USOO691.9184B2 (12) United States Patent (10) Patent No.: US 6,919,184 B2 ISSakani et al. (45) Date of Patent: Jul.19, 2005

(54) ASSAYS FOR IDENTIFYING UBIQUITIN (51) Int. Cl." ...... G01N 33/53 AGENTS AND FOR IDENTIFYING AGENTS (52) U.S. Cl...... 435/7.92; 435/7.1; 435/7.4; THAT MODIFY THE ACTIVITY OF 435/7.6; 435/7.9; 435/7.91; 435/7.93; 435/7.94; UBIQUITIN AGENTS 435/7.95; 435/21; 435/14; 435/28; 436/546; 436/544; 436/164; 436/172; 436/805 (75) Inventors: Sarkiz, D. Issakani, San Jose, CA (US); (58) Field of Search ...... 435/7.92, 7.1, Jianing Huang, Foster City, CA (US); 435/7.4, 7.6, 7.9, 7.91, 7.93, 7.94, 21, 7.95, Julie Sheung, San Francisco, CA (US); 14, 28; 436/546, 544, 164, 172, 805 Todd R. Pray, San Francisco, CA (US) (73) Assignee: Rigel Pharmaceuticals, Inc., South San (56) References Cited Francisco, CA (US) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 5,861,249 A 1/1999 Beach et al. patent is extended or adjusted under 35 5,976,849 A 11/1999 Hustad et al. U.S.C. 154(b) by 419 days. 6,331,396 B1 12/2001 Silverman et al. Primary Examiner-Christopher L. Chin (21) Appl. No.: 10/108,767 ASSistant Examiner-Pensee T. Do (74) Attorney, Agent, or Firm Townsend and Townsend (22) Filed: Mar. 26, 2002 and Crew LLP (65) Prior Publication Data (57) ABSTRACT US 2003/0104474 A1 Jun. 5, 2003 Provided are methods and compositions for assaying for Related U.S. Application Data ubiquitin agents that are enzymatic components of ubiquitin mediated proteolysis and, more particularly, methods and (63) Continuation of application No. 10/091,139, filed on Mar. 4, compositions for assaying for agents that modulate the 2002, now abandoned, which is a continuation-in-part of activity of Such ubiquitin agents. application No. 09/826,312, filed on Apr. 3, 2001, now Pat, No. 6,737,244, which is a continuation-in-part of application No. 09/542,487, filed on Apr. 3, 2000. 17 Claims, 55 Drawing Sheets

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U.S. Patent Jul.19, 2005 Sheet 10 of 55 US 6,919,184 B2

FIGURE 9A

FIGURE 9B MARSK SOAROPP PNDSPYOGGVFFITHFPTDYPFKPPKWAFTTRLYE.PNINS NGSCDIRSQSPATsKVLSCSICOPNPDDPVPE SREWTOKYAM2, FIGURE 10 MRVs KCNew WAOCG WWGOCSHCFEMECIKEACOVROHCPKCRTW

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MADWEDGEENSNCCMS CALASKSGSSGST8GGDKMFSLKKNWAMWSWDVECDTcArcRVOVMDACLRCOAKNKQEDCVWWWGBCRex U.S. Patent Jul. 19, 2005 Sheet 11 0f 55 US 6,919,184 B2

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U.S. Patent Jul.19, 2005 Sheet 13 of 55 US 6,919,184 B2

FIGURE 15A MQIFVKTTGKTITLEVRPSDTIENVKAKIQDKEGIPPDOORLIFAGKQEDGRTISDYNIQKESTLELVLRRGG

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FIGURE 51 UBC7 FIGURE SA l tatelqsall rrqlaelnkn pvegfsagli ddindlyrwev liigppditly eggvfikahlt es fpkdyplpp kmkfi teiwh pnvdkingdvc isillhepged kygyekpeer wlpihtveti 2. isvismlad pngdspanvd aakeWredral gefkrkvarc vrksqetafe FIRE SL3 l atgacggagc tgCag toggc actgotactg cgaagacago tggCag?aact (Caacaaaaat 61 Coagtggaag gottt totgc aggtttaata gatgacaatg atctotaccg atgggaagttc 2. cttattattg gCCCtcCaga tacacttitat galaggtggtg tttttaaggc tCatct tact 8. ttoccCaaaag attatcCCct ccgacctoct aaaatgaaat toattacaga aatctggcac 24 Ccaaatgttg ataaaaatgg tgatgttgttgc atttoctatto ttocatgagec tggggaagat 3O1 alagtatggitt atgaaaagcC agaggaacgc tggctCCCta to Cacactgt ggaaacCatc 35l. atgattagtg toatttctat gotggcagac CCtaatggag actoacctgc taatgttgat 42 9.Ctgcgaaag. aatggaggga agatagalaat ggagaattta aaagaaaagt tgccogctgt 48 9taagaaaaa gCCaagagaC tgcttttgag tga U.S. Patent Jul. 19, 2005 Sheet 50 of 55 US 6,919,184 B2

FIGURE S2 C7 O GetR: 52A l tnagtalkirlin aeykltlap pegivagpan eenffewteal ingpeditcfe fgvfpailsf 6. pldyplapplk mrf toenfhp aiypdgrvci silhapgddlp mgyessaerw spvgsvekil 121 lev vstulaep indesganvda skmwrddreq fylkilakqiva ksilgil

FCGRC S281 l cgc.gcggCtg aggotgaggto gCtcggogca gCtgttgcgg ggcCatggcg gggaCCgOSC 61 totaagaggct gatggCOgag tacaaacaat taacactgaa toCtcCggaa ggaattgtag l2l CaggccoCat gaatgaagag aacttittttg aatgggaggo attgatcatg ggccCagaag 183 acacctgett tgagtttggit gtttittoctg cCatcotgag tttcCCaCtt gattacCCgt 24 taagttcc.ccc aaagatgaga. tttacctgttg agatgtttca tCCCala Catc. talCCCtgatg 30l ggagingtotg CattitCoatc. cticcacgc.gc. Caggogatga CoCCatgggc taC9aggca 36 gC9C9gagcg gtggagtc.ct gtgcagagtg tggagaagat Cotgctgtc.g gtggtgagea 421 totggCaga gCCCaatgac gaaagtggag Ctaacgtgga tgcgtCCaala atgttggogo.g 48 atgaCCggga gCagttctat aagattgcCa agoagat.cgt. Coagaagttct Ctgggactgt 541 gagacctggc CtcgCaCagg cgc.gcacaca CCgCoaa.gca gotcagoatt Ctc.ccc.cggc 601 acact tagtg acagtgatgc totgttgctgg tecCalaacsa ggCagaCttg Caagaaccat 661 ggcatct ttt ttttitt ttee acct tect &Ott0aaa.ca ggcttctett Ctgaaatgat 721 gaCttaatgt Cgaatattga Cagottactg Cagttttaca gtattoctea Calaagggott 78 Cagg tagatt atcagagctg toagcactac ctcteccc.gc tgaalaccago agttcatggC 841 ttcCtgtgga ttocCCtCCC CCCtggagtg ttgagggggt. tgtacctgcc agactitCcag 90 gggacgatgg aataCCCAga acgctoctitc tgaagaaatg gggCCCtgital gotgcagorac 96l aggggaaggg CCC.ggCacCC tttctgggtc Cttoctggitt CCCtgttgggC CCCatgagga. 102 gtCOattact togetttctic Ottoatattt tacaggcaga tgcttittett ataatctaat O8 tacatcttitt Catttgttat atattacaaa. CCatCalcact tagaaatact to Caggalaat ill4l gottttittga agtgttgaatt aataagaaat gggg taalata gaaaagaaat ttattgotga l2Ol ttggccaggit gCggtggttc gtgcctgtaa teccaget.ct ttgggaggcC aaggcaggta 261 gatoacalagg totaggalaatt gagaccatcc tggctaataC agtgaaacCC catgtctgct l32l aaaattacala aaaattagot gggCgtggtg gtgCalCgCCt gtag totCag Ctacticagga l38 ggotgaggca ggagaatcgc ttgaacCCgg Saggoagagg tag cagtgag Ct9aagttccc U.S. Patent Jul.19, 2005 Sheet 51 0f 55 US 6,919,184 B2

FIGURR 52B2 (CONTINUED) l44 gocactgcac tCcagotctgg gCaacagagc gagactCagt Ctcaaaaaga aaaaagaaat 150l titattgctga toacaaggac agacagttitt tt.cccgacca tactCatcala, agatttacgt. lS61 ttgtatatta gtaaCtagtg Cattactaga 9, Caggtgcag gtgaggtott taaagtttca l62l atgaaagttt Cttctggatc tacagaaaaa. attitt titt ttcaatctaa aaactggaea. l681 ttetagggitt tttgtacatt ttggatgcac tgggaattta ttagcacalaa, atcattettt i.74l gCaactcaaa. attocagalagg gactctacca taticttagct Cagagcacag aggagtgcCt BOL tatc.cccaca Cttgactggg Ctgttggaggit 999 Catgttgg gcc.cctgggc cCaggctggg 1861 gacagagcc.c ttgttttgttg act taggatt ttgatgttggit teccatgttc totaa.caggg 92. CCagctgagc ago.acaggcc. aggaggccac agtgitalagca atalacagatc tgccacatgc 981 agaa.gcaaat at Caggcctg togcacacgg goggcattta aataggalatt totatttttg 204 aaataaggga tggtotatga ggCatacagt agatttgatg tgatcettitt etc.coccot 20 togataatgg atcgtggtet gtgttgactga accoacacag agtgtcatgg gtgacagttt 261 ctggttgaag tagctCcacg cctggcttct gtggaCagca gattottt to Cttctoacala 222l ggggctCatt taaaatttgg aggctgggtg ctgttggctca cgc.ctgcaat cc.ca.gcaott 2281 tgggagactg agg.cgggegg atcatgaggt. Caggagat.cg cgaccatect ggctaaCagt 234 galaa.ccctgt. cc cactaaa. aata Caaaala attagc.cggg C9tggtggCg ggcgCCtgta 240 gttcc.cagota citctgaagaC tgaggcagga. gaatggcgtg aaCCCaggag goggagcttg 246 cagtgagetg agatcacgcC actgcactCC agc.ctgggoal acagagtgag actctgtoto 252 aaaaaaaaaa. Ellesla tggaacgCag ggcaagaact cg tatttgga aggagatggg 258l ggaaaggage 99tattataC Ctatgttgta tittgcaggca abatgagatgg agccctotot 264 gtaaagaaga, gtCatttgttg CaagtagaCg gggtotgttgg gtgcaggcCC tggagggg.ca 27Ol Cacaattgcc tggaggcttic tgtgagatcg ggagagggag ge gaggcagt Ctctgacas 2761 aataaagtat ttt tattocat ttgtatttat taaatgaaala aacaatcCCa tggtgtc.cct 282 gttgttgttggit ggala Cctaat gactgttgaa ataaagttct gtgttttccc.tgcoctgc

U.S. Patent Jul.19, 2005 Sheet 53 of 55 US 6,919,184 B2

FGRE SEB2 144 gattogctga attgcc.cgtt tgcatacagg gtctottcct toggtCttitt gtatttittga 1501 ttgttatgta aaacticgctt ttattittaat attgatgtca g tatttcaac tgctgtaaaa. 56 tta taalactt. tta tacttgg gtaagttcc.cc Cagg.cgagtt cctogctotg ggatgcaggc 1621 atgcttctoa CCgtgcagag CtgCaCttgg cctcagctgg Ct9tatggaa atgcaccotic l681 cctoctg.cgc tectctotag aacetggget gtgctgctitt tgagccteag accCCagggc l74l agcatctogg ttctgcgc.ca CttCctttgt gttctatatgg cgttttgttct gtgttgctgt 180l ttaggtaaat aaactgttta tataaaaaaa alaaaaaaala alaaaaaaaala aaaaaa.

U.S. Patent Jul.19, 2005 Sheet 55 0f 55 US 6,919,184 B2

FGR. SS UBC13 FIGURE S5A l maglprriik etcarillaepv pgikaepdes naryfhvvia gpqdspfegg tekhelflipe 6l eyptiaapkvr fratkiyhpnv dklgriclai likdikwspalq irtvillsiqa llsapnpddp 12 landvaeqwk time agaieta raw trlyamn ni

