BACTERIAL SYSTEMS FOR ANALYZING UBIQUITYLATED POLYPEPTIDES

Abstract

Bacterial systems for analyzing ubiquitination of proteins is disclosed herein. Kits for analyzing the ubiquitination and methods for carrying out the analysis are also disclosed.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 16/322,956, filed on Feb. 3, 2019, which is a National Phase of PCT Patent Application No. PCT/IL2017/050876 having International Filing Date of Aug. 8, 2017, which claims the benefit of priority under 35 USC .sctn. 119(e) of U.S. Provisional Patent Application No. 62/371,881 filed on Aug. 8, 2016.

[0002] The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

[0003] The ASCII file, entitled 86367SequenceListing.txt, created on Mar. 22, 2021 comprising 264,259 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.

FIELD AND BACKGROUND OF THE INVENTION

[0004] The present invention, in some embodiments thereof, relates to bacterial systems for analyzing ubiquitylated polypeptides.

[0005] Ubiquitin (Ub) plays a pivotal role in numerous aspects of cellular processes. Therefore, aberrations in the Ub system are involved in a large number of pathologies, including various forms of cancer such as breast and colon cancer, neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and infectious diseases such as HIV and Ebola. Consequently, there is a critical need for a detailed understanding of the Ub system. Although there have been significant advances in understanding the ubiquitination process, much less is known about the downstream processes. These include substrate recognition by specific enzymatic interactions in the Ub system, and specific interactions between these enzymes and their substrates. In humans, for example, there are 2 E1 Ub-activating enzymes, 34 Ub-conjugating enzymes and more than 600 E3 Ub-ligases. These enzymes work on presumably several thousands of protein substrates, where specificity is mainly achieved by the E2:E3 and E3:Substrates interactions.

[0006] Another factor which impedes the researchers' efforts to fully characterize Ub cascades is the presence of deubiquitinating enzymes (DUBs) which rapidly reverse the ubiquitination signal. The half-life time of ubiquitylated proteins is thus extremely short. Specifically, it has been shown that about 100 DUBs that exist reverse the modification in a highly specific manner.

[0007] Background art includes Keren-Kaplan et al., The EMBO Journal (2012) 31, 378-390 and Su et al., J Immunol 2006; 177; 7559-7566.

SUMMARY OF THE INVENTION

[0008] According to an aspect of some embodiments of the present invention there is provided a method of identifying an agent which regulates the activity or amount of a ubiquitinating enzyme or deubiquitinating enzyme comprising:

[0009] (a) contacting a bacterial cell with the agent, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the ubiquitination level of a substrate; and

[0010] (b) measuring the level or the rate of accumulation of the detectable or selectable signal, wherein a change in the level as compared to the level in the absence of the agent, is indicative of an agent which regulates the activity or amount of the ubiquitinating or deubiquitinating enzyme.

[0011] According to an aspect of some embodiments of the present invention there is provided a method of determining whether an enzyme is capable of ubiquitinating a test substrate, the method comprising

[0012] (a) expressing the enzyme in a bacterial cell;

[0013] (b) expressing ubiquitin in the bacterial cell, wherein the ubiquitin is attached to a first polypeptide fragment;

[0014] (c) expressing the test substrate in the bacterial cell, wherein the substrate is attached to a second polypeptide fragment, wherein the first polypeptide fragment associates with the second polypeptide fragment to generate a reporter polypeptide on ubiquitination of the test substrate; and

[0015] (d) analyzing for the presence of the reporter polypeptide in the bacterial cell, wherein a presence of the reporter polypeptide is indicative that the enzyme is capable of ubiquitinating the test substrate.

[0016] According to an aspect of some embodiments of the present invention there is provided a method of identifying a polypeptide substrate for a ubiquitinating enzyme, the method comprising:

[0017] (a) expressing a plurality of candidate polypeptide substrates in a bacterial cell population, wherein each of the candidate polypeptide substrates is attached to an identical first polypeptide fragment;

[0018] (b) expressing the ubiquitinating enzyme in the bacterial cell population;

[0019] (c) expressing ubiquitin in the bacterial cell population, wherein the ubiquitin is attached to a second polypeptide fragment, wherein the second polypeptide fragment associates with the first polypeptide fragment to generate a reporter polypeptide on ubiquitination of the substrate; and

[0020] (d) analyzing in bacterial colonies of the bacterial cell population for a presence or absence of the reporter polypeptide, wherein a presence of the reporter polypeptide is indicative of expression of a substrate for the ubiquitinating enzyme.

[0021] According to an aspect of some embodiments of the present invention there is provided a kit comprising:

[0022] (i) a first polynucleotide which encodes a first polypeptide fragment which is operably linked to a bacterial regulatory sequence, and a cloning site, wherein a position of the cloning site is selected such that upon insertion of a sequence which encodes a test polypeptide into the cloning site, following expression in a bacterial cell, a fusion protein is generated which comprises the test polypeptide in frame with the first polypeptide fragment; and

[0023] (ii) a second polynucleotide comprising a second nucleic acid sequence encoding a second polypeptide fragment which is attached to ubiquitin, the second nucleic acid sequence being operably linked to a bacterial regulatory sequence, wherein the first polypeptide fragment associates with the second polypeptide fragment to generate a reporter polypeptide dependent on ubiquitination of the test polypeptide.

[0024] According to one embodiment, the ubiquitinating enzyme is a ubiquitin E3-ligase.

[0025] According to another embodiment, the ubiquitin E3-ligase is selected from the group consisting of Siah2, Smurf1,MDM2, BRCA1, PARKIN, UBE3A, TRIM5, NEDD4, UBR5 and Huwe1.

[0026] According to one embodiment, the ubiquitin E3-ligase is selected from the group consisting of Siah2, PARKIN, Smurf1, MDM2, BRCA1, MURF1, TRIM32 ITCH, UBE3B and UBE3A.

[0027] According to one embodiment, the substrate is selected from the group consisting of PHD3, SPROUTY2, Mitofusin 1, 2, MIRO, NEMO, SMADs, T.beta.R-I, P53, S5A, HHR23, EPHEXIN5, ARC, PPAR.alpha., cyclin-B, Cdc25C, Calmodulin.

[0028] According to one embodiment, the regulates comprises downregulates.

[0029] According to one embodiment, the regulates comprises upregulates.

[0030] According to one embodiment, the ubiquitinating enzyme further comprises an E1 ligase and an E2 ligase.

[0031] According to one embodiment, the bacterial cell expresses:

[0032] (a) the ubiquitinating enzyme or the deubiquitinating enzyme;

[0033] (b) ubiquitin attached to a first polypeptide fragment; and

[0034] (c) the substrate attached to a second polypeptide fragment, wherein the first polypeptide fragment associates with the second polypeptide fragment to generate a reporter polypeptide on ubiquitination of the substrate.

[0035] According to one embodiment, the reporter polypeptide comprises a selectable polypeptide.

[0036] According to one embodiment, the selectable polypeptide is a split antibiotic resistance polypeptide.

[0037] According to one embodiment, the antibiotic resistance polypeptide is DHFR or B lactamase.

[0038] According to one embodiment, the first polypeptide fragment is attached to the ubiquitin via a linker.

[0039] According to one embodiment, the second polypeptide fragment is attached to the substrate via a linker.

[0040] According to one embodiment, the reporter polypeptide is an optically detectable signal.

[0041] According to one embodiment, the detectable polypeptide is selected from the group consisting of a split fluorescent polypeptide, a split luminescent polypeptide and a split phosphorescent polypeptide.

[0042] According to one embodiment, the analyzing is effected by bimolecular complementation of an antibiotic resistance protein.

[0043] According to one embodiment, the method further comprises expressing all the enzymes of the ubiquitinating enzyme cascade of the enzyme.

[0044] According to one embodiment, the reporter polypeptide is a detectable polypeptide or a selectable polypeptide.

[0045] According to one embodiment, the enzyme is selected from the group consisting of E3 ligase, ubiquitin E1-activating enzyme and ubiquitin E2 conjugating enzyme.

[0046] According to one embodiment, the test substrate comprises an E3 ligase or Rpn10.

[0047] According to one embodiment, the enzyme is a ubiquitin E1-activating enzyme, the method further comprises expressing in the bacterial cell a ubiquitin E2 conjugating enzyme and ubiquitin E3-ligase.

[0048] According to one embodiment, the selectable polypeptide is a split antibiotic resistance polypeptide.

[0049] According to one embodiment, the antibiotic resistance polypeptide is DHFR or B lactamase.

[0050] According to one embodiment, the first polypeptide fragment is attached to the ubiquitin via a linker.

[0051] According to one embodiment, the second polypeptide fragment is attached to the substrate via a linker.

[0052] According to one embodiment, the detectable polypeptide is an optically detectable signal.

[0053] According to one embodiment, the detectable polypeptide is selected from the group consisting of a split fluorescent polypeptide, a split luminescent polypeptide and a split phosphorescent polypeptide.

[0054] According to one embodiment, the analyzing is effected by bimolecular complementation of an antibiotic resistance protein.

[0055] According to one embodiment, the first and the second polynucleotide comprise a bacterial origin of replication.

[0056] According to one embodiment, the reporter polypeptide is a selectable polypeptide.

[0057] According to one embodiment, the reporter polypeptide comprises a selection or detectable polypeptide.

[0058] According to one embodiment, the kit further comprises a third polynucleotide which encodes at least one ubiquitinating enzyme.

[0059] According to one embodiment, the first polynucleotide and/or the second polynucleotide comprises a sequence which encodes at least one ubiquitinating enzyme.

[0060] According to one embodiment, the at least one ubiquitinating enzyme comprises ubiquitin E1-activating enzyme or ubiquitin E2-conjugating enzyme.

[0061] According to one embodiment, the at least one ubiquitinating enzyme comprises ubiquitin E1-activating enzyme and ubiquitin E2-conjugating enzyme.

[0062] According to one embodiment, the at least one ubiquitinating enzyme comprises E3 ligase.

[0063] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0064] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0065] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0066] In the drawings:

[0067] FIGS. 1A-1D--Bacterial genetics approach for selection of ubiquitylated proteins.

[0068] (A) A scheme demonstrates the constructed system for selection in bacteria. The ubiquitination apparatus is expressed from two compatible plasmids each harbors different antibiotic resistance and origin of replication, facilitating co-transformation and selection of the vectors regardless of ubiquitination. pND-Ub denotes the N-terminal fragment of DHFR fused to Ub. pCD-Sub denotes the C-terminal fragment of DHFR fused to a ubiquitination substrate. A complete system confers antibiotic resistance and bacterial growth in the presence of trimethoprim (TRIM). Red cross represents bacteria that express an incomplete system and therefore are not resistant to the selective media. (B) Demonstrates the system functionality. Antibiotic resistance (10 .mu.gr/ml TRIM) is attained only when a complete ubiquitination cascade of Vps9 including wheat Uba1, yeast Ubc4, and yeast Rsp5 are co-expressed with split DHRF fragments fused to Ub and Vps9. (C) Shows that cognate E3-ligase (Rsp5) is necessary for Vps9 ubiquitination and antibiotic resistance. (D) Non-covalent interaction between Vps9 and Ub does not confer resistance for TRIM concentrations above 0.1 .mu.g/ml. Ubiquitination--refers to complete ubiquitination cascade; binding--refers to incomplete ubiquitination cascade (.DELTA.E1,.DELTA.E2).

[0069] FIGS. 2A-2G--Identification and characterization of NleG6-3 as E3-ligase.

[0070] (A) Bacteria that co-express cDHFR fusion to the E3-ligases Rsp5 (representative of the HECT family) with its cognate ubiquitination apparatus including Ubc4 is labeled `complete`. Deletions of E1, E2 or Ub are indicated. (B) Bacteria that co-express cDHFR fusion to the E3-ligases Siah2 (representative of the RING family) with its cognate ubiquitination apparatus including UbcH5a is labeled `complete`. Deletions of E1, E2, Ub or substitution of the UbcH5a with Cdc34 are indicated. The right panel shows Siah2 inhibition by menadione. (C) Shows sequence alignment derived from PSI-BLAST search using human CHIP as probe against the EHEC proteome. ECs3488 CSLYDKDTLVQLVETGGAHPLSREPITESMIMR--SEQ ID NO: 1; ECs2156 CTLFDAAAFSRLVGEGLPHPLTREPITASIIVK--SEQ ID NO: 2; HS_CHIP ITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIP--SEQ ID NO: 3. The full length sequence of the NleG6-3 ligase is as set forth by SEQ ID NO: 4.

[0071] (D) Yeast E2s scan for the E3-ligase ECs3488 (NleG6-3). (E) Self-ubiquitination of NleG6-3 by human UbcH5b/c. (F) Shows homology based model of NleG6-3 (orang) super imposed on the structure of EHEC NleG2-3 (blue) and Human CHIP:Ubch5b complex grey and magenta (respectively). Zooming into the predicted interface with highlighted critical binding residues. (G) Employment of the selection system for mutational analysis of the predicted E2:E3 interface.

[0072] FIGS. 3A-3I--The bacterial selection system facilitates the identification and characterization of Ub-receptors. UBDs promote self-ubiquitination and therefore can be detected by the bacterial selection system. The cDHFR was fused to various UBDs in the pCD-Sub, and co-expressed in the selection system without E3-ligase. (A) Ubiquitination promoted by Hrs was used to validate the system performance. (B) Structure of the Hrs dsUIM:Ub complex (PDB code 2D3G) shows the interacting residues. (C) Western blot analysis demonstrates the formation of a covalent bond between Ub and GST-dsUIM fusion. His.sub.6-Ub was co-expressed with E1 and Ubc4 along with GST-dsUIM. The protein was purified on GSH beads and subjected for western blot with anti GST (red) and anti-Histag (green). The yellow band that migrated at the molecular size of the ubiquitylated protein was detected by both antibodies. (D) The E1 inhibitor PYR41 attenuates bacterial growth only in the non-permissive conditions. Bacteria were grown in 96-well plates supplemented with PYR41 and/or TRIM as indicated. The relative growth rates at the log phase are shown with standard deviation (n=9). (E-H) Show the growth phenotypes of assorted UBDs in the selection system. e. Yeast Rpn10; f. Human STAM1-UIM; g. Human Alix-V domain; h. Yeast Hse1-VHS domain. (I) The binding of Ub to the VHS domains of human STAM1, GGA1 and GGA2 were examined in the selection system. Critical STAM1-VHS residues known to bind Ub were mutated and the growth phenotype was tested.

[0073] FIGS. 4A-4J--Structural insight into a predicted ENTH:Ub interface.

[0074] (A-B) The selection system identified Epsin1-ENTH domains from yeast (Saccharomyces cerevisiae) Ent1 and from zebrafish (Danio rerio) Epn1 function as UBDs. Co-expression of these ENTH domains with the ubiquitination apparatus in the selection system conferred antibiotic resistance and promoted bacterial growth under non-permissive conditions. Structures of the zebrafish and yeast Epsinl ENTH domains are presented. (C-D) Show the 2mFo-DFc sigma-A maps of the fish and the yeast proteins to 2.2 and 1.8.sigma. (respectively) of the final refined models. (E) The superimposition of the fish and the yeast ENTH structures on top of the STAM1-VHS:Ub complex (pdb code 3LDZ). Average C.alpha. RMSD are 1.5 .ANG. and 1.7 .ANG., respectively. The models were energetically minimized and carefully inspected. (F) Zoom in into crystal structure of the VHS:Ub interface (3LDZ). (G-H) Model of the ENTH:Ub complexes. (I) and (J) show zoom in into the interface of the model complexes.

[0075] FIGS. 5A-5I--Characterization of the ENTH:Ub binding interface.

[0076] Structure-based mutants at the predicted ENTH:Ub interface were constructed and characterized. (A) Growth phenotypes of the zebrafish ENTH and Ub mutants in the selection system. (B) Growth curves of Ub wild type and mutants derived from the time-lapse scanning of the spots (density was analyzed by Fiji). (C) As shown in b, but for ENTH mutants. (D) As shown in b and c but for reciprocal mutations. (E) Growth phenotypes of the yeast ENTH and Ub mutants in the selection system. (F) As shown in b, but for yeast ENTH and Ub mutants. (G) The ubiquitination yield for wild-type and indicated mutants of the yeast His.sub.6-MBP-ENTH were evaluated. The apo and ubiquitylated proteins were purified on amylose beads as described.sup.17, resolved on SDS-PAGE and detected by western blot with anti His-tag antibody. (H) Quantification of the ubiquitination yields shown in (G). The ubiquitylated/apo ratio is presented as a percentage of the wild-type ratio. Values were averaged from four independent experiments, and standard deviation error bars are presented. (I) Surface Plasmon Resonance (SPR) analysis of the yeast ENTH:Ub binding affinity of wild-type and mutant proteins. Fitting to binding curves was carried out with a single-site-binding model using the OriginLab software. Standard errors derived from three independent measurements are indicated.

[0077] FIGS. 6A-6D--Sem1 is a ubiquitination substrate of Rsp5.

[0078] Screening of pCD-Sub yeast fusion library revealed Sem1 as potential ubiquitination substrate of Rsp5 in the bacterial selection system. (A) Spots growth at the indicated hours post seeding shows that Rsp5 significantly promotes Sem1 ubiquitination (compare complete system that contains Rsp5 with a .DELTA.Rsp5 cascades). A 30 minutes intervals time-lapse movie of the scans can be found in the supplementary data. (B) Growth curves derived from quantification of the scans using `Time Series Analyzer` in Fiji. Values are average of eight spots with standard deviation bars. (C) Detection of Sem1 ubiquitination in E. coli. Purified His6-MBP-Semi from E. coli that co-express ubiquitination apparatus .sup.17 was resolved on SDS-PAGE and detected by western-bolt with anti-Histag antibody. (D) Sem1 is a ubiquitination substrate of Rsp5 in vivo in yeast. His6-Semi was expressed from Gal inducible promoter in wild-type or rsp5-1 yeast cells. Cultures grew at 25.degree. C. (permissive conditions). Prior induction temperature was shift to 37.degree. C. or remained as indicated. His.sub.6-Sem1 was purified under denatured conditions, resolved and detected as in (C).

[0079] FIGS. 7A-7F--Functional analysis of the vWA:Ub binding patch. (A) Scheme showing a bacterial genetic selection system for ubiquitination (B) Ubiquitination addicted bacterial growth on selective (+Trimethoprim) or non-selective plates. A single scan of the plates 98 hours post seeding is shown. (C) Shows quantitation of ubiquitination dependent bacterial growth. Average density of individual spots monitored by scanning the plates in 1 hour intervals was plotted. Efficiency was calculated as the max growth density divided by the time of half max growth. NSG--no significant growth. (D) Shows a representative SPR response curves for the vWA:Ub complex. (E) Single model binding analysis of SPR affinity measurements of Rpn10:Ub wt and the indicated mutants. Kd values are indicated (right; NB--no binding). (F) Comparison between the relative growth of wt or mutant spots and the SPR measured association constants (K.sub..alpha.). Pearson product-moment correlation coefficient is r=0.99 (p<0.001).

[0080] FIGS. 8A-8B--Schemes show two of the major hurdles that pose challenges in assigning specific associations of components along ubiquitin cascades.

[0081] (A) Ubiquitination is rapidly reversed by deubiquitylases. In human there are about hundred deubiquitinating enzymes (DUBs) which efficiently and rapidly remove the ubiquitin moiety from targeted proteins.

[0082] (B) Ubiquitination cascades are multiplex. For example eight different substrates were found for the BRCA1 E3-ligase. Similarly, 7 different E3-ligases were demonstrated to ubiquitylate p53.

[0083] FIG. 9--Architecture and sequence of the linkers connecting the DHFR fragments to Ub and substrates. Schematic illustration of the constructs used in the selection system. pND-Ub vectors contain a N-Terminal DHFR fragment fused through the flexible linker (linker 1) to Ub. In the pCD-Sub the C-Terminal fragment of the DHFR was fused through the flexible linker (linker 2) to the His.sub.6-MBP-substrate or directly to the substrate.

TABLE-US-00001 Linker1: SEQ ID NO: 5 LIKAAQRAREAERDLAAAVAQAAAGQAVPRAAPRQ Linker2: SEQ ID NO: 6 GGSAGSGSGAGSGSGGSAGSSGSSGASSG.

[0084] FIG. 10--Domain architecture of membrane associated Ub-receptors. Ub moieties mark the binding sites (orange circled U's).

[0085] FIGS. 11A-11E--Ent1-ENTH domain directly binds ubiquitin. (A) Shows crosslinking assay of Ent1-ENTH domain with increased concentrations of Ub. A mild crosslinker, disuccinimidyl suberate (DSS) was used. Reactions were resolved by SDS-PAGE and Coomassie blue stained. (B) Shows crosslinking assay like in (A) of Ent1-ENTH domain with wild-type and Ub I44E mutant for various incubation times (as indicated). (C) Scheme of yeast and zebrafish Epsin-1 derivative constructs. (D). Ubiquitination of yeast Ent1 derivatives. (E). Ubiquitination of zebrafish Epn1 derivatives. His.sub.6-Ub was co-expressed with E1 and Ubc4 along with GST-Epsin1 derivatives. Proteins were purified on GSH-beads and ubiquitination was detected by western blot using anti-Histag antibody as described in.sup.17.

