Novel cyclin-selective ubiquitin carrier polypeptides
AssigneeHarvard University
InventorsJoan V. Ruderman, Avram Hershko, Marc W. Kirschner, Fiona Townsley, Alexander Aristarkhov, Esther Eytan, Hongtao Yu
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Abstract
Disclosed are novel human and clam ubiquitin carrier polypeptides involved in the ubiquitination of cyclins A and/or B. Also disclosed are inhibitors of such polypeptides, nucleic acids encoding such polypeptides and inhibitors, antibodies specific for such polypeptides, and methods of their use.
Claims85
An isolated and purified, non-xenopal, ubiquitin carrier polypeptide (Ubc) involved in the ubiquitination of cyclin A and/or B, and having a Ubc-specific N-terminal extension.
A selective inhibitor of ubiquitin carrier polypeptide (Ubc) function.
The dominant negative mutant of claim 39 which inhibits 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.
A kit useful for the ubiquitination and degradation of a cyclin comprising:
- (a) a ubiquitin-ubiquitin carrier polypeptide complex, wherein the ubiquitin carrier polypeptide is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof;
- and
- (b) a ubiquitin ligase (E3).
A kit useful for the ubiquitination and degradation of a cyclin comprising:
- (a) ubiquitin;
- (b) a ubiquitin activating enzyme (E1);
- (c) ATP;
- (d) a ubiquitin carrier polypeptide selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active portion thereof;
- and
- (e) a ubiquitin ligase (E3).
A method of ubiquitinating a cyclin and/or targeting a cyclin for destruction, comprising the step of contacting the cyclin with:
- (a) a ubiquitin-ubiquitin carrier protein complex, wherein the ubiquitin carrier protein is selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof;
- and
- (b) a ubiquitin ligase (E3).
A method of inhibiting the proliferation of a cell comprising the step of contacting the cell with an inhibitor of ubiquitin carrier polypeptide function, in an amount sufficient to inhibit the ubiquitination of a cyclin.
A method of screening for compounds which inhibit Ubc function, comprising the steps of:
- (a) providing 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;
- (b) performing the assay in the presence and absence of a compound to-be-tested;
- and
- (c) determining 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,
- a reduction of Ubc function measured in the presence of the compound indicating that the compound is an inhibitor of Ubc function.
A method of screening for compounds which inhibit the ubiquitination of a cyclin, comprising the steps of:
- (a) incubating the cyclin with:
- (i) ubiquitin;
- (ii) a ubiquitin activating enzyme (E1);
- (iii) ATP;
- (iv) a ubiquitin carrier polypeptide selected from the group consisting of clam E2-C, human UbcH10, and an enzymatically active fragment thereof;
- (v) a ubiquitin ligase (E3);
- and
- (vi) Cdc2,
- in the presence and in the absence of a compound-to-be-tested;
- and
- (b) measuring the amount of cyclin-ubiquitin-Cdc2 complex formed in step (a) in the presence and in the absence of the compound,
- a reduction in the amount of complex formed in the presence of the compound indicating that the compound inhibits cyclin ubiquitination.
A therapeutic formulation 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.
Dependencies are inferred from the claim text.