Methods and compositions for modulating ubiquitin dependent proteolysis

Abstract

The invention relates to methods and compositions for modulating ubiquitin dependent proteolysis.

Description

FIELD OF THE INVENTION

[0001] The invention relates to methods and compositions for modulating ubiquitin dependent proteolysis.

BACKGROUND OF THE INVENTION

[0002] 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.

[0003] 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).

[0004] 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 (Komitzer 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).

[0005] 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

[0006] 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 ubiquitination complex. The present inventors have also identified a specific region on Cdc53 that binds to Skp1.

[0007] 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.

[0008] 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.

[0009] In a preferred embodiment of the invention a method is provided for modulating ubiquitin dependent proteolysis comprising administering an effective amount of one or more of the following: (a) a complex comprising Cdc34-Cdc53-Skp1; (b) a complex comprising Cdc34-Cdc53-Ckp1-protein containing an F-box motif; (c) a complex comprising Cdc53-protein containing an F-box motif; (d) a peptide comprising the binding domain of Cdc34 that interacts with Cdc53 or the binding domain of Cdc53 that interacts with Cdc34; (e) a peptide comprising the binding domain of Cdc53 that interacts with Skp1 or the binding domain of Skp1 that interacts with Cdc53; or, (f) inhibitors or enhancers of the interaction of Cdc34 or Skp1, with Cdc53.

[0010] The invention still further provides a method for identifying a substance that binds to a complex comprising an E2 ubiquitin conjugating enzyme and a protein of the Cullin family, 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, or a complex comprising a protein of the Cullin family and an F-box binding protein, comprising: (a) reacting the complex with at least one substance which potentially can bind with the interacting molecules in the complex, under conditions which permit the formation of conjugates between the substance and complex and (b) assaying for conjugates, for free substance, or for non-conjugated complexes. The invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with the complexes of the invention.

[0011] Still further the invention provides a method for evaluating a compound for its ability to modulate ubiquitin dependent proteolysis. For example a substance which inhibits or enhances the interaction of the molecules in a complex of the invention, or a substance which binds to the molecules in a complex of the invention may be evaluated. In an embodiment, the method comprises providing a known concentration of a complex of the invention, with a substance which binds to the complex, and a test compound under conditions which permit the formation of conjugates between the substance and complex, and removing and/or detecting conjugates.

[0012] The present invention also contemplates a peptide of the formula I which interferes with the interaction of Cdc53 and Skp1

A-Tyr-Met-X1-X2-Tyr-X3-X4-X5-Tyr-X6-X7-Cys-X8  I

[0013] wherein A represents one to ten amino acids, X1 represents Met, Arg, Thr, or Glu, X2 represents Leu, Phe, or Val, X3 represents Asp or Thr, X4 represents Ala, Ser, His, or Thr, X5 represents Ile or Val, X6 represents Asn or Asp, X7 represents Tyr, Ile, or Met, and X8 represents Thr, Val, or Ala.

[0014] In an embodiment of the present invention a peptide of the formula Ia which interferes with the interaction of Cdc53 and Skp1 is provided:

A1-A2-A3-A4-A5-A6-Tyr- Met-X1-X2-Tyr-X3-X4-X5-Tyr-X6-X7-Cys-X8  Ia

[0015] wherein A1 represents Ile, Asn, His, Ser, or Ala, A2 represents Leu, Met or Phe, A3 represents Ser, Ala, Thr, or Asp, A4 represents Pro, Lys, Arg, or Ser, A5 represents Thr, Lys, Ser, or Glu, A6 represents Met, Asp, Tyr, Gln, or Arg, X7 represents Met, Arg, Thr, or Glu, X2 represents Leu, Phe, or Val, and X3 represents Asp or Thr, X4 represents Ala, Ser, His, or Thr, X5 represents Ile or Val, X6 represents Asn or Asp, X7 represents Tyr, Ile, or Met, and X8represents Thr, Val or Ala.

[0016] The invention also relates to truncations and analogs of the peptides of the invention. The invention also relates to the use of a peptide of the formula I or Ia to interfere with the interaction of a protein of the Cullin family preferably Cdc53 and an F-box binding protein preferably Skp1; and, pharmaceutical compositions for inhibiting the interaction of a protein of the Cullin family preferably Cdc53 and an F-box binding protein preferably Skp1.

[0017] Further, the invention relates to a method of modulating the interaction of Cdc53 and Skp1 comprising changing the amino acid Tyr at position 48 and/or Met at position 49 in Cdc53.

[0018] The peptides and antibodies of the invention, and substances and compounds identified using the methods of the invention may be used to modulate ubiquitin dependent proteolysis, and they may be used to modulate cellular processes of cells (such as proliferation, growth, and/or differentiation, in particular glucose and methionine biosynthesis, gene expression, cell division, and transcription) in which the compounds or substances are introduced.

[0019] Accordingly, the antibodies, peptides, substances and compounds may be formulated into compositions for adminstration to individuals suffering from a proliferative or differentiative condition. Therefore, the present invention also relates to a composition comprising one or more of a peptide or antibody of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, excipient or diluent. A method for modulating proliferation, growth, and/or differentiation of cells is also provided comprising introducing into the cells a peptide or antibody of the invention, a compound or substance identified using the methods of the invention or a composition containing same. Methods for treating proliferative and/or differentiative disorders using the compositions of the invention are also provided.

[0020] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[0021] The invention will be better understood with reference to the drawings in which:

[0022] FIG. 1. Cdc53 two hybrid interactions. (A) Cdc53 two hybrid screens were carried out with three Cdc53 fusion proteins: Gal4DBD-Cdc53, Gal4DBD-Cdc53&Dgr;N, and Gal4DBD-Cdc53&Dgr;K. (B) Interaction of isolates with Gal4DBD-Cdc53 and Gal4DBD-Cdc53&Dgr;K in a &bgr;-galactosidase filter assay. (C) Schematic of Cdc53 interacting proteins. (D) Two hybrid interactions of LexADBD-Met30 derivatives with VP16AD-Skp1 Interactions quantitated by liquid &bgr;-galactosidase assay in Miller units.

[0023] FIG. 2. Genetic interaction between CDC53 and SKP1. (A) cdc53 Skp1 double mutants are inviable at the semi-permissive temperature. Spore clones of a representative tetratype tetrad were grown at 30° C. for two days. (B) Photomicrographs of cells from a representative cdc53-1 Skp1-12 tetratype grown at 25° C.

[0024] FIG. 3. Characterization of Cdc53 complexes in yeast lysates. (A) Effects of temperature sensitive mutations on the composition of Cdc53 immune complexes. The indicated strains containing <CDC53 CEN> or <CDC53M CEN> plasmids were arrested at 37° C. for 2 h. 9E10 anti-MYC immunoprecipitates from each strain were immunoblotted and sequentially probed with anti-Cdc4, anti-Cdc34, anti-Skp1 and anti-MYC antibodies. The anti-Cdc4 antibody did not reliably detect Cdc4 in lysates and so the panels were omitted (see part C, below). (B) Effects of temperature sensitive mutations on the composition of Skp1 immune complexes. Analysis was as above except that strains contained either vector or <SKP1HA CEN> plasmids. Anti-HA immunoprecipitates were probed with anti-Cdc4, anti-Cdc34, anti-Cdc53 and anti-HA antibodies. (C) Abundance of Cdc4 and Met30 in Skp1 mutants. Wild type, Skp1-11, Skp1-12 strains containing either vector, <CDC4F CEN> or <pADH1-MET30HA 2 &mgr;m> plasmids, were analyzed as above. Anti-FLAG and anti-HA immunoprecipitates were probed with anti-Cdc4 polyclonal antibody and anti-HA antibody respectively.

[0025] FIG. 4. Cdc53 interacts with multiple F-box proteins. (A) The indicated immunoprecipitates from wild type cells containing either vector, <CDC53M CEN>, <pADH1-MET30HA 2 &mgr;m>, <PADH1-GRR1HA 2 &mgr;m> and <CDC4F CEN> plasmids were probed with anti-Cdc53, anti-Skp1, anti-Cdc4 and anti-HA antibodies. IgG light chain is indicated by an asterisk. (B) Effects of MET30 or GRR1 overexpression. Strains of the indicated genotype containing an empty vector plasmid <pADH1-MET30HA> (left panel), or <pADH1-GRR1HA> (right panel) were grown at 30° C. for 3 days and photographed.

[0026] FIG. 5. Mutational analysis of CDC53. (A) Deletion analysis of Cdc53 protein-protein interaction domains. Cells were transformed with untagged (lane 1) or MYC-tagged (lane 2) CDC53 or MYC-tagged versions of the indicated CDC53 mutants (lanes 3-8) all expressed from the wild type promoter on CEN plasmids. Lysates from each strain were immunoprecipitated with 9E10 anti-MYC antibodies, immunoblotted and probed with 9E10 (top panel) and polyclonal antibodies specific to each of the indicated proteins (lower panels). (B) Schematic representation of Cdc53 mutant proteins and their ability to rescue a cdc53 deletion strain. Regions of amino acid sequence conservation in the Cdc53 family are indicated in black (see Methods). The positions of the cdc53-1 (R488C) and cdc53-2 (G340D) point mutations, and the regions required for binding to Skp1/F-box proteins and Cdc34 are also indicated. (C) Cdc53 does not contain essential cysteine residues. A cdc53 deletion strain containing a <CDC53HA URA3 CEN> plasmid was transformed with <CDC536C TRP CEN>, <CDC53M TRP CEN>, or an empty vector plasmid, plated on 5-FOA medium to select for Ura cells, and photographed after 2 days.