[0086] FIGS. 12A-12B--Structural divergence within the ENTH domains. The coordinates of several ENTH domains including yeast (magenta), zebrafish (white), and three structures of rat ENTH domains (cyan, red and yellow), were superposed. The axis of helix-8 from each of the structures were calculated. Then the angles among the derived helices were compared (A). Structural differences between the loops tethering helices number 3 and 4 in yeast and zebrafish ENTH domains are presented (B).

[0087] FIG. 13--ENTH/VHS domains can simultaneously associate with membranes and ubiquitin. Superimposing the ENTH complex with the lipid phosphatidylinositol-4,5-bisphosphate, GGA3-VHS complex with the Mannose-6P Receptor tail and the STAM1-VHS:Ub shows that membrane and Ub associations occur at opposite sides of the domain and therefore can occur simultaneously.

[0088] FIGS. 14A-14C--Surface Plasmon Resonance (raw data). Sonograms of the SPR responses of the WT and the indicated mutants are presented.

[0089] FIGS. 15A-15D--a schematic view of the split CAT bacterial selection system for ubiquitination.

[0090] A. The reaction executed by CAT (i.e. transfer of acetyl group from acetyl-CoA to chloramphenicol is shown.

[0091] B. Structure representation of assembled split CAT with chloramphenicol.

[0092] C. Linear representation of the split-CAT system.

[0093] D. cartoon view of the split CAT bacterial selection system.

[0094] FIGS. 16A-16D--Split-CAT system detects ubiquitination.

[0095] A. Shows E3 independent uniquitination of the Ub-binding domain VHS of yeast Hse1.

[0096] B. The split CAT system shows significant higher growth efficiency that shortens the experimental time. Shown is UBE3A dependent ubiquitination of Rpn10 in the split CAT and DHFR selection system.

[0097] C. Mutation in UBE3A E3 ligase (G738E) that causes Angelman-syndrome phenotype.

[0098] D. The split CAT system facilitates the study of E3 ligases that cannot be purified from E. coli such as UBE3B. Shown is UBE3B dependent ubiquitination of Rpn10 in the split CAT system. Kaufman syndrome mutation (G781R) in UBE3B shows a phenotype.

[0099] FIGS. 17A-17B illustrate the DNA sequence and the translation products of CAT.sub.I (FIG. 17 A--SEQ ID NO: 63 and SEQ ID NO: 64) and the split-CAT.sub.I fragments (FIG. 17 B--SEQ ID NOs: 65-68). Arrow marks the cleavage site that was chosen for the split protein fragments (top). The stop codon after residue Q30 and the initiation codon prior residue C31 are shown in the split protein fragments (bottom).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0100] The present invention, in some embodiments thereof, relates to bacterial systems for analyzing the ubiquitination of polypeptides.

[0101] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0102] About one-third of the eukaryotic proteome undergoes ubiquitination, but most of the enzymatic cascades leading to substrate modification are still unknown. The present inventors have invented a genetic selection tool that utilizes E. coli, which lack deubiquitylases, to identify interactions along ubiquitination cascades. Co-expression of split antibiotic resistance protein tethered to ubiquitin and ubiquitination target together with a functional ubiquitination apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. The capability of the screening tool for small molecule modulators, in a high-throughput format is demonstrated herein. In addition, using a complete E2-library in the selection system, the present inventors identified and characterized an E3-ligase from the pathogenic bacteria EHEC. Furthermore, the present inventors identified ENTH as an ultraweak ubiquitin-binding domain, demonstrating the tool's high sensitivity. X-ray crystallography combined with bacterial selection studies facilitates structure-function analysis of the ENTH:ubiquitin interface. Moreover, study of the Rpn10:Ub non-covalent interaction (FIGS. 7A-7F) demonstrated an excellent correlation between the growth efficiency under restrictive conditions of wild-type and mutants in the genetic selection system and the binding affinities measured by Surface Plasmon Resonance (SPR). FIG. 7F shows a comparison between the relative growth efficiency on selective media and the relative SPR association constants (K.sub..alpha.). Linear regression provides a Pearson correlation coefficient of r=0.99 (p<0.001).

[0103] These finding demonstrate that one can employ the system for drug discovery and improvements (hit-to-lead) as the ubiquitination dependent growth is highly correlated with the affinities along the cascade. Whilst further reducing the present invention to practice, the present inventors constructed and screened a yeast fusion library and discovered a novel physiological ubiquitination. Collectively, the developed system provides a robust high-throughput approach for genetic studies of ubiquitination cascades.

[0104] Thus, according to one aspect of the present invention there is provided a method of identifying an agent which regulates the activity or amount of a ubiquitinating enzyme or deubiquitinating enzyme comprising:

[0105] (a) contacting a bacterial cell with the agent, wherein the bacterial cell outputs a detectable or selectable signal which correlates with the ubiquitination level of a substrate; and

[0106] (b) measuring the level or the rate of accumulation of the detectable or selectable signal, wherein a change in the level as compared to the level in the absence of the agent, is indicative of an agent which regulates the activity or amount of the ubiquitinating or deubiquitinating enzyme.

[0107] The method of this aspect of the present invention can screen for agents that upregulate or downregulate the activity and/or amount of the enzyme.

[0108] Agents that may be screened include small molecule agents, peptide agents, nucleic acid agents, antibodies, proteins, chemotherapeutic agents etc.

[0109] The screening assay of this aspect of the present invention uses bacteria that have been genetically modified to output a detectable or selectable signal which correlates with the ubiquitination level of a substrate.

[0110] Ubiquitination takes place by a cascade of enzyme activity (i.e. a plurality of enzymes which work together to bring about the same function--ubiquitination). For example, E1 activates the Ub; then Ub is transferred to E2. E2 together with E3 (or in many cases transfer the Ub to E3) recognize a specific target and ligate the Ub to the target protein.

[0111] Below is a list of the components of the assay which are expressed by the genetically modified bacteria of this aspect of the present invention, each of which will be described in detail herein below.

[0112] 1. Ubiquitin;

[0113] 2. Detectable signal;

[0114] 3. At least one ubiquitinating or deubiquitinating enzyme; and

[0115] 4. Substrate (target for ubiquitylation).

[0116] Any bacteria can be used for this assay so long as it lacks endogenous deubiquitinase activity and preferably also endogenous ubitquitinase activity. In one embodiment, the bacteria has at least 10 fold less endogenous deubiquitinase activity and endogenous ubiquitinase activity than a human cell. In another embodiment, the bacteria has at least 20 fold less endogenous deubiquitinase activity and endogenous ubiquitinase activity than a human cell.

[0117] Preferably the bacteria lack resistance to the selection markers in the current system. Examples of such bacteria include, but are not limited to E. coli K-12 derivatives including W3110, MG1655, DH5.alpha., JM101, JM19, BL21, B834, XL1-Blue; also other non E. coli bacteria may be used.

[0118] According to a particular embodiment, the bacteria used in the system are of the genus Escherichia, such as for example E. coli.

[0119] In order to express the components of the assay, a polynucleotide sequence encoding the elements described above is preferably ligated into a nucleic acid construct suitable for bacterial cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0120] The phrase "an isolated polynucleotide" refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

[0121] As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase; or synthetically synthesized by assembled from short oligonucleotide.

[0122] As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

[0123] As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

[0124] The nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.

[0125] Exemplary promoters contemplated by the present invention include, but are not limited to polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and cytomegalovirus promoters. According to a particular embodiment, the promoter is a bacterial promoter.

[0126] Constitutive promoters suitable for use with the present invention are promoter sequences which are active under most environmental conditions and most types of bacterial cells such as an unregulated bacteriophage lambda left promoter (pL) or pTac which presents high leakiness.

[0127] Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancing elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.

[0128] In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0129] Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.

[0130] In addition to the elements already described, the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

[0131] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

[0132] In a preferred embodiment, the vector comprises a bacterial replication of origin.

[0133] The expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0134] The use of bacterial operon architecture for multi-gene expression, where a single promoter is followed by several open reading frames (ORFs) each contains a ribosome-binding site (Shine-Dalgarno sequence) facilitates the co-expression of the multi-protein complex of the ubiquitination apparatus.

[0135] It will be appreciated that the individual elements comprised in the expression vector can be arranged in a variety of configurations. For example, enhancer elements, promoters and the like, and even the polynucleotide sequence(s) encoding the fusion protein can be arranged in a "head-to-tail" configuration, may be present as an inverted complement, or in a complementary configuration, as an anti-parallel strand. While such variety of configuration is more likely to occur with non-coding elements of the expression vector, alternative configurations of the coding sequence within the expression vector are also envisioned.

[0136] Exemplary methods of introducing the polynucleotides of the present invention into prokaryotic cells are well known in the art--these include, but are not limited to, transforming with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the relevant gene sequences.

[0137] Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production or isolation of the expressed peptide fragments.

[0138] Examples of bacterial constructs include the pET series of E. coli expression vectors (see for example Studier et al (1990) Methods in Enzymol 185:60-89) in which their T7 promoter was replaced with the constitutive active bacteriophage pH (left promoter). Other vectors that may be used are those that belong to the pZE vector family (e.g. pZE21), those that belong to the pCloDF (containing a pCloDF13 origin) and pACYC (containing a p15A origin of replication).

Ubiquitin

[0139] The term "ubiquitin" as used herein refers to either mammalian ubiquitin having a sequence as set forth in SEQ ID NO: 7 or yeast ubiquitin having a sequence as set forth in SEQ ID NO: 8.

Detectable signal

[0140] In one example, the detectable signal is a fluorescent protein or an enzyme producing a colorimetric reaction. Exemplary proteins that generate a detectable signal include, but are not limited to green fluorescent protein (Genbank Accession No. AAL33912), alkaline phosphatase (Genbank Accession No. AAK73766), peroxidase (Genbank Accession No. NP_568674), histidine tag (Genbank Accession No. AAK09208), Myc tag (Genbank Accession No. AF329457), biotin ligase tag (Genbank Accession No. NP_561589), orange fluorescent protein (Genbank Accession No. AAL33917), beta galactosidase (Genbank Accession No. NM_125776), Fluorescein isothiocyanate (Genbank Accession No. AAF22695) and strepavidin (Genbank Accession No. S11540).

[0141] In another example, the detectable signal is a luminescent protein such as products of bacterial luciferase genes, e.g., the luciferase genes encoded by Vibrio harveyi, Vibrio fischeri, and Xenorhabdus luminescens, the firefly luciferase gene FFlux, and the like.

[0142] In one embodiment, the selection is dominant selection, which typically uses a drug to arrest growth of a host cell. Those cells which would express a protein conveying drug resistance would survive the selection. The use of split marker allows the detection of ubiquitination events as further described below.

[0143] In order to output a detectable or selectable signal which correlates with the ubiquitination level of a substrate, the present inventors contemplate using a split polypeptide, wherein one fragment of the polypeptide is linked to ubiquitin and the other fragment of the polypeptide is linked to the substrate. When the polypeptide is expressed as two "split" fragments, there is no detectable or selectable signal. However, when the two fragments are brought close enough together (i.e. on ubiquitination of the substrate) they form a functional protein that emits a detectable or selectable signal--i.e. generate a reporter polypeptide.

[0144] According to a particular embodiment, the split polypeptide combines to generate a reporter polypeptide which is fluorescent, luminescent, phosphorescent or one that confers antibiotic resistance.

[0145] Examples of split polypeptides contemplated by the present invention include, but are not limited to beta lactamase, dihydrofolate reductase (DHFR), focal adhesion kinase, enhanced GFP, horseradish peroxidase, Infrared fluorescent protein IFP1.4 (an engineered chromophore-binding domain (CBD) of a bacteriophytochrome from Deinococcus radiodurans) LacZ (beta-galactosidase)` Luciferase, TEV (Tobacco etch virus protease).

[0146] According to a particular embodiment the split polypeptide provides resistance to an antibiotic when combined, but the bacteria is susceptible to the antibiotic when split. Preferably, the split polypeptide provides resistance to a bacteristatic antibiotic when combined. Examples of bacteriostatic antibiotics include but are not limited to trimethoprim and chloramphenicol.

[0147] In the case of trimethoprim, a split DHFR protein may be expressed. Specifically the use of selective media which lack thymidine, glycine, serine or methionine and contains the trimethoprim antibiotic allows the selection of genes required for the ubiquitination process.

[0148] In the case of chloramphenicol, a split chloramphenicol acetyl transferase (CAT) enzyme can be expressed.

[0149] As used herein, the term CAT refers to an enzyme (EC 2.3.1.28) that catalyzes the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group.

[0150] The CAT of this embodiment may have an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous or identical to the sequence as set forth in SEQ ID NO: 64, as determined using the Standard protein-protein BLAST [blastp] software of the NCBI. An exemplary DNA sequence that encodes full length CAT is set forth in SEQ ID NO: 63.

[0151] In one embodiment the N-terminal fragment comprises a first portion of the catalytic active site of the CAT--e.g. the N terminal fragment typically contains the first 28 or 30 amino acids of the native CAT. The C-terminal fragment comprises the second portion of the catalytic active site of the CAT--for example, the C terminal fragment typically contains the rest of the sequence of the native CAT. The N-terminal fragment associates with the C-terminal fragment to generate an active CAT that is capable of acetylating chloramphenicol.

[0152] In one embodiment, the N terminus of the N-terminal fragment is linked to the C terminus of the substrate or ubiquitin (preferably via a linker).

[0153] In another embodiment, the C terminus of the C-terminal fragment is linked to the N terminus of the substrate or ubiquitin (preferably via a linker)--see for example FIG. 15C.

[0154] Preferably, the first amino acid of the C-terminal fragment is a small amino acid residue--for example cysteine or alanine. Thus, the C terminal fragment may begin with cysteine 31 (wherein the numbering is according to SEQ ID NO: 64), or alanine 29 (wherein the numbering is according to SEQ ID NO: 64). Other small amino acid residues include glycine, alanine, serine, proline, threonine, aspartate and asparagine.

[0155] By being small, the first amino acid of the C-terminal fragment after the formyl-methionine is causes the latter to be posttranslationally removed from the N-terminus (of the C-terminus fragment) hence salvaging the active site arrangement as seen by the activity.

[0156] In one embodiment, the N-terminal fragment comprises/consists of the amino acid sequence as set forth in SEQ ID NO: 67.

[0157] The C-terminal fragment comprises/consists of the amino acid sequence as set forth in SEQ ID NOs: 68.

[0158] The N-terminal fragment may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 65.

[0159] The C-terminal fragment may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 66.

[0160] The DNA and protein sequence of an exemplary split CAT is illustrated in FIG. 17B.

[0161] As mentioned, in order to generate the recombinant bacteria of the above described embodiment, the bacteria are genetically modified to express at least two polypeptide fragments--one of the polypeptide fragments being the split polypeptide fragment linked to ubiquitin and the other polypeptide fragment being the conjugate pair of the split polypeptide which is linked to the substrate.

[0162] In one embodiment, the split polypeptide fragment is directly linked to ubiquitin or substrate. In another embodiment, the split polypeptide fragment is linked to the ubiquitin or substrate via a peptide linker. The linker should be of a length and flexibility that allows functional stability of the reporter polypeptide. The linker is preferably between 10-500 amino acids, more preferably between 20 and 200 amino acids and more preferably between 20-100 amino acids. Exemplary peptide linkers that can be used are set forth in SEQ ID NOs: 5 or 6.

[0163] The first fragment of the reporter polypeptide which is linked to ubiquitin may be encoded on the same nucleic acid construct as the second fragment of the reporter polypeptide which is linked to the substrate. Alternatively, the first fragment of the reporter polypeptide which is linked to ubiquitin may be encoded on a different nucleic acid construct as the second fragment of the reporter polypeptide which is linked to the substrate. This embodiment is illustrated in FIG. 1A. Care should be taken when building the constructs such that the expression level of the first fragment is similar to the expression level of the second fragment. Thus for example, the promoter which is used to express the first fragment may be identical to the promoter used to express the second fragment.

[0164] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. Specifically, the use of different selection markers such as Kan.sup.R and Strep.sup.R) and different origin of replications (such as ColiE1 and CloDF13) is contemplated.

Ubiquitinating Enzyme

[0165] As used herein, the term "ubiquitinating enzyme" refers to ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) or ubiquitin ligases (E3s). Collectively they have the EC number EC 6.3.2.19.

[0166] In one embodiment, the ubiquitinating enzyme is a human ubiquitinating enzyme.

[0167] Ubiquitin-activating enzymes (E1s) have the EC number EC 6.2.1.45, ubiquitin-conjugating enzymes have the EC number EC 2.3.2.23 and ubiquitin ligases have the EC number 2.3.2.27.

[0168] Amino acid sequences of exemplary E1s contemplated by the present invention include, but are not limited to SEQ ID NOs: 9-16.

[0169] Amino acid sequences of exemplary E2s contemplated by the present invention are set forth by SEQ ID NOs: 17-36.

[0170] Table 1, herein below provides nomenclature and most common synonyms used for E2 ubiquitin conjugating enzymes. The E2 nomenclature is in accordance with that used by the Human Genome Organization.

TABLE-US-00002 TABLE 1 Human Genome Organization Nomenclature Synonym UBE2V2 UEV2/MMS2 UBE2D1 UBC4/5/UBCH5A UBE2D2 UBC4/5/UBCH5B UBE2D4 HBUCE1 UBE2D3 UBC4/5 UBE2W FLJ11011 UBE2B UBC2/HHR6B/RAD6B/E217K UBE2L6 RIGB/UBCH8 UBE2N UBC13 UBE2L3 UBCH7 UBE2G1 UBC7/E217K UBE2H UBC8/E220K UBE2M UBC12 UBE2F NCE2 UBE2E2 UBCH8 UBE2E3 UBCH9/UBCM2 UBE2S E224K UBE2U MGC35130 UBE2R1 CDC34 UBE2R2 UBC3B/CDC34B UBE2Z HOYS7 UBE2J2 NCUBE2 Probable ubiquitin- LOC134111/FLJ25076 conjugating enzyme E2 FLJ25076 AKTIP FTS/FT1 UBE2J1 NCUBE1 UBE2V1 UEV1/CROC1 UBE2Q2 DKFZ/UBCI UBE2Q1 NICE5 TSG101/VPS23/SG10 UEVLD UEV3

[0171] In one embodiment, the ubiquitinating enzyme is an E3 ligase.

[0172] Exemplary E3 ligases contemplated by the present invention include, but are not limited to Siah2, Smurf1, MDM2, BRCA1, PARKIN, UBE3A, TRIM5, NEDD4, UBR5, Huwe1, Arkadia, ITCH, MuRF1, TRAF6, Trim32, UBR4, UBE3B and UBE3D.

[0173] According to a particular embodiment, the E3 ligase is selected from the group consisting of Siah2, Smurf1, MDM2, BRCA1, PARKIN, UBE3A, UBE3B, MURF1, TRIM32, TRIM5, NEDD4, UBR5 and Huwe1.

[0174] In another embodiment the E3 ligase is Siah2, Smurf1, MDM2, BRCA1, PARKIN or UBE3A.

[0175] Amino acid sequences of exemplary E3s contemplated by the present invention are set forth in SEQ ID NOs: 37-62.

[0176] Below is a brief description of exemplary E3 ligases contemplated by the present invention and some of their exemplary substrates.

Seven in Absentia Homolog 2 (SIAH2)

[0177] SIAH2 is a RING finger type ubiquitin ligases with a catalytic RING domain on its N-terminus, followed by two zinc fingers and a C-terminal substrate binding domain.

[0178] Siah2 is an E3 ubiquitin ligase implicated in diverse biological processes including p38/JNK/NF-kB signaling pathways, DNA damage, estrogen signaling, programmed cell death, Ras/Raf pathway, mitosis, and hypoxia.

[0179] Siah2 targets numerous substrates for degradation including TRAF2 (ketoglutarate dehydrogenase), Spry2 (Sprouty2), and the prolyl hydroxylase PHD3.

[0180] Siah2 also limits its own availability through self-ubiquitination and degradation.

[0181] Siah2 play a key role in hypoxia, through the regulation of HIF-1.alpha. transcription stability and activity via regulation of PHD3 stability.

Smad Ubiquitination Regulatory Factor-1 (Smurf1)

[0182] Smurf1 is a NEDD4-like Class IV HECT (homologous to E6-AP carboxyl terminus) family E3 ligase with catalytic activity.

[0183] Smurf1 has been linked to several important biological pathways, including the bone morphogenetic protein pathway, the non-canonical Wnt pathway, and the mitogen-activated protein kinase pathway.

[0184] Smurfs possess three functional domains: an N-terminal protein kinase C (PKC)-related C2 domain which binds to phospholipids, targeting Smurfs to intracellular membranes, a central region containing two to four WW (tryptophan residues) protein-interacting domains which mediate ligase-substrate associations through interactions with a variety of proline-rich (PPXY) motifs and proline-containing phosphoserine/phosphothreonine sequences of the protein substrate, and a C-terminal HECT domain, responsible for ubiquitin transfer from a conserved cysteine residue at position 716 to a lysine residue in a substrate protein.

[0185] Smurf1 promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. Smurf1 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1 interacts with MDM2 and MDMX. Smurf1 is also a key negative regulator of transforming growth factor (TGF)-.beta.3/bone morphogenetic protein (BMP) signaling pathway.