FGR. SSB l actC9tgcgt gaggcgagag gag.ccggaga. Cgagaccaga ggCogalactC ggsyttoctgaC 6. aagatggcc.g ggctgccCCg CaggatcatC alaggaaacCC agogtttgct grg Cagalacca 21 gttcCtggca totaaag.ccga. accagatgag agcaa.cgCCC gttatttitca tgttggtotatt i81 gotggcc.ctC aggatt0CCC Ctttgaggga gggaetta aacttgaact atticct tcca 241 gaagaatacc Caatggcago ccctaaagta Cgtttocatga cCaalaattite tCatCctaat 30 gtaga Caagt tgggaagaat atgtttagat attttgaaag ataagtggtc CCCageactg 36 Cagatcc.gca cagttctgct atcgatcCag gocttgttcaa. gtgctoccaa to cagatgat 42. CCattageaa atgatgtagc ggagcagtgg aagaCCaacg. aa.gc.cCaagc Catagaaaca 48 gCtagagcat ggactaggct a tatgcCatg alatala tattt alaattgatac gatcatCaag 541 tstgcatcac ttctectgtt CtgcCaagac Etcetectet ttgtttgcat ttaatggaca 60l cagtcttaga aa Cattacag aataaaaaag cc.ca.gacatc ttcagtoctt tggtgattaa. 661 atgcacatta gCalaatctat gtcttgtc.ct gattcactgt Cataaa.gcat gag cagaggc 721 tagaagitatc atctggattg ttgtgaaacg. tittaaaag.ca gtggcccCtC CCtgCttitta 78 ttcatt tocc Ccatcctggit ttalagtataa. agcactgttga atgaagg tag ttgtCaggitt 84 agctgcaggg gtgtgggtgt ttt tatttta ttt tattitta ttittatttitt Saggggggag 90 gtagtttaat tttatggget cott teccCC ttttttggrtg atctaattgc attggittata 96 agcagctaac Cagg, tctitta galatatgctC tagcCaagttc talactittatt tagaCgCtgt 102 agatggacala gottgattgt tggalaccalaa atgggaacat taatcaaa.ca to acago'cct 108 Cactaataac attgctgtca agtgtagatt occecett.ca aaaaaagott gtgaccattt 14 tgtatggctt gtctggalaac ttctgtaaat ct tatgtttt agtaaaatat, tttttgttat 20 tot US 6,919,184 B2 1 2 ASSAYS FOR IDENTIFYING UBIQUITIN In eukaryotes, Some ubiquitin ligating agents contain AGENTS AND FOR DENTIFYING AGENTS multiple subunits that form a complex called the SCF having THAT MODIFY THE ACTIVITY OF ubiquitin ligating activity. SCFS play an important role in UBIQUITIN AGENTS regulating G1 progression, and consists of at least three subunits, SKP1, Cullins (having at least seven family This is a continuation of application Ser. No. 10/091,139, members) and an F-box protein (of which hundreds of filed Mar. 4, 2002, now abandoned which is a continuation Species are known) which bind directly to and recruit the in-part application of U.S. patent application Ser. No. Substrate to the complex. The combinatorial interactions 09/826,312, filed Apr. 3, 2001 now U.S. Pat. No. 6,737,244 between the SCF's and a recently discovered family of which is a continuation-in-part application U.S. patent appli RING finger proteins, the ROC/APC11 proteins, have been shown to be the key elements conferring ligase activity to cation Ser. No. 09/542,487, filed Apr. 3, 2000 (pending). ubiquitin ligating agents. Particular ROC/Cullin combina FIELD OF THE INVENTION tions can regulate Specific cellular pathways, as exemplified The invention relates to the field of ubiquitin-mediated by the function of APC11-APC2, involved in the proteolytic proteolysis. In particular, the invention relates to methods 15 control of Sister chromatid Separation and exit from telo and compositions for assaying for ubiquitin agents that are phase into G1 in mitosis (see King et al., Supra; Koepp et al., enzymatic components of ubiquitin-mediated proteolysis Cell 97:431-34 (1999)), and ROC1-Cullin 1, involved in the and, more particularly, to methods and compositions for proteolytic degradation of IB in NF-B/IB mediated tran assaying for agents that modulate the activity of Such scription regulation (Tan et al., Mol. Cell 3(4):527–533 ubiquitin agents. (1999); Laney et al., Cell 97:427–30 (1999)). The best characterized ubiquitin ligating agent is the APC BACKGROUND OF THE INVENTION (anaphase promoting complex), which is multi-component Ubiquitin is a highly conserved 76 amino acid protein complex that is required for both entry into anaphase as well expressed in all eukaryotic cells. The levels of many intra as exit from mitosis (see King et al., Science 274:1652-59 cellular proteins are regulated by a ubiquitin-mediated pro 25 (1996) for review). The APC plays a crucial role in regu teolytic process. This process involves the covalent ligation lating the passage of cells through anaphase by promoting of ubiquitin to a target protein, resulting in a poly ubiquitin-mediated proteolysis of many proteins. In addition ubiquitinated target protein which is rapidly detected and to degrading the mitotic B-type cyclin for inactivation of degraded by the 26S proteasome. CDC2 kinase activity, the APC is also required for degra The ubiquitination of these target proteins is known to be dation of other proteins for Sister chromatid Separation and mediated by the enzymatic activity of three ubiquitin agents. Spindle disassembly. Most proteins known to be degraded by Ubiquitin is first activated in an ATP-dependent manner by the APC contain a conserved nine amino acid motif known a ubiquitin activating agent, for example, an E1. The as the “destruction box” that targets them for ubiquitin C-terminus of a ubiquitin forms a high energy thiolester ubiquitination and Subsequent degradation. However, pro bond with the ubiquitin activating agent. The ubiquitin is 35 teins that are degraded during G1, including G1 cyclins, then transferred to a ubiquitin conjugating agent, for CDK inhibitors, transcription factors and Signaling example, an E2 (also called ubiquitin moiety carrier intermediates, do not contain this conserved amino acid protein), also linked to this second ubiquitin agent via a motif. Instead, Substrate phosphorylation appears to play an thiolester bond. The ubiquitin is finally linked to its target important role in targeting their interaction with a ubiquitin protein to form a terminal isopeptide bond under the guid 40 ligating agent for ubiquitin ubiquitination (see Hershko et ance of a ubiquitin ligating agent, for example, an E3. In this al., Ann. Rev. Biochem, 67:429-75 (1998)). process, monomers or oligomers of ubiquitin are attached to Two major classes of E3 ubiquitin ligating agents are the target protein. On the target protein, each ubiquitin is known: the HECT (homologous to E6-AP carboxy covalently ligated to the next ubiquitin through the activity terminus) domain E3 ligating agents; and the RING finger of a ubiquitin ligating agent. 45 domain E3 ligating agents. E6AP is the prototype for the The enzymatic components of the ubiquitination pathway HECT domain Subclass of E3 ligating agents and is a have received considerable attention (for a review, See multi-Subunit complex that functions as a ubiquitin ligating Weissman, Nature Reviews 2:169–178 (2001)). The mem agent for the tumor Suppressor p53 which is activated by bers of the E1 ubiquitin activating agents and E2 ubiquitin papillomavirus in cervical cancer (Huang et al. (1999) conjugating agents are structurally related and well charac 50 Science 286:1321-1326). Members of this class are homolo terized enzymes. There are numerous Species of E2 ubiquitin gous to the carboxyl terminus of E6 AP and utilize a Cys conjugating agents, Some of which act in preferred pairs active Site to form a thiolester bond with ubiquitin, analo with Specific E3 ubiquitin ligating agents to confer speci gous to the E1 activating agents and E2 conjugating agents. ficity for different target proteins. While the nomenclature However, in contrast, the members of the RING finger for the E2 ubiquitin conjugating agents is not Standardized 55 domain class of E3 ligating agents are thought to interact acroSS Species, investigators in the field have addressed this with an ubiquitin-conjugated-E2 intermediate to activate the issue and the skilled artisan can readily identify various E2 complex for the transfer of ubiquitin to an acceptor. ubiquitin conjugating agents, as well as Species homologues Examples of the RING domain class of E3 ligating agents (See Haas and Siepmann, FASEB.J. 11:1257–1268 (1997)). are TRAF6, involved in IKK activation; Cbl, which targets Generally, ubiquitin ligating agents contain two Separate 60 insulin and EGF; Sina/Siah, which targets DCC, Itchy, activities: a ubiquitin ligase activity to attach, via an isopep which is involved in haematopoesis (B, T and mast cells); tide bond, monomers or oligomers of ubiquitin to a target IAP, involved with inhibitors of apoptosis; and Mdm2 which protein, and a targeting activity to physically bring the ligase is involved in the regulation of p53. and substrate together. The substrate specificity of different The RING finger domain subclass of E3 ligating agents ubiquitin ligating agents is a major determinant in the 65 can be further grouped into two Subclasses. In one Subclass, Selectivity of the ubiquitin-mediated protein degradation the RING finger domain and the substrate recognition proceSS. domain are contained on different Subunits of a complex US 6,919,184 B2 3 4 forming the ubiquitin ligating agent (e.g., the RBX1 and the processes can be treated by Such up- or down regulation of F-box subunit of the SCF complex). In the second subclass Signal transducers to enhance or dampen Specific cellular of ubiquitin ligating agents, the ligating agents have the responses. This principle has been used in the design of a RING finger domain and Substrate recognition domain on a number of therapeutics, including phosphodiesterase inhibi single subunit. (e.g., Mdm2 and cbl) (Tyers et al. (1999) tors for airway disease and vascular insufficiency, kinase Science 284:601, 603-604; Joazeiro et al. (2000) inhibitors for malignant transformation and Proteasome 102:549-552). A further class of ligating agents are those inhibitors for inflammatory conditions Such as arthritis. having a “PHD” domain and are homologs of the RING finger domain ligating agents (CoScoy et al. (2001) J. Cell Due to the importance of ubiquitin-mediated proteolysis Biol. 155(7): 1265–1273), e.g., MEKK1. The PHD domain in cellular process, for example cell cycle regulation, there ligating agents are a novel class of membrane-bound E3 is a need for a fast and Simple means for identifying ligating agents. ubiquitin agents that are catalytic components of this enzy matic pathway, and for identifying agents that modulate the Mdm2 belongs to the second subclass of single subunit E3 activity of these catalytic components. Thus, an object of the ligating agents and is involved in regulating the function and present invention is to provide methods of assaying for Stability of p53, an important tumor Suppressor. In cells, p53 15 functions as a DNA-binding transcription factor which ubiquitin agents that are catalytic components of ubiquitin induces the expression of genes involved in DNA repair, mediated proteolysis and, more particularly, methods of apoptosis, and the arrest of cell growth. In approximately assaying for agents that modulate the activity of the ubiq 50% of all human cancer p53 is inactivate by deletion or uitin agents. mutation. The level of p53 in the cell is maintained at low SUMMARY OF THE INVENTION Steady-state levels, and is induced and activated post translationally by various signal pathways responsive to In accordance with the above objects, the present inven cellular stress (Lakin et al. (1999) Oncogene 18:7644-7655; tion provides methods and compositions for assaying for Oren, M. (1999) J. Biol. Chem 274:36031-36,034). Stimuli ubiquitin agents that are enzymatic components of ubiquitin that trigger the StreSS response and activate p53 include mediated proteolysis. More particularly, the present inven oxygen StreSS, inappropriate activation of oncogenes and 25 tion provides methods and compositions for assaying for an agents that cause damage to DNA (e.g., ionizing radiation, agent that modulates the activity of a ubiquitin agent that is chemicals, and ultra violet light). an enzymatic component of ubiquitin-mediated proteolysis. The carboxyl terminus of Mdm2 contains a variant of the Specifically, the methods of the present invention are RING finger domain (Saurin et al. (1996) Trends Biochem. directed to identifying ubiquitin agents Such as ubiquitin Sci. 21:208-214) that is critical for the activity of this E3 activating agents, ubiquitin conjugating agents, and ubiq ligating agent. Recent studies have shown that Mdm2 medi uitin ligating agents, and to identifying agents that modulate ates the ubiquitination of itself resulting in the formation of the activity of these ubiquitin agents. In one aspect, the poly-ubiquitin chains on the protein (Zhihong et al. (2001) invention provides assaying methods that do not require a J.B.C. 276:31,357–31,367; Honda et al. (2000) Oncogene 35 ubiquitin target protein. In the methods of the present 19:1473–1476; Shengyun et al. (2000) 275:8945–8951). invention the ubiquitin agents are combined in different Further, the ubiquitin ligating activity of Mdm2 is dependent combinations with a ubiquitin moiety to assay for the on its RING finger domain. attachment of the ubiquitin moiety, or the modulation of this Typically, the ubiquitination of target proteins by E3 in attachment, to at least one of the following Substrate mol cells results in the formation of poly-ubiquitin chains. An 40 ecules: a ubiquitin agent, a target protein, or a mono- or isopeptide bond is formed between the carboxyl terminus of poly-ubiquitin moiety which is preferably attached to a the ubiquitin and the e-amino group of Lys in the target ubiquitin agent or target protein. protein. The extension or formation of ubiquitin chains In aspects of the methods and compositions of the present results from the formation of additional isopeptide bonds invention, the ubiquitin activating agent is an E1; the ubiq with the Lys" (and sometimes Lys) of a previously 45 uitin conjugating agent is an E2, and/or the ubiquitin ligating conjugated ubiquitin and the carboxyl-terminal Gly of an agent is an E3. In other aspects, the target protein is a additional ubiquitin. The efficient recognition of a ubiquiti mammalian target protein, and in further aspects, the target nated target protein by a proteoSome requires at least four protein is a human target protein. In other aspects, the ubiquitins linked in this configuration. However, in the case ubiquitin moiety is a mammalian ubiquitin, and in further of Mdm2-mediated ubiquitination of p53, neither Lys" or 50 aspects, the ubiquitin moiety is a human ubiquitin. In Lys is involved in the formation of poly-ubiquitin chains. another aspect, the ubiquitin moiety is a ubiquitin derivative. Recent studies show that human Mdm2 mediates multiple In Some aspects, the candidate agent a Small molecule, and mono-ubiquitination of p53 by a mechanism requiring in further aspects, the candidate agent is a peptide. In Some enzyme isomerization (Zhihong et al. (2001) J. Biol. Chem. aspects, the ubiquitin moiety comprises a label, and in 276:31,357–31,367). Further, in vitro, the transfer of ubiq 55 further aspects, the label comprises an epitope tag. In other uitin to p53 can occur independent of E1 when using an E2 aspects, at least a first and a Second ubiquitin moiety is used, pre-conjugated with ubiquitin. These results Suggest that the wherein the first and Second ubiquitin moieties comprise pre-conjugated E2 can bind to Mdm2 and thereafter transfer different fluorescent labels, and wherein the labels form a the ubiquitin to the Mdm2 in the absence of an E1. fluorescence resonance energy transfer (FRET) pair. Thus, ubiquitin agents, Such as the ubiquitin activating 60 In one aspect, the invention provides a method of assaying agents, ubiquitin conjugating agents, and ubiquitin ligating for an agent that modulates the attachment of a ubiquitin agents, are key determinants of the ubiquitin-mediated pro moiety to at least one ubiquitin agent involving the Steps of: teolytic pathway that results in the degradation of targeted a) combining a first ubiquitin agent, a candidate agent, and proteins and regulation of cellular processes. Consequently, a ubiquitin moiety; and b) assaying for the attachment of the agents that modulate the activity of Such ubiquitin agents 65 ubiquitin moiety to the first agent. In an additional aspect, may be used to upregulate or downregulate Specific mol the first ubiquitin agent is an ubiquitin activating agent. In a ecules involved in cellular Signal transduction. Disease further aspect, the ubiquitin activating agent is an E1. US 6,919,184 B2 S 6 In another aspect, the method further comprises including In another aspect, the p53 protein comprises a first FRET a Second ubiquitin agent in the combining Step. In a further label and the ubiquitin moiety comprises a second FRET aspect, the first agent is a ubiquitin conjugating agent and the label. In another aspect, the p53 protein comprises an Second agent is a ubiquitin activating agent. In a further attachment tag. In another aspect, the p53 protein is pro aspect, the ubiquitin conjugating agent is an E2 and the Vided on a Solid Support; and in a further aspect, the Solid ubiquitin activating agent is an E1. Also in a further aspect, Support comprises a microtiter plate or a bead. the ubiquitin conjugating agent is an E2 and the ubiquitin In one aspect, the invention provides a method of assaying activating agent is an E1 comprising the ubiquitin moiety. for a candidate agent that modulates the attachment of a In another aspect, the first agent is a ubiquitin ligating ubiquitin moiety to an Mdm2 protein involving the Steps of: agent and the Second agent is a ubiquitin conjugating agent a) combining a first ubiquitin agent comprising at least one comprises the ubiquitin moiety. In a further aspect, the ubiquitin moiety, an Mdm2 protein, and a candidate agent; ubiquitin ligating agent is an E3 and the ubiquitin conjugat and b) assaying for the attachment of the ubiquitin moiety to ing agent is an E2 comprising the ubiquitin moiety. the Mdm2 protein. In an additional aspect, the first ubiquitin In another aspect, the method further comprises a third agent is a ubiquitin conjugating agent. ubiquitin agent in the combining Step. In a further aspect, the 15 In an additional aspect, the method further comprises third agent is a ubiquitin ligating agent. Also in a further combining a ubiquitin activating agent comprising the ubiq aspect, the ubiquitin ligating agent is an E3. uitin moiety, thereby forming the ubiquitin conjugating In the methods where the assaying concerns the attach agent comprising the ubiquitin moiety, in Step a). ment of the ubiquitin moiety to the first ubiquitin agent, the In an additional aspect, the method further comprises following additional aspects are provided. In one aspect, the combining a ubiquitin activating agent and the ubiquitin first ubiquitin agent comprises a tag. In a further aspect, the moiety, thereby forming the ubiquitin conjugating agent first ubiquitin agent comprises an epitope tag. In another comprising the ubiquitin moiety. aspect, first ubiquitin agent comprises a label. Also in a In another aspect, the invention provides a method of further aspect, the first ubiquitin agent comprises an attach assaying for a candidate agent that modulates the attachment ment tag. In another aspect, the first ubiquitin agent is 25 of a ubiquitin moiety to a p53 protein involving the Steps of: attached to a Solid Support, and in a further aspect, the Solid a) combining a conjugating agent comprising at least one Support is a microtiter plate or a bead. ubiquitin moiety, an Mdm2 protein, a p53 protein, and a In the methods where the assaying concerns the attach candidate agent; and b) assaying for the attachment of the ment of the ubiquitin moiety to a Second ubiquitin agent, the ubiquitin moiety to the p53 protein. following additional aspects are provided. In one aspect, the In an additional aspect, the method further comprises Second ubiquitin agent comprises a tag. In a further aspect, combining a ubiquitin conjugating agent and the ubiquitin the Second ubiquitin agent comprises an epitope tag. In moiety, thereby forming the ubiquitin conjugating agent another aspect, Second ubiquitin agent comprises a label. comprising the ubiquitin moiety. Also in a further aspect, the Second ubiquitin agent com In an additional aspect, the method further comprises prises an attachment tag. In another aspect, the Second 35 combining a ubiquitin activating agent comprising the ubiq ubiquitin agent is attached to a Solid Support, and in a further uitin moiety, thereby forming the ubiquitin conjugating aspect, the Solid Support is a microtiter plate or a bead. agent comprising the ubiquitin moiety, in Step a). In another aspect, the invention provides a method of In an additional aspect, the method further comprises assaying for an agent that modulates the attachment of a combining a ubiquitin activating agent and the ubiquitin ubiquitin moiety to at least one ubiquitin agent involving the 40 moiety, thereby forming the ubiquitin conjugating agent Steps of: a) combining a first ubiquitin agent comprising a comprising the ubiquitin moiety. ubiquitin ligating agent, a Second ubiquitin agent, a candi In another aspect, the invention provides a method of date agent, a ubiquitin moiety, and a Substrate; and b) assaying for a candidate agent that modulates the attachment assaying for the attachment of the ubiquitin moiety to the 45 of a ubiquitin moiety to an Mdm2 protein involving the Steps first agent. In an additional aspect, the Second agent is a of: a) combining a ubiquitin activating agent, a ubiquitin ubiquitin conjugating agent comprising the ubiquitin moiety. conjugating agent, an Mdm2 protein, a candidate agent, and In an additional aspect, the method further comprises a a ubiquitin moiety; and b) assaying for the attachment of the third ubiquitin agent in the combining Step, wherein the third ubiquitin moiety to the Mdm2 protein. agent is a ubiquitin activating agent, wherein the Substrate 50 In another aspect, the invention provides a method of and the ubiquitin moiety comprise different fluorescent assaying for a candidate agent that modulates the attachment labels, and wherein the labels form a fluorescence resonance of a ubiquitin moiety to a p53 protein involving the Steps of: energy transfer (FRET) pair. a) combining a ubiquitin activating agent, a ubiquitin con In the following aspects of the present invention, the jugating agent, an Mdm2 protein, a p53 protein, a candidate ubiquitin ligating agent is preferably an Mdm2 protein and 55 agent, and a ubiquitin moiety; and b) assaying for the the target protein is preferably p53. In a preferred attachment of the ubiquitin moiety to the p53 protein. embodiment, the ubiquitin ligating agent is an Mdm2 fusion In another aspect, the invention provides a method of protein, and more preferably an Mdm2-GST fusion protein. assaying for a candidate agent that modulates the attachment In one aspect of the present methods, the Mdm2 protein of a Second ubiquitin moiety to a p53 protein involving the comprises a first FRET label and the ubiquitin moiety 60 Steps of: a) combining a ubiquitin activating agent, a ubiq comprises a second FRET label. In another aspect, the uitin conjugating agent, an Mdm2 protein, a p53 protein Mdm2 protein comprises an attachment tag. In another comprising a first ubiquitin moiety, wherein the first ubiq aspect, the Mdm2 protein is provided on a Solid Support; and uitin moiety is labeled with a first FRET label, a candidate in a further aspect, the Solid Support comprises a microtiter agent, and a Second ubiquitin moiety labeled with a Second plate or a bead. In another aspect, the Mdm2 protein is a 65 FRET label; and b) assaying for the attachment of the second mammalian Mdm2, and in a further aspect, the Mdm2 is a ubiquitin moiety to the p53 protein by detecting a FRET human Mdm2. reaction. US 6,919,184 B2 7 8 In another aspect, the invention provides a method of modulate the attachment of ubiquitin moiety to an E3 by assaying for a candidate agent that modulates the attachment combining ubiquitin moiety, and E1, E2, and E3 ubiquitin of a first ubiquitin moiety to a p53 protein involving the agents and by combining ubiquitin moiety and E1 and E2 Steps of: a) combining a ubiquitin conjugating agent com ubiquitin agents. FIG. 6A shows a candidate agent that only prising a first ubiquitin moiety labeled with a first FRET, an modulates the attachment of ubiquitin moiety to an E3. FIG. Mdm2 protein, a p53 protein comprising a Second ubiquitin 6B ShowS candidate agent that modulates the attachment of moiety, wherein the first ubiquitin moiety is labeled with a ubiquitin moiety to ubiquitin agents other than E3. Second FRET label, and a candidate agent; and b) assaying FIG. 7 shows the concentration-dependent effects of two for the attachment of the first ubiquitin moiety to the p53 candidate agents that modulate the attachment of ubiquitin protein by detecting a FRET reaction. moiety to an E3 and the attachment of ubiquitin moiety to an In another aspect, the invention provides a method of E2. FIG. 7A shows the results of a candidate agent having assaying for a candidate agent that modulates the attachment a concentration-dependent effect on the attachment of ubiq of a first ubiquitin moiety to a p53 protein involving the uitin moiety to an E3 (by combining ubiquitin moiety, E1, Steps of: a) combining a ubiquitin activating agent compris E2, and E3), but does not have an effect on the attachment ing a first ubiquitin moiety labeled with a first FRET, a 15 of ubiquitin moiety to an E2 (by combining ubiquitin ubiquitin conjugating agent, an Mdm2 protein, a p53 protein moiety, E1, and E2), thus affecting only the attachment of comprising a Second ubiquitin moiety, wherein the first ubiquitin moiety to an E3. FIG. 7B shows the results for a ubiquitin moiety is labeled with a second FRET label, and a candidate modulator having a concentration-dependent candidate agent; and b) assaying for the attachment of the effect on both the attachment of ubiquitin moiety to an E2 first ubiquitin moiety to the p53 protein by detecting a FRET and the attachment of ubiquitin moiety to an E3, thus reaction. affecting a component other than the E3. Other aspects of the invention will become apparent to the FIGS. 8A and 8B (SEQ ID NOS:1, 2) show the nucleic skilled artisan from the following description of the inven acid Sequence encoding rabbit E1 ubiquitin activating agent tion. and the amino acid Sequence of rabbit E1, respectively. 25 BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 9A and 9B (SEQ ID NOS:3, 4) show the nucleic acid Sequence encoding the E2 Ubch5c and the amino acid FIG. 1 shows the relative amounts of attachment of sequence of the E2 Ubch5c, respectively. fluorescently labeled ubiquitin moiety to an E2 resulting from combining a ubiquitin activating agent, ubiquitin con FIG. 10 (SEQ ID NO:5) shows the amino acid sequence jugating agent, and ubiquitin moiety. In these experiments, of the RING finger protein APC11. E2 is His-Ubch5.c. FIG. 11 (SEQ ID NO:6) shows the amino acid sequence FIG. 2 shows the relative amounts of attachment of of the RING finger protein ROC1. ubiquitin moiety to an E3 resulting from various combina FIGS. 12A and 12B (SEQ ID NOS:7, 8) show the nucleic tions of ubiquitin agents and ubiquitin moiety. In these acid sequence encoding the RING finger protein ROC2 and experiments, E3 comprises the RING finger protein ROC1 35 the amino acid Sequence of ROC2, respectively. and the Cullin Cul1. FIGS. 13A and 13B (SEQ ID NOS:9-10) show the FIG.3 shows relative amounts of attachment of ubiquitin nucleic acid Sequence encoding the Cullin CUL5 and the moiety to an E3 resulting from combining E1, E2, E3, and amino acid Sequence of CUL5, respectively. ubiquitin moiety. FIG. 3A shows relative amounts of attach FIGS. 14A and 14B (SEQ ID NOS:11-12) show the ment of ubiquitin moiety to an E3 using varying amounts of 40 nucleic acid Sequence encoding the Cullin APC2 and the E1 in the presence and absence of DMSO. FIG. 3B shows amino acid Sequence of APC2, respectively. relative amounts of attachment of ubiquitin moiety to an E3 FIGS. 15A, 15B and 15C (SEQ ID NOS:13–15) show the using varying amounts of ubiquitin moiety and E3. amino acid Sequences of human ubiquitin moiety, Flag FIG. 4 shows the signal to noise ratio of fluorescent label 45 ubiquitin moiety and Flag-Cys-ubiquitin moiety, respec indicative of the relative amounts of attachment of ubiquitin tively. The Flag and Flag-CyS portions of the Sequence are moiety to an E3, in an assay combining Flag-ubiquitin shown in bold. moiety and an anti-Flag/anti-mouse antibody conjugated to FIGS. 16A and 16B show the E3-dependent incorporation HRP and Luminol fluorescent HRP substrate. The signal was of Flag-Ala-Cys-ubiquitin moiety labeled with FRET fluo measured from a reaction composition combining ubiquitin 50 rophores into E3-ubiquitin moiety complex. Isolation by moiety, E1, E2, and E3, where the E3 specifically bound the HPLC shows emissions from free ubiquitin moiety and reaction receptacle Surface Substrate. The background was ubiquitin moiety attached to the E3 ubiquitin ligating agent. measured as the amount of fluorescence present after per The traces show fluorescent emission at the wavelength forming the assay in the absence of E3. described below, under excitation at 336 nm, the optimal FIG. 5 shows the concentration-dependent effect of two 55 excitation wavelength for IAEDANS. FIG. 16A shows the candidate agents that modulate the attachment of ubiquitin fluorescence signals of IAEDANS (490 nm; larger peak) and moiety to an using two different E3 ubiquitin ligating agents. fluorescein (515 nm, Smaller peak) labeled ubiquitin moiety FIG. 5A shows a concentration-dependent reduction in the following combination with E1 and E2 only. The free attachment of ubiquitin moiety to an E3, in assays compris ubiquitin moiety was isolation using high performance liq ing either ROC1/Cul1 or ROC2/Cul5 as the components of 60 uid chromatography (HPLC). FIG. 16B shows the fluores the E3 ubiquitin ligating agent. FIG. 5B shows a slightly cence signals of IAEDANS (490 nm; larger peak at each different pattern of concentration-dependent reduction of elution volume) and fluorescein (515 nm, Smaller peak at attachment of ubiquitin moiety to an E3, by another candi each elution volume) labeled ubiquitin moiety following date agent. combination with E1 and E2 and E3 (Roc1/Cul1). The FIG. 6 shows the proportions of attachment of ubiquitin 65 dashed line shows optical density of the protein Solution moiety to an E3 and attachment of ubiquitin moiety to an E2, (Scale on right), revealing the high Sensitivity of the fluo in the presence and absence of two candidate agents that rophores despite a very low concentration of protein. US 6,919,184 B2 9 10 FIG. 17 shows the fluorescence emission spectra of free 2) assaying for the attachment of the ubiquitin moiety to ubiquitin moiety labeled with the FRET donor/acceptor pair ULA. In another preferred embodiment, the ubiquitin moi EDANS and fluorescein under excitation at 336 mm. The ety comprises a label. In yet another preferred embodiment, dashed line shows the emission Spectra of free labeled ULA comprises a label. In a preferred embodiment the ubiquitin moiety (reactants), while the Solid line shows the ubiquitin ligating agent comprises an Mdm2 protein. emission spectra of labeled ubiquitin moiety bound to E3 FIG.28 schematically depicts a preferred embodiment for (products). The greatly increased 515:490 nm emission ratio assaying for the attachment of ubiquitin moiety to a a of the E3-bound ubiquitin moiety as compared with the free ubiquitin ligating agent that is an E3 where the assay ubiquitin moiety shows the energy transfer from the EDANS comprises: donor to the fluorescein acceptor of this FRET donor/ 1) combining a ubiquitin conjugating agent that is an E2 acceptor pair. and comprising a ubiquitin moiety+E3+CA, and FIG. 18 shows a schematic representation of GST-Mdm2 2) assaying for the attachment of the ubiquitin moiety to and His-p53. E3. In a preferred embodiment, the E3 is an Mdm2 protein. FIG. 19 shows a Western blot analysis of the attachment FIG.29 schematically depicts a preferred embodiment for of ubiquitin moiety to p53 by Mdm2, in vitro. 15 assaying for the attachment of ubiquitin moiety to a ubiq FIG. 20 shows a schematic of a nickel plate based assay uitin conjugating agent that is an E2 where the assay for the attachment of ubiquitin moiety to p53 by Mdm2. comprises: FIG. 21 shows the results of measuring the luminescence 1) combining a ubiquitin activating agent that is an E1 and indicative of the amount of attachment of ubiquitin moiety comprising a ubiquitin moiety+E2+CA, and to p53 by Mdm2 in the nickel plate based assay. 2) assaying for the attachment of the ubiquitin moiety to FIG. 22 depicts the key for the ubiquitin activating agent E2. (UAA), ubiquitin conjugating agent (UCA), ubiquitin ligat FIG. 30 schematically depicts a preferred embodiment for ing agent (ULA), ubiquitin moiety (U), and candidate agent assaying for the attachment of ubiquitin moiety to a third (CA) used in the Schematics in Figures 25 ubiquitin agent (UA-3) where the assay comprises: FIG.23 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first 1) combining a ubiquitin activating agent that is an E1 and ubiquitin agent (UA-1) where the assay comprises: comprising a ubiquitin moiety+a ubiquitin conjugating 1) combining a UA-1+CA+U; and agent that is an E2+UA-3+CA, and 2) assaying for the attachment of the ubiquitin moiety to 2) assaying for the attachment of the ubiquitin moiety to UA-1. In another preferred embodiment UA-1 is a UAA. In UA-3. In a preferred embodiment, UA-3 comprises an another preferred embodiment, UAA is an E1. In yet another Mdm2 protein. preferred embodiment, UA-1 comprises a label. In another FIG.31 schematically depicts a preferred embodiment for preferred embodiment, the ubiquitin moiety comprises a assaying for the attachment of ubiquitin moiety to a third label. 35 ubiquitin agent that is a ubiquitin ligating agent (ULA) FIG. 24 schematically depicts a preferred embodiment for where the assay comprises: assaying for the attachment of ubiquitin moiety to a Second 1) combining a ubiquitin activating agent that is an E1 and ubiquitin agent (UA-2) where the assay comprises: comprising a ubiquitin moiety+a ubiquitin conjugating 1) combining a first ubiquitin agent that is UAA+UA agent that is an E2+ULA+CA, and 2+CA+U, and 40 2) assaying for the attachment of the ubiquitin moiety to 2) assaying for the attachment of the ubiquitin moiety to ULA. In a preferred embodiment the ubiquitin ligating UA-2. In another preferred embodiment, UA-2 comprises a agent comprises an Mdm2 protein. label. In yet another preferred embodiment, UA-2 comprises FIG. 32 schematically depicts a preferred embodiment for a label. assaying for the attachment of ubiquitin moiety to a ubiq FIG.25 schematically depicts a preferred embodiment for 45 uitin ligating agent that is an E3 where the assay comprises: assaying for the attachment of ubiquitin moiety to a first 1) combining an E1 comprising a ubiquitin moiety+an ubiquitin agent that is a ubiquitin conjugating agent UCA E2+an E3+CA; and where the assay comprises: 2) assaying for the attachment of the ubiquitin moiety to 1) combining a second ubiquitin agent that is UAA+ E3. In a preferred embodiment, the E3 is Mdm2. UCA+CA+U, and 50 FIG.33 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to assaying for the attachment of ubiquitin moiety to a first UCA. ubiquitin agent (UA-1) that is attached to a Solid Support FIG. 26 schematically depicts a preferred embodiment for where the assay comprises: assaying for the attachment of ubiquitin moiety to ubiquitin 1) combining a UA-1 (that is attached to a Solid Support)+ conjugating agent that is an E2 where the assay comprises: 55 CA+U, and 1) combining a ubiquitin activating agent that is an 2) assaying for the attachment of the ubiquitin moiety to E1+E2+CA+U, and UA-1. In another preferred embodiment UA-1 is a UAA. In 2) assaying for the attachment of the ubiquitin moiety to another preferred embodiment, UAA is an E1. In another E2. 60 preferred embodiment, the Solid Support is a microtiter plate. FIG.27 schematically depicts a preferred embodiment for In another preferred embodiment, the Solid Support is a bead. assaying for the attachment of ubiquitin moiety to a first FIG.34 schematically depicts a preferred embodiment for ubiquitin agent that is a ubiquitin ligating agent (ULA) assaying for the attachment of ubiquitin moiety to a Second where the assay comprises: ubiquitin agent (UA-2) that is attached to a Solid Support) 1) combining a second ubiquitin agent that is a ubiquitin 65 where the assay comprises: conjugating agent and comprising a ubiquitin moiety UCA 1) combining a first ubiquitin agent that is UAA+UA-2 U+ULA+CA; and (attached to a Solid Support)+CA+U, and US 6,919,184 B2 11 12 2) assaying for the attachment of the ubiquitin moiety to 2) assaying for the attachment of the ubiquitin moiety to UA-2. In another preferred embodiment, the Solid Support is ULA. In a preferred embodiment the ubiquitin ligating a microtiter plate. In another preferred embodiment, the agent comprises an Mdm2 protein. Solid Support is a bead. In a preferred embodiment, UA-2 FIG. 42 schematically depicts a preferred embodiment for comprises an Mdm2 protein. assaying for the attachment of ubiquitin moiety to a Second FIG. 35 schematically depicts a preferred embodiment for ubiquitin agent (UA-2) that comprises a label where the assaying for the attachment of ubiquitin moiety to a first assay comprises: ubiquitin agent that is a ubiquitin ligating agent (ULA) that 1) combining a first ubiquitin agent that is UAA+UA-2 is attached to a Solid Support where the assay comprises: (plus label)+CA+U; and 1) combining a second ubiquitin agent that is a ubiquitin 2) assaying for the attachment of the ubiquitin moiety to conjugating agent UCA comprising a ubiquitin moiety+ UA-2. In a preferred embodiment, UA-2 comprises an ULA (attached to a Solid Support)+CA; and Mdm2 protein. 2) assaying for the attachment of the ubiquitin moiety to FIG. 43 schematically depicts a preferred embodiment for ULA. In a preferred embodiment the ubiquitin ligating assaying for the attachment of ubiquitin moiety to a first agent comprises an Mdm2. 15 ubiquitin agent (UA-1) that comprises an attachment tag (or FIG. 36 schematically depicts a preferred embodiment for attachment moiety) where the assay comprises: assaying for the attachment of ubiquitin moiety to a first 1) combining a UA-1 (plus attachment tag)+CA+U, and ubiquitin agent (UA-1) that comprises a label where the 2) assaying for the attachment of the ubiquitin moiety to assay comprises: UA-1. 1) combining a UA-1 (plus label)+CA+U; and FIG. 44 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to assaying for the attachment of ubiquitin moiety to a first UA-1. ubiquitin agent that is a ubiquitin ligating agent (ULA) that FIG. 37 schematically depicts a preferred embodiment for comprises an attachment tag (or attachment moiety) where assaying for the attachment of ubiquitin moiety to a first 25 the assay comprises: ubiquitin agent that is a ubiquitin ligating agent (ULA) that 1) combining a second ubiquitin agent that is a ubiquitin comprises a label where the assay comprises: conjugating agent UCA comprising a ubiquitin moiety+ 1) combining a second ubiquitin agent that is a ubiquitin ULA (plus attachment tag)+CA; and conjugating agent UCA comprising a ubiquitin moiety+ 2) assaying for the attachment of the ubiquitin moiety to ULA (plus label)+CA; and ULA. In a preferred embodiment the ubiquitin ligating 2) assaying for the attachment of the ubiquitin moiety to agent comprises an Mdm2 protein. ULA. In a preferred embodiment the ubiquitin ligating FIG. 45 schematically depicts a preferred embodiment for agent comprises an Mdm2 protein. assaying for the attachment of ubiquitin moiety to a Second FIG.38 schematically depicts a preferred embodiment for ubiquitin agent (UA-2) that comprises an attachment tag (or 35 attachment moiety) where the assay comprises: assaying for the attachment of ubiquitin moiety that com 1) combining a first ubiquitin agent that is UAA+UA-2 prises a label, to a first ubiquitin agent (UA-1) where the (plus attachment tag)+CA+U, and assay comprises: 2) assaying for the attachment of the ubiquitin moiety to 1) combining a UA-1+CA+U (plus label); and UA-2. 2) assaying for the attachment of the ubiquitin moiety to 40 FIG. 46 schematically depicts a preferred embodiment for UA-1. assaying for the attachment of ubiquitin moiety to a first FIG. 39 schematically depicts a preferred embodiment for ubiquitin agent (UA-1) that comprises an epitope tag (or assaying for the attachment of ubiquitin moiety that com epitope label) where the assay comprises: prises a label, to a second ubiquitin agent (UA-2) where the 1) combining a UA-1 (plus epitope tag)+CA+U, and assay comprises: 45 2) assaying for the attachment of the ubiquitin moiety to 1) combining a first ubiquitin agent that is UAA+UA UA-1. 2+CA+U (plus label); and FIG. 47 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to assaying for the attachment of ubiquitin moiety to a Second UA-2. ubiquitin agent (UA-2) that comprises an epitope tag (or 50 epitope label) where the assay comprises: FIG. 40 schematically depicts a preferred embodiment for 1) combining a first ubiquitin agent that is UAA+UA-2 assaying for the attachment of ubiquitin moiety that com (plus epitope tag)+CA+U, and prises a label, to a first ubiquitin agent that is a ubiquitin 2) assaying for the attachment of the ubiquitin moiety to ligating agent (ULA) where the assay comprises: UA-2. 1) combining a second ubiquitin agent that is a ubiquitin 55 FIG. 48 schematically depicts a preferred embodiment for conjugating agent UCA comprising a ubiquitin moiety assaying for the attachment of ubiquitin moiety to a Sub (plus label)+ULA+CA; and Strate molecule (S) where the assay comprises: 2) assaying for the attachment of the ubiquitin moiety to 1) combining a first ubiquitin agent that is a ubiquitin ULA. In a preferred embodiment the ubiquitin ligating ligating agent ULA+a Second ubiquitin agent-i-Substrate agent comprises an Mdm2 protein. 60 molecule+CA+U, and FIG. 41 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to assaying for the attachment of ubiquitin moiety to a first the substrate molecule. In a preferred embodiment the ubiquitin agent that is a ubiquitin ligating agent (ULA) ubiquitin ligating agent comprises an Mdm2 protein and the which comprises a label where the assay comprises: Substrate molecule comprises p53. 1) combining a second ubiquitin agent that is a ubiquitin 65 FIG. 49 schematically depicts a preferred embodiment for conjugating agent UCA comprising a ubiquitin moiety+ assaying for the attachment of ubiquitin moiety to a Sub ULA (plus label)+CA; and Strate molecule (S) where the assay comprises: US 6,919,184 B2 13 14 1) combining a first ubiquitin agent that is a ubiquitin In the methods of the present invention the ubiquitin ligating agent ULA+a Second ubiquitin agent that is a agents are combined in different combinations with a ubiq ubiquitin conjugating agent and comprising a ubiquitin uitin moiety to assay for the attachment of the ubiquitin moiety+Substrate molecule+CA, and moiety, or the modulation of this attachment, to at least one 2) assaying for the attachment of the ubiquitin moiety to of the following Substrate molecules: a ubiquitin agent, a the substrate molecule. In a preferred embodiment the target protein, or a mono- or poly-ubiquitin moiety which is ubiquitin ligating agent comprises an Mdm2 protein and the preferably attached to a ubiquitin agent or target protein. For Substrate molecule comprises p53. example, the invention provides the following combination FIG. 50 schematically depicts a preferred embodiment for of ubiquitin agents, plus or minus a target protein, for use in assaying for the attachment of ubiquitin moiety to a Sub methods of: Strate molecule (S) where the assay comprises: 1O 1) combining a first ubiquitin agent that is a ubiquitin 1) assaying for the attachment of a ubiquitin moiety to a ligating agent ULA+a Second ubiquitin agent--a third ubiq ubiquitin activating agent by combining a ubiquitin uitin agent that is a ubiquitin activating agent--a ubiquitin activating agent and a ubiquitin moiety; or moiety comprising a first FRET tag--Substrate molecule 2) assaying for the attachment of a ubiquitin moiety to a comprising a Second FRET tag-CA, and 15 ubiquitin conjugating agent by combining a ubiquitin 2) assaying for the attachment of the ubiquitin moiety to activating agent, ubiquitin conjugating agent, and ubiq the substrate molecule. In a preferred embodiment the uitin moiety; or ubiquitin ligating agent comprises an Mdm2 protein and the 3) assaying for the attachment of a ubiquitin moiety to a Substrate molecule comprises p53. ubiquitin conjugating agent by combining a ubiquitin FIG. 51 depicts the amino acid sequence (FIG. 51A (SEQ activating agent comprising a ubiquitin moiety and a ID NO:16)) and the nucleic acid sequence (FIG. 51B (SEQ ubiquitin conjugating agent; or ID NO:17)) of an E2 in a preferred embodiment. 4) assaying for the attachment of a ubiquitin moiety to a FIG. 52 depicts the amino acid sequence (FIG. 52A (SEQ ubiquitin ligating agent by combining a ubiquitin con ID NO:18)) and the nucleic acid sequence (FIG. 52B1 and jugating agent comprising a ubiquitin moiety and a FIG. 52B2 (SEQ ID NO:19)) of an E2 in a preferred 25 ubiquitin ligating agent; or embodiment. 5) assaying for the attachment of a ubiquitin moiety to a FIG. 53 depicts the amino acid sequence (FIG. 53A (SEQ ubiquitin ligating agent by combining a ubiquitin acti ID NO:20)) and the nucleic acid sequence (FIG. 53B1 and Vating agent, ubiquitin conjugating agent, ubiquitin FIG. 53B2 (SEQ ID NO:21)) of an E2 in a preferred ligating agent, and ubiquitin moiety; or embodiment. 6) assaying for the attachment of a ubiquitin moiety to a FIG. 54 depicts the amino acid sequence (FIG. 54A (SEQ ubiquitin ligating agent by combining a ubiquitin acti ID NO:22)) and the nucleic acid sequence (FIG. 54B (SEQ Vating agent comprising a ubiquitin moiety, a ubiquitin ID NO:23)) of an E2 in a preferred embodiment. conjugating agent, and ubiquitin ligating agent; or FIG.55 depicts the amino acid sequence (FIG. 55A (SEQ 7) assaying for the attachment of a ubiquitin moiety to a ID NO:24)) and the nucleic acid sequence (FIG. 55B (SEQ 35 target protein by combining a ubiquitin activating ID NO:25)) of an E2 in a preferred embodiment. agent, a ubiquitin conjugating agent, a ubiquitin ligat DETAILED DESCRIPTION OF THE ing agent, a ubiquitin moiety, and a target protein; or INVENTION 8) assaying for the attachment of a ubiquitin moiety to a The present invention provides methods and composi 40 target protein by combining a ubiquitin activating agent tions for assaying for ubiquitin agents that are enzymatic comprising a ubiquitin moiety, a ubiquitin conjugating components of ubiquitin-mediated proteolysis. More agent, ubiquitin ligating agent, and target protein; or particularly, the present invention provides methods and 9) assaying for the attachment of a ubiquitin moiety to a compositions for assaying for an agent that modulates the target molecule by combining a ubiquitin activating activity of a ubiquitin agent that is an enzymatic component 45 agent comprising a ubiquitin moiety, a ubiquitin con of ubiquitin-mediated proteolysis. Specifically, the methods jugating agent, and a target protein. of the present invention are directed to identifying ubiquitin In particular, in the methods of the present invention, to agents Such as ubiquitin activating agents, ubiquitin conju assay for a candidate agent that modulates the attachment of gating agents, and ubiquitin ligating agents, and to identi a ubiquitin moiety to a Substrate molecule of interest, a fying agents that modulate the activity of these ubiquitin 50 candidate agent is included in the above examples of com agents. binations. The advantages of the present invention include providing The invention provides a variety of approaches using methods for assaying for the activity of ubiquitin agents in above the combinations of ubiquitin agents to assay for the one reaction vessel thus obviating the need for Subsequent attachment of a ubiquitin moiety to a Substrate molecule of Steps, for example, for Separating and purifying the products 55 interest, or to assay for an agent that modulates the attach of the reaction. Consequently, this approach allows multi ment of a ubiquitin moiety to a Substrate molecule of well array analysis and high throughput Screening tech interest. Examples of the approaches are as follows: niques for agents that modulate the activity of ubiquitin 1) the components of the assay are combined in Solution agents. In addition, the present invention provides methods phase, and then assayed for the attachment of ubiquitin that allow the analysis of many different combinations of 60 moiety to the Substrate molecule of interest; or ubiquitin agents, without requiring prior identification of 2) the components of the assay are combined in Solid Specific target proteins. In particular, the present invention phase by providing the Substrate molecule of interest on provides methods that allow the analysis of different com a Solid Support, and then assayed for the attachment of binations of ubiquitin agents in the absence of a target ubiquitin moiety to the Substrate molecule of interest; protein. Alternatively, the present invention provides meth 65 O ods that allow the analysis of combinations of ubiquitin 3) the components of the assay are combined in Solution agents in the presence of a target protein. phase, then the Substrate molecule of interest is US 6,919,184 B2 15 16 attached to a Solid Substrate, and then assayed for the ubiquitin moiety that is not pre-conjugated. For example, in attachment of ubiquitin moiety to the Substrate mol the case of a ubiquitin conjugating agent comprising a ecule of interest; or ubiquitin moiety, the attachment of ubiquitin moiety to a 4) the components of the assay are combined in Solution, ubiquitin ligating agent can be performed in the absence of then the Substrate molecule of interest that is attached a ubiquitin activating agent and ubiquitin moiety that is not to ubiquitin moiety is purified, the purified product is pre-conjugated. Also, for example, in the case of a ubiquitin then attached to a Solid Substrate, and assayed for the ligating agent comprising a ubiquitin moiety, the attachment attachment of ubiquitin moiety to the Substrate mol of ubiquitin moiety to a target molecule can be performed in ecule. the absence of a ubiquitin activating agent, ubiquitin con Examples of ubiquitin agents are ubiquitin activating jugating agent, and ubiquitin moiety that is not pre agents, ubiquitin conjugating agents, and ubiquitin ligating conjugated. A pre-conjugated ubiquitin agent Suitable for agents. In preferred embodiments, the ubiquitin activating use in the methods and compositions of the present inven agent is preferably an E1 or a variant thereof; the ubiquitin tion can be prepared using methods known in the art. In a conjugating agent is preferably an E2 or a variant thereof; preferred embodiment, pre-conjugated ubiquitin agents are and the ubiquitin ligating agent is preferably an E3 or variant 15 prepared according to Zhihong et al. (2001) J. Biol. Chem. there of. In a preferred embodiment, the E3 is Mdm2. In 276:31,357-31,367. another preferred embodiment, the Mdm2 is a fusion By “target protein herein is meant a protein other than a protein, and more preferably an Mdm2-GST fusion protein. ubiquitin moiety to which a ubiquitin moiety is bound or Thus, the present invention provides methods of assaying attached through the activity of a ubiquitin agent or by the for agents that modulate ubiquitin activating activity, ubiq process of ubiquitination. In preferred embodiments, the uitin conjugating activity, and ubiquitin ligating activity. target protein is a mammalian target protein, and more More particularly, the present invention provides methods of preferably a human target protein. In a preferred assaying for agents that modulate the attachment of a embodiment, the target protein is p53. ubiquitin moiety to a ubiquitin agent, target protein, or In the following preferred embodiments at least one mono- or poly-ubiquitin moiety preferably attached to a 25 ubiquitin agent is combined with a ubiquitin moiety in the ubiquitin agent or target protein. absence of a target protein. In general, the methods involve combining a ubiquitin In a preferred embodiment, the invention provides a moiety and one or more ubiquitin agents in the presence of method of assaying for an agent that modulates the or in the absence of a target protein and measuring the attachment of a ubiquitin moiety to at least one ubiq amount of ubiquitin moiety attached to at least one of the uitin agent involving the steps of: a) combining a first following Substrate molecules: a ubiquitin agent; a target ubiquitin agent, a candidate agent, and a ubiquitin protein; or a mono- or poly-ubiquitin moiety which is moiety; and b) assaying for the attachment of the preferably attached to a ubiquitin agent or target protein. AS ubiquitin moiety to the first agent. In one preferred used herein, “substrate molecule' or “target substrate” and embodiment the first ubiquitin agent is an ubiquitin grammatical equivalents thereof means a molecule, prefer 35 activating agent, and preferably an E1. In another ably a protein, to which a ubiquitin moiety is bound or embodiment, the method further comprises including a attached through the activity of a ubiquitin agent or by the Second ubiquitin agent in the combining Step, where the process of ubiquitination. AS used herein, the “Substrate first agent is preferably a ubiquitin conjugating agent molecule of interest' is the ubiquitin agent, target protein, or and more preferably and E2, and the Second agent is ubiquitin moiety to which the attachment of a ubiquitin 40 preferably a ubiquitin activating agent and more pref moiety is being assayed for in the methods of the present erably an E1. In another embodiment, the ubiquitin invention. As used herein with reference to the activity of conjugating agent is preferably an E2 and the ubiquitin ubiquitin agents, “attachment” refers to the transfer, binding, activating agent is preferably an E1 comprising the ligation, and/or ubiquitination of a mono- or poly-ubiquitin ubiquitin moiety. ubiquitin moiety to a Substrate molecule. Thus, "ubiquiti 45 In another embodiment, the first agent is a preferably a nation' and grammatical equivalents thereof means the ubiquitin ligating agent and more preferably an E3; and attachment, or transfer, binding, and/or ligation of ubiquitin the Second agent is preferably a ubiquitin conjugating moiety to a Substrate molecule; and "ubiquitination reac agent comprising the ubiquitin moiety and more pref tion' and grammatical equivalents thereof refer to the com erably an E2 comprising a ubiquitin moiety. bining of components under conditions that permit ubiquiti 50 nation (i.e., the attachment or transfer, binding, and/or In another embodiment, the method further comprises a ligation of ubiquitin moiety to a Substrate molecule). third ubiquitin agent in the combining Step. In one In Some preferred embodiments, the ubiquitin agent com embodiment, the third agent is preferably a ubiquitin prises a ubiquitin moiety. AS used herein with reference to ligating agent and more preferably an E2. a ubiquitin agent, the phrase “comprising a ubiquitin moi 55 In the methods where the assaying concerns the attach ety' or grammatical equivalents thereof refers to the pre ment of the ubiquitin moiety to the first ubiquitin agent, loading, pre-conjugation, or pre-attachment of a ubiquitin the following preferred embodiments are provided. In moiety to a ubiquitin agent (forming a "pre-conjugated one embodiment, the first ubiquitin agent preferably ubiquitin agent” or "pre-loaded ubiquitin agent”) Such that comprises a tag and more preferably an epitope tag or the attachment of a ubiquitin moiety to a Substrate molecule 60 a label. In another embodiment, the first ubiquitin agent of interest does not require combining all three ubiquitin preferably comprises an attachment tag. In another agents (i.e., an ubiquitin activating agent, ubiquitin conju embodiment, the first ubiquitin agent is preferably gating agent, and ubiquitin ligating agent) and/or combining attached to a Solid Support and more preferably is ubiquitin moiety that is not pre-conjugated. For example in attached to a microtiter plate or a bead. the case of a ubiquitin activating agent comprising a ubiq 65 In the methods where the assaying concerns the attach uitin moiety, the attachment of ubiquitin moiety to a ubiq ment of the ubiquitin moiety to the Second ubiquitin uitin conjugating agent can be performed in the absence of agent, the following preferred embodiments are pro US 6,919,184 B2 17 18 Vided. In one embodiment, the Second ubiquitin agent agent and the ubiquitin moiety, thereby forming the preferably comprises a tag and more preferably an ubiquitin conjugating agent comprising the ubiquitin epitope tag or a label. In another embodiment, the moiety. Second ubiquitin agent preferably comprises an attach In an additional preferred embodiment, the method fur ment tag. In another embodiment, the Second ubiquitin 5 ther comprises combining a ubiquitin activating agent agent is preferably attached to a Solid Support and more comprising the ubiquitin moiety, thereby forming the preferably is attached to a microtiter plate or a bead. ubiquitin conjugating agent comprising the ubiquitin In a preferred embodiment, the invention provides a moiety, in Step a). method of assaying for a candidate agent that modul In an additional preferred embodiment, the method fur lates the attachment of a ubiquitin moiety to an MdM2 ther comprises combining a ubiquitin activating agent protein involving the steps of: a) combining a first and the ubiquitin moiety, thereby forming the ubiquitin ubiquitin agent comprising at least one ubiquitin conjugating agent comprising the ubiquitin moiety. moiety, an MdM2 protein, and a candidate agent; and In another preferred embodiment, the invention provides b) assaying for the attachment of the ubiquitin moiety a method of assaying for a candidate agent that modul to the MdM2 protein. In an additional embodiment, the 15 lates the attachment of a ubiquitin moiety to a p53 first ubiquitin agent is preferably a ubiquitin conjugat protein involving the steps of: a) combining a ubiquitin ing agent. activating agent, a ubiquitin conjugating agent, an MdM2 protein, a p53 protein, a candidate agent, and a In another preferred embodiment, the method further ubiquitin moiety; and b) assaying for the attachment of comprises combining a ubiquitin activating agent com the ubiquitin moiety to the p53 protein. prising the ubiquitin moiety, thereby forming the ubiq In another preferred embodiment, the invention provides uitin conjugating agent comprising the ubiquitin a method of assaying for a candidate agent that modul moiety, in Step a). lates the attachment of a Second ubiquitin moiety to a In another preferred embodiment, the method further comprises combining a ubiquitin activating agent and p53 protein involving the steps of: a) combining a 25 ubiquitin activating agent, a ubiquitin conjugating the ubiquitin moiety, thereby forming the ubiquitin agent, an MdM2 protein, a p53 protein comprising a conjugating agent comprising the ubiquitin moiety. first ubiquitin moiety, wherein the first ubiquitin moiety In another preferred embodiment, the invention provides is labeled with a first FRET label, a candidate agent, a method of assaying for a candidate agent that modul and a Second ubiquitin moiety labeled with a Second lates the attachment of a ubiquitin moiety to an MdM2 FRET label; and b) assaying for the attachment of the protein involving the steps of: a) combining a ubiquitin Second ubiquitin moiety to the p53 protein by detecting activating agent, a ubiquitin conjugating agent, an a FRET reaction. MdM2 protein, a candidate agent, and a ubiquitin In another preferred embodiment, the invention provides moiety; and b) assaying for the attachment of the a method of assaying for a candidate agent that modul ubiquitin moiety to the MidM2 protein. 