[0027] FIG. 6. Specificity of F-box protein function. (A) Methionine repression is mediated by Met30, Cdc34, Cdc53 and Skp1 but not Cdc4. The indicated strains were grown in methionine- free medium and repressed with 1.0 mM methionine for the indicated times. MET25 expression was determined by Northern analysis and normalized to ACT1 expression. (B) Grr1 specifically mediates Cln2 degradation. The indicated strains were incubated at 37° C. for two hours, at which time <pGAL1-CLN2HA> was expressed by the addition of galactose for 1.5 hours, and then repressed with the addition of glucose for the indicated times. Cln2HA was detected by immunoblotting with 12CA5 monoclonal antibody. Exposures were adjusted to give equal Cln2HA signals at time zero. Cln2HA was quantitated and normalized to Cdc28 signals from the same blot probed with anti-Cdc28 antibody.

[0028] FIG. 7 shows the amino acid sequence of a Cdc34 protein.

[0029] FIG. 8 shows the amino acid sequences of a Cdc53 protein and a Cul-2 protein.

[0030] FIG. 9 shows the amino acid sequence of a Skp1 protein.

[0031] FIG. 10 shows the amino acid sequences of a Cdc4 protein, a Met30 protein, and a Grr1 protein.

[0032] FIG. 11 shows that amino acids Y48 and M49 in Cdc53 are required for binding to Skp1. Wild type and Y48W, M49E mutants of Cdc53MYC6 were immunoprecipitated and (A) western blotted for Cdc53MYC6, Cdc53, and Skp1, and (B) silver stained. On the silver-stained gel, bands that exist in the wild type but not the mutant Cdc53 IP are marked with an open triangle.

[0033] FIG. 12A Amino acids Y48 and M49 in Cdc53 are required for binding to Skp1. Wild type and Y48W, M49E mutants of Cdc53MYC6 were immunoprecipitated and western blotted for Cdc53MYC6, Cdc34, and Skp1.

[0034] FIG. 12B Amino acids Y48 and M49 in Cdc53 are required for binding to Skp1. Wild type and Y48W, M49E mutants of Cdc53MYC6 were immunoprecipitated and western blotted for Cdc53MYC6, Cdc34, and Skp1, and (B) silver stained. On the silver-stained gel, bands that exist in the wild type but not the mutant Cdc53 IP are marked with an open triangle.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Definitions

[0036] Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Current Protocols in Molecular Biology (Ansubel) for definitions and terms of the art.

[0037] Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 common L-amino acids. Likewise abbreviations for nucleic acids are the standard codes used in the art.

[0038] “E2 ubiquitin conjugating enzyme” refers to one of the components involved in ubiquitin transfer reactions to form ubiquitin-protein conjugates which are recognized by the 26S proteasome. An example of an E2 ubiquitin conjugating enzyme is Cdc34, and homologs or portions thereof. (See FIG. 7 for a Cdc34 amino acid sequence.)

[0039] “Protein of the Cullin family” refers to the family of proteins involved in the regulation of cell division. The archetypal member of the family is Cdc53. The family also includes, homologs and portions of Cdc53, including the proteins regulating cell division in C. elegans and mammalian cells such as Cul-1, Cul-2, and the metazoan Cdc53 homologs described in Kipreos et al., 1996. (See FIG. 8 for sequences for Cdc53 and Cul-2).

[0040] “F-box binding protein” refers to proteins that bind to proteins containing an F-box motif. Examples of F-box binding proteins are Skp1 and Scon C and homologs, and portions, thereof. (See FIG. 9 for a Skp1 sequence.)

[0041] “Proteins containing an F-box motif” refers to proteins have a characteristic structural motif called the F-box as described in Bai et al, 1996. Examples of the proteins include Cdc4, Grr1, pop1, Met30 , Scon2/Scon3, and several other yeast and mammalian proteins (Bai et al, 1996), and homologs or portions thereof. (See FIG. 10 for a Cdc4 sequence, a Met30 sequence, and a Grr1 sequence.)

[0042] A “binding domain” is that portion of the molecule in a complex of the invention (i.e. E2 ubiquitin conjugating enzyme, protein of the Cullin family, F-box binding protein, or protein containing an F-box motif) which interacts directly or indirectly with another molecule in a complex of the invention. The binding domain may be a sequential portion of the molecule i.e. a contiguous sequence of amino acids, or it may be conformational i.e. a combination of non-contiguous sequences of amino acids which when the molecule is in its native state forms a structure that interacts with another molecule in a complex of the invention.

[0043] By being “derived from” a binding domain is meant any molecular entity which is identical or substantially equivalent to the native binding domain of a molecule in a complex of the invention (i.e. E2 ubiquitin conjugating enzyme, protein of the Cullin family, F-box binding protein; or protein containing an F-box motif). A peptide derived from a specific binding domain may encompass the amino acid sequence of a naturally occurring binding site, any portion of that binding site, or other molecular entity that functions to bind to an associated molecule. A peptide derived from such a binding domain will interact directly or indirectly with an associated molecule in such a way as to mimic the native binding domain. Such peptides may include competitive inhibitors, peptide mimetics, and the like.

[0044] The term “interacting” refers to a stable association between two molecules due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions. Certain interacting molecules interact only after one or more of them has been stimulated. For example, a protein containing an F-box motif may only bind to a substrate if the substrate is phosphorylated (eg. phosphorylated Sic1).

[0045] An enhancer or inhibitor of the interaction of an E2 ubiquitin conjugating enzyme or a F-box binding protein, and a protein of the Cullin family is intended to include a peptide or peptide fragment derived from the binding domain of an E2 ubiquitin conjugating enzyme, an F-box binding protein, or a protein of the Cullin family. The enhancer or inhibitor will not include the full length sequence of the wild-type molecule. Peptide mimetics, synthetic molecules with physical structures designed to mimic structural features of particular peptides, may serve as inhibitors or enhancers. Inhibitors or enhancers affect ubiquitin-dependent proteolysis. The enhancement or inhibition may be direct, or indirect, or by a competitive or non-competitive mechanism.

[0046] “Peptide mimetics” are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports Med. Chem. 24:243-252 for a review ). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Nati. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide, or enhancer or inhibitor of the invention.

[0047] Sequences are “homologous” or considered “homologs” when at least about 70% (preferably at least about 80 to 90%, and most preferably at least 95%) of the nucleotides or amino acids match over a defined length of the molecule. Substantially homologous also includes sequences showing identity to the specified sequence. Preferably, the amino acid or nucleic acid sequences have an alignment score of greater than 5 (in standard deviation units) using the program ALIGN with the mutation gap matrix and a gap penalty of 6 or greater (Dayhoff).

[0048] Peptides of the Invention

[0049] The invention provides peptide molecules which bind to and inhibit the interactions of the molecules in the complexes of the invention. The molecules are derived from the binding domain of an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein; or a protein containing an F-box motif. For example, peptides of the invention include the following amino acids of Cdc53 (see FIG. 8): amino acids 448 to 748 (comprising the binding domain for Cdc34) and amino acids 9 to 280 (comprising the binding domain for Skp1), or portions thereof that bind to Cdc34 and Skp1. Other proteins containing these binding domain sequences may be identified with a protein homology search, for example by searching available databases such as GenBank or SwissProt and various search algorithms and/or programs may be used including FASTA, BLAST (available as a part of the GCG sequence analysis package, University of Wisconsin, Madison, Wis.), or ENTREZ (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.).

[0050] In accordance with an embodiment of the invention; specific peptides are contemplated that mediate the binding of a protein of the Cullin family preferably Cdc53, and an F-box binding protein preferably Skp1.

[0051] Therefore, the invention relates to a peptide of the formula I which interferes with the interaction of Cdc53 and Skp1

A-Tyr-Met-X1-X2-Tyr-X3-X4-X5-Tyr-X6-X7-Cys-X8  I

[0052] wherein A represents one to ten amino acids, X1 represents Met, Arg, Thr, or Glu, X2 represents Leu, Phe, or Val, X3 represents Asp or Thr, X4 represents Ala, Ser, His, or Thr, X5 represents Ile or Val, X6 represents Asn or Asp, X7 represents Tyr, Ile, or Met, and X8 represents Thr, Val, or Ala.

