Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-10-22
2002-07-30
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C530S325000, C530S326000, C530S327000
Reexamination Certificate
active
06426205
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods and compositions for modulating ubiquitin dependent proteolysis.
BACKGROUND OF THE INVENTION
Ubiquitin-dependent proteolysis is a key regulatory mechanism that controls diverse cellular processes (reviewed in Hochstrasser 1996). In this pathway, ubiquitin is transferred via transthioesterification along a cascade of carrier enzymes, E1→E2→E3, and ultimately conjugated in an isopeptide linkage to a lysine residue of a substrate protein. Reiteration of the ubiquitin transferase reaction results in formation of a polyubiquitin chain on the substrate, which is then recognized by the 26S proteasome, and rapidly degraded. Specificity in protein ubiquitination derives from E3 enzymes, also known as ubiquitin-ligases (Hershko et al. 1983). In some cases, an E3 facilitates recognition of the target protein by an E2, while in others an E3 accepts a ubiquitin thioester from an E2 and directly transfers ubiquitin to the substrate (Scheffner et al. 1995). Although substrate recognition is a key aspect of ubiquitin dependent proteolysis, the identification of E3 enzymes has been problematic because the few known E3 families bear no sequence relationship to each other.
Ubiquitin-dependent proteolysis is essential for two major cell cycle transitions, the G1 to S phase transition and the metaphase to anaphase transition (reviewed in King et al. 1996). These transitions mediate alteration between states of high and low cyclin-dependent kinase (Cdk) activity, which in turn ensures that DNA replication origins fire only once per cell cycle and that chromosome segregation follows DNA replication (reviewed in Nasmyth 1996). Key targets of the ubiquitin proteolytic pathway at these transitions include positive regulators of Cdks, the cyclins, and negative regulators of Cdks, the Cdk inhibitors. In budding yeast, a single Cdk, Cdc28 (or Cdk1) is activated in G1 phase by the G1 cyclins Cln1-Cln3, and in S through M phase by the mitotic cyclins, Clb1-Clb6 (reviewed in Nasmyth, 1996). A motif called the destruction box targets mitotic cyclins and other proteins to a cell cycle-regulated E3 ubiquitin-ligase called the Anaphase Promoting Complex (APC) or cyclosome (reviewed in King et al. 1996). In contrast, phosphorylation targets G1 cyclins and Cdk inhibitors for degradation via a constitutive ubiquitination pathway (reviewed in Deshaies, 1997). Genetic analysis in budding yeast has revealed several components of this pathway: Cdc4, a WD40 repeat protein (Yochem and Byers, 1987), Cdc34, an E2 ubiquitin conjugating enzyme (Goebl et al. 1988), Cdc53 a protein that forms a tight complex with phosphorylated Clns (Willems et al. 1996), Grr1, a leucine rich repeat protein (Flick and Johnston, 1981), and Skp1, a protein that binds to a motif called the F-box (Bai et al. 1996). The F-box motif occurs in Cdc4, Grr1 and several other yeast and mammalian proteins (Bai et al. 1996). Cells lacking functional Cdc4, Cdc34, Cdc53 or Skp1 arrest in G1 because the Cdk inhibitor Sic1 is not degraded, which prevents the onset of Clb-Cdc28 activity and initiation of DNA replication (Nugroho and Mendenhall 1994; Schwob et al. 1994; Bai et al. 1996). In late G1 phase, Sic1 is phosphorylated by the Cln-Cdc28 kinases and thus targeted for ubiquitin dependent proteolysis (Schwob et al. 1994; Schneider et al. 1996; Tyers 1996). Recently, a requirement for Cdc4, Cdc34 and Cln2-Cdc28 activity in Sic1 ubiquitination has been demonstrated in an in vitro yeast extract system (Verma et al. 1997). Cdc34, Cdc53 and Skp1 are also required for Cln degradation (reviewed in Deshaies 1997), as is Grr1 (Barral at al. 1995), although this protein was originally identified because of its role in glucose repression (Flick and Johnston 1991).