35 lates the attachment of a first ubiquitin moiety to a p53 Alternatively, the invention provides assays including a protein involving the steps of: a) combining a ubiquitin target protein. In the following preferred embodiments a conjugating agent comprising a first ubiquitin moiety target protein is combined with ubiquitin moiety and at least labeled with a first FRET, an MdM2 protein, a p53 one ubiquitin agent. protein comprising a Second ubiquitin moiety, wherein In another preferred embodiment, the invention provides 40 the first ubiquitin moiety is labeled with a second FRET a method of assaying for an agent that modulates the label, and a candidate agent; and b) assaying for the attachment of a ubiquitin moiety to at least one ubiq attachment of the first ubiquitin moiety to the p53 uitin agent involving the steps of: a) combining a first protein by detecting a FRET reaction. ubiquitin agent comprising a ubiquitin ligating agent, a In another preferred embodiment, the invention provides Second ubiquitin agent, a candidate agent, a ubiquitin 45 a method of assaying for a candidate agent that modul moiety, and a target protein; and b) assaying for the lates the attachment of a first ubiquitin moiety to a p53 attachment of the ubiquitin moiety to the first agent. In protein involving the steps of: a) combining a ubiquitin an additional embodiment, the Second agent is a ubiq activating agent comprising a first ubiquitin moiety uitin conjugating agent comprising the ubiquitin moi labeled with a first FRET, a ubiquitin conjugating ety. 50 agent, an MdM2 protein, a p53 protein comprising a In another preferred embodiment, the method further Second ubiquitin moiety, wherein the first ubiquitin comprises a third ubiquitin agent in the combining Step, moiety is labeled with a second FRET label, and a wherein the third agent is a ubiquitin activating agent; candidate agent; and b) assaying for the attachment of wherein the Substrate and the ubiquitin moiety com the first ubiquitin moiety to the p53 protein by detecting prise different fluorescent labels, and wherein the labels 55 a FRET reaction. form a fluorescence resonance energy transfer (FRET) In a preferred embodiment, the substrate molecule of pair. interest is attached to the Surface of a reaction vessel, Such In another preferred embodiment, the invention provides as the well of a multi-well plate. This embodiment facilitates a method of assaying for a candidate agent that modul the Separation of the ubiquitin moiety that is attached to the lates the attachment of a ubiquitin moiety to a p53 60 Substrate molecule of interest from the unattached or free protein involving the steps of: a) combining a conju ubiquitin moiety. Means for attaching ubiquitin agents or gating agent comprising at least one ubiquitin moiety, target proteins to the Surface of a reaction vessel are an Mdm2 protein, a p53 protein, and a candidate agent; described below. The present methods permits the entire and b) assaying for the attachment of the ubiquitin assay to occur in one vessel, making the assay useful for moiety to the p53 protein. 65 high-throughput Screening applications. In an additional preferred embodiment, the method fur In a preferred embodiment, the ubiquitin moiety is ther comprises combining a ubiquitin conjugating labeled, either directly or indirectly, as further described US 6,919,184 B2 19 20 below, and the amount of label is measured and indicative of acid sequence depicted in FIG. 15A (SEQ ID NO:13) of the amount of attachment of ubiquitin moiety to a Substrate greater than about 80%, more preferably greater than about molecule of interest. Thus, the invention provides methods 85%, even more preferably greater than about 90% and most that permit for easy and rapid detection and measurement of preferably greater than 93%. In some embodiments the the activity of ubiquitin agents, making the assay useful for sequence identity will be as high as about 95 to 98 or 99%. high-throughput Screening applications. In one preferred AS is known in the art, a number of different programs can embodiment, the signal of the label varies with the extent of be used to identify whether a protein (or nucleic acid as the attachment of ubiquitin moiety to the Substrate molecule discussed below) has sequence identity or similarity to a of interest, such as in the FRET system described below. One of ordinary skill in the art will recognize the applica known Sequence. Sequence identity and/or Similarity is bility of the present invention to Screening for agents which determined using Standard techniques known in the art, modulate ubiquitin ubiquitination. including, but not limited to, the local Sequence identity AS used herein, "ubiquitin moiety' refers to a polypeptide algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 which is transferred or attached to another polypeptide by a (1981), by the sequence identity alignment algorithm of ubiquitin agent. The ubiquitin moiety can comprise a ubiq Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the uitin from any species of organism, preferably a eukaryotic 15 search for similarity method of Pearson & Lipman, PNAS Species. In preferred embodiments the ubiquitin moiety USA 85:2444 (1988), by computerized implementations of comprises is a mammlian ubiquitin, and more preferably a these algorithms (GAP, BESTFIT, FASTA, and TFASTA in human ubiquitin. In a preferred embodiment, the ubiquitin the Wisconsin Genetics Software Package, Genetics Com moiety comprises a 76 amino acid human ubiquitin. In a puter Group, 575 Science Drive, Madison, Wis.), the Best preferred embodiment, the ubiquitin moiety comprises the Fit Sequence program described by Devereux et al., Nucl. amino acid sequence depicted in FIG. 15A. Other embodi Acid Res. 12:387–395 (1984), preferably using the default ments utilize variants of ubiquitin, as further described Settings, or by inspection. Preferably, percent identity is below. calculated by FastDB based upon the following parameters: AS used herein, "poly-ubiquitin moiety' refers to a chain mismatch penalty of 1, gap penalty of 1, gap Size penalty of of ubiquitin moieties comprising more than one ubiquitin 25 0.33; and joining penalty of 30, “Current Methods in moiety. AS used herein, "mono-ubiquitin moiety' refers to a Sequence Comparison and Analysis,” Macromolecule Single ubiquitin moiety. In the methods of the present Sequencing and Synthesis, Selected Methods and invention, a mono- or poly-ubiquitin moiety can Serve as a Applications, pp 127-149 (1988), Alan R. Liss, Inc. Substrate molecule for the transfer or attachment of ubiquitin An example of a useful algorithm is PILEUP, PILEUP moiety (which can itself be a mono- or poly-ubiquitin creates a multiple Sequence alignment from a group of moiety). related Sequences using progressive, pairwise alignments. It In a preferred embodiment, when ubiquitin moiety is can also plot a tree showing the clustering relationships used attached to a target protein, that protein is targeted for to create the alignment. PILEUP uses a simplification of the degradation by the 26S proteasome. progressive alignment method of Feng & Doolittle, J. Mol. AS used herein, "ubiquitin moiety' encompasses naturally 35 Evol. 35:351–360 (1987); the method is similar to that occurring alleles and man-made variants of Such a 76 amino described by Higgins & Sharp CABIOS 5:151-153 (1989). acid polypeptide. In a preferred embodiment, the ubiquitin Useful PILEUP parameters including a default gap weight of moiety comprises an amino acid Sequence or nucleic acid 3.00, a default gap length weight of 0.10, and weighted end Sequence corresponding to a sequence of GENBANK acces gapS. sion number P02248, incorporated herein by reference. In 40 Another example of a useful algorithm is the BLAST other preferred embodiments, the ubiquitin moiety com algorithm, described in Altschul et al., J. Mol. Biol. 215, prises an amino acid Sequence or nucleic acid Sequence of a 403-410, (1990) and Karlin et al., PNAS USA sequence corresponding to one of the following GENBANK 90:5873-5787 (1993). A particularly useful BLAST pro accession numbers: NM 006156 (NEDD8); NM 003352 gram is the WU-BLAST-2 program which was obtained (SUMO-1, aka, UBL1); XM 048691 (SUMO-1, aka, 45 from Altschul et al., Methods in Enzymology, 266: 460-480 UBL1); NM 006936 (smt3a); XM 009805 (smt3a); (1996); http://blast.wustl/edu/blast/README.html). XM 095400 (smt3b); NM 006937 (smt3b); XM 041583 WU-BLAST-2 uses several search parameters, most of (smt3b); NM 015783 (ISG15); or NM 005101 (ISG15), which are Set to the default values. The adjustable param each incorporated herein by reference. eters are Set with the following values: overlap span=1, GENBANK accession numbers and their corresponding 50 overlap fraction=0.125, word threshold (T)=11. The HSPS amino acid Sequences or nucleic acid Sequences are found in and HSPS2 parameters are dynamic values and are estab the Genbank data base. Sequences corresponding to Gen lished by the program itself depending upon the composition Bank accession numbers cited herein are incorporated herein of the particular Sequence and composition of the particular by reference. GenBank is known in the art, See, e.g., Benson, database against which the Sequence of interest is being D A, et al., Nucleic Acids Research 26:1-7 (1998) and 55 Searched; however, the values may be adjusted to increase http://www.ncbi.nlm.nih.gov/. Preferably, the ubiquitin moi Sensitivity. ety has the amino acid sequence depicted in FIG. 15A (SEQ An additional useful algorithm is gapped BLAST as ID NO:13). In a preferred embodiment, variants of ubiquitin reported by Altschul et al. Nucleic Acids Res. moiety have an overall amino acid Sequence identity of 25:3389–3402. Gapped BLAST uses BLOSUM-62 substi preferably greater than about 75%, more preferably greater 60 tution scores; threshold T parameter set to 9; the two-hit than about 80%, even more preferably greater than about method to trigger ungapped extensions, charges gap lengths 85% and most preferably greater than 90% of the amino acid of k a cost of 10+k; X, set to 16, and X set to 40 for sequence depicted in FIG. 15A (SEQ ID NO:13). In some database Search Stage and to 67 for the output Stage of the embodiments the Sequence identity will be as high as about algorithms. Gapped alignments are triggered by a Score 93 to 95 or 98%. 65 corresponding to ~22 bits. In another preferred embodiment, a ubiquitin moiety A percent amino acid Sequence identity value is deter protein has an overall Sequence Similarity with the amino mined by the number of matching identical residues divided US 6,919,184 B2 21 22 by the total number of residues of the “longer” sequence in a given Sample. A Substantially pure protein comprises at the aligned region. The "longer Sequence is the one having least about 75% by weight of the total protein, with at least the most actual residues in the aligned region (gaps intro about 80% being preferred, and at least about 90% being duced by WU-Blast-2 to maximize the alignment score are particularly preferred. The definition includes the production ignored). of a protein from one organism in a different organism or The alignment may include the introduction of gaps in the host cell. Alternatively, the protein may be made at a Sequences to be aligned. In addition, for Sequences which Significantly higher concentration than is normally Seen, contain either more or fewer amino acids than the amino through the use of an inducible promoter or high expression acid sequence depictd in FIG. 15A (SEQ ID NO:13), it is promoter, Such that the protein is made at increased con understood that in one embodiment, the percentage of centration levels. Alternatively, the protein may be in a form sequence identity will be determined based on the number of not normally found in nature, as in the addition of an epitope identical amino acids in relation to the total number of amino tag or amino acid Substitutions, insertions and deletions, as acids. Thus, for example, Sequence identity of Sequences discussed below. shorter than that of the sequence depicted in FIG. 15A (SEQ AS used herein and further defined below, “nucleic acid” ID NO:13), as discussed below, will be determined using the 15 may refer to either DNA or RNA, or molecules which number of amino acids in the Shorter Sequence, in one contain both deoxy- and ribonucleotides. The nucleic acids embodiment. In percent identity calculations relative weight include genomic DNA, cDNA and oligonucleotides includ is not assigned to various manifestations of Sequence ing Sense and anti-Sense nucleic acids. Such nucleic acids variation, Such as, insertions, deletions, Substitutions, etc. may also contain modifications in the ribose-phosphate In one embodiment, only identities are Scored positively backbone to increase stability and half life of such molecules (+1) and all forms of Sequence variation including gaps are in physiological environments. assigned a value of “0”, which obviates the need for a The nucleic acid may be double Stranded, Single Stranded, weighted Scale or parameters as described below for or contain portions of both double Stranded or Single Sequence Similarity calculations. Percent Sequence identity Stranded Sequence. AS will be appreciated by those in the art, can be calculated, for example, by dividing the number of 25 the depiction of a single strand (“Watson') also defines the matching identical residues by the total number of residues Sequence of the other Strand (“Crick'); thus the Sequences of the “shorter Sequence in the aligned region and multi depicted in the figures also include the complement of the plying by 100. The "longer Sequence is the one having the Sequence. By the term “recombinant nucleic acid herein is most actual residues in the aligned region. meant nucleic acid, originally formed in vitro, in general, by Ubiquitin moieties of the present invention are polypep the manipulation of nucleic acid by endonucleases, in a form tides that may be shorter or longer than the amino acid not normally found in nature. Thus an isolated nucleic acid, sequence depicted in FIG. 15A (SEQ ID NO:13). Thus, in a in a linear form, or an expression vector formed in vitro by preferred embodiment, included within the definition of ligating DNA molecules that are not normally joined, are ubiquitin moiety are portions or fragments of the amino acid both considered recombinant for the purposes of this inven sequence depicted in FIG. 15A (SEQ ID NO:13). In one 35 tion. It is understood that once a recombinant nucleic acid is embodiment herein, fragments of ubiquitin moiety are con made and reintroduced into a host cell or organism, it will sidered ubiquitin moieties if they are attached to another replicate non-recombinantly, i.e. using the in Vivo cellular polypeptide by a ubiquitin agent. machinery of the host cell rather than in Vitro manipulations, In addition, as is more fully outlined below, ubiquitin however, Such nucleic acids, once produced recombinantly, moieties of the present invention are polypeptides that can 40 although Subsequently replicated non-recombinantly, are be made longer than the amino acid Sequence depicted in Still considered recombinant for the purposes of the inven FIG. 15A (SEQ ID NO:13); for example, by the addition of tion. tags, the addition of other fusion Sequences, or the elucida The terms “polypeptide' and “protein' may be used tion of additional coding and non-coding Sequences. AS interchangeably throughout this application and mean at described below, the fusion of a ubiquitin moiety to a 45 least two covalently attached amino acids, which includes fluorescent peptide, Such as Green Fluorescent Peptide proteins, polypeptides, oligopeptides and peptides. The pro (GFP), is particularly preferred. tein may be made up of naturally occurring amino acids and The ubiquitin moiety, as well as other proteins of the peptide bonds, or Synthetic peptidomimetic Structures. Thus present invention, are preferably recombinant proteins. A “amino acid', or "peptide residue', as used herein means “recombinant protein' is a protein made using recombinant 50 both naturally occurring and Synthetic amino acids. For techniques, i.e. through the expression of a recombinant example, homo-phenylalanine, citrulline and noreleucine nucleic acid as described below. In a preferred embodiment, are considered amino acids for the purposes of the invention. the ubiquitin moiety of the invention is made through the “Amino acid” also includes imino acid residues Such as expression of a nucleic acid Sequence corresponding to proline and hydroxyproline. The Side chains may be in either GENBANK accession number M26880 or AB003730, or a 55 the (R) or the (S) configuration. In the preferred fragment thereof. In a most preferred embodiment, the embodiment, the amino acids are in the (S) or nucleic acid encodes the amino acid Sequence depicted in L-configuration. If non-naturally occurring Side chains are FIG. 15A (SEQ ID NO:13). A recombinant protein is used, non-amino acid Substituents may be used, for example distinguished from naturally occurring protein by at least to prevent or retard in Vivo degradation. one or more characteristics. For example, the protein may be 60 In one embodiment, the present invention provides com isolated or purified away from Some or all of the proteins and positions containing protein variants, for example ubiquitin compounds with which it is normally associated in its wild moiety, E1, E2 and/or E3 variants. These variants fall into type host, and thus may be Substantially pure. For example, one or more of three classes: Substitutional, insertional or an isolated protein is unaccompanied by at least Some of the deletional variants. These variants ordinarily are prepared by material with which it is normally associated in its natural 65 Site Specific mutagenesis of nucleotides in the DNA encod State, preferably constituting at least about 0.5%, more ing a protein of the present compositions, using cassette or preferably at least about 5% by weight of the total protein in PCR mutagenesis or other techniques well known in the art, US 6,919,184 B2 23 24 to produce DNA encoding the variant, and thereafter than those shown in the above list. For example, substitu expressing the DNA in recombinant cell culture as outlined tions may be made which more significantly affect: the above. However, variant protein fragments having up to Structure of the polypeptide backbone in the area of the about 100-150 residues may be prepared by in vitro syn alteration, for example the alpha-helical or beta-sheet Struc thesis using established techniques. Amino acid Sequence ture; the charge or hydrophobicity of the molecule at the variants are characterized by the predetermined nature of the target site; or the bulk of the side chain. The Substitutions variation, a feature that Sets them apart from naturally which in general are expected to produce the greatest occurring allelic or interspecies variation of the protein changes in the polypeptide's properties are those in which amino acid Sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring (a) a hydrophilic residue, e.g. Seryl or threonyl, is Substituted analogue, although variants can also be Selected which have for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, modified characteristics as will be more fully outlined phenylalanyl, Valyl or alanyl; (b) a cysteine or proline is below. Substituted for (or by) any other residue; (c) a residue having While the Site or region for introducing an amino acid an electropositive Side chain, e.g. lysyl, arginyl, or histidyl, Sequence variation is predetermined, the mutation per Se is Substituted for (or by) an electronegative residue, e.g. need not be predetermined. For example, in order to opti 15 glutamyl or aspartyl; or (d) a residue having a bulky side mize the performance of a mutation at a given Site, random chain, e.g. phenylalanine, is Substituted for (or by) one not mutagenesis may be conducted at the target codon or region having a side chain, e.g. glycine. and the expressed variants Screened for the optimal desired The variants typically exhibit the same qualitative bio activity. Techniques for making Substitution mutations at logical activity and will elicit the Same immune response as predetermined sites in DNA having a known Sequence are the naturally-occurring analogue, although variants also are well known, for example, M13 primer mutagenesis and PCR Selected to modify the characteristics of the proteins as mutagenesis. Rapid production of many variants may be needed. Alternatively, the variant may be designed Such that done using techniqueS Such as the method of gene Shuffling, the biological activity of the protein is altered. For example, whereby fragments of Similar variants of a nucleotide glycosylation sites may be altered or removed. Sequence are allowed to recombine to produce new variant 25 Covalent modifications of polypeptides are included combinations. Examples of Such techniques are found in within the scope of this invention. One type of covalent U.S. Pat. Nos. 5,605,703; 5,811,238; 5,873,458; 5,830,696; modification includes reacting targeted amino acid residues 5,939,250; 5,763,239; 5,965,408; and 5,945,325, each of of a polypeptide with an organic derivatizing agent that is which is incorporated by reference herein in its entirety. capable of reacting with Selected Side chains or the N- or Screening of the mutants is performed using the activity C-terminal residues of a polypeptide. Derivatization with assays of the present invention. bifunctional agents is useful, for instance, for crosslinking a Amino acid substitutions are typically of single residues; protein to a water-insoluble support matrix or surface for use insertions usually will be on the order of from about 1 to 20 in the method for Screening assays, as is more fully amino acids, although considerably larger insertions may be described below. Commonly used crosslinking agents tolerated. Deletions range from about 1 to about 20 residues, 35 include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, although in Some cases deletions may be much larger. glutaraldehyde, -hydroxy-Succinimide esters, for example, Substitutions, deletions, insertions or any combination esters with 4-azidosalicylic acid, homobifunctional thereof may be used to arrive at a final derivative. Generally imidoesters, including disuccinimidyl esterS Such as 3,3'- these changes are done on a few amino acids to minimize the dithiobis(Succinimidylpropionate), bifunctional maleimides alteration of the molecule. However, larger changes may be 40 Such as bis-N-maleimido-1,8-Octane and agents Such as tolerated in certain circumstances. When Small alterations in methyl-3-(p-azidophenyl)dithiopropioimidate. the characteristics of the protein are desired, Substitutions of Other modifications include deamidation of glutaminyl an original residue are generally made in accordance with and asparaginyl residues to the corresponding glutamyl and exemplary substitutions listed below. aspartyl residues, respectively, hydroxylation of proline and 45 lysine, phosphorylation of hydroxyl groups of Seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains T. E. Creighton, Pro Original Residue Exemplary Substitutions teins. Structure and Molecular Properties, W.H. Freeman & Ala Ser Co., San Francisco, pp. 79-86 (1983)), acetylation of the Arg Lys 50 N-terminal amine, and amidation of any C-terminal car Asn Gln, His boxyl group. Asp Glu Cys Ser, Ala Another type of covalent modification of a polypeptide Glin Asn included within the Scope of this invention comprises alter Glu Asp ing the native glycosylation pattern of the polypeptide. Gly Pro 55 “Altering the native glycosylation pattern' is intended for His ASn, Gln Ile Leu, Val purposes herein to mean deleting one or more carbohydrate Leu Ile, Val moieties found in native Sequence polypeptide, and/or add Lys Arg, Gln, Glu ing one or more glycosylation Sites that are not present in the Met Leu, Ile native Sequence polypeptide. Phe Met, Leu, Tyr Ser Thr 60 Addition of glycosylation Sites to polypeptides may be Thr Ser accomplished by altering the amino acid Sequence thereof. Trp Tyr The alteration may be made, for example, by the addition of, Tyr Trp, Phe or Substitution by, one or more Serine or threonine residues Val Ile, Leu to the native sequence polypeptide (for O-linked glycosy 65 lation sites). The amino acid sequence may optionally be Substantial changes in function or immunological identity altered through changes at the DNA level, particularly by are made by Selecting Substitutions that are leSS conservative mutating the DNA encoding the polypeptide at preselected US 6,919,184 B2 25 26 bases Such that codons are generated that will translate into molecule that is preferably a ubiquitin activating agent. In a the desired amino acids. preferred embodiment, the ubiquitin activating agent is an Another means of increasing the number of carbohydrate E1. Preferably, the E1 forms a high energy thiolester bond moieties on a polypeptide is by chemical or enzymatic with the ubiquitin moiety. coupling of glycosides to the polypeptide. Such methods are In a preferred embodiment, the invention is also directed to a method of assaying for ubiquitin conjugating activity. described in the art, e.g., in WO 87/05330 published 11 Sep. By "ubiquitin conjugating activity”, “ubiquitin moiety con 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., jugation' and grammatical equivalents thereof is meant the pp. 259-306 (1981). binding or attachment of an activated ubiquitin moiety to a Removal of carbohydrate moieties present on the ubiquitin conjugating agent. AS will be appreciated by those polypeptide may be accomplished chemically or enzymati in the art, due to the presence of the high energy thiolester cally or by mutational Substitution of codons encoding for bond in the conjugate of the ubiquitin moiety-ubiquitin amino acid residues that Serve as targets for glycosylation. conjugating agent, the attached ubiquitin moiety may be Chemical deglycosylation techniques are known in the art joined to other ubiquitin moiety at a low rate in the absence and described, for instance, by Hakimuddin, et al., Arch. of the catalytic activity of a ubiquitin ligating agent (e.g., Biochem. BiophyS., 259:52 (1987) and by Edge et al., Anal. 15 E3). Therefore, some of the ubiquitin moiety will be Biochem., 118:131 (1981). Enzymatic cleavage of carbohy attached in the form of poly-ubiquitin moiety. drate moieties on polypeptides can be achieved by the use of In a preferred embodiment, the invention is directed to a a variety of endo- and eXO-glycosidases as described by method of assaying ubiquitin ligating activity. By "ubiquitin Thotakura et al., Meth. Enzymol., 138:350 (1987). ligating activity”, “ubiquitin moiety ligation' and grammati Another type of covalent modification of a protein com cal equivalents thereof is meant the transfer or attachment of prises linking the polypeptide to one of a variety of non ubiquitin moiety to a Substrate molecule that is preferably a proteinaceous polymers, e.g., polyethylene glycol, polypro target protein or mono- or poly-ubiquitin moiety preferably pylene glycol, or polyoxyalkylenes, in the manner Set forth attached to a target protein. Preferably, each ubiquitin moi in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670, ety is covalently attached by the ubiquitin ligating agent 417; 4,791,192 or 4,179,337. 25 Such that a Subsequent ubiquitin moiety may be attached to Polypeptides of the present invention may also be modi it, to form chains (poly-ubiquitin moieties) comprising a fied in a way to form chimeric molecules comprising a first plurality of ubiquitin moiety molecules. polypeptide fused to another, heterologous polypeptide or The present invention provides methods and composi amino acid Sequence. In one embodiment, Such a chimeric tions comprising combining ubiquitin moiety with other molecule comprises a fusion of a Substrate molecule (e.g., a components. By “combining” is meant the addition of the ubiquitin moiety, ubiquitin agent, or target protein) with a various components into a reaction vessel under conditions tag polypeptide which provides an epitope to which an in which attachment of ubiquitin moiety to a substrate anti-tag antibody can Selectively bind. The epitope tag is molecule interest can occur. In a preferred embodiment, the generally placed at the amino- or carboxyl-terminus of the reaction vessel is a well of a 96 well plate or other com polypeptide. The presence of Such epitope-tagged forms of 35 mercially available multiwell plate. In an alternate preferred a polypeptide can be detected using an antibody against the embodiment, the reaction vessel is in a FACS machine. tag polypeptide. Also, providing an epitope tag enables the Other reaction vessels useful in the present invention polypeptide to be readily purified by affinity purification include, but are not limited to 384 well plates and 1536 well using an anti-tag antibody or another type of affinity matrix plates. Still other reaction vessels useful in the present that binds to the epitope tag. In an alternative embodiment, 40 invention will be apparent to the skilled artisan. the chimeric molecule may comprise a fusion of a polypep The addition of the components may be sequential or in tide disclosed herein with an immunoglobulin or a particular a predetermined order or grouping, as long as the conditions region of an immunoglobulin. For a bivalent form of the amenable to the attachment of ubiquitin to a Substrate chimeric molecule, Such a fusion could be to the Fc region molecule of interest are obtained. Such conditions are well of an IgG molecule. Tags for components of the invention 45 known in the art, and further guidance is provided below. are defined and described in detail below. In a preferred embodiment, one or more components of The present invention provides methods for assaying for the present invention comprise a tag. By “tag” is meant an the attachment of ubiquitin moiety to a Substrate molecule of attached molecule or molecules useful for the identification interest. Preferred embodiments of the invention involve or isolation of the attached molecule(s), which are preferably combining ubiquitin moiety and ubiquitin agents, plus or 50 Substrate molecules. For example, a tag can be an attach minus target protein; and further in the presence or absence ment tag or a label tag. Components having a tag are referred of a candidate agent, under conditions where ubiquitin to as "tag-X”, wherein X is the component. For example, a moiety can attach to a Substrate molecule of interest; and ubiquitin moiety comprising a tag is referred to herein as assaying for the attachment of the ubiquitin moiety to the "tag-ubiquitin moiety'. Preferably, the tag is covalently Substrate molecule of interest, for example, by measuring 55 bound to the attached component. When more than one the amount of ubiquitin moiety (mono- or poly-ubiquitin component of a combination has a tag, the tags will be moiety) attached to the Substrate molecule. In these assays, numbered for identification, for example “tag1-ubiquitin the activity resulting from the combination of different moiety'. Components may comprise more than one tag, in ubiquitin agents and combination of different Subunits of which case each tag will be numbered, for example “tag individual ubiquitin agents, plus or minus target protein; and 60 1,2-ubiquitin moiety'. Preferred tags include, but are not further, in the presence or absence of a candidate ubiquitin limited to, a label, a partner of a binding pair, and a Surface agent, can be observed and measured. Substrate binding molecule (or attachment tag). AS will be In a preferred embodiment, the invention is additionally evident to the skilled artisan, many molecules may find use directed to a method of assaying for ubiquitin activating as more than one type of tag, depending upon how the tag activity. By "ubiquitin activating activity, "ubiquitin moiety 65 is used. activation' and grammatical equivalents thereof is meant the By “label” is meant a molecule that can be directly (i.e., binding or attachment of ubiquitin moiety to a Substrate a primary label) or indirectly (i.e., a secondary label) US 6,919,184 B2 27 28 detected; for example a label can be visualized and/or be employed. In this application, fluorescent emission of the measured or otherwise identified So that its presence or donor will increase when quencher is displaced from close absence can be known. AS will be appreciated by those in the proximity to the donor and fluorescent emission will art, the manner in which this is performed will depend on the decrease when the quencher is brought into close proximity label. Preferred labels include, but are not limited to, fluo to the donor. Useful quenchers include, but are not limited rescent labels, label enzymes and radioisotopes. to, DABCYL, QSY 7 and OSY 33. Useful fluorescent By “fluorescent label' is meant any molecule that may be donor/quencher pairs include, but are not limited to detected via its inherent fluorescent properties. Suitable EDANS/DABCYL, Texas Red/DABCYL, BODIPY/ fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL coumarin, methyl-coumarins, pyrene, Malacite green, and fluorescein/QSY 7 dye. stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, The skilled artisan will appreciate that FRET and fluo IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy rescence quenching allow for monitoring of binding of 5.5, LC Red 705 and Oregon green. Suitable optical dyes are labeled molecules over time, providing continuous informa described in the 1996 Molecular Probes Handbook by tion regarding the time course of binding reactions. Richard P. Haugland, hereby expressly incorporated by 15 It is important to remember that ubiquitin moiety is reference. Suitable fluorescent labels also include, but are ligated to a Substrate molecule by its terminal carboxyl not limited to, green fluorescent protein (GFP; Chalfie, et al., group to a lysine residue, including lysine residues on other Science 263(5148):802–805 (Feb. 11, 1994); and EGFP; ubiquitin moiety. Therefore, attachment of labels or other Clontech-Genbank Accession Number U55762), blue fluo tags should not interfere with either of these active groups on rescent protein (BFP; 1. Quantum Biotechnologies, Inc. the ubiquitin moiety. Amino acids may be added to the 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal Sequence of protein, through means well known in the art (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques and described herein, for the express purpose of providing a 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. point of attachment for a label. In a preferred embodiment, Biol. 6:178-182 (1996)), enhanced yellow fluorescent pro one or more amino acids are added to the Sequence of a tein (EYFP; 1. Clontech Laboratories, Inc., 1020 East 25 component for attaching a tag thereto, preferably a fluores Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, cent label. In a preferred embodiment, the amino acid to et al., J. Immunol. 150(12):5408-5417 (1993)), galactosi which a fluorescent label is attached is Cysteine. dase (Nolan, et al., Proc Natl Acad Sci USA 85(8) By “label enzyme” is meant an enzyme which may be :2603–2607 (April 1988)) and Renilla WO92/15673; WO reacted in the presence of a label enzyme Substrate which 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; produces a detectable product. Suitable label enzymes for U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. use in the present invention include but are not limited to, No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. horseradish peroxidase, alkaline phosphatase and glucose 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; oxidase. Methods for the use of Such Substrates are well U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558) All of known in the art. The presence of the label enzyme is the above-cited references are expressly incorporated herein 35 generally revealed through the enzyme’s catalysis of a by reference. reaction with a label enzyme Substrate, producing an iden In Some instances, multiple fluorescent labels are tifiable product. Such products may be opaque, Such as the employed. In a preferred embodiment, at least two fluores reaction of horseradish peroxidase with tetramethyl cent labels are used which are members of a fluorescence benzedine, and may have a variety of colors. Other label resonance energy transfer (FRET) pair. FRET is phenom 40 enzyme Substrates, Such as Luminol (available from Pierce enon known in the art wherein excitation of one fluorescent Chemical Co.), have been developed that produce fluores dye is transferred to another without emission of a photon. cent reaction products. Methods for identifying label AFRET pair consists of a donor fluorophore and an acceptor enzymes with label enzyme Substrates are well known in the fluorophore. The fluorescence emission Spectrum of the art and many commercial kits are available. Examples and donor and the fluorescence absorption spectrum of the 45 methods for the use of various label enzymes are described acceptor must overlap, and the two molecules must be in in Savage et al., Previews 247:6-9 (1998), Young, J. Virol. close proximity. The distance between donor and acceptor at Methods 24:227–236 (1989), which are each hereby incor which 50% of donors are deactivated (transfer energy to the porated by reference in their entirety. acceptor) is defined by the Förster radius (R), which is By “radioisotope' is meant any radioactive molecule. typically 10-100 A. Changes in the fluorescence emission 50 Suitable radioisotopes for use in the invention include, but Spectrum comprising FRET pairs can be detected, indicating are not limited to 'C, H, P, P, S, 'I, and I. The changes in the number of that are in close proximity (i.e., use of radioisotopes as labels is well known in the art. within 100 A of each other). This will typically result from In addition, labels may be indirectly detected, that is, the the binding or dissociation of two molecules, one of which tag is a partner of a binding pair. By "partner of a binding is labeled with a FRET donor and the other of which is 55 pair is meant one of a first and a Second moiety, wherein labeled with a FRET acceptor, wherein such binding brings Said first and Said Second moiety have a specific binding the FRET pair in close proximity. Binding of such molecules affinity for each other. Suitable binding pairs for use in the will result in an increased fluorescence emission of the invention include, but are not limited to, antigens/antibodies acceptor and/or quenching of the fluorescence emission of (for example, digoxigenin/anti-digoxigenin, dinitrophenyl the donor. 60 (DNP)/anti-DNP, dansyl-X-anti-dansyl, Fluorescein/anti FRET pairs (donor/acceptor) useful in the invention fluorescein, lucifer yellow/anti-lucifer yellow, and include, but are not limited to, EDANS/fluorescien, rhodamine anti-rhodamine), biotin/avid (or biotin/ IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, streptavidin) and calmodulin binding protein (CBP)/ fluorescein/LC Red 640, fluorescein/Cy 5, fluorescein/Cy calmodulin. Other Suitable binding pairs include polypep 5.5 and fluorescein/LC Red 705. 65 tides such as the FLAG-peptide Hopp et al., BioTechnology, In another aspect of FRET, a fluorescent donor molecule 6:1204-1210 (1988); the KT3 epitope peptide Martin et and a nonfluorescent acceptor molecule (“quencher') may al., Science, 255:192-194 (1992); tubulin epitope peptide US 6,919,184 B2 29 30 Skinner et al., J. Biol. Chem., 266:15163-15166 (1991); (for example, from Kodak and Sigma). Methods for the and the T7 gene 10 protein peptide tag Lutz-Freyermuth et production and use of FLAG-labeled proteins are found, for al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990) and example, in Winston et al., Genes and Devel. 13:270–283 the antibodies each thereto. Generally, in a preferred (1999), incorporated herein in its entirety, as well as product embodiment, the Smaller of the binding pair partnerS Serves 5 handbooks provided with the above-mentioned kits. as the tag, as Steric considerations in ubiquitin moiety Biotinylation of target molecules and Substrates is well ligation may be important. AS will be appreciated by those known, for example, a large number of biotinylation agents in the art, binding pair partners may be used in applications are known, including amine-reactive and thiol-reactive other than for labeling, as is further described below. agents, for the biotinylation of proteins, nucleic acids, AS will be appreciated by those in the art, a partner of one carbohydrates, carboxylic acids, See chapter 4, Molecular binding pair may also be a partner of another binding pair. Probes Catalog, Haugland, 6th Ed. 1996, hereby incorpo For example, an antigen (first moiety) may bind to a first rated by reference. A biotinylated Substrate can be attached antibody (Second moiety) which may, in turn, be an antigen to a biotinylated component via avidin or Streptavidin. for a second antibody (third moiety). It will be further Similarly, a large number of haptenylation reagents are also appreciated that Such a circumstance allows indirect binding 15 known (Id.). of a first moiety and a third moiety via an intermediary Methods for labeling of proteins with radioisotopes are Second moiety that is a binding pair partner to each. known in the art. For example, Such methods are found in AS will be appreciated by those in the art, a partner of a Ohta et al., Molec. Cell 3:535-541 (1999), which is hereby binding pair may comprise a label, as described above. It incorporated by reference in its entirety. will further be appreciated that this allows for a tag to be Production of proteins having His-tags by recombinant indirectly labeled upon the binding of a binding partner means is well known, and kits for producing Such proteins comprising a label. Attaching a label to a tag which is a are commercially available. Such a kit and its use is partner of a binding pair, as just described, is referred to described in the QIAexpress Handbook from Qiagen by herein as “indirect labeling”. Joanne Crowe et al., hereby expressly incorporated by By “surface substrate binding molecule' or “attachment 25 reference. tag and grammatical equivalents thereof is meant a mol The functionalization of labels with chemically reactive ecule have binding affinity for a Specific Surface Substrate, groupS Such as thiols, amines, carboxyls, etc. is generally which Substrate is generally a member of a binding pair known in the art. In a preferred embodiment, the tag is applied, incorporated or otherwise attached to a Surface. functionalized to facilitate covalent attachment. Suitable Surface Substrate binding molecules and their Sur The covalent attachment of the tag may be either direct or face Substrates include, but are not limited to poly-histidine via a linker. In one embodiment, the linker is a relatively (poly-his) or poly-histidine-glycine (poly-his-gly) tags and short coupling moiety, that is used to attach the molecules. Nickel substrate; the Glutathione-S Transferase tag and its A coupling moiety may be Synthesized directly onto a antibody substrate (available from Pierce Chemical); the flu component of the invention, ubiquitin moiety for example, HA tag polypeptide and its antibody 12CA5 substrate Field 35 and contains at least one functional group to facilitate et al., Mol. Cell. Biol., 8:2159–2165 (1988); the c-myc tag attachment of the tag. and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody sub Alternatively, the coupling moiety may have at least two strates thereto Evan et al., Molecular and Cellular Biology, functional groups, which are used to attach a functionalized 5:3610-3616 (1985); and the Herpes Simplex virus glyco component to a functionalized tag, for example. In an protein D (gD) tag and its antibody substrate Paborsky et 40 additional embodiment, the linker is a polymer. In this al., Protein Engineering, 3(6)547-553 (1990)). In general, embodiment, covalent attachment is accomplished either Surface binding Substrate molecules useful in the present directly, or through the use of coupling moieties from the invention include, but are not limited to, polyhistidine component or tag to the polymer. In a preferred Structures (His-tags) that bind nickel Substrates, antigens embodiment, the covalent attachment is direct, that is, no that bind to Surface Substrates comprising antibody, haptens 45 linker is used. In this embodiment, the component preferably that bind to avidin substrate (e.g., biotin) and CBP that binds contains a functional group Such as a carboxylic acid which to Surface Substrate comprising calmodulin. is used for direct attachment to the functionalized tag. It Production of antibody-embedded substrates is well should be understood that the component and tag may be known; see Slinkin et al., Bioconj. Chem. 2:342–348 (1991); attached in a variety of ways, including those listed above. Torchilin et al., Supra; Trubetskoy et al., Bioconj. Chem. 50 What is important is that manner of attachment does not 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 Significantly alter the functionality of the component. For (1994); and Wilbur et al., Bioconjugate Chem. 5:220–235 example, in tag-ubiquitin moiety, the tag should be attached (1994) (all of which are hereby expressly incorporated by in Such a manner as to allow the ubiquitin moiety to be reference), and attachment of or production of proteins with covalently attached to another ubiquitin moiety to form antigens is described above. 55 polyubiquitin moiety chains. AS will be appreciated by those Calmodulin-embedded Substrates are commercially in the art, the above description of covalent attachment of a available, and production of proteins with CBP is described label and ubiquitin moiety applies equally to the attachment in Simcox et al., Strategies 8:40-43 (1995), which is hereby of Virtually any two molecules of the present disclosure. incorporated by reference in its entirety. In a preferred embodiment, the tag is functionalized to AS will be appreciated by those in the art, tag-components 60 facilitate covalent attachment, as is generally outlined of the invention can be made in various ways, depending above. Thus, a wide variety of tags are commercially avail largely upon the form of the tag. Components of the inven able which contain functional groups, including, but not tion and tags are preferably attached by a covalent bond. limited to, isothiocyanate groups, amino groups, haloacetyl The production of tag-polypeptides by recombinant groups, maleimides, Succinimidyl esters, and Sulfonyl means when the tag is also a polypeptide is described below. 65 halides, all of which may be used to covalently attach the tag Production of FLAG-labeled proteins is well known in the to a Second molecule, as is described herein. The choice of art and kits for Such production are commercially available the functional group of the tag will depend on the Site of US 6,919,184 B2 31 32 attachment to either a linker, as outlined above or a com moiety, wherein tag1 is a member of a binding pair, pref ponent of the invention. Thus, for example, for direct erably FLAG, tag2 is a fluorescent label and tag3 is either a linkage to a carboxylic acid group of a ubiquitin moiety, fluorescent label Such that tag2 and tag3 are members of a amino modified or hydrazine modified tags will be used for FRET pair or tag3 is a quencher of tag2. coupling via carbodiimide chemistry, for example using In a preferred embodiment, one or more amino acids are 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) added to the ubiquitin moiety Sequence, using recombinant as is known in the art (see Set 9 and Set 11 of the Molecular techniques as described herein, to provide an attachment Probes Catalog, Supra; see also the Pierce 1994 Catalog and point for a tag, preferably a fluorescent label or a quencher. Handbook, pages T-155 to T-200, both of which are hereby In a preferred embodiment, the one or more amino acids are incorporated by reference). In one embodiment, the carbo CyS or Ala-CyS. Preferably, the one or more amino acids are diimide is first attached to the tag, Such as is commercially attached to the N-terminal of the ubiquitin moiety. In a available for many of the tags described herein. preferred embodiment, the one or more amino acids inter In a preferred embodiment, ubiquitin moiety is in the form venes the Sequence of a FLAG tag and the ubiquitin moiety. of tag-ubiquitin moiety, wherein, tag is a partner of a binding In a preferred embodiment, the tag, preferably a fluorescent pair. Preferably in this embodiment the tag is FLAG and the 15 label or a quencher, is attached to the added Cysteine. binding partner is anti-FLAG. Preferably in this In Some embodiments, the methods of the present inven embodiment, a label is attached to the FLAG by indirect tion comprise the use of a ubiquitin activating agent. AS used labeling. Preferably, the label is a label enzyme. Most herein "ubiquitin activating agent” refers to a ubiquitin preferably, the label enzyme is horseradish peroxidase, agent, preferably a protein, capable of transferring or attach which is reacted with a fluorescent label enzyme Substrate. ing a ubiquitin moiety to a ubiquitin conjugating agent. In a Preferably, the label enzyme substrate is Luminol. preferred embodiment, the ubiquitin activating agent forms Alternatively, the label is a fluorescent label. a high energy thiolester bond with ubiquitin moiety, thereby In another preferred embodiment, ubiquitin moiety is in “activating the ubiquitin moiety. In another preferred the form of tag-ubiquitin moiety, wherein the tag is a embodiment, the ubiquitin activating agent binds or attaches fluorescent label. In a particularly preferred embodiment, 25 ubiquitin moiety. In another preferred embodiment, the ubiquitin moiety is in the form of tag1-ubiquitin moiety and ubiquitin activating agent is capable of transferring or tag2-ubiquitin moiety, wherein tag1 and tag2 are the mem attaching ubiquitin moiety to a Substrate molecule that is a bers of a FRET pair. In an alternate preferred embodiment, mono- or poly-ubiquitin moiety. In a preferred embodiment, ubiquitin moiety is in the form of tag1-ubiquitin moiety and the ubiquitin activating agent is capable of transferring or tag2-ubiquitin moiety, wherein tag1 is a fluorescent label attaching ubiquitin moiety to a mono- or poly-ubiquitinated and tag2 is a quencher of the fluorescent label. In either of ubiquitin conjugating agent. these preferred embodiments, when tag1-ubiquitin moiety In a preferred embodiment the ubiquitin activating agent and tag2-ubiquitin moiety are attached to a Substrate mol is an E1. In a preferred embodiment, the E1 is capable of ecule of interest through the activity of a ubiquitin agent, transferring or attaching ubiquitin moiety to an E2, defined preferably tag1 and tag2 are within 100 A of each other, 35 below. more preferable within 70 A, still more preferably within 50 In the methods and compositions of the present invention, A, even more preferably within 40 A, and in Some cases, the ubiquitin activating agent comprises an amino acid preferably within 30 A or less. Sequence or a nucleic acid corresponding to a Sequence of an In yet another preferred embodiment, ubiquitin moiety is Genbank database accession number listed in Table 1 below in the form of tag 1,2-ubiquitin moiety and tag 1,3-ubiquitin and incorporated herein by reference.