[0053] In an embodiment of the present invention a peptide of the formula Ia which interferes with the interaction of Cdc53 and Skp1 is provided:

A1-A2-A3-A4-A5-A6-Tyr- Met-X1-X2-Tyr-X3-X4-X5-Tyr-X6-X7-Cys-X8  Ia

[0054] wherein A1 represents Ile, Asn, His, Ser, or Ala, A2 represents Leu, Met or Phe, A3 represents Ser, Ala, Thr, or Asp, A4 represents Pro, Lys, Arg, or Ser, A5 represents Thr, Lys, Ser, or Glu, A6 represents Met, Asp, Tyr, Gln, or Arg, X1 represents Met, Arg, Thr, or Glu, X2 represents Leu, Phe, or Val, and X3 represents Asp or Thr, X4 represents Ala, Ser, His, or Thr, X5 represents Ile or Val, X6 represents Asn or Asp, X7 represents Tyr, Ile, or Met, and X8represents Thr, Val or Ala.

[0055] All of the peptides of the invention, as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention. In addition to full-length peptides of the invention, truncations of the peptides are contemplated in the present invention. Truncated peptides may comprise peptides of about 7 to 10 amino acid residues.

[0056] The truncated peptides may have an amino group (—NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end. The truncated peptides may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.

[0057] The peptides of the invention may also include analogs of a peptide of the invention, and/or truncations of the peptide, which may include, but are not limited to the peptide of the invention containing one or more amino acid insertions, additions, or deletions, or both. Analogs of the peptide of the invention exhibit the activity characteristic of the peptide e.g. interference with the interaction of Cdc53 with Skp1, and may further possess additional advantageous features such as increased bioavailability, stability, or reduced host immune recognition.

[0058] One or more amino acid insertions may be introduced into a peptide of the invention. Amino acid insertions may consist of a single amino acid residue or sequential amino acids.

[0059] One or more amino acids, preferably one to five amino acids, may be added to the right or left termini of a peptide of the invention. Deletions may consist of the removal of one or more amino acids, or discrete portions from the peptide sequence. The deleted amino acids may or may not be contiguous. The lower limit length of the resulting analog with a deletion mutation is about 7 amino acids.

[0060] It is anticipated that if amino acids are inserted or deleted in sequences outside the Tyr-Met-X1-X2-Tyr-X3-X4-X5-Tyr sequence that the resulting analog of the peptide will exhibit the activity of a peptide of the invention.

[0061] Preferred peptides of the invention include the following: MEVTAIYNYCV, YMEVTAIYNYCVNKS, ILSPTMYMEVYTAIYNYCVNKS, YMTLYTSVYDYCT, YMTLYTSVYDYCTSIT, MAPKDYMTLYTSVYDYCTSIT, YMMLYDAVYNICT, YMMLYDAVYNICTTTT, HMSKKYYMMLYDAVYNICTTTT, YMRFYTHVYDYCT, YMRFYTHVYDYCTSVS, SLTRSQYMRFYTHVYDYCTSVS, YMELYTHVYNYCT, YMELYTHVYNYCTSVH, SMAKSRYMELYTHVYNYCTSVH, YMMLYTTIYNMCT, YMMLYTTIYNMCTQKP, AFDSEQYMMLYTTIYNMCTQKP, YMELYTAIHNTCA, YMELYTAIHNTCADAS, and GMITFYMELYTAIHNTCADAS.

[0062] The invention also includes a peptide conjugated with a selected protein, or a selectable marker (see below) to produce fusion proteins.

[0063] The peptides of the invention may be prepared using recombinant DNA methods. Accordingly, nucleic acid molecules which encode a peptide of the invention may be incorporated in a known manner into an-appropriate expression vector which ensures good expression of the peptide. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses so long as the vector is compatible with the host cell used. The expression vectors contain a nucleic acid molecule encoding a peptide of the invention and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be obtained from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may also be incorporated into the expression vector.

[0064] The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, &bgr;-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0065] The recombinant expression vectors may also contain genes which encode a fusion portion which provides increased expression of the recombinant peptide; increased solubility of the recombinant peptide; and/or aid in the purification of the recombinant peptide by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be inserted in the recombinant peptide to allow separation of the recombinant peptide from the fusion portion after purification of the fusion protein. Examples of fusion expression vectors include pGEX (Amrad Corp., Melboume, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharrnacia, Piscataway, N.J.) which fuse glutathione S-transferase (GSY), maltose E binding protein, or protein A, respectively, to the recombinant protein.

[0066] Recombinant expression vectors may be introduced into host cells to produce a transformant host cell. Transformant host cells include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to include the introduction of nucleic acid (e.g. a vector) into a cell by one of many techniques known in the art. For example, prokaryotic cells can be transformed with nucleic acid by electroporation or calcium-chloride mediated transformation. Nucleic acid can be introduced into mammalian cells using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells may be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

[0067] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the peptides of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991).

[0068] The peptides of the invention may be tyrosine phosphorylated using the method described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992). For example, tyrosine phosphorylation may be induced by infecting bacteria harbouring a plasmid containing a nucleotide sequence encoding a peptide of the invention, with a &lgr;gt11 bacteriophage encoding the cytoplasmic domain of the Elk tyrosine kinase as a LacZ-Elk fusion. Bacteria containing the plasmid and bacteriophage as a lysogen are isolated. Following induction of the lysogen, the expressed peptide becomes phosphorylated by the Elk tyrosine kinase.

[0069] The peptides of the invention may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles fo Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biologu, suprs, Vol 1, for classical solution synthesis.) By way of example, the peptides may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphotyrosine as the N-fluorenylmethoxy-carbonyl-O-dimethyl phosphono-L-tyrosine derivative.

[0070] N-terminal or C-terminal fusion proteins comprising a peptide of the invention conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the peptide, and the sequence of a selected protein or selectable marker with a desired biological function. The resultant fusion proteins contain the peptide fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0071] Cyclic derivatives of the peptides of the invention are also part of the present invention. Cyclization may allow the peptide to assume a more favorable conformation for association with molecules in complexes of the invention. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The side chains of Tyr and Asn may be linked to form cyclic peptides. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides are contemplated that have a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

[0072] It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.

[0073] Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.

[0074] Peptides that interact with an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein; or a protein containing an F-box motif may be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that bind to particular proteins. Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors. (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0075] Peptides of the invention may be used to identify lead compounds for drug development. The structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds which can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein.

[0076] Information about structure-activity relationships may also be obtained from co-crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison may be used to design compounds expected to possess desired activities.

[0077] The peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.

[0078] The peptides of the invention may be used to prepare monoclonal or polyclonal antibodies. Conventional methods can be used to prepare the antibodies. As to the details relating to the preparation of monoclonal antibodies reference can be made to Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2nd Ed., Academic Press, London, 1986. As discussed below the antibodies may be used to identify proteins binding sites for Skp1.

[0079] The peptides and antibodies specific for the peptides of the invention may be labelled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to the skilled artisan. Labeled antibodies specific for the peptides of the invention may be used to screen for proteins with Skp1 binding sites, and labeled peptides of the invention may be used to screen for Skp1 binding site containing proteins such as Cdc53.

[0080] Computer modelling techniques known in the art may also be used to observe the interaction of a peptide of the invention, and truncations and analogs thereof with a molecule in a complex of the invention e.g. Skp1 (for example, Homology Insight II and Discovery available from BioSym/molecular Simulations, San Diego, Calif., U.S.A.). If computer modelling indicates a strong interaction, the peptide can be synthesized and tested for its ability to interfere with the binding of Cdc53 and Skp1 as discussed above.

[0081] Complexes of the Invention

[0082] The complexes of the invention include the following: (a) a complex comprising an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, and a 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. Complexes also containing molecules that bind to a protein containing an F-box motif (eg. Sic1, Cln, Met 4 or activated forms thereof) are also contemplated. It will be appreciated that the complexes may comprise only the binding domains of the interacting molecules and such other flanking sequences as are necessary to maintain the activity of the complexes.

[0083] The invention also contemplates antibodies specific for complexes of the invention. The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g. a Fab or (Fab)2 fragment), an antibody heavy chain, and antibody light chain, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0084] Antibodies specific for the complexes of the invention may be used to detect the complexes in tissues and to determine their tissue distribution. In vitro and in situ detection methods using the antibodies of the invention may be used to assist in the prognostic and/or diagnostic evaluation of proliferative and/or differentiative disorders. Antibodies specific for the complexes of the invention may also be used therapeutically to decrease the degradation of proteins that interact with F-box containing proteins such as Sic1, Cln, and Met4.

[0085] The complexes of the invention play a central role in ubiquitin-dependent proteolysis and some genetic diseases may include mutations at the binding domain regions of the interacting molecules in the complexes of the invention. Therefore, if a complex of the invention is implicated in a genetic disorder, it may be possible to use PCR to amplify DNA from the binding domains to quickly check if a mutation is contained within one of the domains. Primers can be made corresponding to the flanking regions of the domains and standard sequencing methods can be employed to determine whether a mutation is present. This method does not require prior chromosome mapping of the affected gene and can save time by obviating sequencing the entire gene encoding a defective protein.

