグナルソーム
WO2001058476A2 (fr) Procedes et compositions pour le traitement de la maladie d'alzheimer par le renforcement de l'activite de la plasmine ou de l'activite du type de la plasmine
JPH11507823A (ja) 新規なヒト視床下部由来セルピン
AU676721B2 (en) Role of ATP-ubiquitin-dependent proteolysis in MHC-1 restricted antigen presentation and inhibitors thereof
AU732547B2 (en) Novel cyclin-selective ubiquitin carrier polypeptides
WO2000075184A9 (fr) Modulation de taux de proteines au moyen du complexe scf
US6180379B1 (en) Cyclin-selective ubiquitin carrier polypeptides
US20060094013A1 (en) Salt-inducible kinases 2 and use thereof
US7108992B2 (en) ATM kinase compositions and methods
JP2002517998A (ja) ｐ２７（ＫＩＰ１）のＦＫＢＰ−１２との相互作用
US20030228667A1 (en) Nucleotide sequence encoding a modulator of NF-kappaB
WO2004012755A1 (fr) Methodes de regulation de l'activite d'un complexe renfermant brca1-brca2
MXPA98008070A (en) Novedous polypeptides carriers of selected cybinetines of cicl
US20090130112A1 (en) Spatial for altering cell proliferation
WO2005033266A2 (fr) Regulation de l'expression d'acheron
EP1433848A1 (fr) Nouvelle proteine mt0039 inhibitrice de serine protease
WO2006036822A1 (fr) Procede d'identification d'agents qui modulent la croissance ou la survie de cellules

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN

WPC Withdrawal of priority claims after completion of the technical preparations for international publication

Free format text: US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: PA/a/1998/008070

Country of ref document: MX

Ref document number: 1019980708147

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 2250849

Country of ref document: CA

Kind code of ref document: A

Ref document number: 2250849

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 332482

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 1997917760

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997917760

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1019980708147

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1019980708147

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1997917760

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载