TABLE 1.

ORG SYMBOL DESCRIPTION ACCESSION NO. His APPBP amyloid beta precursor protein binding protein 1, 59 kD NM OO3905 His FLJ23251 hypothetical protein FLJ23251 NM O24818 HS GSA7 ubiquitin activating enzyme E1-like protein NM OO6395 HS similar to ubiquitin-activating enzyme E1 (A1S9T and BN75 XM 088743 temperature sensitivity complementing) (H. Sapiens) HS similar to SUMO-1 activating enzyme subunit 1: SUMO-1 XM O901.10 activating enzyme E1 N subunit; sentrin/SUMO-activating protein AOS1; ubiquitin-like protein SUMO-1 activating enzyme HS SAE1 SUMO-1 activating enzyme subunit 1 NM OO5500 and XM OO9036 Dm Uba1 Ubiquitin activating enzyme 1 NG OOO652 and NM O57962 Dm Uba2 Smt3 activating enzyme 2 NM 08OO17 HS UBA2 SUMO-1 activating enzyme subunit 2 NM OO5499 HS UBE1 ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature NM OO3334 sensitivity complementing) and XM 033895 HS UBE1C ubiquitin-activating enzyme E1C (UBA3 homolog, yeast) NM OO3968 Rn Ube1c Ubiquitin-activating enzyme E1C NM 0572.05 Mm Ube1 Ubiquitin-activating enzyme E1-like HS UBE1L Ubiquitin-activating enzyme E1-like NM OO3335 Mm Ube1x ubiquitin-activating enzyme E1, Chr X NM OO9457 Mm Ube1y1 ubiquitin-activating enzyme E1, Chr Y 1 NM 011667 US 6,919,184 B2 33 34

TABLE 1-continued

ORG SYMBOL DESCRIPTION ACCESSIONNO. Mm Ube1y1- ubiquitin-activating enzyme E1, Chr Y. pseudogene 1 M884.81 and ps1 UO9053 Mm Ube1y1- ubiquitin-activating enzyme E1, Chr Y-1, pseudogene 2 UO9054 ps2

Sequences encoding a ubiquitin activating agent may also used herein "ubiquitin conjugating agent” refers to a ubiq be used to make variants thereof that are Suitable for use in uitin agent, preferably a protein, capable of transferring or the methods and compositions of the present invention. The attaching ubiquitin moiety to a ubiquitin ligating agent. In ubiquitin activating agents and variants Suitable for use in Some cases, the ubiquitin conjugating agent is capable of the methods and compositions of the present invention may 15 directly transferring or attaching ubiquitin moiety to lysine be made as described herein. In a preferred embodiment, E1 proteins useful in the residues in a target protein (Hershko et al. (1983) J. Biol. invention include the polypeptides encoded by the amino Chem. 258:8206-8214). In a preferred embodiment, the acid Sequence corresponding to GENBANK accession num ubiquitin conjugating agent is capable of transferring or bers A38564, S23770, AAA61246, P22314, CAA40296 and attaching ubiquitin moiety to a mono- or poly-ubiquitin BAA33144, incorporated herein by reference. In a preferred moiety preferably attached to a ubiquitin agent or target embodiment, E1 has the amino acid Sequence shown in FIG. protein. In a preferred embodiment, the ubiquitin conjugat 8B (SEQ ID NO:2) or is encoded by a nucleic acid com ing agent is capable of transferring ubiquitin moiety to a prising the sequence shown in FIG. 8A (SEQ ID NO:1). mono- or poly-ubiquitinated ubiquitin ligating agent. Preferably E1 is human E1. E1 is commercially available 25 In a preferred embodiment the ubiquitin conjugating from Affiniti Research Products (Exeter, U.K.). agent is an E2. In a preferred embodiment, ubiquitin moiety In a preferred embodiment, nucleic acids which may be used for producing E1 proteins for the invention include, but is transferred from E1 to E2. In a preferred embodiment, the are not limited to, those disclosed by GENBANK accession transfer results in a thiolester bond formed between E2 and numbers M58028, X56976 and AB012.190, incorporated ubiquitin moiety. In a preferred embodiment, E2 is capable herein by reference. In a preferred embodiment, E1 is of transferring or attaching ubiquitin moiety to an E3, encoded by a nucleic acid having a Sequence consisting defined below. essentially of the sequence shown in FIG. 8A (SEQ ID In the methods and compositions of the present invention, NO:1). Variants of the cited E1 proteins, also included in the the ubiquitin activating agent comprises an amino acid term “E1’’, can be made as described herein. 35 Sequence or a nucleic acid Sequence corresponding to a In Some embodiments, the methods of the present inven Sequence of an Genbank data base accession number listed tion comprise the use of a ubiquitin conjugating agent. AS in Table 2 below and incorporated herein by reference.

