Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-01-29
2003-03-04
Nashed, Nashaat T. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C435S193000, C530S350000
Reexamination Certificate
active
06528633
ABSTRACT:
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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.
269:4940-4946). Ubiquitinated cyclins are then rapidly degraded by the 26S proteasome (Hershko et al. (1994)
J. Biol. Chem.
269:4940-4946). This process is catalyzed by a cyclin-specific ubiquitin ligase, E3-C, which is part of a 20S particle, the cyclosome (Sudakin et al (1995)
Mol. Biol. Cell.
6:185-198). Cyclosome activation is initiated by Cdc2 (Félix et al. (1990)
Nature
346:379-382; Sudakin et al. (1995)
Mol. Biol. Cell.
6:185-198) and terminated by an okadaic acid-sensitive phosphatase (Lahav-Baratz et al. (1995)
Proc. Nat. Acad. Sci. USA
, in press). This particle contains homologs of two yeast proteins, Cdc16 and Cdc27 (King et al. (1995)
Cell
81:279-288), proteins required for the destruction of cyclin B and the metaphase-anaphase transition (Tugendreich et al. (1995)
Cell
81:261-268; Irniger et al (1995)
Cell
81:269-277).
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)
J. Biol. Chem.
269:4940-4946). Multiple species of E2's were first found in animal cells (Pickart et al (1985)
J. Biol. Chem.
260:1573-1581), and at least ten different Ubc's have now been identified in yeast (Jentsch (1992)
Ann. Rev. Genetics
26:179-207).
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.
26:179-207)). Class III enzymes have various N-terminal extensions; however, their function is not known.
Genetic and molecular analysis has revealed that different 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). Ubc3 (Cdc34) is required for the G1/S transition (Goebl et al. (1988)
Science
241:1331-1335), where it appears to participate in the ubiquitin-dependent destruction of the G1 cyclin dependent kinase (cdk) inhibitor, p40
sic1
(Schwob et al (1994)
Cell
79:233-244). 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.
269:4940-4946).
However, heretofore, the Ubc(s) responsible for the ubiquitination of the mitotic cyclins in humans were unidentified and characterized.
SUMMARY OF THE INVENTION
It has been discovered that both clam and human have novel cyclin-selective ubiquitin carrier polypeptides which are involved in the ubiquitination of proteins and ubiquitin-directed protein degradation. These findings have been exploited to develop the present invention, which is directed to human and clam ubiquitin carrier polypeptides and inhibitors thereof, to nucleic acids encoding such polypeptides, and to methods employing such ubiquitin carrier polypeptides and inhibitors.
In a first aspect, the invention provides an isolated and purified, non-xenopal, ubiquitin carrier polypeptide (Ubc) involved in the ubiquitination of cyclin A and/or B.
As used herein, the term “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.
The term “non-xenopal” refers to Ubc's which are not derived from frog cells or encoded by frog nucleic acid.
As used herein, 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 UbcH10, and frog Ubc-x.
In some embodiments, the Ubc is recombinantly produced. In other embodiments, 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. As used herein a “fragment” of a molecule such as E2-C, UbcH10, or inhibitors thereof, refers to any smaller polypeptide subset of that molecule. In some embodiments, the Ubc is a clam or human Ubc. In some embodiments, 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:1 or 3. By “homology” is meant sequence identity or similarity.
By similarity is meant the degree to which amino acid changes are in accordance with the conservative amino acid substitutions exemplified in Table 1 below.
TABLE 1
Original Residue
Exemplary Substitutions
Ala
Gly; Ser
Arg
Lys
Asn
Gln; His
Asp
Glu
Cys
Ser
Gln
Asn
Glu
Asp
Gly
Ala; Pro
His
Asn; Gln
Ile
Leu; Val
Leu
Ile; Val
Lys
Arg; Gln; Glu
Met
Leu; Tyr; Ile
Phe
Met; Leu; Tyr
Ser
Thr
Thr
Ser
Trp
Tyr
Tyr
Trp; Phe
Val
Ile; Leu
In particular embodiments, the Ubc has the amino acid sequence set
Aristarkov Alexander
Eytan Esther
Hershko Avram
Kirschner Marc W.
Ruderman Joan V.
Hale and Dorr LLP
Nashed Nashaat T.
President and Fellows of Havard College
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