[0086] Methods for Identifying or Evaluating Substances/Compounds

[0087] The methods described herein are designed to identify substances that modulate the activity of a complex of the invention thus affecting ubquitin dependent proteolysis. Novel substances are therefore contemplated that bind to molecules in the complexes, or bind to other proteins that interact with the molecules, to compounds that interfere with, or enhance the interaction of the molecules in a complex, or other proteins that interact with the molecules.

[0088] The substances and compounds identified using the methods of the invention include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)2, and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules. The substance or compound may be an endogenous physiological compound or it may be a natural or synthetic compound.

[0089] Substances which modulate the activity of a complex of the invention can be identified based on their ability to bind to a molecule in the complex. Therefore, the invention also provides methods for identifying novel substances which bind molecules in the complex. Substances identified using the methods of the invention may be isolated, cloned and sequenced using conventional techniques.

[0090] Novel substances which can bind with a molecule in a complex of the invention may be identified by reacting one of the molecules with a test substance which potentially binds to the molecule, under conditions which permit the formation of substance-molecule conjugates and removing and/or detecting the conjugates. The conjugates can be detected by assaying for substance-molecule conjugates, for free substance, or for non-complexed molecules. Conditions which permit the formation of substance-molecule conjugates may be selected having regard to factors such as the nature and amounts of the substance and the molecule.

[0091] The substance-molecule conjugate, free substance or non-complexed molecules may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against the molecule or the substance, or labelled molecule, or a labelled substance may be utilized. The antibodies, proteins, or substances may be labelled with a detectable substance as described above.

[0092] A molecule, or complex of the invention, or the substance used in the method of the invention may be insolubilized. For example, a molecule, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc: The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.

[0093] The invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a complex of the invention, by assaying for an agonist or antagonist (i.e.enhancer or inhibitor) of the binding of molecules in the complex. The basic method for evaluating if a compound is an agonist or antagonist of the binding of molecules in a complex of the invention, is to prepare a reaction mixture containing molecules and the substance under conditions which permit the formation of complexes, in the presence of a test compound. The test compound may be initially added to the mixture, or may be added subsequent to the addition of molecules. Control reaction mixtures without the test compound or with a placebo are also prepared. The formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of the molecules. The reactions may be carried out in the liquid phase or the molecules, or test compound may be immobilized as described herein.

[0094] It will be understood that the agonists and antagonists i.e. inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the interacting molecules in the complex including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites.

[0095] The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of molecules in a complex of the invention. Thus, the invention may be used to assay for a compound that competes for the same binding site of a molecule in a complex of the invention.

[0096] The invention also contemplates methods for identifying novel compounds that bind to proteins that interact with a molecule of a complex of the invention. Protein-protein interactions may be identified using conventional methods such as co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Methods may also be employed that result in the simultaneous identification of genes which encode proteins interacting with a molecule. These methods include probing expression libraries with labeled molecules. Additionally, x-ray crystallographic studies may be used as a means of evaluating interactions with substances and molecules. For example, purified recombinant molecules in a complex of the invention when crystallized in a suitable form are amenable to detection of intra-molecular interactions by x-ray crystallography. Spectroscopy may also be used to detect interactions and in particular, Q-TOF instrumentation may be used.

[0097] Two-hybrid systems may also be used to detect protein interactions in vivo. Generally, plasmids are constructed that encode two hybrid proteins. A first hybrid protein consists of the DNA-binding domain of a transcription activator protein fused to a molecule in a complex of the invention, and the second hybrid protein consists of the transcription activator protein's activator domain fused to an unkown protein encoded by a cDNA which has been recombined into the plasmid as part of a cDNA library. The plasmids are transformed into a strain of yeast (e.g. S. cerevisiae) that contains a reporter gene (e.g. lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the transcription activator's binding site. The hybrid proteins alone cannot activate the transcription of the reporter gene. However, interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

[0098] It will be appreciated that fusion proteins and recombinant fusion proteins may be used in the above-described methods. It will also be appreciated that the complexes of the invention may be reconstituted in vitro using recombinant molecules and the effect of a test substance may be evaluated in the reconstituted system.

[0099] The reagents suitable for applying the methods of the invention to evaluate substances and compounds that modulate ubiquitin dependent proteolysis may be packaged into convenient kits providing the necessary materials packaged into suitable containers. The kits may also include suitable supports useful in performing the methods of the invention.

[0100] Compositions and Treatments

[0101] The peptides of the invention, and substances and compounds identified using the methods of the invention may be used to modulate ubiquitin dependent proteolysis, and they may be used to modulate cellular processes such as proliferation, growth, and/or differentiation of cells in which the compounds or substances are introduced. Thus, the substances may be used for the treatment of proliferative disorders including various forms of cancer such as leukemias, lymphomas (Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas, carcinomas of solid tissue, hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx, and lung, genitourinary cancers such as cervical and bladder cancer, breast, ovarian, colon, hematopoietic cancers, head and neck cancers, and nervous system cancers, benign lesions such as papillomas, arthrosclerosis, angiogenesis, and viral infections, in particular HIV infections, psoriasis, bone diseases, fibroproliferative disorders such as involving connective tissue, atherosclerosis and other smooth muscle proliferative disorders, chronic inflammation, and arthropathies such as arthritis. In addition to proliferative disorders, the treatment of differentiative disorders which result from, for example, de-differentiation of tissue which may be accompanied by abnormal reentry into mitosis. Such degenerative disorders that may be treated using the peptides and compositions of the invention include neurodegenerative disorders such as chronic neurodegenerative diseases of the nervous system, including Alzheimer's disease, Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degeneration.

[0102] Accordingly, the peptides, substances, antibodies, and compounds may be formulated into pharmaceutical compositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo. By “biologically compatible form suitable for administration in vivo” is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals. Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0103] The acute substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the compound.

[0104] The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, albeit not exclusively, solutions of the substances or compounds in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[0105] The activity of the substances, compounds, antibodies, and compositions of the invention may be confirmed in animal experimental model systems.

[0106] The invention also provides methods for studying the function of a complex of the invention. Cells, tissues, and non-human animals lacking in the complexes or partially lacking in molecules in the complexes may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations in the molecules. A recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create complex deficient cells, tissues or animals. Null alleles may be generated in cells and may then be used to generate transgenic non-human animals.

[0107] The following non-limiting examples are illustrative of the present invention:

EXAMPLE 1

[0108] The following materials and methods were used in the investigations described in the example.

[0109] Methods

[0110] Plasmids

[0111] Plasmids were constructed using standard molecular cloning techniques (Table 1). For two hybrid screens, the CDC53 open reading frame was cloned into the BamH1 site of pAS2 (provided by S. Elledge) to create a Gal4DBD-Cdc53 fusion. Versions that lacked either the N-terminal 280 residues (Gal4DBD-Cdc53&Dgr;N) or internal residues 581-664 (Gal4DBD-Cdc53&Dgr;K) were created by digestion with NcoI or KpnI respectively and religating. Gal4AD-Cdc4&Dgr;3WD is derived from a truncated CDC4 PCR product (Skowyra et al., 1997) cloned into the BamHI site of pGAD424. To test the Skp1-Met30 interaction in the two hybrid system, a SKP1 fragment was cloned into the BamHI site of pVAD1, to create a VP16-Skp1 fusion. LexA-Met30 derivatives were based on pLEXM30-4 (Thomas et al. 1995). Met30 was tagged at the N-terminus with an HA epitope by insertion of a MET30 fragment encoding amino acids 7-640 from pLEXM30-4 into a pADH1-HA expression plasmid (Li and Johnston 1997). Cdc4 was tagged at the N-terminus with a FLAG epitope by site directed mutagenesis. A CDC53 deletion construct was made by replacing an internal BgIII fragment of pGEM53-8 (Mathias et al. 1996) with an ADE2 fragment. To allow for negative selection of wild type CDC53 in the cdc53&Dgr; shuffle strain a 3.6 kbp EcoRI fragment of CDC53 was cloned into a <URA3 CEN> plasmid. Charged to alanine mutagenesis of Cdc53 was carried out in pMT843, as described previously (Willems et al. 1996). Although none of the mutations caused any obvious phenotype, restriction sites incorporated during mutagenesis were used to construct the deletions shown in FIG. 5. The version of Cdc53 in which all six cysteine residues are replaced by alanines (C59A, C157A, C412A, C606A, C754A, C774A) was created in a single site-directed mutagenesis reaction.

[0112] Yeast Strains and Culture

[0113] Yeast strains are listed in Table 2. All strains were isogenic with the W303 background. Standard methods were used for yeast culture and transformation (Kaiser et al. 1994). A cdc53&Dgr; shuffle strain was constructed by deleting one copy of CDC53 with pMT1514 in K699 a/&agr; transforming with pMT951, sporulating and isolating a Ura+Ade+ segregant. Complementation of the shuffle strain by various <CDC53M CEN> plasmids was tested by plating on 0.1% FOA medium. cdc53 Skp1 double mutants were generated in crosses of MTY871 with Y553 and Y555 (Bai et al. 1996). The cln2::pGAL1-CLN2M -LEU2 strain was created by integrating pMT1111 into K699a. The MET30-1 strain (CC786-1A) was created by crossing W303-1B with CM100-1A (Thomas et al. 1995). MET25 mRNA expression was assayed in cultures grown in B media supplemented with 0.1 mM sulfate as the sulfur source (Thomas et al. 1995). At a density of 0.5×107 cells/ml cultures were shifted to 37° C. for 2 hours, repressed with 1.0 mM methionine, and time points taken for RNA extraction Cln2 halflife was determined in pGAL1-CLN2HA strains as described previously (Willems et al. 1996).