TABLE 2

Accession No. Accession No. (nucleic acid (amino acid Name ALIAS sequences) sequences) UBE2D1 His UBC4/5 UBE2D1, UBCH5A, UBC4/5 homolog NM 003338.1 NP 003329.1 homolog UBC9 Gallus gallus UBC9, SUMO-conjugating enzyme ABO69964.1 BAB68210.1 UBC9 Mus musculus nUB69 U76416.1 AAB18790.1 UBC9/UBE21 Hs?? UBE21 U45328.1 AAA866.62.1 UBC9 isoform/MGC:3994 MGC:3994, IMAGE:2819732, UBC9 BCOO4437.1 AAHO4437.1 HS isoform NM OO3345.1 NP OO3336. UBC9 His UBC9, UBE21 FTS homolog Hs + 1aa fused toes homolog, FLJ13258 NM O22476.1 NP O71921. FLJ13988 HS FLJ13988, clone Y79AA1002027, sim AKO24050.1 BAB148OO1 MGC:13396 His to E2-18 BCO1O9001 AAH10900.1 UBE2V2 His MGC:13396, IMAGE:4081461 NM OO3350.2 NP OO3341. MGC:10481 HS UBE2V2, EDAF-1, MMS2, UEV2, BCOO4862.1 AAHO4862.1 XM 054332.1 Hs DDVIT1, ED XM 054332.1 XP 054332. FLJ13855. His MGC:10481, IMAGE:3838157 XM 03.0444.3 XP 03.0444. E2-230 K homolog Hs FLJ13855 NM O22066.1 NP O71349. UBE2V2 His E2-230 K ortholog, FLJ12878, NM OO3339.1 NO OO3330.1 UBE2D3 Hs 1 SNP KIAA1734 NM OO3340.1 NP OO3331. Non-canon Ub-coni Enz UBE2D2, UBCHSB, UBC4, UBC4/5 NM O16336.2 NP O5742O.2 (NCUBE1) homolog NM 014176.1 NP 054895. HSPC15OHS UBE2D3, UBCH5C, UBC4/5 homolog NM 016252.1 NP 057336. Brain 1AP repeat contain NCUBE1, HSU93243, HSPC153, CGI 6 (BIRC6) BIRC6, KIAA1289, apollon UBC8 Mus E2-20 K, UBE2H NM OO9459.1 NP O33485. UBC8 HS UBE2H, UBCH, UBCH2, UBC8 NM OO3344.1 NP OO3335. UBC8 HS 6SNP homolog NM-OO3344.1 NP-OO3335.1 US 6,919,184 B2 35 36

TABLE 2-continued

Accession No. Accession No. (nucleic acid (amino acid Name ALIAS sequences) sequences) UBC8His no 5 UBE2H, UBCH, UBCH2, UBC8 homolog RAD6 homolog Hs UBE2B, RAD6B, HHR6B, UBC2, NM OO3337.1 NP OO3328. RAD6 homolog UBE2V1 war 3 HS UBE2V1, CIR1, UEV1 UEV1A, NM O22442.2 NP O71887. UBE2V1 var 1 Hs early CROC-1, CRO NM 021988.2 NP O68823. stop, 56aa UBE2V1, CIR1, UEV1 UEV1A, NM OO3349.3 NP OO3340. UBE2V1 war 2 HS CROC-1, CRO UBE2V1, CIR1, UEV1 UEV1A, CROC-1, CRO UBE2L6 His UBE2IL6, UBCHS, RIG-B NM OO4223.1 NP OO4214. UBE2L3 Hs 2 SNP UBE2IL3, UBCH7 NM OO3347.1 NP OO3338. UBE2E1. His UBE2E1, UBCH6, UBC4/5 homolog NM OO3341.1 NP OO3332. RAD6/UBE2A Hs UBE2A, RAD6A, HHR6A, UBC2, NM OO3336.1 NP OO3327. UBE2E3 HS RAD6 homolog NM OO6357.1 NP OO6348. UBC12/UBE2M His UBE2E3, UBCH9, UBC4/5 homolog NM OO3969.1 NP OO3960. UBC7/UBE2G1. His UBE2M, HUBC12, UBC12 homolog NM OO3342.1 NP OO3333. UBE2G1, UBC7 homolog Huntingtin interact prot 2 HIP2, LIG, E2-25 K NM OO5339.2 NP OO5330. (HIP2) Hs LIG, HIP2 alternative splicing form ABO22436.1 BAA78556.1 LIG/HIP2 variant Hs UBC6p Hs UBC6p, UBC6 NM O58167.1 NP 477515. UBC6Hs UBC6 AF296658.1 AAK52609.1 HBUCE1/UBE2D2 war HBUCE1, LOC51619 NM O15983.1 NP O57O67. HS UBE2G2, UBC7 homolog XM 036087.1 XP 036087. UBE2G2/UBC7 homolog NCE2 NM 080678.1 NP 542409. HS CDC34, E2-CDC34, E2-32 NM 004359.1 NP OO4.350. NEDD8-con enzyme 2 complementing BCOOO848.1 AAHO0848. (NCE2) Hs IMAGE:3458173 CDC34. His IMAGE:3458173/NICE-5 wa UBE2C His UBE2C, UBCH10 NM OO7019.1 NP OO895.0.1 UBE2C possible short UBE2C, UBCH10 NM OO7019.1 NP OO895.0.1 form. His UBC3/UBE2N Hs UBE2N, UBCH-BEN, UBC13 hom., NM OO3348.1 NP OO3339.1 FLJ25157 His sim to bend AKO57886.1 BABF1605.1 TSG101 Hs 1 SNP FLJ25157, highly similar to E2-23 NM OO6292.1 NP OO6283.1 MGC:21212/NICE-5 war Tumor susceptibility gene 101 BCO17708.1 AAH17708.1 HS MCG:21212, IMAGE:3907760, sim to NICE-5

Sequences encoding a ubiquitin conjugating agent may different E2 proteins and isozymes are known in the filed also be used to make variants thereof that are Suitable for use and may be used in the present invention, provided that the in the methods and compositions of the present invention. E2 has ubiquitin conjugating activity. Also specifically The ubiquitin conjugatin agents and variants Suitable for use 45 included within the term "E2 are variants of E2, which can in the methods and compositions of the present invention be made as described herein. may be made as described herein. In a preferred embodiment, E2 is one of Ubc5 (Ubch5, In a preferred embodiment, the E2 used in the methods preferably Ubch5c), Ubc3 (Ubch 3), Ubc4 (Ubch4) and and compositions of the present invention comprises an UbcX (Ubc10, Ubch10). In a preferred embodiment, E2 is amino acid Sequence or nucleic acid Sequence of a sequence 50 Ubch5c. In a preferred embodiment, E2 has the amino acid corresponding to an Genbank database accession number in sequence shown in FIG. 9B (SEQ ID NO:4) or is encoded the following list: AC37534, P49427, CAA82525, by a nucleic acid consisting essentially of the Sequence AAA58466, AAC41750, P51669, AAA91460, AAA91461, shown in FIG. 9A (SEQ ID NO:3). CAA63538, AAC50633, P27924, AAB36017, Q16763, The E2 used in the methods and compositions of the AAB86433, AAC26141, CAA04156, BAA11675, Q16781, 55 present invention, comprises a nucleic acid Sequence of a NP 003333, BAB 18652, AAHOO468, CAC 16955, Sequence corresponding to Genbank data base accession CAB76865, CAB76864, NP 05536, O00762, XP 009804, number L2205, Z29328, M92670, L401.46, U3.9317, XP 009488, XP 006823, XP 006343, XP 005934, U39318, X92962, U58522, S81003, AF031141, AF075599, XP 002869, XP 003400XP 0.09365, XP 010361, AJO00519, XM009488, NM007019, U73379, L40146, or XP 004699, XP 004019, O14933, P27924, P50550, 60 D83004, each of which is incorporated herein by reference. P52485, P51668, P51669, P49459, P37286, P23567, AS described above, variants of these and other E2 encoding P56554, and CAB45853, each of which is incorporated nucleic acids may also be used to make variant E2 proteins. herein by reference. Particularly preferred are Sequences In a preferred embodiment, the nucleic acid used to make corresponding to Genbank data base accession numbers E2 comprises the sequence shown in FIG. 9A (SEQ ID NP003331, NPO03330, NPO03329, P49427, AAB53362, 65 NO:3). NP008950, XPO09488 and AAC41750, also incorporated by In a preferred embodiment, E2 has a tag, as defined above, reference. The skilled artisan will appreciate that many with the complex being referred to herein as “tag-E2. US 6,919,184 B2 37 38 Preferred E2 tags include, but are not limited to, labels, comprises a ring finger Subunit and a Cullin Subunit. partners of binding pairs and Substrate binding elements. In Examples of RING finger polypeptides suitable for use in a most preferred embodiment, the tag is a His-tag or the methods and compositions of the present invention GST-tag. include, but are not limited to, ROC1, ROC2 and APC11. In Some embodiments, the methods of the present inven Examples of Cullin polypeptides Suitable for use in the tion comprise the use of a ubiquitin ligating agent. AS used methods and compositions of the present invention include, herein "ubiquitin ligating agent” refers to a ubiquitin agent, but are not limited to, CUL1, CUL2, CUL3, CUL4A, preferably a protein, capable of transferring or attaching a CUL4B, CUL5 and APC2. In another preferred ubiquitin moiety to a target molecule. In Some cases, the embodiment, the E3 is mdm2. ubiquitin agent is capable of transferring or attaching ubiq In the methods and compositions of the present invention, uitin moiety to itself or another ubiquitin ligating agent. In the ubiquitin ligating agent comprises an amino acid a preferred embodiment, the ubiquitin ligating agent is an Sequence or a nucleic acid Sequence of a Sequence corre E3. sponding to an accession number in the Genbank database, AS used herein “E3’ refers to a ubiquitin ligating agent European Molecular Biology Laboratories (EMBL) data comprising one or more Subunits, preferably polypeptides, 15 base, or ENSEMBL data base (a joint project of the Euro asSociated with the activity of E3 as a ubiquitin ligating pean Molecular Biology Laboratories and the Sanger agent (i.e., associated with the ligation or attachment of Institute) listed in Table 3 below and incorporated herein by ubiquitin moiety to a target protein, and in Some cases, to reference. The accession numbers from the Genbank data itself or another E3). In a preferred embodiment, E3 is a base can be found as Stated above. The accession numbers member of the HECT domain E3 ligating agents. In another from the EMBL data base are found at www.embl preferred embodiment, E3 is a member of the RING finger heidelberg.de. The accession numbers from the ENSEMBL domain E3 ligating agents. In a preferred embodiment, E3 data base are found at www.ensembl.or.

US 6,919,184 B2 41 42

TABLE 3-continued ENSPOOOOO2O3439 ENSPOOOOO259944 ENSPOOOOO253571 ENSPOOOOO265267 ENSPOOOOOO13772 EMSPOOOOO279757 ENSPOOOOO288913 ENSPOOOOO26322O ENSPOOOOO225283 ENSPOOOOO274773 ENSPOOOOO288918 ENSPOOOOO216225 ENSPOOOOO246907 ENSPOOOOO276311 ENSPOOOOO276573 ENSPOOOOO293538 ENSPOOOOO225285 ENSPOOOOO166144 ENSPOOOOO2373O8 ENSPOOOOO229766 ENSPOOOOO225286 ENSPOOOOO292363 ENSPOOOOO2382O3 ENSPOOOOO242239 ENSPOOOOO23O239 ENSPOOOOO264616 ENSPOOOOO227451 ENSPOOOOO274616 ENSPOOOOO286909 ENSPOOOOO272.390 ENSPOOOOO244360 ENSPOOOOO286773 ENSPOOOOO286910 ENSPOOOOO272.396 ENSPOOOOO2443.59 ENSPOOOOO273480 ENSPOOOOO280609 ENSPOOOOO264767 ENSPOOOOO281105 ENSPOOOOO217173 ENSPOOOOO263651 ENSPOOOOO255.499 ENSPOOOOO268907 ENSPOOOOO290337 ENSPOOOOO261.395 ENSPOOOOO264.614 ENSPOOOOO292962 ENSPOOOOO281930 ENSPOOOOO277584. ENSPOOOOO262482 ENSPOOOOO28O804 ENSPOOOOO257575 ENSPOOOOO224833 ENSPOOOOO261.481 ENSPOOOOO287546 ENSPOOOOO287212 ENSPOOOOO2546O4 ENSPOOOOO261658 ENSPOOOOO24898O ENSPOOOOO29078 ENSPOOOOO240395 ENSPOOOOO288774 ENSPOOOOO287559 ENSPOOOOO28245 ENSPOOOOO24.0318 ENSPOOOOO261675 ENSPOOOOO264926 ENSPOOOOO25424 ENSPOOOOO286945 ENSPOOOOO26688O ENSPOOOOO261737 ENSPOOOOO29064 ENSPOOOOO2.81874 ENSPOOOOO243674 ENSPOOOOO170447 ENSPOOOOO27454 ENSPOOOOO2408O2 ENSPOOOOO284.638 ENSPOOOOO27O944 ENSPOOOOO22494 ENSPOOOOO267825 ENSPOOOCO2476.68 ENSPOOOOO289726 ENSPOOOOO2814 ENSPOOOOO254586 ENSPOOOOO285317 ENSPOOOOO23OO99 ENSPOOOOO28.988 ENSPOOOOO293,123 ENSPOOOOO27848O ENSPOOOOO237455 ENSPOOOOO25532 ENSPOOOOO285805 ENSPOOOOO24O159 ENSPOOOOO2635SO ENSPOOOOO25532 ENSPOOOOO257633 ENSPOOOOO294.256 ENSPOOOOO264,198 ENSPOOOOO29254 ENSPOOOOO266119 ENSPOOOOO279766 ENSPOOOOO263464 ENSPOOOOO27753 ENSPOOOOO233630 ENSPOOOOO2882O4 ENSPOOOOO2596O4 ENSPOOOOO26094 ENSPOOOOO264.033 ENSPOOOOO269439 ENSPOOOOO265673 ENSPOOOOO27845 ENSPOOOOO275619 ENSPOOOOO268061 ENSPOOOOO248983 ENSPOOOOO27845 ENSPOOOOO275637 ENSPOOOOO268.058 ENSPOOOOO269,391 ENSPOOOOO27469 ENSPOOOOO28OO63 ENSPOOOOO268059 ENSPOOOOO249007 ENSPOOOOO21.7740 ENSPOOOOO276333 ENSPOOOOO268060 ENSPOOOOO242719 ENSPOOOOO262952 ENSPOOOOO263651 ENSPOOOOO261825 ENSPOOOOO217169 ENSPOOOOO268154 ENSPOOOOO278302 ENSPOOOOO288587 EMSPOOOOO2S3642 ENSPOOOOO265756 ENSPOOOOO264122 EMSPOOOOO275693 ENSPOOOOO227758 ENSPOOOOO277.490 ENSPOOOOO2.84559 ENSPOOOOO244061 ENSPOOOOO2911.90 ENSPOOOOO26,6625 ENSPOOOOO266252 ENSPOOOOO272598 ENSPOOOOO261537 ENSPOOOOO26,6624 ENSPOOOOO27.8350 ENSPOOOOO2898.18 ENSPOOOOO29.1733 ENSPOOOOO258147 ENSPOOOOO25.9847 ENSPOOOOO238349 ENSPOOOOO274,782 ENSPOOOOO258148 ENSPOOOOO274,855 ENSPOOOOO28O266 ENSPOOOOO271287 ENSPOOOOO258149 ENSPOOOOO259930 ENSPOOOOO242855 ENSPOOOOO261445 ENSPOOOOO264512 ENSPOOOOO217214 ENSPOOOOO276.688 ENSPOOOOO245836 ENSPOOOOO261212 ENSPOOOOO2.83330 ENSPOOOOO28O268 ENSPOOOOO267291 ENSPOOOOO262642 ENSPOOOOO263,535 ENSPOOOOO274,811 ENSPOOOOO292.195 ENSPOOOOO264359 ENSPOOOOO291,416 ENSPOOOOO268.363 ENSPOOOOO21642O ENSPOOOOO217537 ENSPOOOOO291,414 ENSPOOOOO274,828 ENSPOOOOO261464 ENSPOOOOO264777 ENSPOOOOO253769 ENSPOOOOO235150 ENSPOOOOO26OO76 ENSPOOOOO28788O ENSPOOOOO274.786 ENSPOOOOO211960 ENSPOOOOO284244 ENSPOOOOO272674 ENSPOOOOO289896 ENSPOOOOO262843 ENSPOOOOO2925.45 ENSPOOOOO272662 ENSPOOOOO289898 ENSPOOOOO266952 ENSPOOOOO242669 ENSPOOOOO293.245 ENSPOOOOO265771 ENSPOOOOO2883OO ENSPOOOOO288848 ENSPOOOOO283875 ENSPOOOOO229866 ENSPOOOOO291134 ENSPOOOOO261809 ENSPOOOOO262642 ENSPOOOOO286475 ENSPOOOOO261947 ENSPOOOOO262952 ENSPOOOOO2598.65 ENSPOOOOO256257 ENSPOOOOO288715 ENSPOOOOO24.5937 ENSPOOOOO217908 ENSPOOOOO253554. ENSPOOOOO222704 ENSPOOOOO275970 ENSPOOOOO2SSOO4 ENSPOOOOO25965.4 ENSPOOOOO293938 ENSPOOOOO238647 ENSPOOOOO2751.84 ENSPOOOOO28O266 ENSPOOOOO266.030 ENSPOOOOO26885O ENSPOOOOO2751.83 ENSPOOOOO259941 ENSPOOOOO287335 ENSPOOOOO291963 ENSPOOOOO200457 ENSPOOOOO259940 EMSPOOOOO256649 ENSPOOOOO2863.49 ENSPOOOOO261537 ENSPOOOOO27OO86 ENSPOOOOO249240 ENSPOOOOO2576OO ENSPOOOOO2571OO ENSPOOOOO28914O ENSPOOOOO253953 ENSPOOOOO281,843 ENSPOOOOO2863.49 ENSPOOOOO225507 ENSPOOOOO267073 ENSPOOOOO26.1245 ENSPOOOOO2524.45 ENSPOOOOO261593 ENSPOOOOO271.813 ENSPOOOOO245888 ENSPOOOOO294213 ENSPOOOOO257847 ENSPOOOOO248492 ENSPOOOOO222704 ENSPOOOOO259939 ENSPOOOOO262881 ENSPOOOOO265981 ENSPOOOOO245419 ENSPOOOOO236892 ENSPOOOOO222O33 ENSPOOOOO27O28O ENSPOOOOO272O23 ENSPOOOOO238OO1 ENSPOOOOO29OO48 ENSPOOOOO270279 ENSPOOOOO274O68 ENSPOOOOO274657 ENSPOOOOO274327 ENSPOOOOO254959 ENSPOOOOO275233 ENSPOOOOO274799