Mouse Double Minute 2 Homolog (MDM2)

[0186] MDM2 also known as E3 ubiquitin-protein ligase Mdm2 is an important negative regulator of the p53 tumor suppressor.

[0187] Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation.

[0188] Mdm2 contains a C-terminal RING domain (amino acid residues 430-480), which contains a Cis3-His2-Cis3 consensus that coordinates two molecules of zinc. These residues are required for zinc binding, which is essential for proper folding of the RING domain. The RING domain of Mdm2 confers E3 ubiquitin ligase activity and is sufficient for E3 ligase activity in Mdm2 RING autoubiquitination. The RING domain of Mdm2 is unique in that it incorporates a conserved Walker A or P-loop motif characteristic of nucleotide binding proteins, as well as a nucleolar localization sequence.

[0189] Mdm2 is capable of auto-polyubiquitination, and in complex with p300, a cooperating E3 ubiquitin ligase, is capable of polyubiquitinating p53. In this manner, Mdm2 and p53 are the members of a negative feedback control loop that keeps the level of p53 low in the absence of p53-stabilizing signals.

BRCA1

[0190] BRCA1-BARD1 constitutes a heterodimeric RING finger complex of the BRCA1/BRCA2-containing complex (BRCC) that contains significant ubiquitin ligase activity.

[0191] BRCA1 plays critical roles in the repair of chromosomal damage (error-free repair of DNA double-strand breaks), cell cycle checkpoint control, and genomic stability.

[0192] BRCA1 forms several distinct complexes through association with different adaptor proteins, and each complex forms in a mutually exclusive manner.

[0193] BRCA1 combines with other tumor suppressors, DNA damage sensors and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC).

[0194] The BRCA1 protein associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase complexes. Thus, this protein plays a role in transcription, DNA repair of double-strand breaks ubiquitination, transcriptional regulation as well as other functions

Parkin

[0195] Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates.

[0196] It is best known for regulating the disposal of dysfunctional mitochondria (together with PINK1, a serine threonine kinase) via mitochondrial autophagy (i.e., mitophagy). Upon loss of mitochondrial membrane potential, PINK1 becomes stabilized and activated on the outer mitochondrial membrane (OMM), resulting in recruitment and activation of Parkin.

[0197] Parkin facilitates ubiquitination of a broad number of targets expressed on the OMM (e.g., TOM20, Mitofusins, VDAC, Fis1) resulting in recruitment of the autophagy machinery, autophagosome formation and mitochondrial clearance.

[0198] In addition to its established role in mitophagy and UPS, Parkin impacts other neuroprotective cellular pathways, including TNF.alpha. a signaling, and Wnt/.beta. catenin signaling, and is also a putative tumor suppressor.

UBE3A (Gene Coding for E6-Associated Protein; E6-AP)

[0199] This ligase promotes the ubiquitylation and degradation of p53. E6-AP was subsequently shown to ubiquitylate proteins independent of E6 and to serve an independent secondary function as a transcriptional co-activator of nuclear estrogen receptors. E6-AP has been implicated in a broad range of processes (e.g. cell growth, synaptic formation and function, etc.) and has been shown to have many different target substrates (e.g., HHR23A, CDKN1B, MCM7, etc.).

Tripartite Motif-5 (TRIM5)

[0200] This ligase is a RING finger E3 ligase a key anti-viral restriction factor and directly involved in inhibiting HIV-1 replication.

Neural Precursor Cell Expressed Developmentally Downregulated 4 (NEDD4-1)

[0201] Nedd4-1 ubiquitinates a number of substrates, including ENaC, ADRB2, AMPA, Notch, pAKT, VEGFR2, EPS15, LATS1, and MDM2.

[0202] The vacuolar protein sorting protein Alix recruits NEDD4 to HIV-1 Gag protein to facilitate HIV-1 release via a mechanism that involves Alix ubiquitination.

[0203] NEDD4 also binds and ubiquitinates the latent membrane protein 2A (LMP2A) of the Epstein-Bar virus (EBV) to activate B-cell signal transduction.

Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5)

[0204] This ligase is also known as EDD (E3 identified by Differential Display), EDD1, HHYD, KIAA0896, or DD5.

[0205] URBS acts as a general tumor suppressor by ubiquitinating, which increases p53 levels and induces cell senescence. UBR5 also ubiquitinates TopBP1, a topo-isomerase that intervenes in DNA damage response.

HUWE1

[0206] HUWE1 (also known as ARF-BP1, MULE, LASU1, or HECTH9) is an E3 ligase that regulates the stability of diverse cellular substrates and, in consequence, numerous physiological processes, including DNA replication and damage repair, cell proliferation and differentiation, and apoptosis.

[0207] HUWE1 substrates include both tumor promoters (e.g., N-MYC, C-MYC, MCL1) and suppressors (e.g., p53, MYC, MIZ1).

[0208] HUWE1 has demonstrated both pro-oncogenic and tumor-suppressor functions in different tumor models.

[0209] HUWE1 belongs to the HECT (Homologous to E6AP C-Terminus)-family of ubiquitin E3 ligases.

[0210] Other additional E3 ligases and their substrates are provided in Table 2, herein below.

TABLE-US-00003 TABLE 2 Ligase Substrate Function AMFR KAI1 AMFR is also known as gp78. AMFR is an integral ER membrane protein and functions in ER-associated degradation (ERAD). AMFR has been found to promote tumor metastasis through ubiquitination of the metastasis supressor, KAI1. APC/Cdc20 Cyclin B The anaphase promoting complex/cyclosome (APC/C) is a multiprotein complex with E3 ligase activity that regulates cell cycle progression through degradation of cyclins and other mitotic proteins. APC is found in a complex with CDC20, CDC27, SPATC1, and TUBG1. APC/Cdh1 Cdc20, The anaphase promoting Cyclin B, complex/cyclosome (APC/C) is a Cyclin A, multiprotein complex with E3 ligase Aurora A, activity that regulates cell cycle Securin, progression through degradation of Skp2, cyclins and other mitotic proteins. Claspin The APC/C-Cdh1 dimeric complex is activated during anaphase and telophase, and remains active until onset of the next S phase. C6orf157 Cyclin B C6orf157 is also known as H10BH. C6orf157 is an E3 ubiquitin ligase that has been shown to ubiquitinate cyclin B. Cbl Cbl-b and c-Cbl are members of the Cbl family of adaptor proteins that are highly expressed in hematopoietic cells. Cbl proteins possess E3 ubiquitin ligase activity that downregulates numerous signaling proteins and RTKs in several pathways such as EGFR, T cell and B cell receptors, and integrin receptors. Cbl proteins play an important role in T cell receptor signaling pathways. CBLL1 CDH1 CBLL1 is also known as Hakai. CBLL1 is an E3 ubiquitin ligase that ubiquitinates the phosphorylated form of E-Cadherin, causing its degradation and loss of cell-cell adhesions. CHFR PLK1, CHFR is an E3 ubiquitin ligase that Aurora A functions as a mitotic stress checkpoint protein that delays entry into mitosis in response to stress. CHFR has been shown to ubiquitinate and degrade the kinases PLK1 and Aurora A. CHIP HSP70/90, CHIP is an E3 ubiquitin ligase that iNOS, acts as a co-chaperone protein and Runx1, interacts with several heat shock LRRK2 proteins, including HSP70 and HSP90, as well as the non-heat shock proteins iNOS, Runx1 and LRRK2. DTL (Cdt2) p21 DTL is an E3 ubiquitin ligase that complexes with Cullin4 and DDB1, and promotes p21 degradation after UV damage. E6-AP p53, Dlg E6-AP is also known as UBE3A. E6- AP is a HECT domain E3 ubiquitin ligase that interacts with Hepatitis C virus (HCV) core protein and targets it for degradation. The HCV core protein is central to packaging viral DNA and other cellular processes. E6-AP also interacts with the E6 protein of the human papillomavirus types 16 and 18, and targets the p53 tumor-suppressor protein for degradation. Mutations that decrease UBE3A activity may cause Angelman syndrome. Mutations that increase UBE3A activity may cause Autism syndrome. HACE1 HACE1 is an E3 ubiquitin ligase and tumor suppressor. Aberrant methylation of HACE1 is frequently found in Wilms' tumors and colorectal cancer. HECTD1 HECTD1 is an ubiquitin E3 ligase required for neural tube closure and normal development of the mesenchyme. HECTD2 HECTD2 is a probable E3 ubiquitin ligase and may act as a succeptibility gene for neurodegeneration and prion disease. HECTD3 HECTD3 is a probable E3 ubiquitin ligase and may play a role in cytoskeletal regulation, actin remodeling, and vesicle trafficking. HECW1 DVL1, HECW1 is also known as NEDL1. mutant HECW1 interacts with p53 and the SOD1, p53 Wnt signaling protein DVL1, and may play a role in p53-mediated cell death in neurons. HECW2 p73 HECW2 is also known as NEDL2. HECW2 ubiquitinates p73, which is a p53 family member. Ubiquitination of p73 increases protein stability. HERC2 RNF8 HERC2 belongs to a family of E3 ubiquitin ligases involved in membrane trafficking events. HERC2 plays a role in the DNA damage response through interaction with RNF8. HERC3 HERC3 belongs to a family of E3 ubiquitin ligases involved in membrane trafficking events. HERC3 interacts with hPLIC-1 and hPLIC-2 and localizes to the late endosomes and lysosomes. HERC4 HERC4 belongs to a family of E3 ubiquitin ligases involved in membrane trafficking events. HERC4 is highly expressed in testis and may play a role in spermatogenesis. HERC5 HERC5 belongs to a family of E3 ubiquitin ligases involved in membrane trafficking events. HERC5 is induced by interferon and other pro-inflammatory cytokines and plays a role in interferon-induced ISG15 conjugation during the innate immune response. HUWE1 N-Myc, HUWE1 is also known as Mule. C-Myc, p53, HUWE1 is a HECT domain E3 Mcl-1, ubiquitin ligase that regulates TopBP1 degradation of Mcl-1 and therefore regulates DNA damage-induced apoptosis. HUWE1 also controls neuronal differentiation by destabilizing N-Myc, and regulates p53-dependent and independent tumor suppression via ARF. HYD CHK2 HYD is also known as EDD or UBR5. HYD is a regulator of the DNA damage response and is overexpressed in many forms of cancer. ITCH MKK4, RIP2, ITCH plays a role in T cell receptor Foxp3 activation and signaling through ubiquitination of multiple proteins including MKK4, RIP2 and Foxp3. Loss of ITCH function leads to an abberrant immune response and T helper cell differentiation. LNX1 NUMB LNX1 is an E3 ubiquitin ligase that plays a role in cell fate determination during embryogenesis through regulation of NUMB, the negative regulator of Notch signaling. mahogunin Mahogunin is an E3 ubiquitin ligase involved in melanocortin signaling. Loss of mahogunin function leads to neurodegeneration and loss of pigmentation, and may be the mechanism of action in prion disease. MARCH-I HLA-DR.beta. MARCH1 is an E3 ubiquitin ligase found on antigen presenting cells (APCs). MARCH1 ubiquitinates MHC class II proteins and downregulates their cell surface expression. MARCH-II MARCH-II is a member of the MARCH family of E3 ubiquitin ligases. It associates with syntaxin6 in the endosomes and helps to regulate vesicle trafficking. MARCH-III MARCH-III is a member of the MARCH family of E3 ubiquitin ligases. MARCH-III associates with syntaxin6 in the endosomes and helps to regulate vesicle trafficking. MARCH-IV MHC class I MARCH-IV is a member of the MARCH family of E3 ubiquitin ligases. MARCH-IV ubiquitinates MHC class I proteins and downregulates their cell surface expression. MARCH-VI MARCH-VI is also known as TEB4 and is a member of the MARCH family of E3 ubiquitin ligases. It localizes to the endoplasmic reticulum and participates in ER-associated protein degradation. MARCH-VII gp190 MARCH-VII is also known as axotrophin. MARCH-VII was originally identified as a neural stem cell gene, but has since been shown to play a role in LIF signaling in T lymphocytes through degradation of the LIF- receptor subunit, gp190. MARCH- B7-2, MHC MARCH-VIII is also known as c-MIR. VIII class II MARCH-VIII causes the ubiquitination/ degradation of B7-2, which is a co- stimulatory molecule for antigen presentation. MARCH-VIII has also been shown to ubiquitinate MHC class II proteins. MARCH-X MARCH-X is also known as RNF190. MARCH-X is a member of the MARCH family of E3 ubiquitin ligases. The putative role of MARCH-X is not currently known. MDM2 p53 MDM2, an E3 ubiquitin ligase for p53, plays a central role in regulation of the stability of p53. Akt-mediated phosphorylation of MDM2 at Ser166 and Ser186 increases its interaction with p300, allowing MDM2-mediated ubiquitination and degradation of p53. MEKK1 c-Jun, Erk MEKK1 is a well known protein kinase of the STE11 family. MEKK1 phosphorylates and activates MKK4/7, which in turn activates JNK1/2/3. MEKK1 contains a RING finger domain and exhibits E3 ubiquitin ligase activity toward c- Jun and Erk. MIB1 Delta, Jagged Mindbomb homolog 1 (MIB1) is an E3 ligase that facilitates the ubiquitination and subsequent endocytosis of the Notch ligands, Delta and Jagged. MIB2 Delta, Jagged Mind Bomb 2 (MIB2) is an E3 ligase that positively regulates Notch Signaling. MIB2 has been shown to play a role in myotube differentiation and muscle stability. MIB2 ubiquitinates NMDAR subunits to help regulate synaptic plasticity in neurons. MycBP2 Fbxo45, MycBP2 is an E3 ubiquitin ligase TSC2 also known as PAM. MycBP2 associates with Fbxo45 to play a role in neuronal development. MycBP2 also regulates the mTOR pathway through ubiquitination of TSC2. NEDD4 NEDD4 is an E3 ubiquitin ligase highly expressed in the early mouse embryonic central nervous system. NEDD4 downregulates both neuronal

voltage-gated Na+ channels (NaVs) and epithelial Na+ channels (ENaCs) in response to increased intracellular Na+ concentrations. NEDD4L Smad2 NEDD4L is an E3 ubiquitin ligase highly expressed in the early mouse embryonic central nervous system. NEDD4L has been shown to negatively regulate TGF-.beta. signaling by targeting Smad2 for degradation. Parkin Parkin is an E3 ubiquitin ligase that has been shown to be a key regulator of the autophagy pathway. Mutations in Parkin can lead to Parkinson's Disease. PELI1 TRIP, IRAK PELI1 is an E3 ubiquitin ligase that plays a role in Toll-like Receptor (TLR3 and TLR4) signaling to NF- .kappa.B via the TRIP adaptor protein. PELI1 has also been shown to ubiquitinate IRAK. Pirh2 TP53 Pirh2 is also known as RCHY1. Pirh2 is a RING domain E3 ubiquitin ligase. Pirh2 binds p53 and promotes proteosomal degradation of p53 independent of MDM2. Pirh2 gene expression is controlled by p53, making this interaction part of an autoinhibitory feedback loop. PJA1 ELF PJA1 is also known as PRAJA. PJA1 plays a role in downregulating TGF-.beta. signaling in gastric cancer via ubiquitination of the SMAD4 adaptor protein ELF. PJA2 PJA2 is an E3 ubiquitin ligase found in neuronal synapses. The exact role and substrates of PJA2 are unclear. RFFL p53 RFFL is also known as CARP2 and is an E3 ubiquitin ligase that inhibits endosome recycling. RFFL also degrades p53 through stabilization of MDM2. RFWD2 MTA1, p53, RFWD2 is also known as COP1. FoxO1 RFWD2 is an E3 ubiquitin ligase that ubiquitinates several proteins involved in the DNA damage response and apoptosis including MTA1, p53, and FoxO1. Rictor SGK1 Rictor interacts with Cullin1-Rbx1 to form an E3 ubiquitin ligase complex, and promotes ubiquitination and degradation of SGK1. RNF5 JAMP, RNF5 is also known as RMA5. RNF5 paxillin plays a role in ER-associated degradation of misfolded proteins and ER stress response through ubiquitination of JAMP. RNF5 also plays a role in cell motility and has been shown to ubiquitinate paxillin. RNF8 H2A, H2AX RNF8 is a RING domain E3 ubiquitin ligase that plays a role in the repair of damaged chromosomes. RNF8 ubiquitinates Histone H2A and H2A.X at double-strand breaks (DSBs) which recruits 53BP1 and BRCA1 repair proteins. RNF19 SOD1 RNF19 is also known as Dorfin. Accumulation and aggregation of mutant SOD1 leads to ALS disease. RNF19 ubiquitinates mutant SOD1 protein, causing a decrease in neurotoxicity. RNF190 see MARCH-X RNF20 Histone H2B RNF20 is also known as BRE1. RNF20 is an E3 ubiquitin ligase that monoubiquitinates Histone H2B. H2B ubiquitination is associated with areas of active transcription. RNF34 Caspase- RNF34 is also known as RFI. RNF34 8, -10 inhibits death receptor mediated apoptosis through ubiquitination/ degradation of caspase-8 and -10. RNF40 Histone H2B RNF40 is also known as BRE1-B. RNF40 forms a protein complex with RNF20 resulting in the ubiquitination of Histone H2B. H2B ubiquitination is associated with areas of active transcription. RNF125 RNF125 is also known as TRAC-1. RNF125 has been shown to positively regulate T cell activation. RNF128 RNF128 is also known as GRAIL. RNF128 promotes T cell anergy and may play a role in actin cytoskeletal organization in T cell/APC interactions. RNF138 TCF/LEF RNF138 is also known as NARF. RNF138 is associated with Nemo-like Kinase (NLK) and suppresses Wnt/.beta.- Catenin signaling through ubiquitination/degradation of TCF/LEF. RNF168 H2A, H2A.X RNF168 is an E3 ubiquitin ligase that helps protect genome integrity by working together with RNF8 to ubiquitinate Histone H2A and H2A.X at DNA double-strand breaks (DSB). SCF/.beta.- I.kappa.B.alpha., Wee1, SCF/.beta.-TrCP is an E3 ubiquitin ligase TrCP Cdc25A, .beta.- complex composed of SCF (SKP1- Catenin CUL1-F-box protein) and the substrate recognition component, .beta.-TrCP (also known as BTRC). SCF/.beta.-TrCP mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction, and transcription. SCF/ .beta.-TrCP also regulates the stability of .beta.-catenin and participates in Wnt signaling. SCF/FBW7 Cyclin E, c- SCF/FBW7 is an E3 ubiquitin ligase Myc, c-Jun complex composed of SCF (SKP1-CUL1-F-box protein) and the substrate recognition component, FBW7. SCF/FBW7 mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction, and transcription. Target proteins for SCF/FBW7 include the phosphorylated forms of c-Myc, Cyclin E, Notch intracellular domain (NICD), and c-Jun. Defects in FBXW7 may be a cause of breast cancer. SCF/Skp2 p27, p21, SCF/Skp2 is an E3 ubiquitin ligase Fox01 complex composed of SCF (SKP1-CUL1-F-box protein) and the substrate recognition component, Skp2. SCF/Skp2 mediates the ubiquitination of proteins involved in cell cycle progression (specifically the G1/S transition), signal transduction and transcription. Target proteins for SCF/Skp2 include the phosphorylated forms of p27Kip1, p21Waf1/Cip1, and FoxO1. SHPRH PCNA SHPRH is an E3 ubiquitin ligase that plays a role in DNA replication through ubiquitination of PCNA. PCNA ubiquitination prevents genomic instability from stalled replication forks after DNA damage. SIAH1 .beta.-catenin, SIAH1 is an E3 ubiquitin ligase that Bim, TRB3 plays a role in inhibition of Wnt signaling through ubiquitination of .beta.-catenin. SIAH1 has also been shown to promote apoptosis through upregulation of Bim, and to ubiquitinate the signaling adaptor protein TRB3. SIAH2 HIPK2, SIAH2 is an E3 ubiquitin ligase that PHD1/3 plays a role in hypoxia through ubiquitination and degradation of HIPK2. SIAH2 also ubiquitinates PHD1/3, which regulates levels of HIF-1.alpha. in response to hypoxia. SMURF1 Smads SMURF1 is an E3 ubiquitin ligase that interacts with BMP pathway Smad effectors, leading to Smad protein ubiquitination and degradation. Smurf1 negatively regulates osteoblast differentiation and bone formation in vivo. SMURF2 Smads, Mad2 SMURF2 is an E3 ubiquitin ligase that interacts with Smads from both the BMP and TGF-.beta. pathways. SMURF2 also regulates the mitotic spindle checkpoint through ubiquitination of Mad2. TOPORS p53, NKX3.1 TOPORS is an E3 ubiquitin ligase and a SUMO ligase. TOPORS ubiquitinates and sumoylates p53, which regulates p53 stability. TOPORS has also been shown to ubiquitinate the tumor supressor NKX3.1. TRAF6 NEMO, Akt1 TRAF6 is an E3 ubiquitin ligase that functions as an adaptor protein in IL- 1R, CD40, and TLR signaling. TRAF6 promotes NF-.kappa.B signaling through K63 polyubiquitination of IKK, resulting in IKK activation. TRAF6 has also been shown to ubiquitinate Akt1, causing its translocation to the cell membrane. TRAF7 TRAF7 is an E3 ubiquitin ligase and SUMO ligase that functions as an adaptor protein in TNF Receptor and TLR signaling. TRAF7 has been shown to be capable of self- ubiquitination and plays a role in apoptosis via MEKK3-mediated activation of NF-.kappa.B. TRIM63 Troponin I, TRIM63 is also known as Murf-1. MyBP-C, TRIM63 is a muscle-specific E3 MyLC1/2 ubiquitin ligase whose expression is upregulated during muscle atrophy. TRIM63 has been shown to ubiquitinate several important muscle proteins including troponin I, MyBP- C, and MyLC1/2. UBE3B UBE3B is an E3 ubiquitin ligase identified through sequence analysis. The specific substrates and cellular function of UBE3B is currently unknown. UBE3C UBE3C is an E3 ubiquitin ligase also known as KIAA10. UBE3C is highly expressed in muscle and may interact with the transcriptional regulator TIP120B. UBR1 UBR1 is an E3 ubiquitin ligase responsible for proteasomal degradation of misfolded cytoplasmic proteins. UBR1 has also been shown to be a ubiquitin ligase of the N-end rule proteolytic pathway, which regulates degradation of short-lived proteins. UBR2 Histone H2A UBR2 is an E3 ubiquitin ligase that has been shown to ubiquitinate histone H2A, resulting in transcriptional silencing. UBR2 is also part of the N-end rule proteolytic pathway. UHRF2 PCNP UHRF2 is also known as NIRF. UHRF2 is a nuclear protein that may regulate cell cycle progression through association with Chk2. UHRF2 also ubiquitinates PCNP and has been shown to play a role in degradation of nuclear aggregates containing polyglutamine repeats. VHL HIF-1.alpha. VHL is the substrate recognition component of the ECV (Elongin B/C, Cullen-2, VHL) E3 ubiquitin ligase complex responsible for degradation of the transcription factor HIF-1.alpha.. Ubiquitination and degradation of HIF-1.alpha. takes place only during periods of normoxia, but not during hypoxia, thereby playing a central

role in the regulation of gene expression by oxygen. WWP1 ErbB4 WWP1 is an E3 ubiquitin ligase commonly found to be overexpressed in breast cancer. WWP1 has been shown to ubiquitinate and degrade ErbB4. Interestingly, the WWP1 homolog in C. elegans was found to increase life expectancy in response to dietary restriction. WWP2 Oct-4 WWP2 is an E3 ubiquitin ligase that has been shown to ubiquitinate/ degrade the stem cell pluripotency factor Oct-4. WWP2 also ubiquitinates the transcription factor EGR2 to inhibit activation-induced T cell death. ZNRF1 ZNRF1 is an E3 ubiquitin ligase highly expressed in neuronal cells. ZNRF1 is found in synaptic vesicle membranes and may regulate neuronal transmissions and plasticity.