[0114] Two Hybrid Analysis

[0115] Strain Y187 expressing a Gal4DBD fusion was transformed with a yeast Gal4AD-cDNA library (provided by S. Elledge) or a Gal4AD genomic DNA library (James et al. 1996) and screened as described (Durfee et al. 1993). With the cDNA library, Gal4DBD-Cdc53 recovered 1 positive clone (A10) from 140,000 transformants, and Gal4DBD-Cdc53&Dgr;K recovered 2 positive clones (C23 and C24) from 225,000 transformants. Gal4DBD-Cdc53&Dgr;N recovered no positive clones from 427,000 transformants. With the genomic DNA library, Gal4DBD-Cdc53 recovered 5 positive clones (F15, F19, F20, F23, F24) from 1 million transformants and Gal4DBD-Cdc53&Dgr;K recovered 7 positive clones (H1, H6, H8. H9, H11, H13, H17) from 500,000 transformants. Some clones were isolated several independent times but all unique clones are represented in FIG. 1.

[0116] Protein and RNA Analysis

[0117] Preparation of yeast lysates and analysis of total RNA were carried out as described previously (Willems et al. 1996). Northern blots were probed with a 1.3 kbp MET25 fragment and a 0.6 kbp ACTI fragment. mRNA abundance was quantitated on a Molecular Dynamics Storm Phosphorlmager. Immunoblots were processed for ECL detection as described (Willems et al. 1996) and where indicated signals were quantitated by densitometry. Affinity purified anti-Cdc4, anti-Cdc34 and anti-Cdc53 antibodies (provided by M. Goebl), and anti-Cdc28 antibodies (Tyers et al. 1992) were used at dilutions between 1:100 to 1:1,000, depending on the particular antibody. Anti-Skp1 antibodies were used at 1:1,000 (Bai et al. 1996). Anti-Grr1 antibodies were adsorbed against polyacrylamide to eliminate background binding and used at 1:100 (Flick and Johnston 1991). Anti-Met30 antibodies were raised against recombinant Gst-Met30 (residues 297-640 encompassing the WD40 repeats), affinity purified and used at a dilution of 1:100. The 9E10 anti-MYC and 12CA5 anti-HA monoclonal antibodies were produced as ascites fluid and used at 1:10,000. Anti-FLAG M2 antibody conjugated to Sepharose beads was from Kodak. HRP-conjugated secondary antibodies (Amersham) were used at a dilution of 1:10,000.

[0118] Sequence Analysis

[0119] Regions of sequence conservation between Cdc53 homologs identified in database searches were determined by amino acid alignment with ClustalW (Thompson 1994). Conserved residues with a weight of 10 or higher were identified by analysis of 15 full length homologs with the Wisconsin Package program Pretty. Black lines in FIG. 5B indicate the central residue of an 11 residue window containing four or more such conserved residues.

[0120] Results

[0121] Interactions of Cdc53, Skp1, Cdc4 and Met30 in the Two Hybrid System

[0122] To identify proteins that interact with Cdc53, two hybrid screens were carried out with full length Cdc53 and two Cdc53 deletion mutants (FIG. 1A). Two Cdc53 fusion proteins, Gal4DBD-Cdc53 and Gal4DBD-Cdc53&Dgr;K, recovered multiple independent isolates of Skp1, Cdc4 and Met30 from Gal4AD genomic and cDNA libraries (FIG. 1B, 1C). None of the positive clones recovered interacted with Gal4DBD-Cdc53&Dgr;N, suggesting that the N-terminal region of Cdc53 was important for these interactions (see below). Met30 was originally isolated as a methionine-dependent repressor of methionine biosynthesis gene expression, and has a similar overall structure as Cdc4, with an N-terminal F-box and C-terminal WD40 repeats (Thomas et al. 1995; Bai et al. 1996). All of the Met30 and Cdc4 isolates that interacted with Cdc53 contained the F-box motif, suggesting the F-box may mediate interactions with Cdc53. In fact, two of three independent Met30 isolates contained just the F-box and a small amount of flanking region (FIG. 1C). Similarly, three independent Cdc4 isolates encompassed the F-box but lacked more N-terminal sequences. Cdc4 and Met30 isolates missing some or all of the WD40 repeats did however interact more weakly with Cdc53 than the full length proteins (FIG. 1B, C), which may reflect an auxiliary role for the WD40 repeats. Since Cdc4 binds Skp1 via the F-box motif (Bai et al. 1996), a Met30-Skp1 interaction was directly tested for in the two hybrid system. The F-box of Met30 was both necessary and sufficient for interaction of Met30 with Skp1 (FIG. 1D). As for the Cdc53-Met30 interaction, the WD40 repeats of Met30 were required for maximal interaction with Skp1. In summary, two hybrid analysis revealed a Cdc53-Skp1 interaction and suggested the possibility that Cdc53-F-box protein interactions may be bridged by Skp1.

[0123] Cdc53 and Skp1 Interact Genetically

[0124] To assess the in vivo relevance of the Cdc53-SkpI two hybrid interaction,-genetic interactions were tested between CDC53 and SKP1. The cdc53-1 mutation was combined with the Skp1-11 and Skp1-12 mutations. At a semi-permissive temperature of 30° C. both tht cdc53-1 Skp1-11 and cdc53-1 Skp1-12 double mutants were inviable, whereas either single mutant grew as well as the wild type strain (FIG. 2A). Even at a permissive temperature of 25° C., cdc53-1 Skp1-12 double mutants had a severe growth defect, and accumulated multiple hyperpolarized buds (FIG. 2B), akin to the arrest phenotype of single mutants in the Cdc34 pathway (Mathias et al. 1996). In addition, overproduction of CDC53 was found to rescue Skp1 temperature sensitive strains (E. Patton, unpublished data), as reported elsewhere (Skowrya et al. 1997). This genetic evidence suggests that the Cdc53-Skp1 two hybrid interaction reflects a common function of Cdc53 and Skp1 in vivo.

[0125] Cdc53 Associates with Skp1 and Cdc4 in Yeast Lysates

[0126] Next it was determined whether endogenous levels of Cdc53 and Skp1 form a complex in yeast lysates. To minimize possible disruption of complexes by antibodies, epitope-tagged versions of Cdc53 and Skp1 were used. Immunoprecipitation of MYC-tagged Cdc53, followed by immunoblotting with polyclonal antibodies directed against Skp1, revealed a specific association between Cdc53 and Skp1 (FIG. 3A, lane 2). Cdc4 and Cdc34 were also present in the Cdc53 complexes, consistent with the observation that Cdc4 and Cdc53 cofractionate with polyhistidine tagged Cdc34 (Mathias et al. 1996). In the reciprocal coimmunoprecipitation experiment, Cdc53 specifically associated with HA-tagged Skp1, as did Cdc4 and Cdc34 (FIG. 3B, lane 2). Taken together, these results indicate that Cdc53 likely forms a multiprotein complex in vivo with Skp1, Cdc4 and Cdc34.

[0127] To determine if any of these protein-protein interactions correlated with function in vivo, the composition of the Cdc53 complex was examined in various temperature sensitive strains. In one set of experiments, Cdc53 immune complexes were immunoblotted with anti-Cdc4, anti-Cdc34 and anti-Skp1 antibodies (FIG. 3A). In cdc4-1 and Skp1-11 mutants, Cdc4 was not detected in Cdc53 immune complexes. Although this observation was consistent with a bridging role for Skp1, the absence of Cdc4 from the complexes was due at least in part to decreased Cdc4 abundance in the mutants (see FIG. 3C). The Skp1-12 mutation severely decreased the abundance of Cdc4, Cdc53 and Skp1 itself, and so the absence of associated proteins in Cdc53 complexes from Skp1-12 cells was not informative.

[0128] In another set of experiments, Skp1 immune complexes from temperature sensitive strains were immunoblotted with anti-Cdc4, anti-Cdc34 and anti-Cdc53 antibodies (FIG. 3B). In this configuration, the amount of Cdc4 in the complex was also reduced by the cdc4-1 mutation. In contrast, the amount of Cdc4 in the complex was increased by both the cdc34-2 and cdc53-1 mutations. Relative to the abundance of Cdc34 in lysates, the amount of Cdc34 in Skp1 complexes was severely compromised by the cdc53-1 mutation. Cdc53 may therefore bridge the Cdc34-Skp1 interaction (see below).