Sequences encoding a ubiquitin activating agent may also 60 Sequence corresponding to Genbank accession numbers be used to make variants thereof that are Suitable for use in AAD301.47, AAD301.46, or 6320196, incorporated herein the methods and compositions of the present invention. The by reference. In a more preferred embodiment, the ring ubiquitin ligating agents and variants Suitable for use in the finger protein has a Sequence Selected from the group methods and compositions of the present invention may be consisting of that shown in FIGS. 10, 11 and 12B (SEQ ID made as described herein. 65 NOS:5-8). In a preferred embodiment, RING finger subunits include, In a preferred embodiment, Cullins include, but are not but are not limited to, polypeptides having an amino acid limited to, polypeptides having an amino acid Sequence US 6,919,184 B2 43 44 corresponding to Genbank accession number 4503161, Same host cell. Co-expression may be achieved by trans AAC50544, AAC36681, 4503163, AAC51190, AAD23581, forming the cell with a vector comprising nucleic acids 4503165, AAC36304, AAC36682, AAD45191, AAC50548, encoding two or more of the E3 components, or by trans Q13620, 4503167, or AAF05751, each of which is incor forming the host cell with Separate vectors, each comprising porated herein by reference. In addition, in the context of the a single component of the desired E3 protein complex. In a invention, each of the RING finger proteins and Cullins preferred embodiment, the RING finger protein and Cullin encompass variants of the known or listed Sequences, as are expressed in a single host transfected with two vectors, described herein. each comprising nucleic acid encoding one or the other In a preferred embodiment, the Cullin has a Sequence as polypeptide, as described in further detail in the Examples. shown in FIG. 13B or 14B (SEQ ID NOS:10, 12). In a preferred embodiment, E3 has a tag, and this complex These E3 ligating agents and variants may be made as is referred to herein as “tag-E3”. Preferably, the tag is described herein. In a preferred embodiment, nucleic acids attached to only one component of the E3. Preferred E3 tags used to make the RING finger proteins include, but are not include, but are not limited to, labels, partners of binding limited to, those having the nucleic acid Sequences disclosed pairs and Substrate binding elements. More preferably, the in Genbank accession numbers AF142059, AF142060 and 15 tag is a Surface Substrate binding molecule. Most preferably, nucleic acids 433493 to 433990 of NC 001136. In a pre the tag is a His-tag or GST-tag. ferred embodiment, Cullins are made from nucleic acids Ubiquitin moieties, ubiquitin agents, and target molecules including, but not limited to, those having nucleic acid Suitable for use in the methods and compositions of the Sequences disclosed in Genbank accession numbers NM present invention can be cloned and expressed as described 003592, U58087, AF062536, AF126404, NM 003591, below. U83410, NM 003590, AB014517, AF062537, AF064087, Thus, probe or degenerate polymerase chain reaction AF077188, U58091, NM 003478, X81882 and AF191337, (PCR) primer sequences may be used to find other related or each of which is incorporated herein by reference. AS variant ubiquitin moieties, ubiquitin agents, and target pro described above, variants of these Sequences are also encom teins from humans or other organisms. AS will be appreci passed by the invention. 25 ated by those in the art, particularly useful probe and/or PCR In a preferred embodiment, nucleic acid used to produce primer Sequences include the unique areas of a nucleic acid ROC2 comprises the sequence depicted in FIG. 12A (SEQ Sequence. AS is generally known in the art, preferred PCR ID NO:7). In a preferred embodiment, nucleic acid used to primers are from about 15 to about 35 nucleotides in length, produce CUL5 comprises the sequence depicted in FIG. 13A with from about 20 to about 30 being preferred, and may (SEQ ID NO:9). In a preferred embodiment, nucleic acid contain inosine as needed. The conditions for the PCR used to produce APC2 comprises the Sequence depicted in reaction are well known in the art. It is therefore also FIG. 14A (SEQ ID NO:11). understood that provided along with the sequences in the In a preferred embodiment, E3 comprises the RING finger Sequences cited herein are portions of those Sequences, protein/Cullin combination APC11/APC2. In another pre wherein unique portions of 15 nucleotides or more are ferred embodiment, E3 comprises the RING finger protein/ 35 particularly preferred. The skilled artisan can routinely Syn Cullin combination ROC1/CUL1. In yet preferred thesize or cut a nucleotide Sequence to the desired length. embodiment, E3 comprises the RING finger protein/Cullin Once isolated from its natural Source, e.g., contained combination ROC1/CUL2. In still another preferred within a plasmid or other vector or excised therefrom as a embodiment, E3 comprises the RING finger protein/Cullin linear nucleic acid Segment, the recombinant nucleic acid combination ROC2/CUL5. However, the skilled artisan will 40 can be further-used as a probe to identify and isolate other appreciate that any combination of E3 components may be nucleic acids. It can also be used as a “precursor nucleic produced and used in the invention described herein. acid to make modified or variant nucleic acids and proteins. In an alternate embodiment, E3 comprises the ligase Using the nucleic acids of the present invention which E3-alpha, E3A (E6-AP), HERC2, SMURF1, TRAF6, encode a protein, a variety of expression vectors are made. Mdm2, Cbl, Sina/Siah, Itchy, IAP or NEDD-4. In this 45 The expression vectors may be either Self-replicating extra embodiment, the ligase has the amino acid Sequence of that chromosomal vectors or vectors which integrate into a host disclosed in Genbank accession number AAC39845, genome. Generally, these expression vectors include tran Q05086, CAA66655, CAA66654, CAA66656, AAD08657, Scriptional and translational regulatory nucleic acid operably NP 002383, XP 0.06284, AAC51970, XP 013050, linked to the nucleic acid encoding the protein. The term BAB39389, Q00987, AAFO8298 or P46934, each of which 50 “control Sequences' refers to DNA sequences necessary for is incorporated herein by reference. AS above, variants are the expression of an operably linked coding Sequence in a also encompassed by the invention. Nucleic acids for mak particular host organism. The control Sequences that are ing E3 for this embodiment include, but are not limited to, Suitable for prokaryotes, for example, include a promoter, those having the Sequences disclosed in Genbank accession optionally an operator Sequence, and a ribosome binding numbers AF061556, XMOO6284, U76247, XMO13050, 55 Site. Eukaryotic cells are known to utilize promoters, poly X898.032, X98031, X98033, AF071172, Z 12020, adenylation signals, and enhancers. AB056663, AF199364 and D42055 and variants thereof. Nucleic acid is “operably linked' when it is placed into a E3 may also comprise other components, Such as SKP1 functional relationship with another nucleic acid Sequence. and F-box proteins. The amino acid and nucleic acid For example, DNA for a presequence or Secretory leader is sequences for SKP1 correspond to GENBANK accession 60 operably linked to DNA for a polypeptide if it is expressed numbers AAC50241 and U33760, respectively. Many F-box as a preprotein that participates in the Secretion of the proteins are known in the art and their amino acid and polypeptide; a promoter or enhancer is operably linked to a nucleic acid Sequences are readily obtained by the skilled coding Sequence if it affects the transcription of the artisan from various published Sources. Sequence; or a ribosome binding site is operably linked to a In a preferred embodiment, the E3 components are pro 65 coding Sequence if it is positioned So as to facilitate trans duced recombinantly, as described herein. In a preferred lation. As another example, operably linked refers to DNA embodiment, the E3 components are co-expressed in the Sequences linked So as to be contiguous, and, in the case of US 6,919,184 B2 45 46 a Secretory leader, contiguous and in reading fram. However, cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa enhancers do not have to be contiguous. Linking is accom cells. Of greatest interest are Pichia pastoris and P. plished by ligation at convenient restriction sites. If Such methanolica, E. coli, SF9 cells, SF21 cells and Hi-5 cells. Sites do not exist, the Synthetic oligonucleotide adapters or In a preferred embodiment, the proteins are expressed in linkers are used in accordance with conventional practice. mammalian cells. Mammalian expression Systems are also The transcriptional and translational regulatory nucleic acid known in the art, and include retroviral Systems. A mam will generally be appropriate to the host cell used to express malian promoter is any DNA sequence capable of binding the protein; for example, transcriptional and translational mammalian RNA polymerase and initiating the downstream regulatory nucleic acid Sequences from Bacillus are prefer (3) transcription of a coding sequence for a protein into ably used to express the protein in Bacillus. Numerous types mRNA. A promoter will have a transcription initiating of appropriate expression vectors, and Suitable regulatory region, which is usually placed proximal to the 5' end of the Sequences are known in the art for a variety of host cells. In coding Sequence, and a TATA box, using a located 25-30 general, the transcriptional and translational regulatory base pairs upstream of the transcription initiation site. The Sequences may include, but are not limited to, promoter TATA box is thought to direct RNA polymerase II to begin Sequences, ribosomal binding sites, transcriptional Start and 15 RNA Synthesis at the correct site. A mammalian promoter Stop Sequences, translational Start and Stop Sequences, and will also contain an upstream promoter element (enhancer enhancer or activator Sequences. In a preferred embodiment, element), typically located within 100 to 200 base pairs the regulatory Sequences include a promoter and transcrip upstream of the TATA box. An upstream promoter element tional Start and Stop Sequences. determines the rate at which transcription is initiated and can Promoter Sequences encode either constitutive or induc act in either orientation. Of particular use as mammalian ible promoters. The promoters may be either naturally promoters are the promoters from mammalian viral genes, occurring promoters or hybrid promoters. Hybrid promoters, Since the viral genes are often highly expressed and have a which combine elements of more than one promoter, are also broad host range. Examples include the SV40 early known in the art, and are useful in the present invention. promoter, mouse mammary tumor virus LTR promoter, In addition, the expression vector may comprise addi 25 adenovirus major late promoter, herpes simplex virus tional elements. For example, the expression vector may promoter, and the CMV promoter. have two replication Systems, thus allowing it to be main Typically, transcription termination and polyadenylation tained in two organisms, for example in mammalian or Sequences recognized by mammalian cells are regulatory insect cells for expression and in a prokaryotic host for regions located 3' to the translation Stop codon and thus, cloning and amplification. Furthermore, for integrating together with the promoter elements, flank the coding expression vectors, the expression vector contains at least sequence. The 3' terminus of the mature mRNA is formed by one sequence homologous to the host cell genome, and site-specific post-translational cleavage and polyadenyla preferably two homologous Sequences which flank the tion. Examples of transcription terminator and polyadeny expression construct. The integrating vector may be directed lation signals include those derived form SV40. to a specific locus in the host cell by Selecting the appro 35 The methods of introducing exogenous nucleic acid into priate homologous Sequence for inclusion in the vector. mammalian hosts, as well as other hosts, is well known in Constructs for integrating vectors are well known in the art. the art, and will vary with the host cell used. Techniques In addition, in a preferred embodiment, the expression include dextran-mediated transfection, calcium phosphate vector contains a Selectable marker gene to allow the Selec precipitation, polybrene mediated transfection, protoplast tion of transformed host cells. Selection genes are well 40 fusion, electroporation, Viral infection, encapsulation of the known in the art and will vary with the host cell used. polynucleotide(s) in liposomes, and direct microinjection of A preferred expression vector System is a retroviral vector the DNA into nuclei. system such as is generally described in PCT/US97/01019 In a preferred embodiment, proteins are expressed in and PCT/US97/01048, both of which are hereby expressly bacterial Systems. Bacterial expression Systems are well incorporated by reference. 45 known in the art. Proteins of the present invention are produced by cultur A Suitable bacterial promoter is any nucleic acid Sequence ing a host cell transformed with an expression vector con capable of binding bacterial RNA polymerase and initiating taining nucleic acid encoding the protein, under the appro the downstream (3) transcription of the coding sequence of priate conditions to induce or cause expression of the a protein into mRNA. A bacterial promoter has a transcrip protein. The conditions appropriate for protein expression 50 tion initiation region which is usually placed proximal to the will vary with the choice of the expression vector and the 5' end of the coding Sequence. This transcription initiation host cell, and will be easily ascertained by one skilled in the region typically includes an RNA polymerase binding site art through routine experimentation. For example, the use of and a transcription initiation site. Sequences encoding meta constitutive promoters in the expression vector will require bolic pathway enzymes provide particularly useful promoter optimizing the growth and proliferation of the host cell, 55 Sequences. Examples include promoter Sequences derived while the use of an inducible promoter requires the appro from Sugar metabolizing enzymes, Such as galactose, lactose priate growth conditions for induction. In addition, in Some and maltose, and Sequences derived from biosynthetic embodiments, the timing of the harvest is important. For enzymes Such as tryptophan. Promoters from bacteriophage example, the baculoviral Systems used in insect cell expres may also be used and are known in the art. In addition, Sion are lytic viruses, and thus harvest time Selection can be 60 Synthetic promoters and hybrid promoters are also useful; crucial for product yield. for example, the tac promoter is a hybrid of the trp and lac Appropriate host cells include yeast, bacteria, promoter Sequences. Furthermore, a bacterial promoter can archaebacteria, fungi, and insect and animal cells, including include naturally occurring promoters of non-bacterial ori mammalian cells. Of particular interest are Drosophila gin that have the ability to bind bacterial RNA polymerase melanogaster cells, Pichia pastoris and P. methanolica, 65 and initiate transcription. Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus In addition to a functioning promoter Sequence, an effi Subtilis, SF9 cells, SF21 cells, C129 cells, Saos-2 cells, Hi-5 cient ribosome binding site is desirable. In E. coli, the US 6,919,184 B2 47 48 ribosome binding site is called the Shine-Delgarno (SD) graphic techniques, including ion eXchange, hydrophobic, Sequence and includes an initiation codon and a sequence affinity, and reverse-phase HPLC chromatography, and chro 3–9 nucleotides in length located 3-11 nucleotides upstream matofocusing. For example, the ubiquitin moiety protein of the initiation codon. may be purified using a Standard anti-ubiquitin moiety The expression vector may also include a signal peptide antibody column. Ultrafiltration and diafiltration techniques, Sequence that provides for Secretion of the protein in bac in conjunction with protein concentration, are also useful. teria. The Signal Sequence typically encodes a Signal peptide For general guidance in Suitable purification techniques, See comprised of hydrophobic amino acids which direct the Scopes, R., Protein Purification, Springer-Verlag, NY Secretion of the protein from the cell, as is well known in the (1982). The degree of purification necessary will vary art. The protein is either Secreted into the growth media depending on the use of the protein. In Some instances no (gram-positive bacteria) or into the periplasmic space, purification will be necessary. located between the inner and outer membrane of the cell Once made, the compositions find use in a number of (gram-negative bacteria). applications, including, but not limited to, assaying for The bacterial expression vector may also include a Select agents that modulate the activity of a ubiquitin agent. In able marker gene to allow for the Selection of bacterial 15 particular, the compositions can be used to assay for agents Strains that have been transformed. Suitable Selection genes that modulate the transfer or attachment of ubiquitin moiety include genes which render the bacteria resistant to drugs to a Substrate molecule. The term “modulate” as used herein Such as amplicillin, chloramphenicol, erythromycin, with reference to the activity of a ubiquitin agent refers to kanamycin, neomycin and tetracycline. Selectable markers the increase or decrease in an activity of a ubiquitin agent, also include biosynthetic genes, Such as those in the for example, activating activity, conjugating activity, ligat histidine, tryptophan and leucine biosynthetic pathways. ing activity, and more specifically the attachment of ubiq These components are assembled into expression vectors. uitin moiety to a substrate molecule. The term “attachment” Expression vectors for bacteria are well known in the art, as used herein with reference to the activity of a ubiquitin and include vectors for Bacillus Subtilis, E. coli, Strepto agent refers to the binding, transfer, or attachment of a coccuS Cremoris, and StreptococcuS lividans, among others. 25 ubiquitin moiety to a Substrate molecule. The skilled artisan The bacterial expression vectors are transformed into will appreciate that agents that modulate the activity of bacterial host cells using techniques well known in the art, ubiquitin agents (or “modulators') may affect enzyme Such as calcium chloride treatment, electroporation, and activity, enzyme interaction with a Substrate, interaction others. between ubiquitin moiety and the Substrate, or a combina In one embodiment, proteins are produced in insect cells. tion of these. Expression vectors for the transformation of insect cells, and By “candidate”, “candidate agent”, “candidate in particular, baculovirus-based expression vectors, are well modulator”, “candidate ubiquitination modulator” or gram known in the art. matical equivalents herein is meant any candidate molecule, In a preferred embodiment, proteins are produced in yeast e.g. a protein (which herein includes a protein, polypeptide, cells. Yeast expression Systems are well known in the art, 35 and peptide), Small organic or inorganic molecule, and include expression vectors for Saccharomyces polysaccharide, or polynucleotide which are to be tested for cerevisiae, Candida albicans and C. maltosa, Hansenula the ability to modulate the activity of a ubiquitin agent, and polymorpha, Kluyveromyces fragilis and K. lactis, Pichia more specifically, for the ability to modulate the attachment guillerimondii P. methanolica and P. pastoris, Schizosac of ubiquitin moiety to a Substrate molecule. Candidate charomyces pombe, and Yarrowia lipolytica. Preferred pro 40 agents encompass numerous chemical classes. In a preferred moter Sequences for expression in yeast include the induc embodiment, the candidate agents are Small molecules. In ible GAL1,10 promoter, the promoters from alcohol another preferred embodiment, the candidate agents are dehydrogenase, enolase, glucokinase, glucose-6-phosphate organic molecules, particularly Small organic molecules, isomerase, glyceraldehyde-3-phosphate-dehydrogenase, comprising functional groups necessary for Structural inter hexokinase, phosphofructokinase, 3-phosphoglycerate 45 action with proteins, particularly hydrogen bonding, and mutase, pyruvate kinase, and the acid phosphatase gene. typically include at least an amine, carbonyl, hydroxyl or Yeast selectable markers include ADE2, HIS4, LEU2, carboxyl group, preferably at least two of the functional TRP1, and ALG7, which confers resistance to tunicamycin; chemical groups. The candidate agents often comprise cycli the neomycin phosphotransferase gene, which conferS resis cal carbon or heterocyclic Structures and/or aromatic or tance to G418; and the CUP1 gene, which allows yeast to 50 polyaromatic structures Substituted with one or more chemi grow in the presence of copper ions. cal functional groups. The protein may also be made as a fusion protein, using Candidate agents are obtained from a wide variety of techniques well known in the art. Thus, for example, the Sources, as will be appreciated by those in the art, including protein may be made as a fusion protein to increase libraries of Synthetic or natural compounds. AS will be expression, or for other reasons. For example, when the 55 appreciated by those in the art, the present invention pro protein is a peptide, the nucleic acid encoding the peptide vides a rapid and easy method for Screening any library of may be linked to other nucleic acid for expression purposes. candidate modulators, including the wide variety of known Similarly, proteins of the invention can be linked to protein combinatorial chemistry-type libraries. labels, Such as green fluorescent protein (GFP), red fluores In a preferred embodiment, candidate agents are Synthetic cent protein (RFP), blue fluorescent protein (BFP), yellow 60 compounds. Any number of techniques are available for the fluorescent protein (YFP), etc. random and directed Synthesis of a wide variety of organic In a preferred embodiment, the protein is purified or compounds and biomolecules, including expression of ran isolated after expression. Proteins may be isolated or puri domized oligonucleotides. See for example WO94/24314, fied in a variety of ways known to those skilled in the art hereby expressly incorporated by reference, which discusses depending on what other components are present in the 65 methods for generating new compounds, including random Sample. Standard purification methods include chemistry methods as well as enzymatic methods. AS electrophoretic, molecular, immunological and chromato described in WO 94/24314, one of the advantages of the US 6,919,184 B2 49 SO present method is that it is not necessary to characterize the herein, has the potential to code for 207 (10) to 20'. Thus, candidate agent prior to the assay. Using the methods of the with libraries of 107 to 10 different molecules the present present invention, any candidate agents can be Screened for methods allow a “working” Subset of a theoretically com the ability to increase or decease the activity of a ubiquitin plete interaction library for 7 amino acids, and a Subset of agent, or more Specifically for the ability to increase or shapes for the 20' library. Thus, in a preferred embodiment, decrease the attachment of ubiquitin moiety to a Substrate. In at least 10, preferably at least 10, more preferably at least addition, as is known in the art, coding tags using Split 10 and most preferably at least 10 different sequences are Synthesis reactions may be used to essentially identify the simultaneously analyzed in the subject methods. Preferred chemical moieties tested. methods maximize library size and diversity. Alternatively, a preferred embodiment utilizes libraries of In one embodiment, the library is fully randomized, with natural compounds, as candidate agents, in the form of no Sequence preferences or constants at any position. In a bacterial, fungal, plant and animal extracts that are available preferred embodiment, the library is biased. That is, some or readily produced. positions within the Sequence are either held constant, or are Additionally, natural or Synthetically produced libraries Selected from a limited number of possibilities. For example, and compounds are readily modified through conventional 15 in a preferred embodiment, the nucleotides or amino acid chemical, physical and biochemical means. Known pharma residues are randomized within a defined class, for example, cological agents may be Subjected to directed or random of hydrophobic amino acids, hydrophilic residues, Sterically chemical modifications, including enzymatic modifications, biased (either Small or large) residues, towards the creation to produce Structural analogs. of cysteines, for cross-linking, prolines for SH-3 domains, In a preferred embodiment, candidate agents include Serines, threonines, tyrosines or histidines for phosphoryla proteins, nucleic acids, and chemical moieties. tion Sites, etc., or to purines, etc. In a preferred embodiment, the candidate agents are In a preferred embodiment, the bias is towards peptides or proteins, as defined above. In a preferred embodiment, the nucleic acids that interact with known classes of molecules. candidate agents are naturally occurring proteins or frag For example, when the candidate agent is a peptide, it is ments of naturally occurring proteins. Thus, for example, 25 known that much of intracellular Signaling is carried out via cellular extracts containing proteins, or random or directed Short regions of polypeptides interacting with other polypep digests of proteinaceous cellular extracts, may be tested, as tides through Small peptide domains. For instance, a short is more fully described below. In this way libraries of region from the HIV-1 envelope cytoplasmic domain has prokaryotic and eukaryotic proteins may be made for Screen been previously shown to block the action of cellular ing against any number of candidate agents. Particularly calmodulin. Regions of the Fas cytoplasmic domain, which preferred in this embodiment are libraries of bacterial, shows homology to the mastoparan toxin from Wasps, can fungal, viral, and mammalian proteins, with the latter being be limited to a short peptide region with death-inducing preferred, and human proteins being especially preferred. apoptotic or G protein inducing functions. Magainin, a In a preferred embodiment, the candidate agents are natural peptide derived from Xenopus, can have potent peptides of from about 2 to about 50 amino acids, with from 35 anti-tumor and anti-microbial activity. Short peptide frag about 5 to about 30 amino acids being preferred, and from ments of a protein kinase C isozyme (BPKC), have been about 8 to about 20 being particularly preferred. The pep shown to block nuclear translocation of BPKC in Xenopus tides may be digests of naturally occurring proteins as is oocytes following Stimulation. And, Short SH-3 target pep outlined above, random peptides, or "biased' random pep tides have been used as pSuedoSubStrates for Specific binding tides. By "randomized' or grammatical equivalents herein is 40 to SH-3 proteins. This is of course a short list of available meant that each nucleic acid and peptide consists of essen peptides with biological activity, as the literature is dense in tially random nucleotides and amino acids, respectively. this area. Thus, there is much precedent for the potential of Since generally these random peptides (or nucleic acids, Small peptides to have activity on intracellular Signaling discussed below) are chemically Synthesized, they may cascades. In addition, agonists and antagonists of any num incorporate any nucleotide or amino acid at any position. 45 ber of molecules may be used as the basis of biased The Synthetic process can be designed to generate random randomization of candidate modulators as well. ized proteins or nucleic acids, to allow the formation of all Thus, a number of molecules or protein domains are or most of the possible combinations over the length of the Suitable as Starting points for the generation of biased Sequence, thus forming a library of randomized candidate randomized candidate modulators. A large number of Small bioactive proteinaceous agents. 50 molecule domains are known, that confer a common The library should provide a sufficiently structurally function, Structure or affinity. In addition, as is appreciated diverse population of randomized agents to effect a proba in the art, areas of weak amino acid homology may have bilistically Sufficient range of diversity to allow interaction Strong Structural homology. A number of these molecules, with a particular ubiquitin ligating agent enzyme. domains, and/or corresponding consensus Sequences, are Accordingly, an interaction library must be large enough So 55 known, including, but are not limited to, SH-2 domains, that at least one of its members will have a structure that SH-3 domains, PleckStrin, death domains, protease interacts with a ubiquitin agents or other components of a cleavage/recognition sites, enzyme inhibitors, enzyme ubiquitin reaction, for example, ubiquitin moiety or target Substrates, and Traf. protein. Although it is difficult to gauge the required absolute In a preferred embodiment, the candidate agents are Size of an interaction library, nature provides a hint with the 60 nucleic acids. With reference to candidate agents, by immune response: a diversity of 107-10 different antibodies "nucleic acid' or "oligonucleotide' or grammatical equiva provides at least one combination with sufficient affinity to lents herein means at least two nucleotides covalently linked interact with most potential antigens faced by an organism. together. A nucleic acid of the present invention will gen Published in vitro selection techniques have also shown that erally contain phosphodiester bonds, although in Some a library size of 107 to 10 is sufficient to find structures with 65 cases, as outlined below, nucleic acid analogs are included affinity for a target. A library of all combinations of a peptide that may have alternate backbones, comprising, for example, 7 to 20 amino acids in length, Such as generally proposed phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925 US 6,919,184 B2 S1 52 (1993) and references therein; Letsinger, J. Org. Chem. AS described above generally for proteins, nucleic acid 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 candidate agent may be naturally occurring nucleic acids, (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); random nucleic acids, or "biased' random nucleic acids. For Sawaietal, Chem. Lett. 805 (1984), Letsinger et al., J. Am. example, digests of prokaryotic or eukaryotic genomes may Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica be used as is outlined above for proteins. Where the ultimate Scripta 26:141 91986)), phosphorothioate (Mag et al., expression product is a nucleic acid, at least 10, preferably Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. at least 12, more preferably at least 15, most preferably at 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. least 21 nucleotide positions need to be randomized, with Soc. 111:2321 (1989), O-methylphophoroamidite linkages more preferable if the randomization is less than perfect. (see Eckstein, Oligonucleotides and Analogues: A Practical Similarly, at least 5, preferably at least 6, more preferably at Approach, Oxford University Press), and peptide nucleic least 7 amino acid positions need to be randomized; again, acid backbones and linkages (see Egholm, J. Am. Chem. more are preferable if the randomization is less than perfect. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. In a preferred embodiment, the candidate agents are 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carisson organic moieties. In this embodiment, as is generally et al., Nature 380:207 (1996), all of which are incorporated 15 described in WO94/24314, candidate agents are synthesized by reference). Other analog nucleic acids include those with from a Series of Substrates that can be chemically modified. positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. “Chemically modified’ herein includes traditional chemical USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. reactions as well as enzymatic reactions. These Substrates 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; generally include, but are not limited to, alkyl groups Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (including alkanes, alkenes, alkynes and heteroalkyl), aryl (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); groups (including arenes and heteroaryl), alcohols, ethers, Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); amines, aldehydes, ketones, acids, esters, amides, cyclic Chapters 2 and 3, ASC Symposium Series 580, “Carbohy compounds, heterocyclic compounds (including purines, drate Modifications in Antisense Research”, Ed. Y. S. San pyrimidines, benzodiazepins, beta-lactams, tetracylines, ghui and P. Dan Cook, MeSmaeker et al., Bioorganic & 25 cephalosporins, and carbohydrates), Steroids (including Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomo estrogens, androgens, cortisone, ecodySone, etc.), alkaloids lecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (including ergots, Vinca, curare, pyrollizdine, and (1996)) and non-ribose backbones, including those mitomycines), organometallic compounds, hetero-atom described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and bearing compounds, amino acids, and nucleosides. Chemi Chapters 6 and 7, ASC Symposium Series 580, “Carbohy cal (including enzymatic) reactions may be done on the drate Modifications in Antisense Research”, Ed. Y. S. San moieties to form new Substrates or candidate agents which ghui and P. Dan Cook. Nucleic acids containing one or more can then be tested using the present invention. carbocyclic Sugars are also included within the definition of AS will be appreciated by those in the art, it is possible to nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) Screen more than one type of candidate agent at a time. Thus, pp.169-176). Several nucleic acid analogs are described in 35 the library of candidate agents used may include only one Rawls, C & E News Jun. 2, 1997 page 35. All of these type of agent (i.e. peptides), or multiple types (peptides and references are hereby expressly incorporated by reference. organic agents). The assay of Several candidates at one time These modifications of the ribose-phosphate backbone may is further discussed below. be done to facilitate the addition of additional moieties Such The present invention provides methods and composi as labels, or to increase the stability and half-life of Such 40 tions comprising combining different combinations of ubiq molecules in physiological environments. uitin agents, with ubiquitin moiety, in the presence or As will be appreciated by those in the art, all of these absence of a target protein. In preferred embodiments, a nucleic acid analogs may find use in the present invention. candidate agent is included in the combining to assay for an In addition, mixtures of naturally occurring nucleic acids agent that modulates the attachment of a ubiquitin moiety to and analogs can be made. Alternatively, mixtures of different 45 a Substrate molecule. In preferred embodiments the ubiq nucleic acid analogs, and mixtures of, naturally occurring uitin moiety and/or the Substrate molecule of interest in the nucleic acids and analogs may be made. Particularly pre assay comprises a tag. ferred are peptide nucleic acids (PNA) which includes Preferably the tag is a label, a partner of a binding pair, or peptide nucleic acid analogs. These backbones are Substan a Substrate binding molecule (or attachment tag). In a tially non-ionic under neutral conditions, in contrast to the 50 preferred embodiment, the tag is an epitope tag. In another highly charged phosphodiester backbone of naturally occur preferred embodiment, the tag is a label. More preferably, ring nucleic acids. the tag is a fluorescent label or a binding pair partner. In a The nucleic acids may be single Stranded or double preferred embodiment, the tag is a binding pair partner and Stranded, as Specified, or contain portions of both double the ubiquitin moiety is labeled by indirect labeling. In the Stranded or Single Stranded Sequence. The nucleic acid may 55 indirect labeling embodiment, preferably the label is a be DNA, both genomic and cDNA, RNA or a hybrid, where fluorescent label or a label enzyme. In an embodiment the nucleic acid contains any combination of deoxyribo- and comprising a label enzyme, preferably the Substrate for that ribo-nucleotides, and any combination of bases, including enzyme produces a fluorescent product. In a preferred uracil, adenine, thymine, cytosine, guanine, inosine, Xatha embodiment, the label enzyme Substrate is luminol. In a nine hypoxathanine, isocytosine, isoguanine, etc. AS used 60 preferred embodiment, combining Specifically excludes herein, the term “nucleoside' includes nucleotides and combining the components with a target protein. nucleoside and nucleotide analogs, and modified nucleo In another preferred embodiments, a preferred combina SideS Such as amino modified nucleosides. In addition, tion is Tag1-ubiquitin moiety, tag2-ubiquitin moiety. "nucleoside' includes non-naturally occurring analog struc Preferably, tag1 and tag2 are labels, preferably fluorescent tures. Thus for example the individual units of a peptide 65 labels, most preferably tag1 and tag2 constitute a FRET pair. nucleic acid, each containing a base, are referred to herein as In a preferred embodiment, a preferred combination is a nucleoside. tag1-ubiquitin moiety and tag2-Substrate molecule of inter US 6,919,184 B2 S3 S4 est. Preferably, tag1 is a label, a partner of a binding pair, or reagents that otherwise improve the efficiency of the assay, a Substrate binding molecule and tag2 is a different label, Such as protease inhibitors, nuclease inhibitors, anti partner of a binding pair, or Substrate binding molecule. microbial agents, etc., may be used. The compositions will More preferably, tag1 is a fluorescent label or a member of also preferably include adenosine tri-phosphate (ATP). a binding pair. When tag1 is a member of a binding pair, The mixture of components may be added in any order preferably tag1 is indirectly labeled. Still more preferably, that promotes the activity ubiquitin agents, and more spe tag-1 is indirectly labeled with a label enzyme. Preferably cifically promotes the attachment of ubiquitin moiety to a the label enzyme Substrate used to reveal the presence of the Substrate molecule of interest in the assay, or optimizes enzyme produces a fluorescent product, and more preferably identification of the modulating activity of a candidate is luminol. In the presently described combination, prefer agent. In a preferred embodiment, ubiquitin moiety is pro ably tag2 is a Surface Substrate binding element, more vided in a reaction buffer solution, followed by addition of preferably a His-tag. the ubiquitin ubiquitination enzymes. In an alternate pre In a preferred embodiment, the methods of the invention ferred embodiment, ubiquitin moiety is provided in a reac do not comprise a target protein. In a preferred embodiment, tion buffer Solution, a candidate agent is then added, fol a mono- or poly-ubiquitin moiety is a Substrate molecule, as 15 lowed by the addition of ubiquitin agents. discussed above. Because the different combinations of Once combined, in a preferred embodiment, the amount ubiquitin agents are specific for particular target proteins, the of ubiquitin moiety attached to a Substrate molecule of present assays are much more versatile then conventional interest in an assay of the present invention, is measured. AS assays which require a target protein. However, the activity will be understood by one of ordinary skill in the art, the of these ubiquitin agents can be assayed in the methods of mode of measuring may depend on the Specific tag attached the present invention because the methods permit the use of to the ubiquitin moiety. AS will also be apparent to the any variation of Such combinations without first identifying skilled artisan, the amount of ubiquitin moiety attached to a the Specific target protein to which the combination is Substrate molecule will encompass not only the particular directed. ubiquitin moiety bound directly to the Substrate molecule, The components of the present assays may be combined 25 but also a mono- or poly-ubiquitin moiety preferably in varying amounts. In a preferred embodiment, ubiquitin attached to the Substrate molecule. moiety is combined at a final concentration of from 20 to In a preferred embodiment, the tag attached to the ubiq 200 ng per 100 ul reaction solution, most preferable at about uitin moiety is a fluorescent label. In a preferred 100 ng per 100 ul reaction solution. embodiment, the tag attached to ubiquitin moiety is an In a preferred embodiment, the ubiquitin activating agent, enzyme label or a binding pair member which is indirectly preferably an E1, is combined at a final concentration of labeled with an enzyme label. In this latter preferred from 1 to 50 ng per 100 ul reaction solution, more preferably embodiment, the enzyme label substrate produces a fluo from 1 ng to 20 ng per 100 ul reaction Solution, most rescent reaction product. In these preferred embodiments, preferably from 5 ng to 10 ng per 100 ul reaction solution. the amount of ubiquitin moiety bound is measured by In a preferred embodiment, the ubiquitin conjugating 35 luminescence. agent, preferably an E2, is combined at a final concentration In other preferred embodiments, at least a first and a of 10 to 100 ng per 100 ul reaction solution, more preferably Second ubiquitin moiety is used, wherein the first and Second 10-50 ng per 100 ul reaction solution. ubiquitin moieties comprise different fluorescent labels, and In a preferred embodiment, the ubiquitin ligating agent, wherein the labels form a FRET pair. preferably an E3, is combined at a final concentration of 40 AS used herein, “luminescence' or "fluorescent emission' from 1 ng to 500 ng per 100 ul reaction solution, more means photon emission from a fluorescent label. In an preferably from 50 to 400 ng per 100 ul reaction solution, embodiment where FRET pairs are used, fluorescence mea still more preferably from 100 to 300 ng per 100 ul reaction Surements may be taken continuously or at time-points solution, and still more preferably about 100 ng per 100 ul during the ligation reaction. Equipment for Such measure reaction Solution. In a preferred embodiment, the ubiquitin 45 ment is commercially available and easily used by one of ligating agent is combined at a final concentration of from 50 ordinary skill in the art to make Such a measurement. to 100 ng per 100 ul reaction solution, still more preferably Other modes of measuring the attachment of ubiquitin from 20 to 50 ng per 100 ul reaction solution, and still more moiety to a Substrate molecule of are well known in the art preferably about 10 ng to 20 ng per 100 ul reaction solution. and easily identified by the skilled artisan for each of the The components of the present assays are combined under 50 labels described herein. For example, radioisotope labeling reaction conditions that favor the activity of the ubiquitin may be measured by Scintillation counting, or by densito agents of the present invention, and more Specifically favir metry after exposure to a photographic emulsion, or by using the attachment of ubiquitin moiety to a Substrate molecule of a device Such as a Phosphorimager. Likewise, densitometry interest in the assay. Generally, this will be under physi may be used to measure the attachment of ubiquitin moiety ological conditions. Incubations may be performed at any 55 following a reaction with an enzyme label Substrate that temperature which facilitates optimal activity, typically produces an opaque product when an enzyme label is used. between 4 and 40 C. Incubation periods are selected for In a preferred embodiment, the substrate molecule of optimum activity, but may also be optimized to facilitate interest in the assays of the present invention is bound to a rapid high through put Screening. Typically between 0.5 and surface Substrate. This may be achieved as described above 1.5 hours will be sufficient. 