[0211] The term "deubiquitinating" enzyme refers to an enzyme that cleaves ubiquitin from proteins.

[0212] According to a specific embodiment, the deubiquitinating enzyme is a cysteine protease or a metalloprotease.

[0213] Exemplary deubiquitinating enzymes which may be expressed in the system include USP7 that is known to deubiquitinate MDM2, USP47, USP2, USP7, USP15, USP9X, USP28, USP30.

[0214] The ubiquitinating or deubiquitinating enzymes may be expressed from the same expression constructs as the substrate and the ubiquitin or on separate constructs.

Substrates

[0215] Examples of substrates include polypeptides that are known to be ubiquitinated in vivo in humans by E3 ligase or deubiquitinated in vivo by deubiquitinating enzymes.

[0216] According to a specific embodiment, the substrate is one that is known to be ubiquitinated differentially in a disease such as cancer.

[0217] Exemplary substrates that may be expressed in the bacteria have been described herein above.

[0218] According to a particular embodiment, the substrate is selected from the group consisting of PHD3, SPROUTY2, Mitofusin 1, 2, MIRO, NEMO, SMADs, T.beta.R-I, P53, S5A, HHR23, EPHEXIN5, ARC, PPAR.alpha., cyclin-B, Cdc25C and Calmodulin.

[0219] It will be appreciated that as well as expressing the substrate and the ubiquitin (together with the split reporter polypeptide), the recombinant bacteria should also express the ubiquitinating or deubiquitinating enzyme.

[0220] In one embodiment, the bacteria express at least one E1 enzyme, at least one E2 enzyme and at least one E3 enzyme.

[0221] Preferably, the bacteria expresses the E2 enzyme that is a cognate pair for the E3 enzyme.

[0222] In another embodiment, the bacteria expresses at least one deubiquitinating enzyme.

[0223] An exemplary system is illustrated in FIG. 1A, whereby the E1 and E2 enzyme are expressed from the same construct as the ubiquitin and the E3 enzyme is expressed from the same construct as the substrate.

[0224] In another embodiment, the E1 and E2 enzyme are expressed from the same construct as the substrate and the E3 enzyme is expressed from the same construct as the ubiquitin.

[0225] In still a further embodiment, the E1 and E2 enzyme are expressed from the same construct as the substrate and/or the ubiquitin and the E3 enzyme is expressed from an additional expression construct.

[0226] The additional expression construct from which the E3-ligase is expressed may use a different selection marker as that used for the other constructs (e.g. Amp.sup.R selection marker). It may also use a different origin of replication such as p15A. The promoter for this expression construct may be inducible or constitutive. In one embodiment, the promoter is a weak constitutive promoter such as the pTac promoter which is leaky without the addition of inducer (IPTG).

The present inventors contemplate using a kit for easy preparation of the expression constructs.

[0227] Such a kit may comprise:

[0228] (i) a first polynucleotide which encodes a first polypeptide fragment which is operably linked to a bacterial regulatory sequence, and a cloning site, wherein a position of the cloning site is selected such that upon insertion of a sequence which encodes a test polypeptide (i.e. the substrate) into the cloning site, following expression in a bacterial cell, a fusion protein is generated which comprises the test polypeptide in frame with the first polypeptide fragment; and

[0229] (ii) a second polynucleotide comprising a second nucleic acid sequence encoding a second polypeptide fragment which is attached to ubiquitin, the second nucleic acid sequence being operably linked to a bacterial regulatory sequence, wherein the first polypeptide fragment associates with the second polypeptide fragment to generate a reporter polypeptide (e.g. selectable polypeptide, as further described herein above) dependent on ubiquitination of the test polypeptide.

[0230] The first polynucleotide and the second polynucleotide may be on the same expression vector or on a separate expression vector. If present on different expression vectors, then preferably each polynucleotide sequence comprises a bacterial origin of replication.

[0231] The kit may comprise a third polynucleotide which encodes at least one ubiquitinating enzyme. Alternatively, the first polynucleotide and/or the second polynucleotide may comprise a sequence which encodes the ubiquitinating enzyme (e.g. E1, E2 and/or E3).

[0232] Once an agent has been identified as a regulator of a ubiquitinating or deubiquitinating enzyme, therapeutic potential thereof may be tested using other known in-vitro tests. The candidate agent's therapeutic potential may also be tested in animal models the related disease (e.g. cancer).

[0233] Once its therapeutic potential has been corroborated, pharmaceutical compositions comprising same may be synthesized.

[0234] It will be appreciated that the system described herein can be manipulated to determine whether an enzyme is capable of ubiquitinating a test substrate.

[0235] In this embodiment, the enzyme is expressed in a bacterial cell, as well as the ubiquitin (which is attached to the first polypeptide fragment (as described herein above) and the test substrate (which is attached to the second fragment (as described herein above). The method may be used to test many different E3 enzymes in various combinations with E2 and E1.

[0236] The method proceeds by analyzing for the presence of the reporter polypeptide in the bacterial cell--a presence or amount of the reporter polypeptide is indicative that the enzyme is capable of ubiquitinating the test substrate.

[0237] In another aspect, the system described herein can be used to determine whether an enzyme is capable of ubiquitinating a test substrate.

[0238] The method of this aspect of the present invention comprises: (a) expressing a plurality of candidate polypeptide substrates in a bacterial cell population, wherein each of the candidate polypeptide substrates is attached to an identical first polypeptide fragment (as described herein above);

[0239] (b) expressing the ubiquitinating enzyme in the bacterial cell population;

[0240] (c) expressing ubiquitin in the bacterial cell population, wherein the ubiquitin is attached to a second polypeptide fragment (as described herein above), wherein the second polypeptide fragment associates with the first polypeptide fragment to generate a reporter polypeptide on ubiquitination of the substrate; and

[0241] (d) analyzing in bacterial colonies of the bacterial cell population for a presence or absence of the reporter polypeptide, wherein a presence of the reporter polypeptide is indicative of expression of a substrate for the ubiquitinating enzyme.

[0242] As used herein the term "about" refers to .+-.10%

[0243] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

[0244] The term "consisting of" means "including and limited to".

[0245] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0246] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0247] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0248] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0249] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0250] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0251] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0252] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

[0253] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0254] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, biophysical, bacterial genetics and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Materials and Methods

Plasmid Construction

[0255] Selection system for ubiquitination: Two sets of E. coli-compatible expression vectors were constructed. Ubiquitin and different substrates were fused to two fragments of the murine DHFR, under control of a constitutive lambda phage promoter pLtetO1 (see FIG. 1A).

[0256] The pND-Ub vector is based on a modified pZE21 vector, contacting the pLtetO1. The mouse cDNA of the nDHFR fragment (residues 1-108) was PCR-amplified and sub-cloned between the KpnI and PacI sites (the PacI site was inserted into the vector by PCR) into the pZE21 vector. Then a flexible linker was constructed using a two-step PCR method and digested with PacI and NotI (the NotI site was inserted into the vector by PCR) and cloned into the vector downstream to the pLtetO1-nDHFR. Ubiquitination apparatus cassettes containing a His.sub.6-Ub-E2-E1 were PCR-amplified from pGEN plasmids expressing different E2s or Ub mutants and sub-cloned as fusion to the C-terminus of the nDHFR-linker1 into the NotI and AvrII endonuclease recognition sites.

[0257] The pCD-Sub vector was constructed based on a pCDF-duet vector. The T7 promoter was substituted with pLtetO1. The cDHFR fragment (residues 109-187) was PCR-amplified and subcloned under the pLtetO1 promoter at the MluI and AscI endonuclease recognition sites (the AscI site was inserted by PCR). A second linker was fused to the N-terminus of MBP and cloned into the AscI site. Substrates were PCR-amplified and sub-cloned into SacII and SpeI sites downstream and in-frame with the cDHFR-linker2-MBP. In some vectors the MBP was removed. Some vectors were prepared by complete chemical synthesis assembly.sup.48.

[0258] Cloning into expression, purification and detection vectors: The ENTH domain of zebrafish Epn1.sub.(18-157) and the dsUIM of human Hrs.sub.(257-276) were subcloned into the pGST-parallel2 vector between BamHI and EcoRI endonuclease recognition sites. The ENTH domain of yeast Ent1.sub.(1-152) was subcloned into the pCDF-duet vector fused to His.sub.6-MBP as previously described (pCOG21.sup.17). For detection of ubiquitination in bacteria, yeast Sem1 was subcloned into pCDF-duet vector fused to His.sub.6-MBP. For yeast expression and purification His.sub.6-Sem1 was subcloned into pGREG600 by recombineering that also removed the GFP from the vector.

[0259] The structure and sequences of all vectors were confirmed by restriction endonuclease and DNA-sequencing analyses.

[0260] Site-directed mutagenesis: Point mutations were introduced using the ExSite approach (Stratagene). The entire vector was amplified using Phusion DNA polymerase. Parental DNA was digested by DpnI and the DNA was blunt ligated. All mutants were sequenced to ensure that the desired mutations were introduced and that no other mutations occurred.

[0261] Ubiquitination-dependent E. coli growth assay: E. coli W3110 (from Ezra Yagil laboratory at TAU) were co-transformed with the pND-Ub and the pCD-Sub plasmids and plated on LB agar supplemented with 34 .mu.g/ml kanamycin and 25 .mu.g/ml streptomycin. 5 ml of liquid LB medium supplemented with the same antibiotic concentrations were inoculated with a single colony and left to grow overnight at 37.degree. C. The culture was harvested and washed twice with 5 ml of minimal Davis medium. The optical density (OD.sub.600 nm) was measured and adjusted to 0.2. Two and a half microliters of the diluted cultures from each sample were spotted on agar Davis plates containing 0, 0.5, 5, 10, 20 or 50 .mu.g/ml of TRIM. The plates were incubated for 2-3 days at 30.degree. C. and photographed with a UV camera in identical conditions. Each spotting assay was repeated at least 6 times.

[0262] Selection experiments in solution growth media: Overnight cultures in LB medium (supplemented with 30 .mu.g/ml kanamycin and 25 .mu.g/ml streptomycin) were harvested and re-suspended in Davis minimal medium. Diluted cells (OD.sub.600, 0.2) were grown at 30.degree. C. in 96-well plates containing 0.2 ml Davis medium supplemented with 0, 1, 5, 7, 10 and 12 .mu.g/ml TRIM and with or without 100 .mu.M Pyr-41. Growth rates were monitored by measuring the optical density (OD.sub.595) using a microplate spectrophotometer. Doubling time was calculated for early logarithmic growth (OD.sub.595 between 0.02 and 0.2). All experiments were performed at least 9 times (n=9).

Genetic Selection Assays for Characterization of Structural Based Mutants

[0263] Data collection: E. coli W3110 expressing the pND-Ubs, pCD-Subs (and sometimes also an Ampicillin resistance plasmid that constitutively expresses E3-ligase) grew to logarithmic phase at 37.degree. C. in 5m1 of LB medium supplemented with 23 .mu.g/ml Kanamycin, 16 .mu.g/ml Streptomycin and 33 .mu.g/ml Ampicillin. The culture was harvested and washed once with 5 ml of minimal Davis medium. The bacterial density was adjusted to OD600nm value of 0.3. Culture samples (2.5 .mu.l each) were spotted on Davis agar Petri dishes containing 10 .mu.g/ml trimethoprim. Culture in each experiment was spotted usually three to four times. Most experiments were repeated at least three to four times (therefore 9<n>16). Time-lapse (30 or 60 minutes) scanning took place in 26.degree. C. incubator using a regular A4/US-letter office scanner (Epson Perfection V37).sup.40.

[0264] Image analysis: Images were read into Fiji.sup.41 as a stack using `import->image sequence`. The spots density were measured using the Time Series Analyzer V3 (Balaji J 2007; a Java ARchive ImageJ/Fiji plugin that can be downloaded and installed from www(dot)rsb(dot)info(dot)nih(dot)gov/ij/plugins/time-series(dot)html). Regions Of Interest (ROIs) were specified (typically as 20.times.20 ovals) of and their total intensities (bacterial densities) were integrated and plotted where `Z-axis` is the image time index. Similarly, the background was measured and calculated and subtracted from the collected data. Logistic regressions of growth curves were calculated using Origin. A single parameter that describes growth efficiency was calculated as follows: the growth curve slope at the `half max density` was extracted and divided by its time index.

[0265] Protein purification: Proteins were purified from E. coli using affinity tags as previously described.sup.17. For crystallization purposes, proteins were concentrated to 5-20 mg/ml using centricon (Amicon Ultra), in a final solution of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5 mM dithiothreitol (DTT).

[0266] Western-blot analysis: Following separation on SDS-PAGE, samples were transferred onto a nitrocellulose membrane and incubated with rabbit anti-His epitope tag antibody (1:20 000 dilution, Rockland) or mouse anti-GST antibody (1:200, Santa Cruz), and infrared dye coupled goat anti-mouse secondary antibody (1:12 000, LI-COR). Blots were scanned using an Odyssey system (LI-COR Biosciences) at 700 and 800 nm.

[0267] Crystallization and data collection: Crystallization and data collection and processing of yeast Ent1-ENTH have been reported.sup.60. Purified Zebrafish Epn1-ENTH was concentrated to 10 mg/ml and was crystallized in 0.1M KPO.sub.4 pH 7.0 and 19% PEG 3,350 at 20.degree. C. Crystals were cryo-protected with 25% ethylene glycol and were frozen in liquid nitrogen. Data were collected at the ID29 beamline (ESRF, Grenoble) and processed with HKL2000 software.sup.61

[0268] Structure determination and refinement: Structure of ENTH domains were solved by molecular replacement (MR); 1H0A.sup.37 was used for searching model with PHASER.sup.62. To facilitate the MR search, the first 17 residues were removed and alanine reduction was exerted to the non-conserved residues. Model building and refinement were carried out with PHENIX.sup.63, Refmac5.sup.64 and COOT.sup.65.

[0269] Ubiquitylated-Rpn10 was purified from E. coli and crystallized as previously described 5, 35. It was found that the extremely thin crystals (1-3 .mu.m) were highly sensitive to radiation damage. BEST software was used, which precisely predicted an efficient data-collection strategy to achieve a full completeness dataset for these C2 space-group crystals, with only 95 images at oscillation steps of 1.75.degree.. Data were collected at the ID14-4 beamline (ESRF) at wavelength of .lamda.=0.93930 .ANG. under cryo conditions of 100.degree. K. The structure was determined by molecular replacement using PHASER 42, where the Rpn10-vWA domain from S. pombe (PDB 2X5N) and Ub (PDB 1UBQ) 41 were used as initial search models. PHENIX auto-build was used to provide an initial model. Model-building and refinement were carried out with PHENIX 43, Refmac5 44 and COOT 45. The geometry parameters of the isopetide bond were restrained within the refinement process in PHENIX. The structure was validated with PROCHECK 46 and the PDB validation tool. Statistics of Ramachandran analysis yielded 98.7% of the residues were found in the most favored or additional allowed regions and 1.3% were found in the generously allowed regions. None of the residues were found in disallowed regions. It was found that the linker tethering the vWA domain to the UIM has an intrinsic propensity for cleavage at the beginning of the hinge. Indeed, the linker spontaneously clipped-off during crystallization. Consequently, the crystallized protein contained the vWA domain (residues 1-191) and its conjugated Ub at K84. Interestingly, the structures of apo Rpn10 from S. cerevisiae and from S. pombe.sup.18, 24 also present truncated forms of the protein at the same location.

[0270] Crosslinking assays: Crosslinking was carried out using 0.5 mM disuccinimidyl substrate (DSS) as described by.sup.66.

[0271] Surface plasmon resonance: Purified His.sub.6-MBP-ENTH or mutants were immobilized on an Ni.sup.+2 chip at a density of .about.800 U. Untagged Ub (analyte) was injected at a flow rate of 20 ml per min in 150 mM NaCl, 10 mM HEPES pH 7.0 and 0.010% poly-sorbate 20 at 24.degree. C.; 500 mM imidazole was used for surface regeneration. To avoid immobilization of aggregated analyte the purified Ub was chromatographed by gel filtration and briefly sonicated immediately before SPR experiments. Data were processed with BioEvaluation and fitting carried out as a single-site-binding model with OriginLab. Standard errors derived from at least three samples.

[0272] SPR analysis of Rpn10-vWA:Ub and mutants non-covalent interactions. The experimental set-up comprised .alpha.-GST antibody immobilized on a CMS chip according to the manufacturer's protocol (GE Healthcare). The ligands (Rpn10 derivatives) were expressed and purified as GST-fusion-proteins and captured on the chip. Free mono-Ub was the analyte. Prior to each experiment the ligand and the analyte proteins were subjected to size exclusion chromatography. Each measurement was taken in triplicate. The experiment comprised 90-100 seconds for binding, 300-350 s for dissociation. Wild-type and mutant Ub analytes were injected at a flow rate of 10 ml/min in 10 mM HEPES pH 7.0, 150 mM NaCl, and 0.005% polysorbate-20 at 25.degree. C. At the end of each experiment, the ligands were removed and surface regeneration was achieved by flowing 10 mM glycine-HCl pH 2.6, followed by sequential washing steps of 1-2 min with 0.1% SDS, 10 mM NaOH. Data were processed using Biacore BIAevaluation software. A single-site-binding model was used for curve fitting of the binding data (Sigma Plot). For plotting, data were scaled such that Rmax=100.

[0273] Detection of ubiquitination in vivo: pScHis.sub.6-Sem1 plasmid was transformed into the SEY6210, rsp5::HIS3, pDsRed415-rsp5.sup.WT and rsp5-1 (rsp5.sup.L733S) MATa ura3-52, his3-200, trp1-901, lys2-801, suc2-9, leu2-3 strains and grew at 26 in 50 ml of YPD medium (2% glucose) supplemented with 200 .mu.g/ml G418. At log phase the cultures were harvested and the pastes were washed with DDW and transferred to 50 ml YPD medium (2% galactose) supplemented with 200 .mu.g/ml G418. Each culture was divided into two flasks and grew at permissive (26.degree. C.) and restrictive (37.degree. C.) temperatures for additional 4 hours. The cultures were harvested and lysed in 2 ml cold 1.85 N NaOH, 7.5% .beta.-mercaptoethano for 10 min on ice. Proteins were precipitated with the addition of half volume of 50% TCA (final concentration of 25% TCA) and collected by centrifugation (18,000 rpm 20 min). The pellet was resuspended and washed with 3 ml ice-cold 80% acetone, and collected by centrifugation (18,000 rpm 5 min). Then the pellet was re-suspended in 1.5 ml resuspension buffer (6 M guanidine HCl, 100 mM Tris-HCl, 100 mM NaCl, 0.1% triton X-100, pH 8.8) and incubated at 25.degree. C. for 1 hour while rotating. The protein fraction incubated 20 min at 4.degree. C. with 70 .mu.l Ni+2 resin and washed twice with resuspension buffer followed by two washes with buffer 2 (8M urea, 100 mM Tris-HCl pH 8.8, 100 mM NaCl, 0.1% triton X-100) and two washes with buffer 3 (50 mM Tris-HCl pH 7.5, 150 mM NaCl). The Ni+2 beads were boiled in Laemmli buffer and separated by SDS-PAGE followed by western blot analysis with anti-His6 antibody.