[0129] As the anti-Cdc4 antibodies used could not reliably detect Cdc4 in yeast lysates, it was not possible to determine directly if the Skp1 mutations reduced the abundance of Cdc4. However, immunoprecipitation of a FLAG-tagged version of Cdc4 followed by immunoblotting with anti-Cdc4 polyclonal antibody revealed that Cdc4 abundance is greatly diminished in Skp1-11 and Skp1-12 strains (FIG. 3C). The abundance of another F-box protein, Met30, was similarly reduced by the Skp1-11 and Skp1-12 mutations (FIG. 3C). As noted above, the abundance of Cdc53 is also decreased by the Skp1-12 mutation. Thus, Skp1 may function at least in part to stabilize both Cdc53 and F-box proteins. Overall, each of temperature sensitive mutations perturbs the mutual interactions, by altering the abundance of a given component in lysates and/or the immune complexes.

[0130] Cdc53 Interacts with Two Other F Box Proteins, Met30 and Grr1

[0131] To corroborate the Cdc53-Met30 and Skp1-Met30 two hybrid interactions, studies were carried out to determine if Met30 formed complexes with Cdc53 and Skp1 in yeast lysates. For this purpose an HA-tagged version of Met30 expressed from the constitutive ADH1 promoter was used. Immunoprecipitation of Met30 followed by immunoblotting against Cdc53 and Skp1 revealed the presence of both Cdc53 and Skp1 in Met30 immune complexes (FIG. 4A).

[0132] Because several lines of evidence suggest that Grr1 may function with Skp1 and Cdc53 to mediate Cln1/2 degradation (Barral et al. 1995; Bai et al. 1996; Willems et al. 1996), studies were carried out to test if Grr1 interacts with Cdc53. Indeed, both Cdc53 and Skp1 were specifically immunoprecipitated with an HA-tagged version of Grr1 (FIG. 4A). In a control experiment, FLAG-tagged Cdc4 immune complexes also contained Cdc53 and Skp1, thereby completing the set of pairwise coimmunoprecipitations between Cdc4, Cdc34, Cdc53 and Skp1 (FIG. 3A, B; Mathias et al. 1996). It was not possible to reproducibly detect Cdc34 in the F-box protein immune complexes, perhaps because each of these complexes necessarily contains only a fraction of the total Cdc34, Cdc53 and Skp1. Within the limits of the antibodies it was not possible to detect Cdc4 in Met30 and Grr1 immune complexes, suggesting that F-box proteins form mutually exclusive complexes (data not shown). Thus, Skp1 and Cdc53 form independent complexes with at least three different F-box proteins in vivo.

[0133] The ability of Cdc53 to interact with multiple F-box proteins raised the possibility that different F-box proteins may compete for binding to a Cdc34-Cdc53-Skp1 core complex. This possibility was tested by overexpressing MET30 or GRR1 in cdc4-1, cdc34-2 and cdc53-1 temperature sensitive strains. Overexpression of MET30 dramatically impaired growth of a cdc4-1 strain at 30° C., and caused a mild growth defect in cdc53 and cdc34 strains (FIG. 4B) but had no effect on either Skp1-11 or Skp1-12 strains (data not shown). Although overexpression of GRR1 did not affect growth of a cdc4-1 strain, the growth of cdc34-2 and cdc53-1 strains was retarded at 30° C. (FIG. 4B). It has been noted previously that high level expression of GRR1 is lethal in Skp1-12 strains at 30° C. (Li and Johnston 1997), and high level expression of Cdc4 causes inviability of cdc34 and cdc53 strains at 23° C. (Mathias et al. 1996). Taken together, the above results suggest various F-box proteins may compete for binding to a core Cdc34-Cdc53-Skp1 complex in vivo, and that the relative stoichiometry of the various complexes is critical for viability.

[0134] Cdc53 is a Scaffold Protein for Cdc34 and Skp1-F-Box Protein Complexes

[0135] To identify potential protein-protein interaction domains of Cdc53, a series of Cdc53 deletion mutants were constructed using natural and engineered restriction sites (see Methods). Each of the mutant proteins was expressed to similar levels as wild type Cdc53 (FIG. 5A). The ability of each Cdc53 mutant protein to interact with Cdc34. Skp1 and the three F-box proteins Cdc4, Grr1, Met30 was assessed by immunoblot analysis of MYC-tagged Cdc53 immune complexes with specific polyclonal antibodies (FIG. 5A). In this experiment, each of the interactions detected involved approximately wild type levels of Cdc53 (which was expressed from a low copy plasmid) and endogenous levels of each of the associated proteins. Deletion of an N-terminal region of Cdc53 (residues 9-280) completely disrupted Skp1 binding. In parallel, the binding of all three F-box proteins was specifically disrupted. Importantly, Cdc34 still interacted with Cdc53&Dgr;9-280, eliminating the possibility that the truncated protein was simply misfolded and entirely non-functional. Conversely, deletion of an internal region of Cdc53 (residues 448-748) abrogated Cdc34 binding but did not affect binding of Skp1 or any of the F-box proteins. The strict correlation between the Cdc53-Skp1 interaction and Cdc53-F-box protein interactions is most easily explained by a bridging function for Skp1. Furthermore, the independent non-overlapping binding regions in Cdc53 indicate that the protein-protein interactions within Cdc53 complexes occur in a modular fashion.

[0136] Importantly, Cdc53 mutants that were unable to bind either Skp1/F-box proteins or Cdc34 could not complement a cdc53 deletion strain, while mutants unaffected in protein-protein interactions could complement (FIG. 5B). In order to determine if the Skp1/F-box protein and Cdc34 binding domains of Cdc53 corresponded to conserved regions of Cdc53, 15 different members-of the Cdc53 family were aligned (FIG. 5B, see Methods for details of the sequence alignment). Sequence similarity within the Cdc53 family is restricted to a broad internal region and a narrow region at the extreme C-terminus. Surprisingly, the latter region is not required for binding to Skp1/F-box proteins or Cdc34, nor for viability (FIG. 5A, B). However, the internal conserved region overlaps with the Cdc34 binding site. There is relatively poor conservation in the N-terminus of Cdc53, despite the fact that this region contains the Skp1 binding site. The interaction with Skp1 may possibly be limited to a subset of the Cdc53 family.

[0137] Based on the sequence alignment many conserved charged residues in Cdc53 were mutated to alanines but none of the mutants had any overt phenotype. For instance, mutation of the most conserved stretch in the entire protein, IVRIMK (residues 755-760), to polyalanine did not cause an obvious defect in Cdc53 function or in binding to Skp1/F-box proteins or Cdc34 (FIG. 5A, B). To further explore the structure/function relationship of Cdc53, the sequence of two temperature sensitive alleles of CDC53 were determined. The cdc53-1 mutation causes an R488C substitution while the cdc53-2 mutation causes a G340D substitution. Both mutations alter highly conserved residues, even though G340 does not lie within a window of conserved residues. Interestingly, the cdc53-1 mutation occurs within the Cdc34 binding region. In conjunction with the defective Skp1-Cdc34 interaction in cdc53-1 strains (FIG. 3B), this result strongly suggests that the cdc53-1 mutation specifically perturbs the Cdc34 binding site.

[0138] In addition to target protein recognition, some E3 ligases form ubiquitin thioester intermediates on catalytic cysteine residues (Scheffner et al. 1995). As Cdc53 is a component of an E3 ligase complex, studies were carried out to determine whether any of the cysteine residues in the Cdc53 sequence were required for function in vivo. Simultaneous mutation of all six cysteine residues in Cdc53 to alanine did not impair complementation of a cdc53 deletion strain (FIG. 5C). Although this mutational analysis does not rule out thioester formation on Cdc53, such reactions cannot be essential for viability. The primary function of Cdc53 is therefore to act as a scaffold protein for Skp1/F-box proteins and Cdc34.

[0139] Cdc34, Cdc53 and Skp1 are Mediators of Methionine Repression

[0140] To assess the biological significance of the Cdc53-Met30 and Skp1-Met30 interactions, experiments were carried out to determined if Cdc34, Cdc53, or Skp1 were required for proper regulation of methionine biosynthesis genes. The regulation of MET25, which encodes homocysteine synthase and is representative of methionine regulated genes was examined. MET25 is activated by the Cbf1-Met4-Met28 transcriptional complex and repressed by Met30 (Thomas et al. 1995; Kuras et al. 1996). As expected, methionine repressed MET25 expression in wild type cells (FIG. 6A). As MET30 is an essential gene, an antimorphic allele called MET30-1 was used as a positive control for methionine derepression (Thomas et al. 1995). As shown previously, MET25 is incompletely repressed by methionine in MET30-1 cells. Strikingly, repression of MET25 by methionine was severely compromised in cdc53-1 cells and completely defective in cdc34-2, Skp1-11 and Skp1-12 cells (FIG. 6A). In contrast, MET25 was effectively repressed with wild type kinetics in cdc4-1 cells, thereby demonstrating the specificity of F-box protein function in methionine biosynthesis gene regulation. The derepression of MET25 observed in cdc34, cdc53 and Skp1 mutants did not depend on G1 phase cell cycle arrest because derepression did not occur in cdc4-1 cells which arrest at the identical point in G1, and yet did occur in Skp1-12 mutants which arrest in G2 phase.