60 for the binding of a label to ubiquitin moiety. A variety of other reagents may be included in the In another preferred embodiment, a ubiquitin activating compositions. These include reagents like Salts, Solvents, agent is bound to a Surface Substrate in the absence of a buffers, neutral proteins, e.g. albumin or detergents which ubiquitin conjugating agent and ubiquitin ligating agent. may be used to facilitate optimal activity of ubiquitin agents, This may be achieved, as described above for the binding of and more Specifically facilitate the attachment of ubiquitin 65 a label to ubiquitin moiety. This may also be accomplished moiety to a Substrate molecule of interest in the assay; and/or using tag-ubiquitin activating agent, wherein the tag is a reduce non-specific or background interactions. Also Surface Substrate binding molecule. US 6,919,184 B2 SS S6 In another preferred embodiment, a ubiquitin conjugating protein comprises an attachment tag. In another agent is bound to a Surface Substrate in the absence of a embodiment, the MidM2 protein is preferably provided on a ubiquitin ligating agent. This may be achieved, as described Solid Support, and more preferably the Solid Support com above for the binding of a label to ubiquitin moiety. This prises a microtiter plate or a bead. In another embodiment, may also be accomplished using tag-ubiquitin conjugating the mdm2 protein is preferably a mammalian mdm2 and agent, wherein the tag is a Surface Substrate binding mol more preferably a human mdm2. ecule. In another preferred embodiment, the target protein is p53 In another preferred embodiment, a ubiquitin ligating and comprises a first FRET label and the ubiquitin moiety agent is bound to a Surface Substrate in the absence of a comprises a second FRET label. target protein. This may be achieved, as described above for In another embodiment, the p53 protein preferably com the binding of a label to ubiquitin moiety. This may also be prises an attachment tag. In another embodiment, the p53 accomplished using tag-ubiquitin ligating agent, wherein the protein is preferably provided on a Solid Support, and more tag is a Surface Substrate binding molecule. preferably the Solid Support comprises a microtiter plate or In general, any Substrate binding molecule can be used. In a bead. a preferred embodiment, the tag is a His-tag and the Surface 15 In a preferred embodiment, the compositions of the inven Substrate is nickel. In a preferred embodiment, the nickel tion are used to identify agents that modulate the attachment Surface Substrate is present on the Surface of the Wells of a of ubiquitin moiety to a Substrate molecule. In this multi-well plate, such as a 96 well plate. Such multi-well embodiment, the composition includes a candidate agent. In plates are commercially available. The binding of the a preferred embodiment, the measured amount and/or rate of enzyme to a Surface Substrate facilitates the Separation of tag-ubiquitin moiety binding to the Substrate molecule is bound ubiquitin moiety from unbound ubiquitin moiety. In compared with the that when the candidate agent is absent the present embodiment, the unbound ubiquitin moiety is from the composition, whereby the presence or absence of easily washed from the receptacle following the ligation the agent's effects on the attachment of ubiquitin moiety to reaction. AS will be appreciated by those of skill in the art, a Substrate molecule is determined. In this embodiment, the use of any Surface Substrate binding element and recep 25 whether the agent enhances or inhibits, or reduces or tacle having the surface substrate to which it binds will be increases the attachment of ubiquitin moiety to the Substrate effective for facilitating the Separation of bound and molecule is determined. unbound ubiquitin moiety. In a preferred embodiment, the composition of the inven In an alternative embodiment, the Substrate molecule of tion containing a candidate agent lackS E3 and the amount interest in the assays of the present invention comprise a and/or rate of ubiquitin moiety attached to E2 is measured. bead that is attached to the Substrate molecule directly or via This embodiment may also comprise the Step of comparing a substrate binding element. Following ligation, the beads the amount and/or rate of ubiquitin moiety attached to E2 in may be separated from the unbound ubiquitin moiety and the a composition lacking both E3 and a candidate agent, bound ubiquitin moiety measured. In a preferred whereby the modulating activity of the candidate agent is embodiment, the Substrate molecule of interest in the assay 35 determined. In a preferred embodiment, the percentage of the present invention comprises a bead and the ubiquitin difference in the amount of ubiquitin moiety attached to E2 agents in the assay are combined with a tag-ubiquitin moiety in the presence and absence of the candidate agent is wherein the tag is a fluorescent label. In this embodiment, compared with the percentage difference in the amount the beads with bound ubiquitin moiety may be separated attached to E3 in the presence and absence of candidate using a fluorescence-activated cell Sorting (FACS) machine. 40 agent, whereby the point of effect of the candidate agent in Methods for Such use are described in U.S. patent applica the cascade of enzymatic activity and attachment of ubiq tion Ser. No. 09/047,119, which is hereby incorporated in its uitin moiety to a Substrate molecule is determined. That is, entirety. The amount of bound ubiquitin moiety can then be it is determined whether the candidate agent affects the measured. attachment of ubiquitin moiety to E1, E2, and/or E3. In another embodiment, none of the ubiquitin agents are 45 In another preferred embodiment, the compositions of the bound to a surface substrate. Preferably in this embodiment, invention are used to identify agents that modulate the the assays comprise a tag1-ubiquitin moiety and tag2 attachment of ubiquitin moiety to a Substrate molecule of ubiquitin moiety. Preferably, tag1 and tag2 are labels, pref interest in the assay. In this embodiment, the present assays erably fluorescent labels, most preferably tag1 and tag2 include a candidate agent. In a preferred embodiment, where constitute a FRET pair. In this embodiment, the attachment 50 tag1 and tag2 constitute a FRET pair, the measured amount of ubiquitin moiety to the Substrate molecule of interest is and/or rate of tag1-ubiquitin moiety and tag2-ubiquitin measured by measuring the fluorescent emission Spectrum. moiety binding to a Substrate molecule (as a poly-ubiquitin This measuring may be continuous or at one or more times moiety and/or ubiquitin moiety attached to a Substrate following the combination of the components. Alteration in molecule) is compared with the amount or rate of Such the fluorescent emission spectrum of the combination as 55 attachment in the absence of the candidate agent, whereby compared with unligated ubiquitin moiety indicates the the presence or absence of the candidate agent's effect on the amount of ubiquitin ubiquitination. The skilled artisan will attachment of ubiquitin moiety to a Substrate molecule is appreciate that in this embodiment, alteration in the fluo determined. In this embodiment, whether the candidate rescent emission spectrum results from ubiquitin moiety agent enhances or inhibits, or increases or decreases, the bearing different members of the FRET pair being brought 60 attachment of ubiquitin moiety to the Substrate molecule is into close proximity, either through the formation of poly also determined. ubiquitin moiety and/or by binding nearby locations on a In a preferred embodiment, multiple assays are performed protein, preferably a target protein. Simultaneously in a high throughput Screening System. In In one preferred embodiment of the present methods, the this embodiment, multiple assays may be performed in ubiquitin ligating agent is an MdM2 protein and comprises 65 multiple receptacles, Such as the Wells of a 96 well plate or a first FRET label and the ubiquitin moiety comprises a other multi-well plate. As will be appreciated by one of skill Second FRET label. In another embodiment, the MidM2 in the art, Such a System may be applied to the assay of US 6,919,184 B2 57 58 multiple candidate agents and/or multiple combinations of Materials and Methods ubiquitin agents with ubiquitin moiety. In a preferred Plasmid is transformed in BL21 DE3 competent E. coli embodiment, the present invention is used in a high (Stratagene, cat # 230132). Cells are grown at 37° C. in throughput Screening System for determining the attachment TB+100 ug/ml amplicillin and 0.4% glucose to an OD600 of of ubiquitin moiety to a Substrate molecule of interest, by about 0.6, induced with addition of 320 uM IPTG and combining different combinations of ubiquitin agents, in the allowed to grow for another 3 h before harvest. The pellets presence or absence of a target protein. In an alternate are washed once with cold PBS, then resuspended in about preferred embodiment, the present invention is used in a 6 volumes of lysis buffer (20 mM Tris, 10% glycerol, 0.5 M high throughput Screening System for Simultaneously testing Nacil, 2.5 mM EDTA, 1 mM TCEP plus Complete-EDTA the effect of individual candidate agents by additionally Free Protease inhibitor tablets, 1 tablet/25 ml of resuspended combinining a candidate agent. cells, pH 8.0). The Suspension is homogenized and Sonicated In another aspect, the invention provides a method of 3x30 Sec. NP40, then added to a final concentration of 0.5% assaying for the attachment of a ubiquitin moiety to a and the tubes are rocked for 30 min at 4 C. Following Substrate molecule of in a mixture. Ubiquitin moiety is centrifugation at 11000 rpm for 25 to 30 min, the Supernatant introduced into a cell or mixture of protein, preferably a cell 15 is incubated with Glutathione Sepharose 4B (Amersham, cat lysate, under conditions in which the attachment of ubiquitin # 17-0756-01) at a ratio of 1 ml of beads per 100 ml of moiety to a Substrate molecule of interest can take place. In original culture volume for 1 to 2 hours at 4 C. with gentle this embodiment, the ubiquitin moiety is in the form of rocking. The beads are pelleted and washed once with 10 tag1-ubiquitin moiety and tag2-2-ubiquitin moiety, wherein bed volumes of the lysis buffer, then twice with 10 bed tag1 and tag2 constitute a FRET pair or tag1 is a fluorescent volumes of Prescission Protease buffer (50 mM Tris-HCL, label and tag2 is a quencher of tag1. Fluorescent emission 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% NP-40, pH Spectrum is measured as an indication of whether ubiquitin 7.0.). Prescission Protease (Amersham, product #27-0843) ubiquitination activity is present in the mixture or cell. In a is added at a ratio of 80 ul (160 Units) per ml of GST resin, preferred embodiment, the ubiquitin moiety also comprises and allowed to incubate for 4 h at 4 C. The Supernatant a member of a binding pair, Such as FLAG. In this latter 25 containing the cleaved E2 protein is collected, and the resin embodiment, components involved in ubiquitin ubiquitina is washed twice with one bed volume of Prescission buffer. tion can be isolated from the mixture using any one of a All three fractions are analyzed by SDS-PAGE and pooled number of affinity-based Separation means Such as fluores when appropriate. cent beads coated with anti-FLAG antibody or amino pre Ubiquitin Moiety Production cipitation using anti-FLAG antibodies, or using anti-FLAG Ubiquitin moiety was cloned into the pFlag-Mac Expres antibody attached to a Solid Support. Other means of Sepa sion Vector (Sigma) as a HindIII-EcoRI fragment by PCR. rating ubiquitin moiety attached components of the cell or This results in expression of amino-terminal Flag fusion mixture will be readily apparent to the skilled artisan. ubiquitin moiety in E. Coli. Ubiquitin moiety attached components So Separated in this Materials and Methods method may include ubiquitin agents and target proteins. 35 The induction of protein expression and cell lysis is The skilled artisan will appreciate that Separation of these similar to the above GST-E2 preparation, except that the components for individual identification or Subsequent Supernatant is loaded over a FLAG-affinity resin (VWR, cat investigation may be obtained by Several means well known # IB 13020) at a ratio of 15 ml of beads per 1 L of original in the art, such as by HPLC or electrophoresis. culture. The resin is then washed with 10 bed volumes of It is understood by the skilled artisan that the steps of the 40 lysis buffer. The protein is eluted from the column with: 100 assays provided herein can vary in order. It is also mM Acetic acid, 10% glycerol, 200 mM NaCl, 2.5 mM understood, however, that while various options (of EDTA, 0.1% NP-40, pH 3.5. The elutions are collected as 1 compounds, properties selected or order of Steps) are pro bed volume fractions into tubes that contain /10" volume of Vided herein, the options are also each provided individually, 2M Tris, 80 mM B-ME, pH 9.0 to neutralize the pH. The and can each be individually Segregated from the other 45 elution fractions are analyzed by SDS-PAGE and the appro options provided herein. Moreover, Steps which are obvious priate fractions are pooled and dialyzed against 400 volumes and known in the art that will increase the sensitivity of the of 20 mM Tris, 10% glycerol, 200 mM NaCl, 2.5 mM assay are intended to be within the Scope of this invention. EDTA, pH 8.0. For example, there may be additionally washing Steps, Production of E3 blocking Steps, etc. 50 Coding Sequences for E3 complex were also amplified by The following examples serve to more fully describe the PCR and baculoviruses were generated using the Bac-to-Bac manner of using the above-described invention, as well as to system (GibcoBRL). E3 contains two subunits, which are Set forth the best modes contemplated for carrying out expressed by co-infection of the two baculovirus in the same various aspects of the invention. It is understood that these Hi-5 insect cells. One of the subunit is His-tagged, with the examples in no way Serve to limit the true Scope of this 55 other associating Subunit untagged. The detail procedure invention, but rather are presented for illustrative purposes. was done following the Bac to Bac Baculovirus Expression All references cited herein are expressly incorporated by system by GibcoBRL. For example, ROC1 was cloned into reference in their entirety. the pFastBachtb vector with a N-terminal His6-tag, while EXAMPLES CUL1 was insert into the pFastBac1 vector without any 60 fusing tag. After transposition and Bacmid DNA transfection Example 1 into SF-9 cells, Baculoviruses were harvested, amplified, Production of E2, E3, and Ubiquitin Moiety and used to co-infect Hi-5 cells for protein expression. E2 Production Materials and Methods The open reading frame of E2 (Ubch5c) was amplified by Cells are harvested, washed once with cold PBS, and PCR and cloned into the pGex-6p-1 E. Coli. expression 65 resuspended in about 6 volumes of lysis buffer (20 mM Tris, vector (Amersham Pharmacia) as BgIII-EcoRI fragments, 20% glycerol, 0.5 M Nacil, 15 mM imidazole, 1 mM TCEP with N-terminus in frame fused to the GST-tag. plus Complete-EDTA Free Protease inhibitor tablets, 1 US 6,919,184 B2 59 60 tablet/25 ml of resuspended cells, pH 358.0). The suspen Results sion is then Sonicated 3x30 sec, followed by addition of Attachment of Ubiquitin Moiety to E1 and Attachment of NP40 to a final concentration of 0.5% and incubation for 30 Ubiquitin Moiety to E2 min at 4 C. The lysate is then centrifuged and the Super FIG. 1A shows the luminescence measured for E1 alone natant is incubated with pre-equilibrated (lysis buffer+ and for E1+his-E2, as described above. NP40) Ni-NTA Agarose beads (Qiagen, cat # 1000632) for 1 to 2 hrs. The pelleted beads are washed 2 times with lysis Example 3 buffer, resuspended in 1 to 2 volumes of lysis buffer and transferred to a disposable column for elution. Elution is accomplished using 5x1-bed Volume aliquots of Lysis The attachment of ubiquitin moiety to E3, by combining buffer+250 mM imidazole. Elution fractions are analyzed by E1+E2+E3 and ubiquitin moiety, was assayed using the SDS-PAGE and appropriate fractions are pooled. The elu following protocol with Flag-ubiquitin, purified from E. tion pool is then desalted using either a desalting column or coli, the E2 Ubch5c, purified as GST-Ubch5c from E. coli a centrifugal concentration device (more often used for large with the GST tag removed, and the E3 His-ROC1/Cul1 Volumes.) When using centrifugal devices, the eluted pool is 15 complex purified from Hi-5 cells by Baculovirus diluted 1:1 with lysis buffer that has no imidazole and spun co-infection. This assay was also used to Show the effects of at the appropriate Speed until the Volume is reduced by half. candidate agents on the attachment of ubiquitin moiety to At this point an equal volume of fresh buffer is added and the E3. device is respun. This is done a total of four times resulting Materials and Methods in a 32 fold exchange. The wells of Nickel-substrate 96-well plates (Pierce Chemical) are blocked with 100 ul of 1 casein/phosphate Example 2 buffered saline (PBS) for 1 hour at room temperature, then washed with 200 ul of PBST (0.1% Tween-20 in PBS) 3 E1+E2 Assay times. To each well is added the following Flag-ubiquitin 25 moiety (See above) reaction Solution: The attachment of ubiquitin moiety to an E2, by combin Final Concentration ing E1+E2 and ubiquitin moiety, was assayed using the 62.5 mM Tris pH 7.5 following protocol with Flag-ubiquitin, purified from E. 6.25 m MgCl, coli, and the E2 Ubch5c, purified as His-Ubch5c from E. O.75 mM, DTT coli. 2.5 mM ATP Materials and Methods 2.5 mM NaF The following procedures were used for assays measuring 12.5 nM Okadaic acid the attachment of ubiquitin moiety to E2. The wells of 100 ng Flag-ubiquitin moiety (made as described above). Nickel-substrate 96-well plates (Pierce Chemical) are 35 The buffer solution is brought to a final volume of 80 ul blocked with 100 ul of 1% casein/phosphate buffered saline with millipore-filtered water. (PBS) for 1 hour at room temperature, then washed with 200 For assays directed to identifying agents that modulate the ul of PBST (0.1% Tween-20 in PBS) 3 times. To each well attachment of ubiquitin moiety to E3, 10 ul of a candidate is added the following Flag-ubiquitin moiety (See above) agent in DMSO is then added to the solution. If no candidate reaction Solution: 40 agent is added, 10 ul of DMSO is added to the solution. Final Concentration To the above solution is then added 10 ul containing the 62.5 mM Tris pH 7.5 ubiquitin agents in 20 mM Tris buffer, pH 7.5, and 5% 6.25 m MgCl glycerol. E2 -Ubch5c and E3-HisROC1/Cul1 are made as O.75 mM, DTT described above. E1 is obtained commercially (Affiniti 2.5 mM ATP 45 Research Products, Exeter, U.K.). The following amounts of 2.5 mM NaF each enzyme are used for these assays: 5 ng/well of E1, 25 12.5 nM Okadaic acid nl/well E2; and 100 ng/well His-E3. The reaction is then 100 ng Flag-ubiquitin moiety (made as described above). allowed to proceed at room temperature for 1 hour. The buffer solution is brought to a final volume of 80 ul Following the ubiquitin ubiquitination reaction, the Wells with millipore-filtered water, followed by the addition of 10 50 are washed with 200 ul of PBST 3 times. For measurement ul of DMSO. of the E3-attached ubiquitin moiety, 100 ul of Mouse To the above solution is then added 10 ul of E1.His-E2 in anti-Flag (1:10,000) and ant-Mouse Ig-HRP (1:15,000) in 20 mM Tris buffer, pH 7.5, and 5% glycerol. His-E2 is made PBST are added to each well and allowed to incubate at as described above. E1 is obtained commercially (Affiniti room temperature for 1 hour. The wells are then washed with Research Products, Exeter, U.K.). The following amounts of 55 200ul of PBST 3 times, followed by the addition of 100 ul each enzyme are used for these assays: 5 ng/well of E1, 25 of luminol Substrate (/S dilution). Luminescence for each nl/well E2. The reaction is then allowed to proceed at room well is then measured using a fluorimeter. temperature for 1 hour. Results Following the ubiquitin reaction, the Wells are washed Attachment of Ubiquitin Moiety to E3 with 200 ul of PBST 3 times. For measurement of the 60 FIG. 2 shows the luminescence measured for several E2-attached ubiquitin moiety, 100 ul of Mouse anti-Flag different combinations of ubiquitin agents. In these (1:10,000) and ant-Mouse Ig-HRP (1:15,000) in PBST are experiments, only E3 was in the form His-E3. The lumines added to each well and allowed to incubate at room tem cence measurements show that the assay Specifically mea perature for 1 hour. The wells are then washed with 200 ul Sures the activity of the entire cascade of activity or attach of PBST 3 times, followed by the addition of 100 ul of 65 ment of ubiquitin moiety by the ubiquitin agents, which luminol substrate (/S dilution). Luminescence for each well requires the presence of all three ubiquitin agents (i.e., is then measured using a fluorimeter. E1+E2+E3) in this reaction. US 6,919,184 B2 61 62 Varying the Amounts of Ubiquitin Agents FLAG-Ala-Cys-ubiquitin moiety. Protein was expressed FIG. 3A shows the relative effect of varying the amount and purified as described above. of E1 on the attachment of ubiquitin moiety to E3 in the Either fluorescein 5-maleimide (peak emission at 515 nm) above procedure, in presence and absence of DMSO. The or 1.5-iodacetamide EDANS (IAEDANS; peak emission at addition of about 10 ng per 100 ul reaction solution provides 490 nm) was reacted with the thiol group on the cysteine of maximum amounts of attachment of ubiquitin moiety to E3 the ubiquitin moiety produced as above to form a thioether. with the other components of the reaction maintained as The labeling was performed in PBS with 1 mM TCEP. detailed above. The presence of DMSO does not signifi Labeled protein was Separated from free label by gel filtra cantly affect the activity of the ubiquitin agents. tion. The relative effect of varying E3 and ubiquitin moiety The ubiquitin assay was performed Substantially as concentration in the ubiquitin reaction is shown in FIG. 3B. described above, with a few modifications. No nickel Sub Generally Speaking, maximum amounts of attachment of Strate was used in the reaction wells, So all of the compo nents were free in Solution. Equal amounts of fluorescein ubiquitin moiety to E3 was obtained with 200 to 300 ng per labeled ubiquitin moiety and IAEDANS labeled ubiquitin 100 ul of E3 at each concentration of ubiquitin moiety, while moiety were used. The reaction was performed at room increasing ubiquitin moiety concentration generally 15 temperature for 2 hours in a volume of 100-150 ul, then increased the amount of attachment of ubiquitin moiety to stopped with 50 ul of 0.5M EDTA, pH 8. E3 at each concentration of E3. Following the reaction, the products were separated in It was also found that blocking of the wells with 1% PBS with 1 mM TCEP by HPLC on a Superdex-75 HR casein improved the Signal to noise ratio over either no 10/30 Size-exclusion column using fluorescence emission blocking or blocking with 5% bovine serum albumen detection. A larger molecular weight cutoff gel-filtration (BSA). Background was determined after combining all of column (e.g., Superdex 200 HR 10/30) could be used to the components as above except His-E3 and measuring the resolve individual ligation Species. resulting fluorescence after pre-treating the Wells with 5% Results BSA, 1% casein or nothing. Results are shown in FIG. 4. FIGS. 16A and 16B show the E3-dependent incorporation Identification of Agents that Modulate the Attachment of 25 of Flag-Ala-Cys-ubiquitin moiety labeled with FRET fluo Ubiquitin Moiety to E3 rophores into E3-ubiquitin moiety complex. Isolation by To show that the assay is useful for identifying agents that HPLC shows emissions from free ubiquitin moiety and modulate the attachment of ubiquitin moiety to E3, Several ubiquitin moiety attached to the E3 ubiquitin ligating agent. candidate agents were combined with the ubiquitin agents The traces show fluorescent emission at the wavelength (E1+E2+E3) and ubiquitin moiety, at varying concentrations described below, under excitation at 336 nm, the optimal as described above. FIG. 5 shows the results from two excitation wavelength for IAEDANS. FIG. 16A shows the identified agents that modulate the attachment of ubiquitin fluorescence signals of IAEDANS (490 nm, larger peak) and moiety to E3. The modulators decreased the attachment of fluorescein (515 nm, Smaller peak) labeled ubiquitin moiety ubiquitin moiety to E3 in a dose-dependent fashion corre following combination with E1 and E2 only. The free sponding to the concentration of the ubiquitin agents present 35 ubiquitin moiety was isolation using high performance liq in the reaction which comprised either ROC1/Cul1 or uid chromatography (HPLC). FIG. 16B shows the fluores ROC2/Cul5 as the E3 component. cence signals of IAEDANS (490 nm; larger peak at each Comparison of the effect of the modulators on the attach elution volume) and fluorescein (515 nm, Smaller peak at ment of ubiquitin moiety to E3, as described above, either each elution volume) labeled ubiquitin moiety following containing E1, E2 and His-E3 or containing E1, His-E2 and 40 combination with E1 and E2 and E3 (Roc1/Cul1). The lacking E3, shows whether the modulator affects E3 or a dashed line shows optical density of the protein Solution ubiquitin agent other than E3. In FIG. 6A, the identified (Scale on right), revealing the high Sensitivity of the fluo modulator decreases the attachment of ubiquitin to E3 in the rophores despite a very low concentration of protein. presence of E3, but does not modulate the attachment in the Fluorescein labeled ubiquitin moiety and IAEDANS absence of E3, showing that the modulator has a specific 45 labeled ubiquitin moiety was attached to E3 in approxi effect on the attachment of ubiquitin moiety to E3. In mately equal amounts. A comparison of the spectral analysis contrast, results shown in FIG. 6B for another modulator of fluorescent emission from the free (unligated) ubiquitin reveals that this agent reduces activity whether or not E3 is moiety labeled with both fluorophores and the E3-attached present, showing that the affects of this agent effect the ubiquitin moiety shows a distinct increase in ratio of emis activity of ubiquitin agents other than E3. 50 sion at 515 nm versus 490 nm (FIG. 17). This shows that in Example 4 the attached ubiquitin moiety, the fluorophores on different ubiquitin moieties are sufficiently close for FRET to be FRET Analysis of Ubiquitin Moiety Attached to E3 measured. Ubiquitin moiety was prepared, labeled with either Example 5 EDANS or fluorescein, and the fluorescence of each of these 55 labels and their interaction as a FRET pair was measured to show attachment of the labeled ubiquitin moiety to E3 and The attachment of ubiquitin moiety to p53, by combining FRET activity of the attached ubiquitin moiety. E1+E2 Ubch5c+Gst-Mdm2+His-p53, and Flag-ubiquitin Materials and Methods moiety, was assayed using the following protocol with: E1 Ubiquitin moiety were produced incorporating CyS resi 60 obtained commercially (Affiniti Research Products, Exeter, dues into the FLAG-ubiquitin moiety Sequence by Site U.K.); Flag-ubiquitin moiety purified from E. coli; E2 directed mutagenesis using either the primer Ubch5c (also called Ubc-5) purified as GST-Ubch5c from E. 5'-CCCCCCAAGCTTTGCATGCAGATTTTCGTGA coli with the GST tag removed; GST-Mdm2 (schematically AGACCCTGACC-3' (SEQ ID NO:26) to produce FLAG depicted in FIG. 18) purified from Hi-5 cells by Baculovirus Cys-ubiquitin moiety, or the primer 65 infection with the GST tag intact; and p53 purified from Hi-5 5'-CCCCCCAAGCTTGCGTGCATGCAGATTTTCG cells by Baculovirus infection (Schematically depicted in TGAAGACCCTGACC-3' (SEQ ID NO:27) to produce FIG. 18). E2 Ubch5c was made as described above. Gst US 6,919,184 B2 63 64 Mdm2 and His-p53 were made as described above for 2.0 mM ATP GST-Ubch5c and E3 His-ROC1/Cul1, respectively. 100 ng Flag-ubiquitin moiety (made as described above) Materials and Methods 100 ng His-p53. The following procedures were used for assays measuring The buffer solution is brought to a final volume of 80 ul the attachment of ubiquitin moiety to p53 by Western blot 5 with millipore-filtered water, followed by the addition of 10 analysis. The following combinations of ubiquitin agents, ul of DMSO. ubiquitin moiety, and p53 were combined in a reaction To the above solution is then added 10 ul of E1, His-E2, mixture: E1+E2 Ubch5c+p53 (as a control); Mdm2+p53 (as and Mdm2 in 20 mM Tris buffer, pH 7.5, and 5% glycerol. a control); E1+E2 Ubch5c (as a control); and E1+E2 The controls contained either Mdm2 alone or His-p53 alone. Ubch5c+Mdm2+p53 (as a control). To each reaction mixture The His-E2 and Mdm2 is made as described above, and E1 is added the following: is obtained commercially (as described above). The follow Final Concentration ing amounts of each enzyme are used for these assays: 5 50 mM Tris pH 7.5 ng/well of E1; 15 ng/well E2 Ubch5c; and 50 ng/well 5 mM MgCl, Mdm2. The reaction is then allowed to proceed at room O.6 mM DTT 15 temperature for 1 hour. 2.0 mM ATP Following the ubiquitin reaction, the Wells are washed 100 ng Flag-ubiquitin moiety (made as described above) with 200 ul of PBS 3 times. For measurement of the 100 ng His-p53. p53-attached ubiquitin moiety, 100 ul of Mouse anti-Flag The buffer solution is brought to a final volume of 80 ul (1:10,000) and ant-Mouse lg-HRP (1:15,000) in PBS are with millipore-filtered water, followed by the addition of 10 added to each well and allowed to incubate at room tem ul of DMSO. perature for 1 hour. The wells are then washed with 200 ul To the above solution is then added 10 ul of E1+E2 of PBS 3 times, followed by the addition of 100 ul of Ubch5c+p53; Mdm2+p53; E1+E2 Ubch5.c; or E1+E2 luminol substrate (/S dilution). Luminescence for each well Ubch5c+Mdm2+p53, in 20 mM Tris buffer, pH 7.5, and 5% is then measured using a fluorimeter. glycerol. The His-E2 and Mdm2 is made as described above, 25 Results and E1 is obtained commercially (as described above). The Attachment of Ubiquitin Moiety to p53 following amounts of each enzyme are used for these assays: FIG. 21 shows the luminescence measured for His-p53 5 ng of E1; 15 ng E2 Ubch5c; and 50 ng Mdm2. The reaction alone, Mdm2 alone, and for Mdm2+His-p53 as described is then allowed to proceed at 37 C. for 1 hour. above. The products of the reaction were then resolved by Examples of preferred embodiments are depicted in the SDS-PAGE; analyzed by Western blot using Mouse anti following figures. Flag and ant-Mouse Ig-HRP FIG. 22 depicts the key for the ubiquitin activating agent Results (UAA), ubiquitin conjugating agent (UCA), ubiquitin ligat Attachment of Ubiquitin Moiety to p53 ing agent (ULA), ubiquitin moiety (U), and candidate agent FIG. 19 shows the attachment of ubiquitin moiety mea 35 (CA) used in the Schematics in Figures sured for E1+E2 Ubch5c+p53; Mdm2+p53; E1+E2 Ubch5c; FIG.23 schematically depicts a preferred embodiment for and E1+E2 Ubch5c+Mdm2+p53, as described above. assaying for the attachment of ubiquitin moiety to a first ubiquitin agent (UA-1) where the assay comprises: Example 6 1) combining a UA-1+CA+U; and 40 2) assaying for the attachment of the ubiquitin moiety to UA-1. In another preferred embodiment UA-1 is a UAA. In The attachment of ubiquitin moiety to p53, by combining another preferred embodiment, UAA is an E1. In yet another E1+E2 Ubch 5c+Gst-Mdm2+His-p53, and Flag-ubiquitin preferred embodiment, UA-1 comprises a label. In another moiety, was assayed using the following protocol with: E1 preferred embodiment, the ubiquitin moiety comprises a obtained commercially (Affiniti Research Products, Exeter, 45 label. U.K.); Flag-ubiquitin moiety purified from E. coli; E2 FIG. 24 schematically depicts a preferred embodiment for Ubch5c (also called Ubc-5) purified as GST-Ubch5c from E. assaying for the attachment of ubiquitin moiety to a Second coli with the GST tag removed; GST-Mdm2 (schematically ubiquitin agent (UA-2) where the assay comprises: depicted in FIG. 18) purified from Hi-5 cells by Baculovirus 1) combining a first ubiquitin agent that is UAA+UA infection with the GST tag intact; and p53 purified from Hi-5 50 2+CA+U, and cells by Baculovirus infection (Schematically depicted in 2) assaying for the attachment of the ubiquitin moiety to FIG. 18). E2 Ubch5c was made as described above. Gst UA-2. In another preferred embodiment, UA-2 comprises a Mdm2 and His-p53 were made as described above for label. In yet another preferred embodiment, UA-2 comprises GST-Ubch5c and E3 His-ROC1/Cul1, respectively. a label. Materials and Methods 55 FIG.25 schematically depicts a preferred embodiment for The following procedures were used for assays measuring assaying for the attachment of ubiquitin moiety to a first the attachment of ubiquitin moiety to p53, and are illustrated ubiquitin agent that is a ubiquitin conjugating agent UCA schematically in FIG. 20. The wells of Nickel-substrate where the assay comprises: 96-well plates (Pierce Chemical) are blocked with 100 ul of 1) combining a second ubiquitin agent that is UAA+ 1% casein/phosphate buffered saline (PBS) for 1 hour at 60 UCA+CA+U, and room temperature, then washed with 200 ul of PBS 3 times. 2) assaying for the attachment of the ubiquitin moiety to To each well is added the following Flag-ubiquitin moiety UCA. (See above) reaction Solution: FIG. 26 schematically depicts a preferred embodiment for Final Concentration assaying for the attachment of ubiquitin moiety to ubiquitin 50 mM Tris pH 7.5 65 conjugating agent that is an E2 where the assay comprises: 5 mM MgCl 1) combining a ubiquitin activating agent that is an O.6 mM DTT E1+E2+CA+U, and US 6,919,184 B2 65 66 2) assaying for the attachment of the ubiquitin moiety to FIG.34 schematically depicts a preferred embodiment for E2. assaying for the attachment of ubiquitin moiety to a Second FIG.27 schematically depicts a preferred embodiment for ubiquitin agent (UA-2) that is attached to a Solid Support) assaying for the attachment of ubiquitin moiety to a first where the assay comprises: ubiquitin agent that is a ubiquitin ligating agent (ULA) 1) combining a first ubiquitin agent that is UAA+UA-2 where the assay comprises: (attached to a Solid Support)+CA+U, and 1) combining a second ubiquitin agent that is a ubiquitin 2) assaying for the attachment of the ubiquitin moiety to conjugating agent and comprising a ubiquitin moiety UCA UA-2. In another preferred embodiment, the Solid Support is U+ULA+CA; and a microtiter plate. In another preferred embodiment, the 2) assaying for the attachment of the ubiquitin moiety to Solid Support is a bead. In a preferred embodiment, UA-2 ULA. In another preferred embodiment, the ubiquitin moi comprises an Mdm2 protein. ety comprises a label. In yet another preferred embodiment, FIG.35 schematically depicts a preferred embodiment for ULA comprises a label. In a preferred embodiment the assaying for the attachment of ubiquitin moiety to a first ubiquitin ligating agent comprises an Mdm2 protein. ubiquitin agent that is a ubiquitin ligating agent (ULA) that FIG.28 schematically depicts a preferred embodiment for 15 is attached to a Solid Support where the assay comprises: assaying for the attachment of ubiquitin moiety to a a 1) combining a second ubiquitin agent that is a ubiquitin ubiquitin ligating agent that is an E3 where the assay conjugating agent UCA comprising a ubiquitin moiety+ comprises: ULA (attached to a Solid Support)+CA; and 1) combining a ubiquitin conjugating agent that is an E2 2) assaying for the attachment of the ubiquitin moiety to and comprising a ubiquitin moiety+E3+CA, and ULA. In a preferred embodiment the ubiquitin ligating 2) assaying for the attachment of the ubiquitin moiety to agent comprises an Mdm2. E3. In a preferred embodiment, the E3 is an Mdm2 protein. FIG. 36 schematically depicts a preferred embodiment for FIG.29 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a first assaying for the attachment of ubiquitin moiety to a ubiq ubiquitin agent (UA-1) that comprises a label where the uitin conjugating agent that is an E2 where the assay 25 assay comprises: comprises: 1) combining a UA-1 (plus label)+CA+U; and 1) combining a ubiquitin activating agent that is an E1 and 2) assaying for the attachment of the ubiquitin moiety to comprising a ubiquitin moiety+E2+CA, and UA-1. 2) assaying for the attachment of the ubiquitin moiety to FIG. 37 schematically depicts a preferred embodiment for E2. assaying for the attachment of ubiquitin moiety to a first FIG. 30 schematically depicts a preferred embodiment for ubiquitin agent that is a ubiquitin ligating agent (ULA) that assaying for the attachment of ubiquitin moiety to a third comprises a label where the assay comprises: ubiquitin agent (UA-3) where the assay comprises: 1) combining a second ubiquitin agent that is a ubiquitin 1) combining a ubiquitin activating agent that is an E1 and conjugating agent UCA comprising a ubiquitin moiety+ comprising a ubiquitin moiety+a ubiquitin conjugating 35 ULA (plus label)+CA; and agent that is an E2+UA-3+CA, and 2) assaying for the attachment of the ubiquitin moiety to 2) assaying for the attachment of the ubiquitin moiety to ULA. In a preferred embodiment the ubiquitin ligating UA-3. In a preferred embodiment, UA-3 comprises an agent comprises an Mdm2 protein. Mdm2 protein. FIG.38 schematically depicts a preferred embodiment for FIG.31 schematically depicts a preferred embodiment for 40 assaying for the attachment of ubiquitin moiety that com assaying for the attachment of ubiquitin moiety to a third prises a label, to a first ubiquitin agent (UA-1) where the ubiquitin agent that is a ubiquitin ligating agent (ULA) assay comprises: where the assay comprises: 1) combining a UA-1+CA+U (plus label); and 1) combining a ubiquitin activating agent that is an E1 and 2) assaying for the attachment of the ubiquitin moiety to comprising a ubiquitin moiety+a ubiquitin conjugating 45 UA-1. agent that is an E2+ULA+CA, and FIG. 39 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to assaying for the attachment of ubiquitin moiety that com ULA. In a preferred embodiment the ubiquitin ligating prises a label, to a second ubiquitin agent (UA-2) where the agent comprises an Mdm2 protein. assay comprises: FIG. 32 schematically depicts a preferred embodiment for 50 1) combining a first ubiquitin agent that is UAA+UA assaying for the attachment of ubiquitin moiety to a ubiq 2+CA+U (plus label); and uitin ligating agent that is an E3 where the assay comprises: 2) assaying for the attachment of the ubiquitin moiety to 1) combining an E1 comprising a ubiquitin moiety+an UA-2. E2+an E3+CA; and FIG. 40 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to 55 assaying for the attachment of ubiquitin moiety that com E3. In a preferred embodiment, the E3 is Mdm2. prises a label, to a first ubiquitin agent that is a ubiquitin FIG.33 schematically depicts a preferred embodiment for ligating agent (ULA) where the assay comprises: assaying for the attachment of ubiquitin moiety to a first 1) combining a second ubiquitin agent that is a ubiquitin ubiquitin agent (UA-1) that is attached to a Solid Support conjugating agent UCA comprising a ubiquitin moiety where the assay comprises: 60 (plus label)+ULA+CA; and 1) combining a UA-1 (that is attached to a Solid Support)+ 2) assaying for the attachment of the ubiquitin moiety to CA+U, and ULA. In a preferred embodiment the ubiquitin ligating 2) assaying for the attachment of the ubiquitin moiety to agent comprises an Mdm2 protein. UA-1. In another preferred embodiment UA-1 is a UAA. In FIG. 41 schematically depicts a preferred embodiment for another preferred embodiment, UAA is an E1. In another 65 assaying for the attachment of ubiquitin moiety to a first preferred embodiment, the Solid Support is a microtiter plate. ubiquitin agent that is a ubiquitin ligating agent (ULA) In another preferred embodiment, the Solid Support is a bead. which comprises a label where the assay comprises: US 6,919,184 B2 67 68 1) combining a second ubiquitin agent that is a ubiquitin 1) combining a first ubiquitin agent that is UAA+UA-2 conjugating agent UCA comprising a ubiquitin moiety+ (plus epitope tag)+CA+U, and ULA (plus label)+CA; and 2) assaying for the attachment of the ubiquitin moiety to 2) assaying for the attachment of the ubiquitin moiety to UA-2. ULA. In a preferred embodiment the ubiquitin ligating FIG. 48 schematically depicts a preferred embodiment for agent comprises an Mdm2 protein. assaying for the attachment of ubiquitin moiety to a Sub FIG. 42 schematically depicts a preferred embodiment for Strate molecule (S) where the assay comprises: assaying for the attachment of ubiquitin moiety to a Second 1) combining a first ubiquitin agent that is a ubiquitin ubiquitin agent (UA-2) that comprises a label where the ligating agent ULA+a Second ubiquitin agent+Substrate assay comprises: molecule+CA+U, and 1) combining a first ubiquitin agent that is UAA+UA-2 2) assaying for the attachment of the ubiquitin moiety to (plus label)+CA+U; and the substrate molecule. In a preferred embodiment the 2) assaying for the attachment of the ubiquitin moiety to ubiquitin ligating agent comprises an Mdm2 protein and the UA-2. In a preferred embodiment, UA-2 comprises an Substrate molecule comprises p53. Mdm2 protein. 15 FIG. 49 schematically depicts a preferred embodiment for FIG. 43 schematically depicts a preferred embodiment for assaying for the attachment of ubiquitin moiety to a Sub assaying for the attachment of ubiquitin moiety to a first Strate molecule (S) where the assay comprises: ubiquitin agent (UA-1) that comprises an attachment tag (or 1) combining a first ubiquitin agent that is a ubiquitin attachment moiety) where the assay comprises: ligating agent ULA+a Second ubiquitin agent that is a 1) combining a UA-1 (plus attachment tag)+CA+U, and ubiquitin conjugating agent and comprising a ubiquitin 2) assaying for the attachment of the ubiquitin moiety to moiety+Substrate molecule+CA, and UA-1. 2) assaying for the attachment of the ubiquitin moiety to FIG. 44 schematically depicts a preferred embodiment for the substrate molecule. In a preferred embodiment the assaying for the attachment of ubiquitin moiety to a first ubiquitin ligating agent comprises an Mdm2 protein and the ubiquitin agent that is a ubiquitin ligating agent (ULA) that 25 Substrate molecule comprises p53. comprises an attachment tag (or attachment moiety) where FIG.50 schematically depicts a preferred embodiment for the assay comprises: assaying for the attachment of ubiquitin moiety to a Sub 1) combining a second ubiquitin agent that is a ubiquitin Strate molecule (S) where the assay comprises: conjugating agent UCA comprising a ubiquitin moiety+ 1) combining a first ubiquitin agent that is a ubiquitin ULA (plus attachment tag)+CA; and ligating agent ULA+a Second ubiquitin agent+a third ubiq 2) assaying for the attachment of the ubiquitin moiety to uitin agent that is a ubiquitin activating agent--a ubiquitin ULA. In a preferred embodiment the ubiquitin ligating moiety comprising a first FRET tag-i-substrate molecule agent comprises an Mdm2 protein. comprising a Second FRET tag--CA, and FIG. 45 schematically depicts a preferred embodiment for 2) assaying for the attachment of the ubiquitin moiety to assaying for the attachment of ubiquitin moiety to a Second 35 the substrate molecule. In a preferred embodiment the ubiquitin agent (UA-2) that comprises an attachment tag (or ubiquitin ligating agent comprises an Mdm2 protein and the attachment moiety) where the assay comprises: Substrate molecule comprises p53. 1) combining a first ubiquitin agent that is UAA+UA-2 As depicted in FIG. 51, in a preferred embodiment, the E2 (plus attachment tag)+CA+U, and has the amino acid Sequence (FIG. 51A) and the nucleic acid 2) assaying for the attachment of the ubiquitin moiety to 40 sequence (FIG. 51B). UA-2. As depicted in FIG. 52, in a preferred embodiment, the E2 FIG. 46 schematically depicts a preferred embodiment for has the amino acid Sequence (FIG. 52A) and the nucleic acid assaying for the attachment of ubiquitin moiety to a first sequence (FIG. 52B1 and FIG. 52B2). ubiquitin agent (UA-1) that comprises an epitope tag (or As depicted in FIG. 53, in a preferred embodiment, the E2 epitope label) where the assay comprises: 45 has the amino acid sequence (FIG. 53A) and the nucleic acid 1) combining a UA-1 (plus epitope tag)+CA+U, and sequence (FIG. 53B1 and FIG. 53B2). 2) assaying for the attachment of the ubiquitin moiety to As depicted in FIG. 54, in a preferred embodiment, the E2 UA-1. has the amino acid Sequence (FIG. 54A) and the nucleic acid FIG. 47 schematically depicts a preferred embodiment for sequence (FIG. 54B). assaying for the attachment of ubiquitin moiety to a Second 50 As depicted in FIG.55, in a preferred embodiment, the E2 ubiquitin agent (UA-2) that comprises an epitope tag (or has the amino acid sequence (FIG.55A) and the nucleic acid epitope label) where the assay comprises: sequence (FIG. 55B).