[0274] Library construction: Yeast GST-tagged ORFs (GE collection) were pooled from 384 pins plated cultures and plasmids were isolated as a pool. The GST fusion genes were PCR amplified. The PCR products were size fractionated (350 to 3000 bp) by gel electrophoresis followed by PCR purification using PureLink, kit (Invitrogen) and sub-cloned in-frame with cDHFR to the pCD-Sub vector by recombineering. The resulted library was transformed to DH5.alpha. cells and kept in -80.degree. C. Isolated plasmids of the library were transformed to W3110 competent cells that contained the pND-Ub and constitutively expressed E3-ligase plasmids. Followed transformation the bacteria were plated on selective media.

[0275] Accession codes: Atomic coordinates and structure factors for the crystal structures of Saccharomyces cerevisiae Ent1-ENTH and Danio rerio Epn1-ENTH domains have been deposited in the Protein Data Bank under ID codes rcsb079376 and rcsb074441 respectively.

Results

Construction of a Selection System for Ubiquitination Events in Bacteria

[0276] A system was generated for genetic screening of ubiquitination events in E. coli. In this system, two fragments of a split reporter gene are tethered to Ub and a ubiquitination substrate and are co-expressed along with ubiquitination apparatus in E. coli (FIG. 1A). Specifically, the N-terminal fragment of murine dihydrofolate reductase (nDHFR) was fused to the N-terminus of Ub (in a plasmid denoted pND-Ub) and the C-terminal fragment of DHFR (cDHFR) to the N-terminus of the substrate (in a plasmid denoted pCD-Sub). Pending on substrate ubiquitination, the two DHFR fragments are assembled into a functional enzyme conferring antibiotic [trimethoprim (TRIM)] resistance and growth on selective media.sup.19. The DHFR fragments were tethered to Ub and to the substrate with long linkers designed to confer flexible but stable characteristics facilitating the functional assembly of the reporter (FIG. 9). The system has a polycistronic architecture in which ubiquitination apparatus and the substrate are co-expressed from two or three compatible vectors (FIG. 1A). Synthetic operons are expressed from a constitutive promoter [an unregulated .lamda.-phage left promoter (pL)], suitable for bacterial genetic studies.

[0277] To assess the functionality of the system, the well-characterized Ub-receptor Vps9.sup.15-17 was fused to cDHFR (pCD-Sub) and co-expressed together with Rsp5 (E3) and the pND-Ub vector that expresses E1 and yeast Ubc4 (E2). It was found that the W3110 strain provided the best genetic background under the described experimental conditions. Bacteria expressing a complete ubiquitination system for Vps9 grew under both permissive and non-permissive conditions (FIG. 1B). However, strains lacking Ub or E1/E2 did not grow on selective media. To demonstrate that the developed system maintains the known E3:substrate specificity, Rsp5 was replaced with Siah2, a non-cognate E3-ligase. Bacteria grew only when the cognate E3 was expressed (FIG. 1C), even though Siah2 was functional in the selection system, as it promoted self-ubiquitination (FIG. 2B).

[0278] The split DHFR system was originally developed for identification of non-covalent protein-protein interactions.sup.19, suggesting that the selection system may detect non-covalent UBDs:Ub interactions. Since UBDs present a fairly low affinity to Ub.sup.14, different antibiotic concentrations were tested to identify suitable growth conditions for Vps9:Ub non-covalent interactions. Although Vps9 presents one of the highest UBD:Ub measured affinities.sup.15, 16, growth was found only at antibiotic concentration of 0.1 .mu.g/ml (FIG. 1D). Since such antibiotic concentration is limited for selection, it seems that the developed system is selective only for ubiquitination events.

The Ubiquitination Selection System Facilitates Identification and Characterization of E3s

[0279] Most E3-ligases undergo self-ubiquitination.sup.20. The developed selection system thus provides a straightforward tool for their identification and characterization as self-ubiquitination is predicted to confer antibiotic resistance. Yeast Rsp5 and human Siah2, representatives of the HECT and the RING ligases, respectively, were fused to cDHFR. Growth phenotypes were observed under non-permissive conditions, only when all the required ubiquitination components were co-expressed (FIGS. 2A-2B).

[0280] Siah2 inhibition by menadione may play an important role in cancer therapy.sup.21. As the engineered bacteria became addicted to self-ubiquitination of Siah2, its inhibition was predicted to present growth arrest phenotype. FIG. 2B (right) shows a growth arrest phenotype in the presence of menadione only under the addictive conditions. Similarly, Cdc34, a non-cognate E2, did not support growth. The developed system could thus be employed for screening of potential drugs. It was suggested that some E3s use one E2 to attach the first Ub and a different E2 for building the Ub-chain.sup.22. The present system may facilitate the identification of such E2s.

Identification of a Novel E3-Ligase and Its Cognate E2s

[0281] To demonstrate the system's ability to identify a novel E3-ligase, the present inventors focused on a U-box family from the pathogenic enterohaemorrhagic E. coli (EHEC). They used the well-characterized human ligase CHIP as a probe in a PSI-BLAST search against the EHEC proteome. The search retrieved an uncharacterized sequence, ECs3488, as a potential ligase containing a conserved 35-amino-acid sequence with 22% identity to a helix-loop-helix-beta region of the CHIP U-box domain.sup.23 (FIG. 2C). ECs3488, also named NleG6-3, has been postulated as an E3-ligase, but its expression and function have never been demonstrated.sup.24, 25. ECs3488 was cloned as a fusion with cDHFR and screened against a full yeast library of E2s, resulting in the identification of Ubc4/5 as cognate E2s for the putative E3-ligase (FIG. 2D). Then, the ligase functionality was examined with the human E2 orthologs UbcH5B/C (FIG. 2E).

[0282] Based on the structures of NleG2-3 and of the CHIP:UbcH5a complex (PDB 2KKX and 20XQ).sup.24 structural model for the ECs3488:E2 interface was built (FIG. 2F) and the selection system was used to assess the model. The results corroborated the structural model, as the ECs3488:E2-binding mutants presented significant growth arrest phenotypes (FIG. 2G).

Selection Approach for Identification and Characterization of UBDs

[0283] Ubiquitin binding domains (UBDs) usually bind mono-Ub with low affinity, ranging from 2 .mu.M to 2100 .mu.M.sup.15, 26, posing a challenge to biochemical and biophysical studies. The selection system stabilizes the dynamic and weak UBD:Ub non-covalent interactions by forming a covalent bond between Ub and Ub-receptor (i.e. ubiquitination) in the bacteria. The present inventors assessed the ability of the system to sense low affinity UBD by tethering the double-sided UIM (dsUIM) of Hrs as substrate.sup.27, 28 (FIG. 3A). The structures of Hrs:STAM (also known as ESCRT-0 complex) and particularly its dsUIM:Ub complex, were determined and facilitated detailed molecular assessment.sup.29, 30. The E3-independent self-ubiquitination of most Ub-receptors and the promiscuous function of Ubc4/5 subfamily members aided in the analysis. Available lysine residues for ubiquitination do not necessarily need to be part of the UBD as was demonstrated for several UIM and UBA proteins.sup.31. Alanine residues at each face of the dsUIM were demonstrated to interact with the Ub 144 hydrophobic patch (FIG. 3B). Indeed, it was found that the A266Q, A268Q double mutant presented a growth arrest phenotype. Similarly, I44-patch mutant abolished bacterial growth. The phenotype of Ub-G76R demonstrated that growth is also dependent on ubiquitination. Furthermore, Western-blot analysis showed that a covalent bond between Ub and Hrs is generated (FIG. 3C). Finally, it was demonstrated that the growth is dependent on a functional ubiquitination apparatus by omitting the E1/E2 enzymes (FIG. 3A) or by administration of the E1 inhibitor, PYR-41.sup.32 (FIG. 3D).

[0284] The system's functionality with other structurally different UBDs, including the proteasomal receptor Rpn10, human STAM1-UIM and ALIX-V domains.sup.33 and the yeast Hse1-VHS domain.sup.26, that together are involved in multivesicular and retroviruses budding, was furthered verified/validated (FIGS. 3E-3H).

[0285] Similar to ESCRT-0 (STAM, HRS and Hse1) components, GGAs proteins also utilize UBDs to transport ubiquitylated-cargo from Golgi to the multivesicular body. An affinity of 2100 .mu.M was demonstrated to the GGA3-VHS:Ub complex.sup.26. Critical tryptophan and leucine residues were identified in STAM1 and other VHS domains that bind Ub at the 144 patch. Moreover, the VHS domains of GGA1 and 2, which naturally lack the critical leucine, do not bind Ub. It was demonstrated that despite the weak affinity, the selection system distinguished between these phenotypes (FIG. 3I).

[0286] One benefit of a bacteriostatic antibiotic like trimethoprim is that it enables the accumulation of functional DHFR assemblies, due to the ubiquitination, up-to a threshold level that is sufficient to confer resistance while not harming the bacteria. Thus, the developed system may provide a super-sensitive readout for genetic identification and characterization of potential UBDs, without the need to purify them.

ENTH is a UBD

[0287] The present inventors next sought to challenge the system to detect a novel ultraweak-affinity UBD. ENTH domains assume a similar fold to that of VHS.sup.34. Moreover, Epsin proteins possess a similar architecture to the Ub-receptors Hrs, STAM and GGAs by harboring VHS/ENTH, two Ub binding patches (dsUIM or 2xUIM or GAT domains) followed by a long flexible linker containing endocytic machinery binding elements including a clathrin-binding box (FIG. 10). Therefore, although ENTH probably lacks the critical tryptophan or leucine residues.sup.26, it was speculated that it too binds Ub. The selection system was employed and it was demonstrated that cDHFR-ENTH domains of yeast and zebrafish promote growth on selective media in an E3-independent manner (FIGS. 4A-4B). This suggests that these ENTH domains directly bind Ub.about.E2. Indeed, biochemical crosslinking assays and purification/detection of ubiquitylated yeast Ent1 derivatives from E. coil.sup.17 strongly support the genetic data suggesting that ENTH directly binds Ub (FIGS. 11A-11E).

Structure of ENTH Domain Provides Insight Into Mechanism of Ub Recognition

[0288] To obtain high resolution information on the ENTH:Ub interaction, the present inventors tried to crystalize the complex. Probably due to the ultraweak affinity (see quantification below), only crystals of apo ENTH or Ub were obtained. The the structures of yeast (Sc_ENTH) and zebrafish (Zf_ENTH) domains were determined by molecular replacement to 1.95 .ANG. and 1.41 .ANG. resolutions respectively (Table 3 and FIGS. 4C-4J).

TABLE-US-00004 TABLE 3 Crystallographic Data and Refinement Statistics Construct Z.f ENTH S.c ENTH Data Collection X-ray source ESRF ID29 ESRF ID29 Wavelength (.ANG.) 0.978 0.978 Space group P1 P 21 21 21 Cell dimensions a, b, c, (.ANG.) a = 42.364, b = 51.724, a = 32.69, b = 35.46, c = 64.639 c = 110.62 .alpha., .beta., .gamma. (.degree.) .alpha. = 89.333, .beta. = 76.373, .alpha. = .beta. = .gamma. = 79.737 .gamma. = 90 Resolution (.ANG.) 1.4-62.79 (1.409-1.425 ).sup.a R-merge.sup. (%) 7.5 (3.2) 0.166 I/.sigma.I 5.3 (4.4) 6.79 (1.50) Completeness (%) 94.0 (88.97) 99.18 (99.58) Redundancy 3.4 (3.0) Observed reflections 325,868 37572 Unique reflections 95136 (9256) 9806 (1429) Refinement Resolution 1.41-62.79 .sup.a(1.41-1.425) 1.95-55.42 .sup.a(1.95-2.0) R.sub.work/R.sub.free 0.1545/0.2015 0.1862/0.2364 Number of atoms 6022 1254 Protein 5189 137 Ligand/ion 10 Water 823 97 average B factor 17.5 19.00 (.ANG..sup.2) Solvent (%) 28.83 29.40 r.m.s. deviations Bond lengths (.ANG.) 0.009 0.018 Bond angles (.degree.) 1.134 1.30 Ramachandran (%) Favored 94.1 96 Additional allowed 5.7 4.0 Outliers 0.2 0.0 Disallowed 0.0 0.0 PDB code 5LP0 5LOZ .sup. R-merge = .SIGMA.hkl .SIGMA.i | Ii(hkl) - (I(hkl)) |/.SIGMA.hkl .SIGMA.i Ii(hkl), where Ii(hkl) is the intensity of the ith observation of reflection hkl and <I(hkl)> is the average intensity of reflection hkl. .sup.aOuter resolution shell

[0289] High-quality electron density maps (FIGS. 4C-4D) showed that although these structures are highly similar, apparent differences can be seen in the loop tethering helices-3 and 4 and the angle between the superhelix structure and helix-8 (FIGS. 12A-12B).

[0290] To generate a structural model of the ENTH:Ub complex, the ENTH domains were superimposed onto the STAM1-VHS:Ub complex.sup.26, 35, 36 The models showed that the STAM1 W26 and L30 Ub-binding residues were naturally substituted with S37 and S41 in Zf_ENTH and K36 and 140 in Sc_ENTH. (FIGS. 4C-4J). Intriguingly, these models suggest that Ub R42 and R72 form electrostatic interactions with E42 and D45 or E41 and E44 of Zf_ENTH and Sc_ENTH, respectively. Both models predicted additional interactions that seemed to contribute little to the binding.

[0291] Intriguingly, superimposing the ENTH:Ub model onto the structure of ENTH complex with the membrane lipid phosphatidylinositol-4,5-bisphosphate.sup.37 shows that ENTH binds Ub and the membrane lipid at opposite sites (FIG. 13), suggesting that ENTH can bind Ub while associated with the membrane. Similarly, superimposing the VHS:Ub complex onto the VHS:M6PR-tail (the acidic-cluster-dileucine sorting signal of the mannose-6-phosphate receptor) complex.sup.38, 39, shows the same phenomenon, suggesting that both VHS and ENTH domains can recognize ubiquitylated-transmembrane-cargo while associated with the membrane.

Genetic Approach for Structural Validation of the ENTH:Ub Interface

[0292] ENTH domains lack the critical Trp or Ile, suggesting that cumulative interactions of peripheral residues compensate for its role in Ub binding. To evaluate the contributions of specific residues to the ENTH:Ub interaction, point mutations were introduced at the predicted interface. The growth rates were monitored by time-lapse scanning the bacteria using a simple A4/US-letter scanner with a modified control software.sup.40. To quantify the growth rates, a simple Fiji.sup.41 based time series analyzer procedure was applied to measure a typical stack of 100-200 scans (see details in the Materials and Methods section) by means of optical density (FIGS. 5B-5F).

[0293] Ala mutagenesis or exchanging the charge of the acidic residues of Zf_ENTH demonstrated growth arrest phenotypes (FIGS. 5A, 5C, 5D). A similar phenotype, though less severe for the Ub R42E, R72E mutant, was found when the acidic residues of the yeast protein were mutated, suggesting slight structural differences between these complexes. Remarkably, a permutation cycle in which the Zf_ENTH E42 and D45 residues were replaced with arginine residues, and Ub R42 and R72 were replaced with glutamic residues, restored growth (FIG. 5D). This result reflects the accuracy level of the structural model and the high sensitivity of the selection system. Moreover, the Ub R42E, R72E mutant could not suppress the Zf_ENTH E42A, D45A mutant, signifying the importance of these electrostatic interactions. The Zf_ENTH S37G and Y83A mutants, predicted to provide lesser contributions to the binding, indeed yielded minor but significant growth phenotypes (FIG. 5A). Taken together, the results of this mutational analysis strongly corroborate the structural model and demonstrate the power of the developed system to detect and quantify relatively minor differences in protein-protein interactions along Ub pathways.

[0294] The yields of ubiquitination of the Sc_ENTH wild-type and mutant proteins were biochemically quantified (FIGS. 5G-5H). The ENTH E41R, E44R and the Ub R42E, R72E double mutants significantly reduced the ubiquitination yield by about 60-80%.

[0295] Finally, to biophysically corroborate the data and to quantify the affinity of the ENTH:Ub complex, Surface Plasmon Resonance (SPR) measurements were performed with immobilized Sc_ENTH and free mono-Ub (FIG. 5I). Ultraweak binding with K.sub.d of 2,300 .mu.M was found for the wild-type complex. This result is compatible with our model and previous measurements of homologous VHS:Ub complexes in Hrs, Vps27 and GGA3, which present affinities of 1,400, 1,500 and 2,100 .mu.M, respectively.sup.26. Interestingly, the Ub R42E, R72E or Sc_ENTH E41R, E44R mutants showed saturation binding curves that could be fit to a single binding model (FIG. 5I and FIGS. 14A-14C) with respective estimated affinities 2.6 fold and 3.5 fold lower than that of the wild-type. Notably, for these mutants, the Ub (analyte) concentrations were too low to obtain accurate K.sub.d values, as reflected in the high standard errors. Together, the correlation between the SPR measurements, the genetic and biochemical data provides a rough estimation for the sensitivity of the selection system in monitoring Ub binding.

[0296] Ultraweak (.about.3,000 .mu.M) protein-protein interactions are significant as they regulate various biological functions.sup.42, 43. Typically, K63-tri-Ub chains constitute the main signal for clathrin-dependent membrane protein trafficking.sup.44-46. Therefore, Ub-receptors decoding this signal usually possess three Ub-binding patches.sup.47 (FIGS. 11A-11E). Avidity and/or cooperative of tandem UBDs render selectivity of these Ub-receptors. The contribution of the VHS domain to the total affinity and selectivity of VHS-UIM proteins has been thoroughly studied26, 35. Therefore, the ultraweak affinity described herein should be considered in the context of full-length Epsin, which contains two additional UIMs.

Identification of Sem1 as Ubiquitination Target of Rsp5

[0297] One of the greatest challenges in the Ub field is to identify association between E3-ligases and their cognate ubiquitination targets (FIGS. 6A-6D). To demonstrate the potential of the developed system to address this challenge, the present inventors constructed a whole genome yeast fusion library in-frame with cDHFR and screened the library against Rsp5. The entire array of yeast GST-tagged ORFs (GE collection) were amalgamated and plasmids were isolated as a pool. The plasmid library was PCR-amplified and the products were subcloned into the developed selection system by Gibson assembly.sup.48. As many Ub-receptors may undergo E3-independent ubiquitination, the present inventors expected to obtain false positive growth of these ORFs, and therefore they employed an assay for E3-independent ubiquitination. They compared the growth rates of the positive colonies with and without Rsp5 using the scanner as described above. Less than 100 positive colonies were identified (from 10 Petri dishes). Most of them showed very similar growth rates in the presence or absence of Rsp5. However, the colony of Sem1 demonstrated significantly higher growth rates when Rsp5 was co-expressed. Sem1 and its human orthologue DSS 1 (Deleted Split-hand/Split-foot) are involved in critical processes including development, proteasome assembly, DNA repair and cancer.sup.49-53. As DSS1 possesses two conserved UBDs.sup.54, Sem1 was tested to see if it underwent E3-independet ubiquitination. It was found that Rsp5 significantly promoted Sem1 ubiquitination (FIGS. 6A-6B). Moreover, detection of ubiquitylated His.sub.6-MBP-Sem1 from E. coli showed highly similar results (FIG. 6C). Since this is the first report for Sem1 ubiquitination, the present inventors tested if Sem1 undergoes Rsp5 dependent ubiquitination in vivo in yeast. Ubiquitination was detected in yeast extracts of wild-type and a temperature sensitive rsp5 allele, rsp5-1. Expression of galactose dependent His.sub.6-Sem1 at permissive and restrictive temperatures showed that Rsp5 is a bona fide ligase of Sem1, as unlike the wt, the ubiquitination in rsp5-1 was detected at 26.degree. C. but not at 37.degree. C. (FIG. 6D).

Use of Chloramphenicol Acetyl Transferase (CAT) as a Selection Marker

[0298] Based on the crystal structure of CAT.sub.I in its apo and complex with chloramphenicol (PDB accessions 3U9B and 3U9F) the present inventors designed a split-CAT system. To test if the newly designed split-CAT system can identify ubiquitination events, they tethered a ubiquitylation target to the nCAT fragment and Ub to the cCAT fragment (FIGS. 15A-15D). The fused proteins were co-expressed with their cognate ubiquitylation apparatus and bacteria were spotted on selective media (rich agar supplemented with 7-10 mg of chloramphenicol per ml). At first, a simple ubiquitylation cascade was tested consisting of Ub-receptor (a Ub-Binding Domain UBD containing protein) as the ubiquitylation target. Specifically, the Ub-receptor of Hse1-VHS domain tethered to the nCAT was co-expressed with nCAT tethered to Ub. Wheat E1 (Uba1) and the yeast E2 (Ubc4) were also expressed. As shown in FIG. 16A expression of the complete ubiquitylation cascade of VHS domain tethered to the split-CAT system presented growth phenotype. However, when the ubiquitylation enzymes E1 and E2 or Ub were removed growth arrest phenotypes were found.

