Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-08-20
2004-06-08
Hutson, Richard (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S252300, C435S254110, C435S320100, C435S219000, C435S183000, C435S325000, C536S023100, C536S023200, C536S023500, C530S350000
Reexamination Certificate
active
06747128
ABSTRACT:
BACKGROUND OF THE INVENTION
The ubiquitin-mediated proteolysis system is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle.
Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.
The conjugation of ubiquitin to protein substrates is a multi-step process. Ubiquitin is a small, highly curved protein which must be activated before it is transferred to a substrate protein. Accordingly, in an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of a ubiquitin activating enzyme, E1. Activated ubiquitin is then transesterified to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes transfer ubiquitin to a lysine residue of a protein substrate. Substrates are recognized either directly by ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases. A major cellular mechanism by which proteins are degraded in eukaryotic cells is by ubiquitinylation of the protein, thereby targeting the protein for degradation by the 26S proteasome (Hochstrasser (1995)
Curr. Opin. Cell Biol.
7:215). Ubiquitin is a small, highly conserved protein, which must be activated before it is transferred to a substrate protein. Activation of ubiquitin occurs through formation of a thioester bond between the COOH terminus of the ubiquitin molecule and a ubiquitin-activating enzyme, E1. Ubiquitin is then transesterified to one member of a family of a ubiquitin conjugating enzymes, E2 enzymes. Ubiquitin is then transferred, either directly or indirectly, to a lysine residue of a substrate protein. Transfer to the substrate protein may require the assistance of a ubiquitin ligase also termed E3 enzyme or complex. An E3 is generally required for the formation of multiubiquitin chains on the substrate, a step that facilitates efficient recognition of the substrate by the proteosome. It has been suggested that E3 is the primary source of substrate specificity in the ubiquitination cascade, as some E3s have been shown to directly bind substrates (Hershko et al. (1986)
J. Biol. Chem.
261:11992; Bartel et al. (1990)
EMBO J.
9:3179). Furthermore, in some situations, a ubiquitin molecule is first transferred from an ubiquitin conjugating enzyme to an E3 enzyme or complex, prior to being transferred to the substrate protein (Willems et al., supra).
Ubiquitination of proteins and subsequent protein degradation plays an important role in various steps of the cell cycle and is thus crucial in the regulation of cell proliferation and differentiation. Briefly, cell-cycle events are thought to be regulated by a series of interdependent biochemical steps. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. In all eukaryotic cells examined to date, the cell cycle appears to be regulated by the sequential activation of a series of the CDK's or Cyclin Dependent Kinases (reviewed in Morgan, (1995)
Nature
374:131-134; King et al., (1994)
Cell
79:563-571; Norbury and Nurse, (1992)
Annu. Rev. Biochem.
61:441-470). Yeast cells contain a single CDK known as cdc2 in
S. pombe
(Beach et al., (1982)
Nature
300:706-709; Booher and Beach, (1986)
Gene
31:129-134; Hindley and Phear, (1984)
Gene
21:129-134; Nurse and Bissett, (1981)
Nature
292:558-560; Simanis and Nurse, (1986)
Cell
45:261-268; and for review see Forsburg and Nurse, (1991b)
Annu. Rev. Cell Biol.
7:227-256) and cdc28 in
S. cerevisiae
. Drosophila and vertebrates have several CDKs, including CDK1, CDK2, CDK4, and CDK6 (Elledge S. J. (1996)
Science
274:1664).
The activity of the CDKs is controlled at least in part by the association of the CDKs with various cyclins during progression through the cell cycle. Cyclins also contribute to substrate specificity The CDK-cyclin complex is both positively and negatively regulated by several mechanisms including phosphorylation, binding to inhibitors (CKIs) and other proteins such as Suc1 (Cks1) that might modify their specificity or accessibility to regulators, and protein degradation by the ubiquitin conjugation pathway (Patra et al. (1996)
Genes Dev.
10:1503).
In addition to their role in activating mitosis, the cyclin-CDKs are required to stimulate the initiation of DNA replication. In yeast, the activity of cyclin-CDKs is blocked specifically by the inhibitor Sic1, which is present in cells from late mitosis until shortly after START. Thus, degradation of Sic1 is necessary for DNA replication. In yeast, it has been shown that degradation of this protein is mediated by the ubiquitin conjugating enzyme (E2) Cdc34 and also requires cdc4 and cdc53 and a Skp1 protein. These proteins are also involved in degradation of other cell cycle regulatory proteins in yeast, including the G1 cyclins, which are required for executing the START of the cell cycle (King et al. (1996)
Science
274:1653; Willens et al. (1996)
Cell
86:453; Bai et al. (1996)
Cell
86: 263; and Mathias et al (1996)
Mol. Cell. Biol.
16:6634).
In particular, molecular cloning of cdc34, a gene required for the G1-S transition in budding yeast, revealed that a ubiquitin conjugation step was required just before the initiation of DNA replication. cdc34 encodes a ubiquitin conjugating enzyme that participates in the destruction of multiple proteins, including the G1 cyclins CLN2 and CLN3, as well as proteins not directly related to cell cycle control. However, accumulation of these substrates does not account for the cell cycle arrest of cdc34
ts
mutants. The nature of the crucial target of cdc34 at the G1-S transition was first implied by genetic studies. A strain deficient in all S-phase and mitotic cyclins recapitulated the cdc34
ts
mutant phenotype, suggesting that the cdc34 pathway might be required for generating S-phase CDK activity. Extracts made from cdc34
ts
mutants inhibit S-phase CDKs, implying that cdc34 may be required for the degradation of a CDK inhibitor. A candidate for this activity was SIC1, a tight-binding S-phase CDK inhibitor (Mendenhall (1993)
Science
259:216; Nugroho et al. (1994)
Mol Cell. Biol.
14:3320). SIC1 is normally degraded as wild-type cells enter S phase, but accumulates in cdc34
ts
mutants. SIC1 appears to be the crucial substrate blocking progression from G1 to S phase in cdc34
ts
mutants, because cdc34
ts
sic1&Dgr; double mutants initiate DNA replication at the nonpermissive temperature (Schwob et al. (1994)
Cell
79:233). As predicted by these findings, expression of a non-degradable form of SIC1 in wild-type strains blocks cell division at the G1-S transition (King et al. (1996)
Science
274: 1652). Ubiquitin-dependent proteolysis of a CDK inhibitor is therefore a crucial mechanism by which the
Caligiuri Maureen
Rolfe Mark
GPC Biotech Inc.
Hutson Richard
Ropes & Gray LLP
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