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 27 SEQ ID NO 1 LENGTH 3177 TYPE DNA ORGANISM: Oryctolagus cuniculus

<400 SEQUENCE: 1

atgtc.cagot cqcc.gctgtc. caagaaacgt cqcgtgtcc g g gcc to atcc aaag.ccgggit 60

US 6,919,184 B2 71

-continued cc caagttctg gcgtoaaaat coacgtttct gaccaggagc tigcagagcgc caatgcttct 2460 gttgacgaca gcc gtttaga ggagctdaag gottacgctgc ctagoccc.ga caagctocct 252O ggatto aaga tigtaccc.cat tdactittgag aag gatgatg atagtaactt to acatggac 258O ttcattgttgg cc.gcatccala cct cogggcc gaaaactato acattcc.ccc to cag accgg 264 O caca agagca agctgattgc agg galagatc atcc.ca.gc.ca ttgcc acgac cacagcagot 27 OO gtogttggcc ttgttgttgtct ggagctgtac aaggtag toc agggacaccg acaccitcgac 276 O. toctacaaga atggitttcct caacctggcc ctg.ccgttitt toggtttcto tdaacctctg 282O gctgcaccac gtcaccagta citatalaccala gagtggacat totgg gatcg citttgaggitt 2880 Cagg gactgc agcc.caacgg to aggagatg accotcaaac aattic citcga citactittaag 2.940 acagag caca aattggagat taccatgctg. tcc cagggtg to tccatgct citattoctitc 3OOO tittatoccag ctg.cgaagct caaggaacgg ttggaccago C gatgacaga gattgtaagc 3060 Cgtgtgtcga agc gaaagct gggcc.gc.cac gtgcggg.cgc tiggtgcttga gctgtgctgc 312 O aacgacgaga gcggcgagga C gtcgaagtc. cccitacgtoc gatataccat cogittaa 3177

<210> SEQ ID NO 2 &2 11s LENGTH 1059 &212> TYPE PRT <213> ORGANISM: Oryctolagus cuniculus <400 SEQUENCE: 2 Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly Pro Asp 1 5 10 15 Pro Llys Pro Gly Ser Asn Cys Ser Pro Ala Glin Ser Val Leu Pro Glin 2O 25 30 Val Pro Ser Ala Pro Thr Asn Gly Met Ala Lys Asn Gly Ser Glu Ala 35 40 45 Asp Ile Asp Glu Gly Lieu. Tyr Ser Arg Glin Leu Tyr Val Lieu Gly His 50 55 60 Glu Ala Met Lys Arg Lieu Glin Thr Ser Ser Val Lieu Val Ser Gly Lieu 65 70 75 8O Arg Gly Lieu Gly Val Glu Ile Ala Lys Asn. Ile Ile Leu Gly Gly Val 85 90 95 Lys Ala Val Thr Lieu. His Asp Glin Gly. Thir Ala Glin Trp Ala Asp Lieu 100 105 110 Ser Ser Glin Phe Tyr Lieu Arg Glu Glu Asp Ile Gly Lys Asn Arg Ala 115 120 125 Glu Val Ser Glin Pro Arg Leu Ala Glu Leu Asn Ser Tyr Val Pro Val 130 135 1 4 0 Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu Ser Gly Phe Glin 145 15 O 155 160 Val Val Val Lieu. Thr Asn. Ser Pro Leu Glu Asp Gln Leu Arg Val Gly 1.65 170 175 Glu Phe Cys His Ser Arg Gly Ile Lys Lieu Val Val Ala Asp Thr Arg 18O 185 19 O Gly Lieu Phe Gly Glin Leu Phe Cys Asp Phe Gly Glu Glu Met Ile Leu 195 200 2O5 Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Thr Met Val Ser Met Val 210 215 220 Thr Lys Asp Asin Pro Gly Val Val Thr Cys Lieu. Asp Glu Ala Arg His 225 230 235 240 US 6,919,184 B2 73 74

-continued

Gly Phe Glu Ser Gly Asp Phe Wall Ser Phe Ser Glu Wall Glin Gly Met 245 250 255

Thr Glu Telu Asn Gly Asn Glin Pro Ile Glu Ile Lys Wall Telu Gly Pro 260 265 27 O

Thr Phe Ser Ile Cys Asp Thr Ser Asn Phe Ser Asp Ile Arg 275 280 285

Gly Gly Ile Wall Ser Glin Wall Lys Wall Pro Lys Ile Ser Phe 29 O 295

Ser Telu Ser Ala Ser Teu Ala Glu Pro Asp Phe Wall Met Thr Asp Phe 305 310 315 320

Ala Phe Ser Arg Pro Ala Glin Telu His Ile Gly Phe Glin Ala Telu 325 330 335

His Phe Cys Ala Glin His Ser Arg Pro Pro Arg Pro Arg Asn Glu 340 345 35 O

Glu Asp Ala Ala Glu Teu Wall Thr Telu Ala Arg Ala Wall Asn Ser 355 360 365

Ala Ser Ser Ala Wall Glin Glin Asp Ser Telu Asp Glu Asp Telu Ile Arg 370 375

Asn Telu Ala Phe Wall Ala Ala Gly Asp Telu Ala Pro Ile Asn Ala Phe 385 390 395 400

Ile Gly Gly Telu Ala Ala Glin Glu Wall Met Ala Ser Gly Lys 405 410 415

Phe Met Pro Ile Met Glin Trp Telu Tyr Phe Ala Teu Glu Telu 420 425 43 O

Pro Glu Asp Glu Ser Teu Thr Glu Asp Teu Pro Arg Glin 435 4 40 4 45

Asn Arg Gly Glin Wall Ala Wall Phe Gly Ser Asp Telu Glin Glu 450 455 460

Lys Telu Gly Glin Lys Phe Telu Wall Gly Ala Gly Ala Ile Gly 465 470 475 480

Glu Telu Telu Lys Asn Phe Ala Met Ile Gly Teu Gly Cys Gly Glu 485 490 495

Asn Gly Glu Ile Ile Wall Thr Met Asp Thr Ile Glu Lys Ser Asn 5 OO 505

Teu Asn Arg Glin Phe Teu Phe Arg Pro Trp Asp Wall Thr Telu 515 525

Ser Asp Thr Ala Ala Ala Ala Wall His Glin Met Asn Pro His Ile Arg 530 535 540

Wall Thr Ser His Glin Asn Arg Wall Gly Pro Asp Thr Glu Arg Ile Tyr 545 550 555 560

Asp Asp Asp Phe Phe Glin Thr Telu Asp Gly Wall Ala Asn Ala Telu Asp 565 570 575

Asn Wall Asp Ala Arg Met Met Asp Arg Arg Wall Tyr Arg 585 59 O

Pro Telu Telu Glu Ser Gly Thr Telu Gly Thr Gly Asn Wall Glin 595 600 605

Wall Wall Ile Pro Phe Teu Thr Glu Ser Tyr Ser Ser Ser Glin Asp Pro 610 615 62O

Pro Glu Lys Ser Ile Pro Ile Cys Thr Telu Lys Asn Phe Pro Asn Ala 625 630 635 640

Ile Glu His Thr Teu Glin Trp Ala Arg Asp Glu Phe Glu Gly Telu Phe 645 650 655

Glin Pro Ala Glu Asn Wall Asn Glin Tyr Teu Thr Asp Pro Phe US 6,919,184 B2 75

-continued

660 665 67 O Val Glu Arg Thr Lieu Arg Lieu Ala Gly Thr Glin Pro Leu Glu Val Lieu 675 680 685 Glu Ala Val Glin Arg Ser Lieu Val Lieu Gln Leu Pro Glin Ser Trp Ala 69 O. 695 7 OO Asp Cys Val Thr Trp Ala Cys His His Trp His Thr Glin Tyr Ser Asn 705 710 715 720 Asn. Ile Arg Glin Leu Lieu. His Asn. Phe Pro Pro Asp Gln Lieu. Thir Ser 725 730 735 Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu 740 745 750 Thr Phe Asp Val Ser Asn Pro Leu. His Leu Asp Tyr Val Met Ala Ala 755 760 765 Ala Asn Lieu Phe Ala Glin Thr Tyr Gly Lieu Ala Gly Ser Glin Asp Arg 770 775 78O

Ala Ala Wall Ala Thr Lieu. Leu Glin Ser Wall Glin Wall Pro Glu Phe Thr 785 790 795 8OO Pro Lys Ser Gly Val Lys Ile His Val Ser Asp Glin Glu Lieu Glin Ser 805 810 815 Ala Asn Ala Ser Val Asp Asp Ser Arg Lieu Glu Glu Lieu Lys Ala Thr 820 825 83O Leu Pro Ser Pro Asp Lys Lieu Pro Gly Phe Lys Met Tyr Pro Ile Asp 835 840 845 Phe Glu Lys Asp Asp Asp Ser Asn Phe His Met Asp Phe Ile Val Ala 85 O 855 860 Ala Ser Asn Lieu Arg Ala Glu Asn Tyr Asp Ile Pro Pro Ala Asp Arg 865 870 875 88O His Lys Ser Lys Lieu. Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala Thr 885 890 895 Thir Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys Val 9 OO 905 910 Val Glin Gly His Arg His Leu Asp Ser Tyr Lys Asn Gly Phe Lieu. Asn 915 920 925 Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro Arg 930 935 940 His Glin Tyr Tyr Asn Glin Glu Trp Thr Leu Trp Asp Arg Phe Glu Val 945 950 955 96.O Glin Gly Lieu Glin Pro Asn Gly Glu Glu Met Thr Lieu Lys Glin Phe Lieu 965 970 975 Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Ile Thr Met Leu Ser Glin 98O 985 99 O Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu Lys 995 10 OO 1005 Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Val Ser Arg Val Ser 1010 1015 1020 Lys Arg Lys Lieu Gly Arg His Val Arg Ala Lieu Val Lieu Glu Lieu 1025 1030 1035 Cys Cys Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pro Tyr Val 1040 1045 105 O Arg Tyr Thr Ile Arg Glx 1055

<210> SEQ ID NO 3 US 6,919,184 B2 77 78

-contin ued

<211& LENGTH 4 44 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 3 atgg.cgctga aacggattaa taaggaactt agtgatttgg cc.cgtgaccc to cago.acaa 60 tgttctgcag gtocagttgg ggatgatatg titt cattggc aagccacaat tatgg gacct 120 aatgacagoc catato aagg cggtgtatto tttittgacaa titcatttitcc. tacagacitac 18O cc cittcaaac caccita aggt tgcatttaca acaagaattit atcatccaaa. tattaacagt 240 aatggcagoa tttgttctoga tattotaaga to acagtggit cgcctgctitt aacaattitct aaagttctitt tatccatttg ttcactgcta tgtgatccala accoagatga cc.cccitagtg 360 ccagagattg cacggatcta taaaacagac agagataagt acaacagaat atctogggaa 420 tggacticaga agtatgcc at gtga 444

<210> SEQ ID NO 4 &2 11s LENGTH 148 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 4 Met Ala Leu Lys Arg Il e Asn Lys Glu Lieu Ser Asp Leu Ala Arg Asp 1 5 10 15

Pro Pro Ala Glin Cys Ser Ala Gly Pro Val Gly Asp Asp Met Phe His 25 30

Trp Glin Ala Thr Ile Met Gly Pro Asn Asp Ser Pro Tyr Glin Gly Gly 35 40 45

Wall Phe Phe Leu. Thir Il e His Phe Pro Thr Asp Tyr Pro Phe Lys Pro 50 55 60

Pro Llys Val Ala Phe Thr Thr Arg Ile Tyr His Pro Asn. Ile Asn. Ser 65 70 75 8O

Asn Gly Ser Ile Cys Lieu. Asp Ile Leu Arg Ser Gln Trp Ser Pro Ala 85 90 95

Leu. Thir Ile Ser Lys Val Lieu Lieu Ser Ile Cys Ser Leu Lleu. Cys Asp 100 105 110

Pro Asn. Pro Asp Asp Pro Leu Val Pro Glu Ile Ala Arg Ile Tyr Lys 115 120 125 Thr Asp Arg Asp Lys Tyr Asn Arg Ile Ser Arg Glu Trp Thr Gln Lys 130 135 1 4 0

Tyr Ala Met Glx 145

<210 SEQ ID NO 5 &2 11s LENGTH 84 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 5 Met Lys Wall Lys Ile Lys Cys Trp Asn Gly Val Ala Thr Trp Leu Trp 1 5 10 15

Val Ala Asn Asp Glu Asn. Cys Gly Ile Cys Arg Met Ala Phe Asn Gly 25 30

Cys Cys Pro Asp Cys Lys Val Pro Gly Asp Asp Cys Pro Leu Val Trp 35 40 45 Gly Glin Cys Ser His Cys Phe His Met His Cys Ile Leu Lys Trp Lieu 50 55 60 US 6,919, 184 B2 79 80

-contin ued

His Ala Glin Glin Val Glin Gln His Cys Pro Met Cys Arg Glin Thr Trp 65 70 75

Lys Phe Lys Glu

<210> SEQ ID NO 6 &2 11s LENGTH 108 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 6 Met Ala Ala Ala Met Asp Val Asp Thr Pro Ser Gly Thr Asn Ser Gly 1 5 10 15

Ala Gly Lys Lys Arg Ph e Glu Wall Lys Lys Trp Asn Ala Wall Ala Leu 2O 25 30 Trp Ala Trp Asp Ile Wa l Val Asp Asn. Cys Ala Ile Cys Arg Asn His 35 40 45

Ile Met Asp Lieu. Cys Il e Glu Cys Glin Ala Asn Glin Ala Ser Ala Thr 50 55 60

Ser Glu Glu Cys Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His 65 70 75 8O

Phe His Cys Ile Ser Arg Trp Lieu Lys Thr Arg Glin Val Cys Pro Leu 85 90 95

Asp Asn Arg Glu Trp Gl u Phe Glin Lys Tyr Gly His 100 105

<210 SEQ ID NO 7 <211& LENGTH 342 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 7 atggc.cgacg tggalaga.cgg agaggaalacc tgcgc.cctgg ccitct cactic Cgggagctca 60 ggctcaacgt. Cgggaggcga. caagatgttc toccitcaaga agtggaacco ggtggcCatg 120 tggagctggg acgtggagtg cgatacgtgc gcc atctgca gggto: Caggit gatggatgcc 18O tgtcttagat gtoaa.gctga aaaaaaaa. gaggactgtg ttgttggtotg gggagaatgt 240 aatcattcct to cacaactg citgcatgtcc Ctgtgggtga aacagaacaa togctg.ccct citctgc.ca.gc aggactgggt ggtocaaaga atcggcaaat ga 342

<210 SEQ ID NO 8 &2 11s LENGTH 113 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 8 Met Ala Asp Wall Glu As p Gly Glu Glu Thr Cys Ala Leu Ala Ser His 1 5 10 15

Ser Gly Ser Ser Gly Ser Thr Ser Gly Gly Asp Lys Met Phe Ser Lieu 25 30

Lys Lys Trp Asin Pro Wall Ala Met Trp Ser Trp Asp Val Glu 35 40 45 Thr Cys Ala Ile Cys Arg Val Glin Val Met Asp Ala Cys Lieu Arg Cys 50 55 60

Glin Ala Glu Asn Lys Gl in Glu Asp Cys Val Val Val Trp Gly Glu Cys 65 70 75 8O

Asn His Ser Phe His As in Cys Cys Met Ser Lieu Trp Val Lys Glin Asn US 6,919,184 B2 81 82

-continued

85 90 95 Asn Arg Cys Pro Leu. Cys Glin Glin Asp Trp Val Val Glin Arg Ile Gly 100 105 110 Lys

<210 SEQ ID NO 9 &2 11s LENGTH 2.343 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 9 atgg.cgacgt. ctaatctgtt aaagaataaa ggttctottc agtttgaaga caaatgg gat 60 tittatgc.gc.c cgattgttitt gaagcttitta cgc.caggaat citgttacaaa acagcagtgg 120 tittgatctgt titt.cggatgt gcatgcagtc tgtc.tttggg atgataaagg cc.ca.gcaaaa 18O attcatcagg citttaaaaga agatattott gagtttatta agCaggCaca ggCacgagta 240 citgagc catc aagatgatac ggctttgcta aaag catata ttgttgaatg gc gaaagttc tittacacaat gtgatattitt accalaalacct ttttgttcaac tagagattac tittaatgggit 360 aaac agggca gcaataaaaa atcaaatgtg gaaga cagta ttgttcgaaa gctitatgctt 420 gata catgga atgagt caat cittittcaaac ataaaaaa.ca gactic caaga tagtgcaatg 480 aagctggtac atgctgagag attgggagaa gcttittgatt citcagotggit tattggagta 540 agagaatcct atgtta acct ttgttctaat cct gaggata aacttcaaat ttatagg gac 600 aattittgaga agg catacitt ggattcaa.ca gagagattitt atagaacaca agcaccotcg 660 tatttacaac caaatggtgt acagaattat atgaaatatg cagatgctaa attaaaagaa 720 gaagaaaaac gag cactacg ttatttagaa acalagacgag aatgtaactic cgttgaagca citcatggaat gctgttgtaaa tgcc.ctggtg acatcattta aagagacitat cittagct gag 840 tgccaaggca tgatcaagag aaatgaaact gaaaaattac atttaatgtt ttcattgatg 9 OO gacaaagttc citaatggitat agagccaatg ttgaaag act tggaggalaca tat cattagt 96.O gctggCCtgg cagatatggit agcagotgct gaalactatta citactgactic tgagaaatac O20 gttgag cagt tact tacact atttaataga tittagtaaac togtoaaaga agcttittcaa gatgatccac gatttcttac tgcaa.gagat aaggc gtata aag cagttgt taatgatgct 14 O accatattta aacttgaatt acctttgaag Cagaaggggg tgggattaaa aactcagoct 200 gaatcaaaat gcc ctdagct gcttgccaat tactgttgaca tgttgctaag aaaaa CaCCa 260 ttaa.gcaaaa alactalacctic tgaagagatt gaa.gcaaag.c ttaaagaagt gct cittggta 320 cittaagtatg tacagaacaa agatgtttitt atgaggitatc ataaagcto a tittgacacga cgtottatat tag acatcto tgc.cgatagt gaaattgaag aaaacatggit agagtggcta 4 40 agagaagttg gtatgc.ca.gc ggattatgta aacaa.gcttg citagaatgtt to aggacata 5 OO aaagtatctg aagatttgaa ccaagcttitt aaggaaatgc acaaaaataa. taaattggca 560 ttaccagotg attcagittaa tataaaaatt citgaatgctg gc gcc togto aagaagttct gagaaagttct ttgttctdact toctactgaa citggagg act tgataccgga agtagaagaa 680 ttctacaaaa. aaaatcatag tggtagaaaa ttacattggc atcatctoat gtoaaatgga 740 attatalacat ttaagaatga agttggtoaa tatgatttgg agg talaccac gtttcagotc 800 gctgtattgt ttgcatggaa ccaaagaccc agagagaaaa tdagctittga aaatcttaag 860 cittgcaactg aactccctga tgctgaactt aggaggacitt tatggtottt agtag ctittc 920 US 6,919,184 B2 83 84

-continued coaaaactica aacggcaagt tttitttgtat gacccitcaag tdaacticacc caaag actitt 1980 acagaaggta coctottctic agtgaaccag gagttcagtt taataaaaaa. tgcaaaggitt 20 40

Cagaaaaggg gtaaaatcaa cittgattgga cgtttgcago to act acaga aaggatgaga 2100 gaagaa.gaga atgaaggaat agttcaacta c gaatactaa galaccCagga agctatoata 216 O caaataatga aaatgagaaa gaaaattagt aatgcto agc tgcag actoga attagtagaa 2220 attittgaaaa acatgttctt gcc acaaaag aaaatgataa aagagcaaat agagtggcta 228O atagag caca aatacatcag alagagatgaa totgatatoa acactitt cat atatatggca 234. O taa 2343

<210> SEQ ID NO 10 &2 11s LENGTH 861 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 10 Met Arg Ser Phe Ala Trp Gly Ser Ser Gly Asp His Wall Gly Asp Lys 1 5 10 15

Ser Glu Glu Ala Pro Gl y Ala Trp Asp Glu Val Ser Ala Wall Gly Ala 2O 25 30

Lieu. Leu Glin Arg Pro Pro His Pro Gly Ala Gly Pro Thr Gly Pro Gly 35 40 45

Pro Trp Trp Glu Lieu Arg Pro Pro Wall Lys Ala Trp Pro Gly Arg Glu 50 55 60

Arg His Glu Phe Ser Arg Arg Lieu Val Ser Arg Glu Ser Lieu Lys 65 70 75 8O

Asn Met Ala Thir Ser As n Telu Telu Lys Asn Lys Gly Ser Telu Glin Phe 85 90 95

Glu Asp Lys Trp Asp Phe Met Arg Pro Ile Wall Teu Telu Leu Arg 100 105 110

Glin Glu Ser Val Thr Lys Glin Glin Trp Phe Asp Teu Phe Ser Asp Wall 115 120 125

His Ala Wall Cys Lieu Trp Asp Asp Lys Gly Pro Ala Ile His Glin 130 135 1 4 0

Ala Lieu Lys Glu Asp Il e Leu Glu Phe Ile Lys Glin Ala Glin Ala Arg 145 15 O 155 160

Wall Leu Ser His Glin As p Asp Thr Ala Leu Lieu Ala Ile Wall 1.65 170 175

Glu Trp Arg Lys Phe Phe Thr Glin Cys Asp Ile Teu Pro Lys Pro Phe 18O 185 19 O

Cys Glin Lieu Glu Ile Thr Leu Met Gly Lys Glin Gly Ser Asn 195 200

Ser Asn. Wall Glu Asp Ser Ile Val Arg Lys Lieu Met Teu Asp Thir Trp 210 215 220

Asn Glu Ser Ile Phe Ser Asn. Ile Lys Asn Arg Teu Glin Asp Ser Ala 225 23 O 235 240

Met Lys Lieu Wal His Al a Glu Arg Leu Gly Glu Ala Phe Asp Ser Glin 245 250 255

Leu Wall Ile Gly Val Arg Glu Ser Tyr Val Asn Teu Ser Asn. Pro 260 265 27 O

Glu Asp Lys Leu Glin Il e Tyr Arg Asp Asn. Phe Glu Lys Ala Tyr Leu 275 280 285

Asp Ser Thr Glu Arg Phe Tyr Arg Thr Glin Ala Pro Ser Leu Glin US 6,919,184 B2 85 86

-continued

29 O 295

Pro Asn Gly Wall Glin Asn Met Ala Asp Ala Telu Lys 305 310 315 320

Glu Glu Glu Lys Arg Ala Teu Telu Glu Thr Arg Arg Glu 325 330 335

Asn Ser Wall Glu Ala Teu Met Glu Cys Wall Asn Ala Telu Wall Thr 340 345 35 O

Ser Phe Lys Glu Thr Ile Teu Ala Glu Glin Gly Met Ile Arg 355 360 365

Asn Glu Thr Glu Lys Teu His Telu Met Phe Ser Teu Met Asp Wall 370 375

Pro Asn Gly Ile Glu Pro Met Telu Asp Teu Glu Glu His Ile Ile 385 390 395 400

Ser Ala Gly Telu Ala Asp Met Wall Ala Ala Ala Glu Thr Ile Thr Thr 405 410 415

Asp Ser Glu Lys Tyr Wall Glu Glin Telu Telu Thr Teu Phe Asn Arg Phe 420 425 43 O

Ser Telu Wall Glu Ala Phe Glin Asp Asp Pro Arg Phe Telu Thr 435 4 40 4 45

Ala Arg Asp Ala Lys Ala Wall Wall Asn Asp Ala Thr Ile Phe 450 455 460

Lys Telu Glu Telu Pro Teu Glin Gly Wall Gly Teu Thr Glin 465 470 475 480

Pro Glu Ser Cys Pro Glu Telu Telu Ala Asn Asp Met Telu 485 490 495

Teu Arg Thr Pro Teu Ser Lys Lys Telu Thr Ser Glu Glu Ile Glu 5 OO 505

Ala Telu Glu Wall Teu Telu Wall Telu Wall Glin Asn 515 525

Asp Wall Phe Met Arg His Lys Ala His Teu Thr Arg Arg Telu Ile 530 535 540

Teu Asp Ile Ser Ala Asp Ser Glu Ile Glu Glu Asn Met Wall Glu Trp 545 550 555 560

Teu Arg Glu Wall Gly Met Pro Ala Asp Tyr Wall Asn Telu Ala Arg 565 570 575

Met Phe Glin Asp Ile Wall Ser Glu Asp Teu Asn Glin Ala Phe 585 59 O

Glu Met His Asn Asn Telu Ala Telu Pro Ala Asp Ser Wall Asn 595 600 605

Ile Lys Ile Telu Asn Ala Gly Ala Trp Ser Arg Ser Ser Glu Wall 610 615

Phe Wall Ser Telu Pro Thr Glu Telu Glu Asp Teu Ile Pro Glu Wall Glu 625 630 635 640

Glu Phe Lys Asn His Ser Gly Arg Teu His Trp His His 645 650 655

Teu Met Ser Asn Gly Ile Ile Thr Phe Lys Asn Glu Wall Gly Glin 660 665 67 O

Asp Telu Glu Wall Thr Thr Phe Glin Telu Ala Wall Teu Phe Ala Trp Asn 675 680 685

Glin Arg Pro Glu Lys Ile Ser Phe Glu Asn Teu Telu Ala Thr 69 O. 695 7 OO

Glu Telu Pro Asp Ala Glu Teu Arg Thr Teu Trp Ser Telu Wall Ala 705 710 715 720