[0299] To compare the growth efficiency between the split-DHFR vs. the split-CAT systems, a UBE3A (also known as E6AP) Rpn10 ubiquitylation dependent cascade was constructed in both systems. A K>R mutation within UBE3A was constructed which results in a constitutive hyperactive E3-ligase. This ligase was expressed from a 3.sup.rd vector under the regulation of the leaky promoter pTac (i.e. without the addition of IPTG) in bacteria that express Ub, Rpn10, Uba1 (E1) and UBCH7 (E2). FIG. 16B shows a significant difference in growth efficiency between the two systems.

[0300] To demonstrate the system application to study the effect of point mutations an Angelman syndrome (AS) mutation was introduced into UBE3A and bacterial growth was analyzed in wild-type (self-arrested), K>R mutant (hyperactive) and AS mutation on the background of the K>R mutation (FIG. 16C). It was found that the K>R mutation resulted in a higher efficient growth phenotype while the AS mutation resulted in a decreased efficient growth phenotype compared with the WT enzyme. Similarly, the function of UBE3B a HECT E3 Ub-ligase that presents a difficulty in purification in its active from and which is involved in Kaufman Syndrome (KS; Flex et al. 2013) was also assessed in the system. Using the selection system, the critical lysine residue that undergoes self-ubiquitylation that lead to allosteric restrain of the enzyme was identified and a K>R unrestrained mutant was constructed. KS mutation on the background of the UBE3B K>R demonstrated a growth phenotype (FIG. 16D).

[0301] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0302] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

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Sequence CWU 1

1

68133PRTArtificial sequencesynthetic peptide 1Cys Ser Leu Tyr Asp Lys Asp Thr Leu Val Gln Leu Val Glu Thr Gly1 5 10 15Gly Ala His Pro Leu Ser Arg Glu Pro Ile Thr Glu Ser Met Ile Met 20 25 30Arg233PRTArtificial sequencesynthetic peptide 2Cys Thr Leu Phe Asp Ala Ala Ala Phe Ser Arg Leu Val Gly Glu Gly1 5 10 15Leu Pro His Pro Leu Thr Arg Glu Pro Ile Thr Ala Ser Ile Ile Val 20 25 30Lys333PRTArtificial sequencesynthetic peptide 3Cys Thr Leu Phe Asp Ala Ala Ala Phe Ser Arg Leu Val Gly Glu Gly1 5 10 15Leu Pro His Pro Leu Thr Arg Glu Pro Ile Thr Ala Ser Ile Ile Val 20 25 30Lys4140PRTArtificial sequencefull length sequence of the NleG6-3 ligase 4Met Glu Arg Arg Ala Val Ala Leu Glu Arg Gln Leu Asn Gly Gly Val1 5 10 15Asp Phe Leu Arg Ser Val Asn Asn Tyr Phe Gln Ser Val Met Ala Glu 20 25 30His Arg Glu Asn Lys Thr Ser Asn Lys Ile Leu Met Glu Lys Ile Asn 35 40 45Ser Cys Val Phe Gly Thr Asp Ser Asn His Phe Ser Cys Pro Glu Ser 50 55 60Phe Leu Thr Cys Pro Ile Thr Leu Asp Thr Pro Ala Asn Gly Val Phe65 70 75 80Met Arg Asn Ser Gln Gly Ala Glu Ile Cys Ser Leu Tyr Asp Lys Asp 85 90 95Thr Leu Val Gln Leu Val Glu Thr Gly Gly Ala His Pro Leu Ser Arg 100 105 110Glu Pro Ile Thr Glu Ser Met Ile Met Arg Lys Asp Glu Cys His Phe 115 120 125Asp Ser Lys Lys Glu Ser Phe Val Ala Ser Asp Ala 130 135 140535PRTArtificial sequenceFlexible linker amino acid sequence 5Leu Ile Lys Ala Ala Gln Arg Ala Arg Glu Ala Glu Arg Asp Leu Ala1 5 10 15Ala Ala Val Ala Gln Ala Ala Ala Gly Gln Ala Val Pro Arg Ala Ala 20 25 30Pro Arg Gln 35629PRTArtificial sequenceFlexible linker amino acid sequence 6Gly Gly Ser Ala Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Gly1 5 10 15Ser Ala Gly Ser Ser Gly Ser Ser Gly Ala Ser Ser Gly 20 25776PRTArtificial sequencemammalian ubiquitin amino acid sequence 7Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70 75875PRTArtificial sequenceyeast ubiquitin amino acid sequence 8Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ser Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser 50 55 60Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70 7591051PRTTriticum aestivum 9Met Leu Pro Arg Lys Arg Glu Ile Val Ala Gly Glu Val Glu Asp Leu1 5 10 15Gln Lys Lys Thr Arg Ala Gly Glu Gly Glu Val Thr Arg Glu Glu Gly 20 25 30Asp Ala Ala Met Ala Gly Arg Gly Asn Glu Ile Asp Glu Asp Leu His 35 40 45Ser Arg Gln Leu Ala Val Tyr Gly Arg Glu Thr Met Lys Arg Leu Phe 50 55 60Gly Ser Asn Val Leu Val Ser Gly Leu Gln Gly Leu Gly Ala Glu Ile65 70 75 80Ala Lys Asn Leu Val Leu Ala Gly Val Lys Ser Val Thr Leu His Asp 85 90 95Asp Gly Asn Val Glu Leu Trp Asp Leu Ser Ser Asn Phe Phe Leu Ser 100 105 110Glu Asn Asp Val Gly Gln Asn Arg Ala Gln Ala Cys Val Gln Lys Leu 115 120 125Gln Glu Leu Asn Asn Ala Val Leu Val Ser Ala Leu Thr Gly Asp Leu 130 135 140Thr Lys Glu His Leu Ser Lys Phe Gln Ala Val Val Phe Thr Asp Ile145 150 155 160Ser Leu Asp Lys Ala Ile Glu Phe Asp Asp Tyr Cys His Ser Gln Gln 165 170 175Pro Pro Ile Ala Phe Ile Lys Ser Glu Val Arg Gly Leu Phe Gly Ser 180 185 190Val Phe Cys Asp Phe Gly Pro Glu Phe Thr Val Leu Asp Val Asp Gly 195 200 205Glu Glu Pro His Thr Gly Ile Val Ala Ser Ile Ser Asn Asp Asn Pro 210 215 220Ala Leu Val Ser Cys Val Asp Asp Glu Arg Leu Glu Phe Gln Asp Gly225 230 235 240Asp Leu Val Val Phe Ser Glu Val His Gly Met Thr Glu Leu Asn Asp 245 250 255Gly Lys Pro Arg Lys Val Lys Asn Ala Arg Pro Tyr Ser Phe Phe Leu 260 265 270Glu Glu Asp Thr Ser Ser Phe Gly Ala Tyr Val Arg Gly Gly Ile Val 275 280 285Thr Gln Val Lys Pro Pro Lys Val Ile Lys Phe Lys Pro Leu Lys Glu 290 295 300Ala Met Ser Glu Pro Gly Glu Phe Leu Met Ser Asp Phe Ser Lys Phe305 310 315 320Glu Arg Pro Pro Leu Leu His Leu Ala Phe Gln Ala Leu Asp Lys Phe 325 330 335Arg Thr Glu Leu Ser Arg Phe Pro Val Ala Gly Ser Thr Asp Asp Val 340 345 350Gln Arg Val Ile Glu Tyr Ala Ile Ser Ile Asn Asp Thr Leu Gly Asp 355 360 365Arg Lys Leu Glu Glu Ile Asp Lys Lys Leu Leu His His Phe Ala Ser 370 375 380Gly Ser Arg Ala Val Leu Asn Pro Met Ala Ala Met Phe Gly Gly Ile385 390 395 400Val Gly Gln Glu Val Val Lys Ala Cys Ser Gly Lys Phe His Pro Leu 405 410 415Tyr Gln Phe Phe Tyr Phe Asp Ser Val Glu Ser Leu Pro Val Asp Pro 420 425 430Leu Glu Pro Gly Asp Leu Lys Pro Lys Asn Ser Arg Tyr Asp Ala Gln 435 440 445Ile Ser Val Phe Gly Ser Lys Leu Gln Asn Lys Leu Glu Glu Ala Lys 450 455 460Ile Phe Met Val Gly Ser Gly Ala Leu Gly Cys Glu Phe Leu Lys Asn465 470 475 480Leu Ala Leu Met Gly Ile Ser Cys Ser Gln Asn Gly Asn Leu Thr Leu 485 490 495Thr Asp Asp Asp Val Ile Glu Lys Ser Asn Leu Ser Arg Gln Phe Leu 500 505 510Phe Arg Asp Trp Asn Ile Gly Gln Pro Lys Ser Thr Val Ala Ala Thr 515 520 525Ala Ala Met Val Ile Asn Pro Lys Leu His Val Glu Ala Leu Gln Asn 530 535 540Arg Ala Ser Pro Glu Thr Glu Asn Val Phe Asn Asp Ala Phe Trp Glu545 550 555 560Asn Leu Asp Ala Val Val Asn Ala Leu Asp Asn Val Thr Ala Arg Met 565 570 575Tyr Ile Asp Ser Arg Cys Val Tyr Phe Gln Lys Pro Leu Leu Glu Ser 580 585 590Gly Thr Leu Gly Ala Lys Cys Asn Thr Gln Met Val Ile Pro His Leu 595 600 605Thr Glu Asn Tyr Gly Ala Ser Arg Asp Pro Pro Glu Lys Gln Ala Pro 610 615 620Met Cys Thr Val His Ser Phe Pro His Asn Ile Asp His Cys Leu Thr625 630 635 640Trp Ala Arg Ser Glu Phe Glu Gly Leu Leu Glu Lys Thr Pro Thr Glu 645 650 655Val Asn Ala Phe Leu Ser Asn Pro Thr Thr Tyr Ile Ser Ala Ala Arg 660 665 670Thr Ala Gly Asp Ala Gln Ala Arg Asp Gln Leu Glu Arg Val Ile Glu 675 680 685Cys Leu Asp Arg Asp Lys Cys Glu Thr Phe Gln Asp Ser Ile Thr Trp 690 695 700Ala Arg Leu Lys Phe Glu Asp Tyr Phe Ser Asn Arg Val Lys Gln Leu705 710 715 720Thr Phe Thr Phe Pro Glu Asp Ser Met Thr Ser Ser Gly Ala Pro Phe 725 730 735Trp Ser Ala Pro Lys Arg Phe Pro Arg Pro Val Glu Phe Ser Ser Ser 740 745 750Asp Gln Ser Gln Leu Ser Phe Ile Leu Ala Ala Ala Ile Leu Arg Ala 755 760 765Glu Thr Phe Gly Ile Pro Ile Pro Glu Trp Ala Lys Thr Pro Asn Lys 770 775 780Leu Ala Ala Glu Ala Val Asp Lys Val Ile Val Pro Asp Phe Gln Pro785 790 795 800Lys Gln Gly Val Lys Ile Val Thr His Glu Lys Ala Thr Ser Leu Ser 805 810 815Ser Ala Ser Val Asp Asp Ala Ala Val Ile Glu Glu Leu Ile Ala Lys 820 825 830Leu Glu Glu Val Ser Lys Thr Leu Pro Ser Gly Phe His Met Asn Pro 835 840 845Ile Gln Phe Glu Lys Asp Asp Asp Thr Asn Phe His Met Asp Val Ile 850 855 860Ala Gly Phe Ala Asn Met Arg Ala Arg Asn Tyr Ser Ile Pro Glu Val865 870 875 880Asp Lys Leu Lys Ala Lys Phe Ile Ala Gly Arg Ile Ile Pro Ala Ile 885 890 895Ala Thr Ser Thr Ala Met Ala Thr Gly Leu Val Cys Leu Glu Leu Tyr 900 905 910Lys Ala Leu Ala Gly Gly His Lys Val Glu Asp Tyr Arg Asn Thr Phe 915 920 925Ala Asn Leu Ala Ile Pro Leu Phe Ser Ile Ala Glu Pro Val Pro Pro 930 935 940Lys Thr Ile Lys His Gln Glu Leu Ser Trp Thr Val Trp Asp Arg Trp945 950 955 960Thr Val Thr Gly Asn Ile Thr Leu Arg Glu Leu Leu Glu Trp Leu Lys 965 970 975Glu Lys Gly Leu Asn Ala Tyr Ser Ile Ser Cys Gly Thr Ser Leu Leu 980 985 990Tyr Asn Ser Met Phe Pro Arg His Lys Glu Arg Leu Asp Arg Lys Val 995 1000 1005Val Asp Val Ala Arg Glu Val Ala Lys Met Glu Val Pro Ser Tyr 1010 1015 1020Arg Arg His Leu Asp Val Val Val Ala Cys Glu Asp Asp Asp Asp 1025 1030 1035Asn Asp Val Asp Ile Pro Leu Val Ser Val Tyr Phe Arg 1040 1045 1050101053PRTTriticum aestivum 10Met Leu Pro Ser Lys Arg Pro Ser Asp Ala Ala Ala Gly Asp Glu Asn1 5 10 15Gly Arg Gly Gly Asp Ala Arg Gly Pro Gly Ser Gly Arg Arg Arg Ala 20 25 30Arg Ala Ala Ala Gly Ala Val Thr Ala Ala Pro Gln Glu Ile Asp Glu 35 40 45Asp Leu His Ser Arg Gln Leu Ala Val Tyr Gly Arg Glu Thr Met Arg 50 55 60Arg Leu Phe Ala Ser Asp Val Leu Val Ser Gly Leu Asn Gly Leu Gly65 70 75 80Ala Glu Ile Ala Lys Asn Leu Ala Leu Ala Gly Val Lys Ser Val Thr 85 90 95Ile His Asp Val Lys Thr Val Lys Met Trp Asp Leu Ser Gly Asn Phe 100 105 110Phe Leu Ser Glu Asp Asp Ile Gly Lys Asn Arg Ala Ala Ala Cys Val 115 120 125Ala Lys Leu Gln Glu Leu Asn Asn Ala Val Leu Ile Ser Ala Leu Thr 130 135 140Glu Glu Leu Thr Thr Glu His Leu Ser Lys Phe Gln Ala Val Val Phe145 150 155 160Thr Asp Ile Asp Leu Asp Lys Ala Tyr Glu Phe Asp Asp Tyr Cys His 165 170 175Asn His Gln Pro Pro Ile Ser Phe Ile Lys Ser Glu Val Cys Gly Leu 180 185 190Phe Gly Ser Val Phe Cys Asp Phe Gly Pro Lys Phe Thr Val Leu Asp 195 200 205Val Asp Gly Glu Asp Pro His Thr Gly Ile Ile Ala Ser Ile Ser Asn 210 215 220Asp Asn Pro Ala Leu Ile Ser Cys Val Asp Asp Glu Arg Leu Glu Phe225 230 235 240Gln Asp Gly Asp Leu Val Val Phe Ser Glu Val His Gly Met Thr Glu 245 250 255Leu Asn Asp Gly Lys Pro Arg Lys Val Lys Asn Ala Arg Pro Phe Ser 260 265 270Phe Ser Ile Glu Glu Asp Thr Ser Asn Phe Gly Ile Tyr Val Lys Gly 275 280 285Gly Ile Val Thr Gln Val Lys Glu Pro Lys Val Leu Cys Phe Lys Ala 290 295 300Leu Arg Asp Ala Met Thr Asp Pro Gly Glu Val Leu Leu Ser Asp Phe305 310 315 320Ser Lys Phe Glu Arg Pro Pro Val Leu His Leu Ala Phe Gln Ala Leu 325 330 335Asp Lys Phe Lys Lys Asp His Gly Arg Cys Pro Ala Ala Gly Cys Glu 340 345 350Glu Asp Ala His Ser Phe Leu Lys Ile Ala Ala Ala Ile Asn Glu Ala 355 360 365Ser Ala Asp Arg Lys Leu Asp Thr Ile Asp Glu Lys Leu Phe Arg Gln 370 375 380Phe Ala Ser Gly Ser Arg Ala Val Leu Asn Pro Met Ala Ala Met Phe385 390 395 400Gly Gly Ile Val Gly Gln Glu Val Val Lys Ala Cys Ser Gly Lys Phe 405 410 415His Pro Leu Asn Gln Phe Phe Tyr Phe Asp Ser Val Glu Ser Leu Pro 420 425 430Thr Tyr Pro Leu Glu Pro Gln Asp Leu Lys Pro Ser Asn Asn Arg Tyr 435 440 445Asp Ala Gln Val Ser Val Phe Gly Ser Lys Leu Gln Lys Lys Met Glu 450 455 460Glu Ala Asn Thr Phe Val Val Gly Ser Gly Ala Leu Gly Cys Glu Phe465 470 475 480Leu Lys Asn Leu Ala Leu Met Gly Val Ser Cys Ser Ser Lys Gly Lys 485 490 495Leu Thr Ile Thr Asp Asp Asp Ile Ile Glu Lys Ser Asn Leu Ser Arg 500 505 510Gln Phe Leu Phe Arg Asp Trp Asn Ile Gly Gln Ala Lys Ser Thr Val 515 520 525Ala Ala Thr Ala Ala Ser Ala Ile Asn Pro Ser Leu His Ile Asp Ala 530 535 540Leu Gln Asn Arg Ala Cys Pro Asp Thr Glu Asn Val Phe His Asp Thr545 550 555 560Phe Trp Glu Gly Leu Asp Val Val Ile Asn Ala Leu Asp Asn Val Asn 565 570 575Ala Arg Met Tyr Met Asp Met Arg Cys Leu Tyr Phe Gln Lys Pro Leu 580 585 590Leu Glu Ser Gly Thr Leu Gly Ala Lys Cys Asn Ile Gln Met Val Ile 595 600 605Pro His Leu Thr Glu Asn Tyr Gly Ala Ser Arg Asp Pro Pro Glu Lys 610 615 620Gln Ala Pro Met Cys Thr Val His Ser Phe Pro His Asn Ile Asp His625 630 635 640Cys Leu Thr Trp Ala Arg Ser Glu Phe Glu Gly Leu Leu Glu Lys Thr 645 650 655Pro Asn Glu Val Asn Ser Phe Leu Ser Asn Pro Ala Gln Tyr Ala Ala 660 665 670Ala Met Arg Lys Ala Gly Asp Ala Gln Ala Arg Glu Leu Leu Glu Arg 675 680 685Val Ser Glu Cys Leu Asn Lys Asp Arg Cys Ser Thr Phe Asp Asp Cys 690 695 700Ile Ser Trp Ala Arg Leu Lys Phe Glu Asp Tyr Phe Ser Asn Arg Val705 710 715 720Lys Gln Leu Thr Phe Thr Phe Pro Glu Asp Ala Ala Thr Ser Met Gly 725 730 735Ala Pro Phe Trp Ser Ala Pro Lys Arg Phe Pro Arg Ala Leu Gln Phe 740 745 750Ser Ala Ala Asp Gln Ser His Leu Asn Phe Ile Met Ser Ala Ser Ile 755 760 765Leu Arg Ala Glu Ser Phe Gly Val Ala Ile Pro Glu Trp Ala Lys Asp 770 775 780Thr Ser Lys Leu Ala Asp Val Val Asn Lys Ile Ala Val Pro Thr Phe785 790 795 800Glu Pro Lys Gln Gly Val Asn Ile Val Thr Asp Glu Lys Ala Ser Asn 805 810 815Leu Ser Ser Thr Ser Val Asp Asp Val Ala Val Ile Glu Asp Leu Leu 820 825 830Ala Lys Leu Gln Glu Tyr Ala Lys Met Leu Leu Pro Gly Phe Gln Met 835 840 845Lys Pro Ile Gln Phe Glu Lys Asp Asp Asp Thr Asn Phe His Met Asp 850 855 860Leu Ile Ser Gly Leu Ala Asn Met Arg Ala Arg Asn Tyr Ser Ile Pro865 870 875 880Glu Val Asp Lys Leu Lys Ala Lys Phe Ile Ala Gly Arg Ile Ile Pro 885 890 895Ala Ile Ala Thr Ser