[0141] Specificity of F-Box Protein Function in Cln2 Degradation

[0142] It has been shown previously that Cln2 is stabilized in Grr1&Dgr;, cdc34-2, cdc53-1 and Skp1-12 strains (Barral et al. 1995; Deshaies et al. 1995; Bai et al. 1996; Willems et al. 1996). To directly assess the specificity of F-box protein function in Cln2 degradation, the halflife of Cln2 in cdc4-1, Grr1&Dgr; and MET30-1 strains was compared. We used glucose repression of a pGAL1-CLN2HA construct to measure Cln2 decay rates, as described previously (Willems et al. 1996). Cln2 was strongly stabilized in Grr1&Dgr;cells, slightly stabilized in cdc4-1 cells and not stabilized at all in MET30-1 cells (FIG. 6B). Thus Grr1 is the primary mediator of Cln2 degradation, at least under the conditions employed in these experiments. In contrast to Cln2 degradation, and consistent with previous results (Schwob et al. 1994; Bai et al. 1996), Sic1 degradation was found to require Cdc4, but not Grr1 or Met30 (E. Patton, unpublished data).

[0143] Discussion

[0144] Modular Protein-Protein Interactions Allow Combinatorial Control of Skp1-Cdc53-F-Box Protein (SCF) Complexes

[0145] Cdc53 was shown to form a multiprotein complex in vivo with Cdc4, Cdc34 and Skp1. Furthermore, two other F-box proteins, Grr1 and Met30 , form analogous complexes with Skp 1 and Cdc53. Consistent with these in vivo observations, recombinant Cdc4 and Grr1 assemble into a complex with Cdc34, Cdc53 and Skp1 (Skowrya et al. 1997). To simplify description of the various F-box containing complexes, the generic term SCF, for Skp1-Cdc53-F-box protein complex has been adopted(Skowrya et al. 1997; Feldman et al. 1997). The specific F-box complexes described above are thus designated SCFCdc4, SCFGrr1 and SCFMet30. Formally, SCF complexes are E3 ubiquitin-ligases, as they interact with both substrates and an E2 enzyme, Cdc34 (Willems et al., 1996; Feldman et al. 1997; Skowrya et al. 1997). In another sense, the Cdc34-Cdc53-Skp1 triad forms a core complex that adapts to various F-box proteins via Skp1; this complex is referred to as the E2/E3 core complex.

[0146] Substantial evidence indicates that Skp1 bridges F-box proteins to Cdc53. First, the F-box of Met30 is sufficient for interaction with Skp1 and Cdc53 in the two hybrid system. Second, analysis of Cdc53 complexes from Skp1 and cdc4 strains shows that Cdc4 is dispensable for the Cdc53-Skp1 interaction. Third, deletion analysis of Cdc53 reveals that the interaction domain for Skp1 matches that of three different F-box proteins, Cdc4, Grr1 and Met30. Furthermore, like the Cdc4-Skp1 interaction, the Cdc53-Skp1 interaction occurs in the absence of other proteins in vitro (Skowrya et al. 1997). However, the interaction of Skp1 with Grr1 in the two hybrid system requires both the F-box and the leucine rich repeats of Grr1 (Li and Johnston 1997). Similarly, the WD40 repeats of Cdc4 and Met30 are required for maximal interaction with Skp1. Overall, it is certain that one function of Skp1 is to help recruit F-box proteins to Cdc53 complexes, perhaps in conjunction with other domains. As noted above, Skp1 may also be required for stabilization of F-box proteins and Cdc53 in vivo.

[0147] In addition to the Skp1-F-box interaction, protein-protein interactions within the E2/E3 core complex are of a modular nature. Skp1 binds to the N-terminal region of Cdc53, whereas Cdc34 binds a conserved internal region of Cdc53. The modular nature of these protein-protein interactions and the absence of cysteine-dependent functions in vivo indicates that Cdc53 is a scaffold protein that may anchor Cdc34, Skp1, F-box protein and substrate in the appropriate orientation for ubiquitin transfer.

[0148] F-Box Proteins Confer Specificity on SCF Function

[0149] Despite identification of Cdc34, Cdc53 and Skp1 through defects in Sic1 degradation, it is now clear that SCF complexes also control Cln degradation, glucose repression and methionine repression. SCFCdc4 regulates the G1 to S phase transition through proteolysis of several key cell cycle regulators. The dramatic cell cycle arrest phenotype caused by loss of SCFCdc4 obscures the pleiotropic functions of the E2/E3 core complex, despite the fact that Met30 and Grr1 play crucial roles in cellular metabolism (Flick and Johnston 1991; Thomas et al. 1995). SCFGrr1 has a role in both nutrient sensing and cell division, through regulation of glucose repression and Cln degradation, respectively (Flick and Johnston 1991; Barral et al. 1995; Li and Johnston 1997). The present inventors have discovered the existence of a third SCF complex, SCFMet30, and demonstrated that in addition to Met30, each component of the E2/E3 core complex is required for regulation of methionine biosynthesis genes.

[0150] The specificity of each SCF complex for different cellular processes is demonstrated by a remarkable absence of cross-talk between some of the pathways. For instance, the cdc4-1 mutation does not affect MET25 repression and conversely, the MET30-1 mutation does not affect Cln2 degradation. Although Cdc4 appears not to mediate Cln2 degradation under the experimental conditions employed here, Cdc4 does interact weakly with Cln2 (Skowrya et al. 1997), so a role for Cdc4 in Cln degradation should not yet be excluded. The growth defects caused by high level expression of CDC4, GRR1 or MET30 in various SCF mutants suggests that different F-box proteins may be in equilibrium with a limiting amount of the E2/E3 core complex. If this is so, then F-box proteins may themselves be subject to stringent regulation. The decreased abundance of Cdc4 and Met30 in Skp1 temperature sensitive strains is consistent with this possibility, as is the regulation of Grr1 abundance by glucose (Li and Johnston 1997).

[0151] It is likely that other SCF complexes regulate yet other processes in yeast. A possible G2 function is suggested by the G2 arrest phenotype of Skp1-12 cells (Bai et al. 1996), and by interactions of Skp1 with the Cbf3 kinetochore complex (Connelly and Hieter 1996; Stemmann and Lechner 1996). Finally, because yeast contains two Cdc53 homologs and one Skp1 homolog, orthologous SCF pathways may also exist.

[0152] Substrates of SCF Complexes

[0153] To date, only Sic1 has been unequivocally identified as a direct target for ubiquitination by a SCF complex. Reconstitution of phosphorylation dependent Sic1 ubiquitination has been achieved in vitro, in both a yeast extract system and in a purified system with recombinant proteins (Verma et al. 1997, Skowrya et al. 1997; Feldman et al. 1997). Strong circumstantial evidence suggests that, in addition to Sic1, SCFCdc4 also targets Fab, Cdc6 and Gcn4 for degradation (Henchoz et al. 1997; McKinney and Cross 1994; Piatti et al. 1996; D. Kornitzer, personal communication). Although ubiquitination of Cln1/2 has not yet been reconstituted, SCFGrr1 specifically binds to phosphorylated Cln1/2, consistent with Grr1-dependent degradation of Cln1/2 in vivo (Skowyra et al., 1997). Genetic analysis suggests that a negative regulator of glucose repressed genes called Rgt1 could be a possible target of the SCFGrr1 complex (Erickson and Johnston 1994; Vallier et al. 1994). However, it is not known if Rgt1 physically interacts with Grr1, nor if Rgt1 is regulated by ubiquitin dependent proteolysis. The requirement for SCFMet30 function in methionine repression implicates ubiquitin-dependent proteolysis. Because Met30 forms a complex with the transactivator Met4, it is possible that Met30 targets Met4 for degradation, although other components of the Met4 transcriptional complex, Cbf1 and Met28, are also candidate targets (Kuras et al. 1996). The mechanisms whereby SCF complex activity is regulated in response to glucose and methionine are unknown, but could involve phosphorylation, subcellular localization, F-box protein abundance and complex assembly (Li and Johnston 1997; Pause et al. 1997).