Other important regulatory proteins are degraded via the Cdc34 pathway, including the Cln-Cdc28 inhibitor Far1 (McKinney et al. 1993; Henchoz et al. 1997), the replication protein Cdc6 (Piatti et al. 1996), and the transcription factor Gcn4 (Kornitzer et al. 1994). Aside from its G1 function, Skp1 also plays a role in G2 because certain conditional alleles of SKP1 arrest cells in G2, and because Skp1 is a component of the Cbf3 kinetochore complex (Bai et al. 1996; Connelly and Hieter 1996; Stemmann and Lechner 1996).
Genetic and biochemical evidence indicates that Cdc53 interacts with Cdc4 and Cdc34 (Willems et al. 1996; Mathias et al. 1996), and that the F-box of Cdc4 binds Skp1 (Bai et al. 1996). These interactions, and the fact that Cdc53 physically associates with phosphorylated forms of Cln2, suggest that Cdc4, Cdc34, Cdc53 and Skp1 may participate in an E2/E3 ubiquitination complex that recognizes and ubiquitinates phosphorylated substrates (Bai et al. 1996; Willems et al. 1996). Divergence of the Sic1 and Cln degradation pathways apparently occurs at the level of the two F-box proteins. Cdc4 is required for degradation of Sic1 (Schwob et al. 1994), whereas Grr1 is required for Cln1/2 degradation (Barral et al. 1995). It was therefore hypothesized that distinct F-box proteins recruit specific substrates to an E3 ubiquitin-ligase complex that contains Skp1 (Bai et al. 1996). The existence of a complex in vivo containing F-box proteins and Cdc34, Cdc53 and Skp1 has yet to be demonstrated.
SUMMARY OF THE INVENTION
Through analysis of Cdc53-interacting proteins the present inventors determined that Cdc53 forms complexes with Skp1, Cdc34, and each of the F-box proteins Cdc4, Grr1 and Met30 in vivo. Each F-box protein confers functional specificity on a core Cdc34-Cdc53-Skp1 complex for Sic1 degradation, Cln degradation and methionine biosynthesis gene regulation, respectively. The present inventors showed that Cdc53 is a scaffold that tethers Skp1/F-box proteins to Cdc34 within an E2/E3 ubiquintination complex. The present inventors have also identified a specific region on Cdc53 that binds to Skp1.
Broadly stated the present invention relates to (a) a complex comprising an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein, and optionally a protein containing an F-box motif; and (b) a complex comprising a protein of the Cullin family and a protein containing an F-box motif. The invention is also directed to (a) a peptide derived from the binding domain of an E2 ubiquitin conjugating enzyme that interacts with a protein of the Cullin family; (b) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an E2 ubiquitin conjugating enzyme; (c) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an F-box binding protein; preferably a peptide of the formula I or Ia or (d) a peptide derived from the binding domain of an F-box binding protein that interacts with a protein of the Cullin family. The invention also contemplates antibodies specific for the complexes and peptides of the invention.
The present invention also provides a method of modulating ubiquitin dependent proteolysis comprising administering an effective amount of one or more of the following: (a) a complex comprising an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein, and optionally a protein containing an F-box motif; (b) a complex comprising a protein of the Cullin family and a protein containing an F-box motif; (c) a peptide derived from the binding domain of an E2 ubiquitin conjugating enzyme that interacts with a protein of the Cullin family; (d) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an E2 ubiquitin conjugating enzyme; (d) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an F-box binding protein; preferably a peptide of the formula I or Ia (e) a peptide derived from the binding domain of an F-box binding protein that interacts with a protein of the Cullin family; or (f) enhancers or inhibitors of the interaction of an E2 ubiquitin conjugating enzyme or an F-box binding protein, with a protein of the Cullin family.
Tyers Mike
Willems Andrew
Merhant & Gould P.C.
Mount Sinai Hospital Corporation
Slobodyansky Elizabeth
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