Thr Ala Met Ala Thr Gly Leu Val Cys Leu Glu 900 905 910Leu Tyr Lys Val Ile Ala Gly Glu His Pro Val Glu Asp Tyr Arg Asn 915 920 925Thr Phe Ala Asn Leu Ala Leu Pro Leu Phe Ser Met Ala Glu Pro Val 930 935 940Pro Pro Lys Val Met Lys His Lys Glu Thr Ser Trp Thr Val Trp Asp945 950 955 960Arg Trp Ser Val Gln Gly Asn Leu Thr Leu Ala Glu Leu Leu Gln Trp 965 970 975Phe Ala Asp Lys Gly Leu Thr Ala Tyr Ser Ile Ser Cys Gly Thr Ser 980 985 990Leu Leu Tyr Asn Asn Met Phe Ala Arg His Lys Asp Arg Leu Thr Lys 995 1000 1005Lys Val Val Asp Ile Ala Arg Glu Val Ala Lys Val Asp Val Pro 1010 1015 1020Glu Tyr Arg Arg His Leu Asp Ile Gly Val Ala Cys Glu Asp Glu 1025 1030 1035Asp Glu Asn Asp Val Asp Ile Pro Leu Val Ser Val Tyr Phe Arg 1040 1045 1050111058PRTHomo sapiens 11Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly Pro Asp1 5 10 15Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala Gln Ser Val Leu Ser Glu 20 25 30Val Pro Ser Val Pro Thr Asn Gly Met Ala Lys Asn Gly Ser Glu Ala 35 40 45Asp Ile Asp Glu Gly Leu Tyr Ser Arg Gln Leu Tyr Val Leu Gly His 50 55 60Glu Ala Met Lys Arg Leu Gln Thr Ser Ser Val Leu Val Ser Gly Leu65 70 75 80Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Ile Ile Leu Gly Gly Val 85 90 95Lys Ala Val Thr Leu His Asp Gln Gly Thr Ala Gln Trp Ala Asp Leu 100 105 110Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Ile Gly Lys Asn Arg Ala 115 120 125Glu Val Ser Gln Pro Arg Leu Ala Glu Leu Asn Ser Tyr Val Pro Val 130 135 140Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu Ser Gly Phe Gln145 150 155 160Val Val Val Leu Thr Asn Thr Pro Leu Glu Asp Gln Leu Arg Val Gly 165 170 175Glu Phe Cys His Asn Arg Gly Ile Lys Leu Val Val Ala Asp Thr Arg 180 185 190Gly Leu Phe Gly Gln Leu Phe Cys Asp Phe Gly Glu Glu Met Ile Leu 195 200 205Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Ala Met Val Ser Met Val 210 215 220Thr Lys Asp Asn Pro Gly Val Val Thr Cys Leu Asp Glu Ala Arg His225 230 235 240Gly Phe Glu Ser Gly Asp Phe Val Ser Phe Ser Glu Val Gln Gly Met 245 250 255Val Glu Leu Asn Gly Asn Gln Pro Met Glu Ile Lys Val Leu Gly Pro 260 265 270Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Phe Ser Asp Tyr Ile Arg 275 280 285Gly Gly Ile Val Ser Gln Val Lys Val Pro Lys Lys Ile Ser Phe Lys 290 295 300Ser Leu Val Ala Ser Leu Ala Glu Pro Asp Phe Val Val Thr Asp Phe305 310 315 320Ala Lys Phe Ser Arg Pro Ala Gln Leu His Ile Gly Phe Gln Ala Leu 325 330 335His Gln Phe Cys Ala Gln His Gly Arg Pro Pro Arg Pro Arg Asn Glu 340 345 350Glu Asp Ala Ala Glu Leu Val Ala Leu Ala Gln Ala Val Asn Ala Arg 355 360 365Ala Leu Pro Ala Val Gln Gln Asn Asn Leu Asp Glu Asp Leu Ile Arg 370 375 380Lys Leu Ala Tyr Val Ala Ala Gly Asp Leu Ala Pro Ile Asn Ala Phe385 390 395 400Ile Gly Gly Leu Ala Ala Gln Glu Val Met Lys Ala Cys Ser Gly Lys 405 410 415Phe Met Pro Ile Met Gln Trp Leu Tyr Phe Asp Ala Leu Glu Cys Leu 420 425 430Pro Glu Asp Lys Glu Val Leu Thr Glu Asp Lys Cys Leu Gln Arg Gln 435 440 445Asn Arg Tyr Asp Gly Gln Val Ala Val Phe Gly Ser Asp Leu Gln Glu 450 455 460Lys Leu Gly Lys Gln Lys Tyr Phe Leu Val Gly Ala Gly Ala Ile Gly465 470 475 480Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gly Leu Gly Cys Gly Glu 485 490 495Gly Gly Glu Ile Ile Val Thr Asp Met Asp Thr Ile Glu Lys Ser Asn 500 505 510Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp Asp Val Thr Lys Leu Lys 515 520 525Ser Asp Thr Ala Ala Ala Ala Val Arg Gln Met Asn Pro His Ile Arg 530 535 540Val Thr Ser His Gln Asn Arg Val Gly Pro Asp Thr Glu Arg Ile Tyr545 550 555 560Asp Asp Asp Phe Phe Gln Asn Leu Asp Gly Val Ala Asn Ala Leu Asp 565 570 575Asn Val Asp Ala Arg Met Tyr Met Asp Arg Arg Cys Val Tyr Tyr Arg 580 585 590Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Val Gln 595 600 605Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser Gln Asp Pro 610 615 620Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys Asn Phe Pro Asn Ala625 630 635 640Ile Glu His Thr Leu Gln Trp Ala Arg Asp Glu Phe Glu Gly Leu Phe 645 650 655Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr Leu Thr Asp Pro Lys Phe 660 665 670Val Glu Arg Thr Leu Arg Leu Ala Gly Thr Gln Pro Leu Glu Val Leu 675 680 685Glu Ala Val Gln Arg Ser Leu Val Leu Gln Arg Pro Gln Thr Trp Ala 690 695 700Asp Cys Val Thr Trp Ala Cys His His Trp His Thr Gln Tyr Ser Asn705 710 715 720Asn Ile Arg Gln Leu Leu His Asn Phe Pro Pro Asp Gln Leu Thr Ser 725 730 735Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu 740 745 750Thr Phe Asp Val Asn Asn Pro Leu His Leu Asp Tyr Val Met Ala Ala 755 760 765Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Thr Gly Ser Gln Asp Arg 770 775 780Ala Ala Val Ala Thr Phe Leu Gln Ser Val Gln Val Pro Glu Phe Thr785 790 795 800Pro Lys Ser Gly Val Lys Ile His Val Ser Asp Gln Glu Leu Gln Ser 805 810 815Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Glu Glu Leu Lys Ala Thr 820 825 830Leu Pro Ser Pro Asp Lys Leu Pro Gly Phe Lys Met Tyr Pro Ile Asp 835 840 845Phe Glu Lys Asp Asp Asp Ser Asn Phe His Met Asp Phe Ile Val Ala 850 855 860Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro Ser Ala Asp Arg865 870 875 880His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala Thr 885 890 895Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys Val 900 905 910Val Gln Gly His Arg Gln Leu Asp Ser Tyr Lys Asn Gly Phe Leu Asn 915 920 925Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro Arg 930 935 940His Gln Tyr Tyr Asn Gln Glu Trp Thr Leu Trp Asp Arg Phe Glu Val945 950 955 960Gln Gly Leu Gln Pro Asn Gly Glu Glu Met Thr Leu Lys Gln Phe Leu 965 970 975Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Ile Thr Met Leu Ser Gln 980 985 990Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu Lys 995 1000 1005Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Val Ser Arg Val Ser 1010 1015 1020Lys Arg Lys Leu Gly Arg His Val Arg Ala Leu Val Leu Glu Leu 1025 1030 1035Cys Cys Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pro Tyr Val 1040 1045 1050Arg Tyr Thr Ile Arg 1055121058PRTOryctolagus cuniculus 12Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly Pro Asp1 5 10 15Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala Gln Ser Val Leu Pro Gln 20 25 30Val Pro Ser Ala Pro Thr Asn Gly Met Ala Lys Asn Gly Ser Glu Ala 35 40 45Asp Ile Asp Glu Gly Leu Tyr Ser Arg Gln Leu Tyr Val Leu Gly His 50 55 60Glu Ala Met Lys Arg Leu Gln Thr Ser Ser Val Leu Val Ser Gly Leu65 70 75 80Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Ile Ile Leu Gly Gly Val 85 90 95Lys Ala Val Thr Leu His Asp Gln Gly Thr Ala Gln Trp Ala Asp Leu 100 105 110Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Ile Gly Lys Asn Arg Ala 115 120 125Glu Val Ser Gln Pro Arg Leu Ala Glu Leu Asn Ser Tyr Val Pro Val 130 135 140Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu Ser Gly Phe Gln145 150 155 160Val Val Val Leu Thr Asn Ser Pro Leu Glu Asp Gln Leu Arg Val Gly 165 170 175Glu Phe Cys His Ser Arg Gly Ile Lys Leu Val Val Ala Asp Thr Arg 180 185 190Gly Leu Phe Gly Gln Leu Phe Cys Asp Phe Gly Glu Glu Met Ile Leu 195 200 205Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Thr Met Val Ser Met Val 210 215 220Thr Lys Asp Asn Pro Gly Val Val Thr Cys Leu Asp Glu Ala Arg His225 230 235 240Gly Phe Glu Ser Gly Asp Phe Val Ser Phe Ser Glu Val Gln Gly Met 245 250 255Thr Glu Leu Asn Gly Asn Gln Pro Ile Glu Ile Lys Val Leu Gly Pro 260 265 270Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Phe Ser Asp Tyr Ile Arg 275 280 285Gly Gly Ile Val Ser Gln Val Lys Val Pro Lys Lys Ile Ser Phe Lys 290 295 300Ser Leu Ser Ala Ser Leu Ala Glu Pro Asp Phe Val Met Thr Asp Phe305 310 315 320Ala Lys Phe Ser Arg Pro Ala Gln Leu His Ile Gly Phe Gln Ala Leu 325 330 335His Lys Phe Cys Ala Gln His Ser Arg Pro Pro Arg Pro Arg Asn Glu 340 345 350Glu Asp Ala Ala Glu Leu Val Thr Leu Ala Arg Ala Val Asn Ser Lys 355 360 365Ala Ser Ser Ala Val Gln Gln Asp Ser Leu Asp Glu Asp Leu Ile Arg 370 375 380Asn Leu Ala Phe Val Ala Ala Gly Asp Leu Ala Pro Ile Asn Ala Phe385 390 395 400Ile Gly Gly Leu Ala Ala Gln Glu Val Met Lys Ala Cys Ser Gly Lys 405 410 415Phe Met Pro Ile Met Gln Trp Leu Tyr Phe Asp Ala Leu Glu Cys Leu 420 425 430Pro Glu Asp Lys Glu Ser Leu Thr Glu Asp Lys Cys Leu Pro Arg Gln 435 440 445Asn Arg Tyr Asp Gly Gln Val Ala Val Phe Gly Ser Asp Leu Gln Glu 450 455 460Lys Leu Gly Arg Gln Lys Tyr Phe Leu Val Gly Ala Gly Ala Ile Gly465 470 475 480Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gly Leu Gly Cys Gly Glu 485 490 495Asn Gly Glu Ile Ile Val Thr Asp Met Asp Thr Ile Glu Lys Ser Asn 500 505 510Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp Asp Val Thr Lys Leu Lys 515 520 525Ser Asp Thr Ala Ala Ala Ala Val His Gln Met Asn Pro His Ile Arg 530 535 540Val Thr Ser His Gln Asn Arg Val Gly Pro Asp Thr Glu Arg Ile Tyr545 550 555 560Asp Asp Asp Phe Phe Gln Thr Leu Asp Gly Val Ala Asn Ala Leu Asp 565 570 575Asn Val Asp Ala Arg Met Tyr Met Asp Arg Arg Cys Val Tyr Tyr Arg 580 585 590Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Val Gln 595 600 605Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser Gln Asp Pro 610 615 620Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys Asn Phe Pro Asn Ala625 630 635 640Ile Glu His Thr Leu Gln Trp Ala Arg Asp Glu Phe Glu Gly Leu Phe 645 650 655Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr Leu Thr Asp Pro Lys Phe 660 665 670Val Glu Arg Thr Leu Arg Leu Ala Gly Thr Gln Pro Leu Glu Val Leu 675 680 685Glu Ala Val Gln Arg Ser Leu Val Leu Gln Leu Pro Gln Ser Trp Ala 690 695 700Asp Cys Val Thr Trp Ala Cys His His Trp His Thr Gln Tyr Ser Asn705 710 715 720Asn Ile Arg Gln Leu Leu His Asn Phe Pro Pro Asp Gln Leu Thr Ser 725 730 735Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu 740 745 750Thr Phe Asp Val Ser Asn Pro Leu His Leu Asp Tyr Val Met Ala Ala 755 760 765Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Ala Gly Ser Gln Asp Arg 770 775 780Ala Ala Val Ala Thr Leu Leu Gln Ser Val Gln Val Pro Glu Phe Thr785 790 795 800Pro Lys Ser Gly Val Lys Ile His Val Ser Asp Gln Glu Leu Gln Ser 805 810 815Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Glu Glu Leu Lys Ala Thr 820 825 830Leu Pro Ser Pro Asp Lys Leu Pro Gly Phe Lys Met Tyr Pro Ile Asp 835 840 845Phe Glu Lys Asp Asp Asp Ser Asn Phe His Met Asp Phe Ile Val Ala 850 855 860Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro Pro Ala Asp Arg865 870 875 880His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala Thr 885 890 895Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys Val 900 905 910Val Gln Gly His Arg His Leu Asp Ser Tyr Lys Asn Gly Phe Leu Asn 915 920 925Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro Arg 930 935 940His Gln Tyr Tyr Asn Gln Glu Trp Thr Leu Trp Asp Arg Phe Glu Val945 950 955 960Gln Gly Leu Gln Pro Asn Gly Glu Glu Met Thr Leu Lys Gln Phe Leu 965 970 975Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Ile Thr Met Leu Ser Gln 980 985 990Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu Lys 995 1000 1005Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Val Ser Arg Val Ser 1010 1015 1020Lys Arg Lys Leu Gly Arg His Val Arg Ala Leu Val Leu Glu Leu 1025 1030 1035Cys Cys Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pro Tyr Val 1040 1045 1050Arg Tyr Thr Ile Arg 1055131058PRTBos taurus 13Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly Pro Asp1 5 10 15Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala His Ser Val Leu Ser Glu 20 25 30Val Pro Ser Val Pro Ala Asn Gly Met Ala Lys Asn Val Ser Asp Ala 35 40 45Asp Ile Asp Glu Gly Leu Tyr Ser Arg Gln Leu Tyr Val Leu Gly His 50 55 60Glu Ala Met Lys Arg Leu Gln Thr Ser Ser Val Leu Val Ser Gly Leu65 70 75 80Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Ile Ile Leu Gly Gly Val 85 90 95Lys Ala Val Thr Leu His Asp Gln Gly Thr Ala Gln Trp Ala Asp Leu 100 105 110Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Ile Gly Lys Asn Arg Ala 115 120 125Glu Val Ser Gln Pro Arg Leu Ala Glu Leu Asn Ser Tyr Val Pro Val 130 135 140Ser Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu Ser Asp Phe Gln145 150 155 160Val Val Val Leu Thr Asn Ser Pro Leu Glu Asp Gln Leu Arg Val Gly 165 170 175Glu Phe Cys His Ser His Gly Ile Lys Leu Val Val Ala Asp Thr Arg 180 185 190Gly