[0154] SCF Complexes in Other Species

[0155] SCF complexes have recently emerged as key regulators in other organisms. In S. pombe, a Cdc4 homolog, pop1, controls the initiation of S phase by targeting the Cdk inhibitor rum1 and the Cdc6 homolog cdc18 for ubiquitin-dependent proteolysis (Kominami and Toda 1997). In C. elegans, null mutants of a Cdc53 homolog called Cul-1 cause hyperplasia in all tissues, suggesting that it too may target activators of division for degradation (Kipreos et al. 1996). In human cells, Skp1 binds to cyclin A-Cdk2 through its associated F-box protein, Skp2, (Zhang et al. 1995) and also forms a specific complex with human Cul-1 (Y. Xiong, personal communication). Another human cullin, Cul-2, physically associates with the VHL tumour suppressor protein, and may thus also regulate cell proliferation (Pause et al. 1997). As in yeast, degradation of mammalian G1 cyclins and Cdk inhibitors is phosphorylation and ubiquitin dependent (Clurman et al. 1996; Won and Reed 1996; Diehl et al. 1997; Sheaff et al. 1997) and so it will be of prime importance to determine the role of SCF complexes in these pathways. The control of gene expression by proteolysis is now well documented in several systems (Pahl and Baeuerle 1996), and by analogy to glucose and methionine regulation in yeast, SCF complexes may prove to be general transcriptional regulators. Indeed, the Met30 homologs Scon2/SconB and the Skp1 homolog SconC regulate sulfur metabolism in other fungi (Natorff et al. 1993; Kumar and Paietta 1995), suggesting that control of methionine biosynthesis by SCF complexes may be conserved. As metazoans contain at least six Cdc53 homologs (Kipreos et al. 1996), and as SCF complexes control multiple processes in yeast, it is likely that analogous SCF complexes will have both cell cycle and non-cell cycle functions in higher species. The combinatorial control of SCF ubiquitin-ligase complexes provides an adaptable regulatory system that controls cell function through ubiquitin-dependent protein degradation.

EXAMPLE 2

Identification of a Skp1 Binding Region in the Cdc53 Protein of the Budding Yeast Saccharomyces cerevisiae

[0156] Amino acids 9-280 of the Cdc53 protein in Saccharomyces cerevisiae have been shown to be necessary for association with Skp1 (Patton et al, 1998 Genes Dev. 12:692). Similar experiments have narrowed the region necessary for the Cdc53/Skp1 interaction to amino acids 9 to 99 of Cdc53. The amino acid sequence of this region of Cdc53 was aligned with other Cdc53 cullin homologues (FIG. 11). The amino acids corresponding to tyrosine (Y) 48 and methionine (M) 49 in Cdc53 were absolutely conserved in a subset of homologues more closely related to Cdc53 but not present in any of the other more distantly related homologues (with the exception of Y50 in C3A11.08 in Schizosaccharomyces pombe). These two amino acids in Cdc53 were mutated by Kunkel mutagenesis to tryptophan (W) and glutamic acid (E) respectively, the homologous residues in Caenorhabditus elegans Cul-3. Five independent TRP1 plasmid isolates (pMT1939-1943) of this mutagenesis reaction, as well as a plasmid carrying a wild type CDC53 gene (pMT843), were transformed into a CDC53 “shuffle strain” (strain Mty1243, genotype ura3 trp1 leu2 his3 ade2 cdc53::ADE2<CDC53HA3 URA3 CEN6 ARSH4>). These mutant and wild type CDC53 genes were tagged with a MYC6 epitope. These strains were grown on complete minimal medium agar plates containing 1 g/L of 5-fluoroorotic acid, which kills cells that produce Ura3, thus killing any cells that do not lose the <CDC53HA3 URA3> plasmid. None of the five CDC53-Y48W,M49EMYC6 mutants conferred viability on the shuffle strain, while the wild type CDC53MYC6 did. Cells containing either the wild type CDC53MYC6 or one of three isolates of the mutant CDC53MYC6 were grown to late-log phase (2×107 cells/ml), harvested, washed, resuspended in lysis buffer (50 mM Tris-Cl pH 7.5, 250 mM NaCl, 50M NaF, 5 mM EDTA, 0.1% NP-40, 1 mM DTT) plus protease inhibitors, snap frozen in liquid nitrogen, and ground into a powder under liquid nitrogen. This powder was thawed, spun to remove cellullar debris, and cleared by spinning in an ultracentrifuge. Protein concentration in the lysate was adjusted to 24 mg/ml in a volume of 1.25 ml lysis buffer +10 mM N-ethyl maleimide, for a final mass of 30 mg protein for each strain. 25 &mgr;l of a 50% slurry in lysis buffer of protein-A beads (Pierce) cross-linked with dimethyl suberimidate to the anti-MYC monoclonal antibody 9E10 was added to each lysate, incubated with gentle rocking at 4° C. for several hours, washed several times with lysis buffer, aspirated, resuspended in protein sample buffer, and run on two 10% polyacrylamide gels. One gel was transferred to polyvinylidene fluoride membrane and western blotted with anti-MYC, anti-Cdc34, and anti-Skp1 antibodies (FIG. 12A). The mutant Cdc53 still binds to Cdc34 but not to Skp1. The second gel was silver stained (see W. Wray et al. 1981, Anal. Biochem. 118:197) (FIG. 12B). A number of bands that are present in the Cdc53MYC6 immunoprecipitation disappear in the Cdc53-Y48W, M49EMYC6 immunoprecipitation.

[0157] Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. All modifications coming within the scope of the following claims are claimed.

[0158] All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. 1 TABLE 1 Plasmids used in this study Plasmid Relevant characteristics Source pMT634 pGAL1-CLN2-HA LEU2 URA3 CEN Willems et al. (1997) pMT817 CDC53-C-NorI TRP1 CEN Willems et al. (1996) pMT843 CDC53M TRP1 CEN Willems et al. (1996) pMT915 GAL4 AD-CDC4&Dgr;3WD LEU2 2&mgr;m This study pMT918 CDC53 in pAS1-CYH2 TRP1 2&mgr;m This study pMT951 CDC53HA URA3 CEN This study pMT954 pGAL4DBD-CDC53 &Dgr;N TRP1 2&mgr;m This study pMT955 pGAL4DBD-CDC53 &Dgr;K TRP1 2&mgr;m This study pMT1111 pUC119 cln2::GAL-CLN2M-LEU2 B. Schneider pMT1511 SKP1HA LEU2 CEN P. Heiter pMT1514 cdc53::ADE2 in pGEM3 This study pBF339 pADH1HA TRP1 2&mgr;m Li and Johnston (1997) pBF494 pADH1HA-GRR1&Dgr;N TRP1 2&mgr;m Li and Johnston (1997) pMT1707 pADH1HA-MET3O TRP1 2&mgr;m This study pMT1850 CDC53M&Dgr;9-280 TRP1 CEN This study pMT1854 CDC53M&Dgr;448-763,H767A TRP1 CEN This study pMT1856 CDC53M&Dgr;448-748 TRP1 CEN This study pMT1857 CDC53M&Dgr;757-815 TRP1 CEN This study pMT1858 CDC53M&Dgr;794-815 TRP1 CEN This study pMT1859 CDC53MIVRIMK TRP1 CEN This study pMT1861 CDC53M6CTRP1 CEN This study pLexM30-4 pLEXADBD-MET30 TRP1 2&mgr;m This study pLexM30-4(297-540) pLEXADBD-MET30 &Dgr;297-540 TRP1 2&mgr;m This study pLexM30-4(158-297) pLEXADBD-MET30 &Dgr;158-297 TRP1 2&mgr;m This study pLexM30-4(158-540) pLEXADBD-MET30 &Dgr;158-540 TRP1 2&mgr;m This study pVAD1-SKP1 pVAD-SKP1 LEU2 2&mgr;m This study pSE1111 GAL4AD-SNF1 LEU2 2&mgr;m S. Elledge pSE1112 GAL4DBD-SNF4 TRP1 2&mgr;m S. Elledge pRS314 TRP1 CEN Sikorski and Hieter (1989)

[0159] 2 TABLE 2 Yeast strains used in this study Strain Relevant genotype Source K699 MATa ade2-1 can1-100 his3-1,15S leu2-3,112 trp1-1 ura3 K. Nasmyth K699 a/&agr; MATa/MAT&agr; ade2-1/ade2-1 can1-100/can1-100 his3-1,15/his3-1,15 K. Nasmyth leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3/ura3 MTY668 MATa cdc4-1 This study MTY670 MATacdc34-2 Willems at el. (1996) MTY871 MATa cdc53-1 Willems at el. (1996) MTY1243 cdc53::ADE2, pMT951 plasmid This study MTY1293 cdc53-1 skp1-11 This study MTY1294 cdc53-1 skp1-12 This study MTY1295 cln2::pGAL1-CLN2M LEU2 This study Y187 MATa ade2-101 his3-&Dgr;200 leu2-3,112 trp1-901 ura3-52 gal4&Dgr;gal80&Dgr; S. Elledge URA3::GAL-lacZ LYS2::GAL-HIS3 Y190 as for Y187 but MAT&agr; S. Elledge Y553 MAT&agr; skp1-11 Bai et al. (1996) Y555 Mat&agr; skp1-12 Bai et al. (1996) WX131-2c MAT&agr; cdc53-2 trp1-7 ura3-52 ade2 M. Goebl CC786-1A ade2 his3 leu2 ura3 trpI MET30-1 This study C170 ade2 his3 leu2 trp1 met4::TRP1 ura3::lexAop-lacZ::URA3 Kuras and Thomas (1995)

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Claims

1. A method for 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, and a 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; (e) a peptide derived from the binding domain of an F-box binding protein that interacts with a protein of the Cullin family; (f) enhancers or inhibitors of the interaction of an E2 ubiquitin conjugating enzyme or a F-box binding protein, and a protein of the Cullin family.