Leu Phe Gly Gln Leu Phe Cys Asp Phe Gly Glu Glu Met Ile Leu 195 200 205Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Ala Met Val Ser Met Val 210 215 220Thr Lys Asp Asn Pro Gly Val Val Thr Cys Leu Asp Glu Ala Arg His225 230 235 240Gly Phe Glu Ser Gly Asp Phe Val Ser Phe Ser Glu Val Gln Gly Met 245 250 255Ile Glu Leu Asn Gly Ser Gln Pro Met Glu Ile Lys Val Leu Gly Pro 260 265 270Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Phe Ser Asp Tyr Ile Arg 275 280 285Gly Gly Ile Val Ser Gln Val Lys Val Pro Lys Lys Ile Ser Phe Lys 290 295 300Ser Leu Pro Ala Ser Leu Ala Glu Pro Asp Phe Val Met Thr Asp Phe305 310 315 320Ala Lys Tyr Ser Arg Pro Ala Gln Leu His Ile Gly Phe Gln Ala Leu 325 330 335His His Phe Cys Ala Gln His Gly Arg Ser Pro Arg Pro His Asn Glu 340 345 350Glu Asp Ala Ala Glu Leu Val Thr Ile Ala Gln Ala Val Asn Ala Arg 355 360 365Ser Leu Pro Ala Val Gln Gln Gly Ser Leu Asp Glu Asp Leu Ile Arg 370 375 380Lys Leu Ala Tyr Val Ala Ala Gly Asp Leu Ala Pro Ile Asn Ala Phe385 390 395 400Ile Gly Gly Leu Ala Ala Gln Glu Val Met Lys Ala Cys Ser Gly Lys 405 410 415Phe Met Pro Ile Met Gln Trp Leu Tyr Phe Asp Ala Leu Glu Cys Leu 420 425 430Pro Glu Asp Lys Glu Ala Leu Thr Glu Asp Lys Cys Leu Pro Arg Gln 435 440 445Asn Arg Tyr Asp Gly Gln Val Ala Val Phe Gly Ser Asp Leu Gln Glu 450 455 460Arg Leu Gly Lys Gln Lys Tyr Phe Leu Val Gly Ala Gly Ala Ile Gly465 470 475 480Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gly Leu Gly Cys Ala Glu 485 490 495Asp Gly Glu Ile Val Val Thr Asp Met Asp Thr Ile Glu Lys Ser Asn 500 505 510Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp Asp Val Thr Lys Leu Lys 515 520 525Ser Asp Thr Ala Ala Ala Ala Val Arg Gln Met Asn Pro His Ile Arg 530 535 540Val Thr Ser His Gln Asn Arg Val Gly Pro Asp Thr Glu Arg Ile Tyr545 550 555 560Asp Asp Asp Phe Phe Gln Asn Leu Asp Gly Val Thr Asn Ala Leu Asp 565 570 575Asn Val Asp Ala Arg Met Tyr Met Asp Arg Arg Cys Val Tyr Tyr Arg 580 585 590Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Val Gln 595 600 605Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser Gln Asp Pro 610 615 620Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys Asn Phe Pro Asn Ala625 630 635 640Ile Glu His Thr Leu Gln Trp Ala Arg Asp Glu Phe Glu Gly Leu Phe 645 650 655Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr Leu Thr Asp Pro Lys Phe 660 665 670Val Glu Arg Thr Leu Arg Leu Ala Gly Thr Gln Pro Leu Glu Val Leu 675 680 685Glu Ala Val Gln Arg Ser Leu Val Leu Gln Arg Pro Gln Thr Trp Ala 690 695 700Asp Cys Val Thr Trp Ala Cys His His Trp His Thr Gln Tyr Ser Asn705 710 715 720Asn Ile Arg Gln Leu Leu His Asn Phe Pro Pro Asp Gln Leu Thr Ser 725 730 735Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu 740 745 750Thr Phe Asp Val Ser Asn Pro Leu His Leu Asp Tyr Val Ile Ala Ala 755 760 765Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Thr Gly Ser Gln Asp Arg 770 775 780Ala Ala Val Ala Thr Leu Leu Gln Ser Val Gln Val Pro Glu Phe Thr785 790 795 800Pro Lys Ser Gly Val Lys Ile His Val Ser Asp Gln Glu Leu Gln Ser 805 810 815Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Glu Glu Leu Lys Ala Thr 820 825 830Leu Pro Ser Pro Glu Lys Leu Pro Gly Phe Lys Met Tyr Pro Ile Asp 835 840 845Phe Glu Lys Asp Asp Asp Thr Asn Phe His Met Asp Phe Ile Val Ala 850 855 860Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro Pro Ala Asp Arg865 870 875 880His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala Thr 885 890 895Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys Val 900 905 910Val Gln Gly His Arg Gln Leu Asn Ser Tyr Lys Asn Gly Phe Leu Asn 915 920 925Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro Arg 930 935 940His Gln Tyr Tyr Asn Gln Glu Trp Thr Leu Trp Asp Arg Phe Glu Val945 950 955 960Gln Gly Leu Gln Pro Asn Gly Glu Glu Met Thr Leu Lys Gln Phe Leu 965 970 975Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Ile Thr Met Leu Ser Gln 980 985 990Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu Lys 995 1000 1005Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Val Ser Arg Val Ser 1010 1015 1020Lys Arg Lys Leu Gly Arg His Val Arg Ala Leu Val Leu Glu Leu 1025 1030 1035Cys Cys Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pro Tyr Val 1040 1045 1050Arg Tyr Thr Ile Arg 1055141024PRTSaccharomyces cerevisiae 14Met Ser Ser Asn Asn Ser Gly Leu Ser Ala Ala Gly Glu Ile Asp Glu1 5 10 15Ser Leu Tyr Ser Arg Gln Leu Tyr Val Leu Gly Lys Glu Ala Met Leu 20 25 30Lys Met Gln Thr Ser Asn Val Leu Ile Leu Gly Leu Lys Gly Leu Gly 35 40 45Val Glu Ile Ala Lys Asn Val Val Leu Ala Gly Val Lys Ser Met Thr 50 55 60Val Phe Asp Pro Glu Pro Val Gln Leu Ala Asp Leu Ser Thr Gln Phe65 70 75 80Phe Leu Thr Glu Lys Asp Ile Gly Gln Lys Arg Gly Asp Val Thr Arg 85 90 95Ala Lys Leu Ala Glu Leu Asn Ala Tyr Val Pro Val Asn Val Leu Asp 100 105 110Ser Leu Asp Asp Val Thr Gln Leu Ser Gln Phe Gln Val Val Val Ala 115 120 125Thr Asp Thr Val Ser Leu Glu Asp Lys Val Lys Ile Asn Glu Phe Cys 130 135 140His Ser Ser Gly Ile Arg Phe Ile Ser Ser Glu Thr Arg Gly Leu Phe145 150 155 160Gly Asn Thr Phe Val Asp Leu Gly Asp Glu Phe Thr Val Leu Asp Pro 165 170 175Thr Gly Glu Glu Pro Arg Thr Gly Met Val Ser Asp Ile Glu Pro Asp 180 185 190Gly Thr Val Thr Met Leu Asp Asp Asn Arg His Gly Leu Glu Asp Gly 195 200 205Asn Phe Val Arg Phe Ser Glu Val Glu Gly Leu Asp Lys Leu Asn Asp 210 215 220Gly Thr Leu Phe Lys Val Glu Val Leu Gly Pro Phe Ala Phe Arg Ile225 230 235 240Gly Ser Val Lys Glu Tyr Gly Glu Tyr Lys Lys Gly Gly Ile Phe Thr 245 250 255Glu Val Lys Val Pro Arg Lys Ile Ser Phe Lys Ser Leu Lys Gln Gln 260 265 270Leu Ser Asn Pro Glu Phe Val Phe Ser Asp Phe Ala Lys Phe Asp Arg 275 280 285Ala Ala Gln Leu His Leu Gly Phe Gln Ala Leu His Gln Phe Ala Val 290 295 300Arg His Asn Gly Glu Leu Pro Arg Thr Met Asn Asp Glu Asp Ala Asn305 310 315 320Glu Leu Ile Lys Leu Val Thr Asp Leu Ser Val Gln Gln Pro Glu Val 325 330 335Leu Gly Glu Gly Val Asp Val Asn Glu Asp Leu Ile Lys Glu Leu Ser 340 345 350Tyr Gln Ala Arg Gly Asp Ile Pro Gly Val Val Ala Phe Phe Gly Gly 355 360 365Leu Val Ala Gln Glu Val Leu Lys Ala Cys Ser Gly Lys Phe Thr Pro 370 375 380Leu Lys Gln Phe Met Tyr Phe Asp Ser Leu Glu Ser Leu Pro Asp Pro385 390 395 400Lys Asn Phe Pro Arg Asn Glu Lys Thr Thr Gln Pro Val Asn Ser Arg 405 410 415Tyr Asp Asn Gln Ile Ala Val Phe Gly Leu Asp Phe Gln Lys Lys Ile 420 425 430Ala Asn Ser Lys Val Phe Leu Val Gly Ser Gly Ala Ile Gly Cys Glu 435 440 445Met Leu Lys Asn Trp Ala Leu Leu Gly Leu Gly Ser Gly Ser Asp Gly 450 455 460Tyr Ile Val Val Thr Asp Asn Asp Ser Ile Glu Lys Ser Asn Leu Asn465 470 475 480Arg Gln Phe Leu Phe Arg Pro Lys Asp Val Gly Lys Asn Lys Ser Glu 485 490 495Val Ala Ala Glu Ala Val Cys Ala Met Asn Pro Asp Leu Lys Gly Lys 500 505 510Ile Asn Ala Lys Ile Asp Lys Val Gly Pro Glu Thr Glu Glu Ile Phe 515 520 525Asn Asp Ser Phe Trp Glu Ser Leu Asp Phe Val Thr Asn Ala Leu Asp 530 535 540Asn Val Asp Ala Arg Thr Tyr Val Asp Arg Arg Cys Val Phe Tyr Arg545 550 555 560Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Thr Gln 565 570 575Val Ile Ile Pro Arg Leu Thr Glu Ser Tyr Ser Ser Ser Arg Asp Pro 580 585 590Pro Glu Lys Ser Ile Pro Leu Cys Thr Leu Arg Ser Phe Pro Asn Lys 595 600 605Ile Asp His Thr Ile Ala Trp Ala Lys Ser Leu Phe Gln Gly Tyr Phe 610 615 620Thr Asp Ser Ala Glu Asn Val Asn Met Tyr Leu Thr Gln Pro Asn Phe625 630 635 640Val Glu Gln Thr Leu Lys Gln Ser Gly Asp Val Lys Gly Val Leu Glu 645 650 655Ser Ile Ser Asp Ser Leu Ser Ser Lys Pro His Asn Phe Glu Asp Cys 660 665 670Ile Lys Trp Ala Arg Leu Glu Phe Glu Lys Lys Phe Asn His Asp Ile 675 680 685Lys Gln Leu Leu Phe Asn Phe Pro Lys Asp Ala Lys Thr Ser Asn Gly 690 695 700Glu Pro Phe Trp Ser Gly Ala Lys Arg Ala Pro Thr Pro Leu Glu Phe705 710 715 720Asp Ile Tyr Asn Asn Asp His Phe His Phe Val Val Ala Gly Ala Ser 725 730 735Leu Arg Ala Tyr Asn Tyr Gly Ile Lys Ser Asp Asp Ser Asn Ser Lys 740 745 750Pro Asn Val Asp Glu Tyr Lys Ser Val Ile Asp His Met Ile Ile Pro 755 760 765Glu Phe Thr Pro Asn Ala Asn Leu Lys Ile Gln Val Asn Asp Asp Asp 770 775 780Pro Asp Pro Asn Ala Asn Ala Ala Asn Gly Ser Asp Glu Ile Asp Gln785 790 795 800Leu Val Ser Ser Leu Pro Asp Pro Ser Thr Leu Ala Gly Phe Lys Leu 805 810 815Glu Pro Val Asp Phe Glu Lys Asp Asp Asp Thr Asn His His Ile Glu 820 825 830Phe Ile Thr Ala Cys Ser Asn Cys Arg Ala Gln Asn Tyr Phe Ile Glu 835 840 845Thr Ala Asp Arg Gln Lys Thr Lys Phe Ile Ala Gly Arg Ile Ile Pro 850 855 860Ala Ile Ala Thr Thr Thr Ser Leu Val Thr Gly Leu Val Asn Leu Glu865 870 875 880Leu Tyr Lys Leu Ile Asp Asn Lys Thr Asp Ile Glu Gln Tyr Lys Asn 885 890 895Gly Phe Val Asn Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Ile 900 905 910Ala Ser Pro Lys Gly Glu Tyr Asn Asn Lys Lys Tyr Asp Lys Ile Trp 915 920 925Asp Arg Phe Asp Ile Lys Gly Asp Ile Lys Leu Ser Asp Leu Ile Glu 930 935 940His Phe Glu Lys Asp Glu Gly Leu Glu Ile Thr Met Leu Ser Tyr Gly945 950 955 960Val Ser Leu Leu Tyr Ala Ser Phe Phe Pro Pro Lys Lys Leu Lys Glu 965 970 975Arg Leu Asn Leu Pro Ile Thr Gln Leu Val Lys Leu Val Thr Lys Lys 980 985 990Asp Ile Pro Ala His Val Ser Thr Met Ile Leu Glu Ile Cys Ala Asp 995 1000 1005Asp Lys Glu Gly Glu Asp Val Glu Val Pro Phe Ile Thr Ile His 1010 1015 1020Leu151080PRTArabidopsis thaliana 15Met Leu His Lys Arg Ala Ser Glu Ala Asn Asp Lys Asn Asp Asn Thr1 5 10 15Ile Ile Gly Ser Asp Leu Ala Ser Ser Lys Lys Arg Arg Ile Asp Phe 20 25 30Thr Glu Ser Ser Ser Asp Lys Ser Ser Ser Ile Leu Ala Ser Gly Ser 35 40 45Ser Arg Gly Phe His Gly Asp Ser Val Val Gln Gln Ile Asp Met Ala 50 55 60Phe Gly Asn Ser Asn Arg Gln Glu Ile Asp Glu Asp Leu His Ser Arg65 70 75 80Gln Leu Ala Val Tyr Gly Arg Glu Thr Met Arg Arg Leu Phe Ala Ser 85 90 95Asn Val Leu Ile Ser Gly Met His Gly Leu Gly Ala Glu Ile Ala Lys 100 105 110Asn Leu Ile Leu Ala Gly Val Lys Ser Val Thr Leu His Asp Glu Arg 115 120 125Val Val Glu Leu Trp Asp Leu Ser Ser Asn Phe Val Phe Ser Glu Asp 130 135 140Asp Val Gly Lys Asn Arg Ala Asp Ala Ser Val Gln Lys Leu Gln Asp145 150 155 160Leu Asn Asn Ala Val Val Val Ser Ser Leu Thr Lys Ser Leu Asn Lys 165 170 175Glu Asp Leu Ser Gly Phe Gln Val Val Val Phe Ser Asp Ile Ser Met 180 185 190Glu Arg Ala Ile Glu Phe Asp Asp Tyr Cys His Ser His Gln Pro Pro 195 200 205Ile Ala Phe Val Lys Ala Asp Val Arg Gly Leu Phe Gly Ser Val Phe 210 215 220Cys Asp Phe Gly Pro Glu Phe Ala Val Leu Asp Val Asp Gly Glu Glu225 230 235 240Pro His Thr Gly Ile Ile Ala Ser Ile Ser Asn Glu Asn Gln Ala Phe 245 250 255Ile Ser Cys Val Asp Asp Glu Arg Leu Glu Phe Glu Asp Gly Asp Leu 260 265 270Val Val Phe Ser Glu Val Glu Gly Met Thr Glu Leu Asn Asp Gly Lys 275 280 285Pro Arg Lys Ile Lys Ser Thr Arg Pro Tyr Ser Phe Thr Leu Asp Glu 290 295 300Asp Thr Thr Asn Tyr Gly Thr Tyr Val Lys Gly Gly Ile Val Thr Gln305 310 315 320Val Lys Gln Pro Lys Leu Leu Asn Phe Lys Pro Leu Arg Glu Ala Leu 325 330 335Lys Asp Pro Gly Asp Phe Leu Phe Ser Asp Phe Ser Lys Phe Asp Arg 340 345 350Pro Pro Leu Leu His Leu Ala Phe Gln Ala Leu Asp His Phe Lys Ala 355 360 365Glu Ala Gly Arg Phe Pro Val Ala Gly Ser Glu Glu Asp Ala Gln Lys 370 375 380Leu Ile Ser Ile Ala Thr Ala Ile Asn Thr Gly Gln Gly Asp Leu Lys385 390 395 400Val Glu Asn Val Asp Gln Lys Leu Leu Arg His Phe Ser Phe Gly Ala 405 410 415Lys Ala Val Leu Asn Pro Met Ala Ala Met Phe Gly Gly Ile Val Gly 420 425 430Gln Glu Val Val Lys Ala Cys Ser Gly Lys Phe His Pro Leu Phe Gln 435 440 445Phe Phe Tyr Phe Asp Ser Val Glu Ser Leu Pro Ser Glu Pro Val Asp 450 455 460Ser Ser Asp Phe Ala Pro Arg Asn Ser Arg Tyr Asp Ala Gln Ile Ser465 470 475 480Val Phe Gly Ala Lys Phe Gln Lys Lys Leu Glu Asp Ala Lys Val Phe 485 490 495Thr Val Gly Ser Gly Ala Leu Gly Cys Glu Phe Leu Lys Asn Leu Ala 500 505 510Leu Met Gly Val Ser Cys Gly Ser Gln Gly Lys Leu Thr Val Thr Asp 515 520 525Asp Asp Ile Ile Glu Lys Ser Asn Leu Ser Arg Gln Phe Leu Phe Arg 530 535 540Asp Trp Asn Ile Gly Gln Ala Lys Ser Thr Val Ala Ala Ser Ala Ala545 550 555 560Ala Val Ile Asn Pro Arg Phe Asn Ile Glu Ala Leu Gln Asn Arg Val 565 570

575Gly Ala Glu Thr Glu Asn Val Phe Asp Asp Ala Phe Trp Glu Asn Leu 580 585 590Thr Val Val Val Asn Ala Leu Asp Asn Val Asn Ala Arg Leu Tyr Val 595 600 605Asp Ser Arg Cys Leu Tyr Phe Gln Lys Pro Leu Leu Glu Ser Gly Thr 610 615 620Leu Gly Thr Lys Cys Asn Thr Gln Ser Val Ile Pro His Leu Thr Glu625 630 635 640Asn Tyr Gly Ala Ser Arg Asp Pro Pro Glu Lys Gln Ala Pro Met Cys 645 650 655Thr Val His Ser Phe Pro His Asn Ile Asp His Cys Leu Thr Trp Ala 660 665 670Arg Ser Glu Phe Glu Gly Leu Leu Glu Lys Thr Pro Ala Glu Val Asn 675 680 685Ala Tyr Leu Ser Ser Pro Val Glu Tyr Thr Asn Ser Met Met Ser Ala 690 695 700Gly Asp Ala Gln Ala Arg Asp Thr Leu Glu Arg Ile Val Glu Cys Leu705 710 715 720Glu Lys Glu Lys Cys Glu Thr Phe Gln Asp Cys Leu Thr Trp Ala Arg 725 730 735Leu Arg Phe Glu Asp Tyr Phe Val Asn Arg Val Lys Gln Leu Ile Tyr 740 745 750Thr Phe Pro Glu Asp Ala Ala Thr Ser Thr Gly Ala Pro Phe Trp Ser 755 760 765Ala Pro Lys Arg Phe Pro Arg Pro Leu Gln Tyr Ser Ser Ser Asp Pro 770 775 780Ser Leu Leu Asn Phe Ile Thr Ala Thr Ala Ile Leu Arg Ala Glu Thr785 790 795 800Phe Gly Ile Pro Ile Pro Glu Trp Thr Lys Asn Pro Lys Glu Ala Ala 805 810 815Glu Ala Val Asp Arg Val Ile Val Pro Asp Phe Glu Pro Arg Gln Asp 820 825 830Ala Lys Ile Val Thr Asp Glu Lys Ala Thr Thr Leu Thr Thr Ala Ser 835 840 845Val Asp Asp Ala Ala Val Ile Asp Asp Leu Ile Ala Lys Ile Asp Gln 850 855 860Cys Arg His Asn Leu Ser Pro Asp Phe Arg Met Lys Pro Ile Gln Phe865 870 875 880Glu Lys Asp Asp Asp Thr Asn Tyr His Met Asp Val Ile Ala Gly Leu 885 890 895Ala Asn Met Arg Ala Arg Asn Tyr Ser Ile Pro Glu Val Asp Lys Leu 900 905 910Lys Ala Lys Phe Ile Ala Gly Arg Ile Ile Pro Ala Ile Ala Thr Ser 915 920 925Thr Ala Met Ala Thr Gly Leu Val Cys Leu Glu Leu Tyr Lys Val Leu 930 935 940Asp Gly Gly His Lys Val Glu Ala Tyr Arg Asn Thr Phe Ala Asn Leu945 950 955 960Ala Leu Pro Leu Phe Ser Met Ala Glu Pro Leu Pro Pro Lys Val Val 965 970 975Lys His Arg Asp Met Ala Trp Thr Val Trp Asp Arg Trp Val Leu Lys 980 985 990Gly Asn Pro Thr Leu Arg Glu Val Leu Gln Trp Leu Glu Asp Lys Gly 995 1000 1005Leu Ser Ala Tyr Ser Ile Ser Cys Gly Ser Cys Leu Leu Phe Asn 1010 1015 1020Ser Met Phe Thr Arg His Lys Glu Arg Met Asp Lys Lys Val Val 1025 1030 1035Asp Leu Ala Arg Asp Val Ala Lys Val Glu Leu Pro Pro Tyr Arg 1040 1045 1050Asn His Leu Asp Val Val Val Ala Cys Glu Asp Glu Asp Asp Asn 1055 1060 1065Asp Val Asp Ile Pro Leu Val Ser Ile Tyr Phe Arg 1070 1075 1080161058PRTRattus norvegicus 16Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly Pro Asp1 5 10 15Pro Lys Pro Gly Ser Asn Cys Ser Ser Ala Gln Ser Val Leu Ser Glu 20 25 30Val Ser Ser Val Pro Thr Asn Gly Met Ala Lys Asn Gly Ser Glu Ala 35 40 45Asp Ile Asp Glu Ser Leu Tyr Ser Arg Gln Leu Tyr Val Leu Gly His 50 55 60Glu Ala Met Lys Met Leu Gln Thr Ser Ser Val Leu Val Ser Gly Leu65 70 75 80Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Ile Ile Leu Gly Gly Val 85 90 95Lys Ala Val Thr Leu His Asp Gln Gly Thr Thr Gln Trp Ala Asp Leu 100 105 110Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Ile Gly Lys Asn Arg Ala 115 120 125Glu Val Ser Gln Pro Arg Leu Ala Glu Leu Asn Ser Tyr Val Pro Val 130 135 140Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu Ser Gly Phe Gln145 150 155 160Val Val Val Leu Thr Asn Ser Pro Leu Glu Glu Gln Leu Arg Val Gly 165 170 175Glu Phe Cys His Ser Arg Gly Ile Lys Leu Val Val Ala Asp Thr Arg 180 185 190Gly Leu Phe Gly Gln Leu Phe Cys Asp Phe Gly Glu Glu Met Val Leu 195 200 205Thr Asp Ser Asn Gly Glu Gln Pro Leu Ser Ala Met Val Ser Met Val 210 215 220Thr Lys Asp Asn Pro Gly Val Val Thr Cys Leu Asp Glu Ala Arg His225 230 235 240Gly Phe Glu Thr Gly Asp Phe Val Ser Phe Ser Glu Val Gln Gly Met 245 250 255Val Gln Leu Asn Gly Cys Gln Pro Ile Glu Ile Lys Val Leu Gly Pro 260 265 270Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Phe Ser Asp Tyr Ile Arg 275 280 285Gly Gly Ile Val Ser Gln Val Lys Val Pro Lys Lys Ile Ser Phe Lys 290 295 300Ser Leu Pro Ala Ser Leu Ala Glu Pro Asp Phe Val Met Thr Asp Phe305 310 315 320Ala Lys Tyr Ser Arg Pro Ala Gln Leu His Ile Gly Phe Gln Ala Leu 325 330 335His Gln Phe Cys Ala Gln His Asn Arg Pro Pro Arg Pro Arg Asn Glu 340 345 350Glu Asp Ala Thr Glu Leu Val Thr Leu Ala Gln Ala Val Asn Ala Arg 355 360 365Ser Pro Pro Ala Val Gln Gln Asp Asn Val Asp Glu Asp Leu Ile Arg 370 375 380Lys Leu Ala Tyr Val Ala Ala Gly Asp Leu Ala Pro Ile Asn Ala Phe385 390 395 400Ile Gly Gly Leu Ala Ala Gln Glu Val Met Lys Ala Cys Ser Gly Lys 405 410 415Phe Met Pro Ile Met Gln Trp Leu Tyr Phe Asp Ala Leu Glu Cys Leu 420 425 430Pro Glu Asp Lys Glu Ala Leu Thr Glu Asp Lys Cys Leu Pro Arg Gln 435 440 445Asn Arg Tyr Asp Gly Gln Val Ala Val Phe Gly Ser Asp Leu Gln Glu 450 455 460Lys Leu Gly Lys Gln Lys Tyr Phe Leu Val Gly Ala Gly Ala Ile Gly465 470 475 480Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gly Leu Gly Cys Gly Glu 485 490 495Gly Gly Glu Val Val Val Thr Asp Met Asp Thr Ile Glu Lys Ser Asn 500 505 510Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp Asp Val Thr Lys Leu Lys 515 520 525Ser Asp Thr Ala Ala Ala Ala Val Arg Gln Met Asn Pro Tyr Ile Gln 530 535 540Val Thr Ser His Gln Asn Arg Val Gly Pro Asp Thr Glu Arg Ile Tyr545 550 555 560Asp Asp Asp Phe Phe Gln Asn Leu Asp Gly Val Ala Asn Ala Leu Asp 565 570 575Asn Val Asp Ala Arg Met Tyr Met Asp Arg Arg Cys Val Tyr Tyr Arg 580 585 590Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Val Gln 595 600 605Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser Gln Asp Pro 610 615 620Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys Asn Phe Pro Asn Ala625 630 635 640Ile Glu His Thr Leu Gln Trp Ala Arg Asp Glu Phe Glu Gly Leu Phe 645 650 655Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr Leu Thr Asp Ser Lys Phe 660 665 670Val Glu Arg Thr Leu Arg Leu Ala Gly Thr Gln Pro Leu Glu Val Leu 675 680 685Glu Ala Val Gln Arg Ser Leu Val Leu Gln Arg Pro Gln Thr Trp Gly 690 695 700Asp Cys Val Thr Trp Ala Cys His His Trp His Thr Gln Tyr Cys Asn705 710 715 720Asn Ile Arg Gln Leu Leu His Asn Phe Pro Pro Asp Gln Leu Thr Ser 725 730 735Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu 740 745 750Thr Phe Asp Val Asn Asn Thr Leu His Leu Asp Tyr Val Met Ala Ala 755 760 765Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Thr Gly Ser Gln Asp Arg 770 775 780Ala Ala Val Ala Ser Leu Leu Gln Ser Val Gln Val Pro Glu Phe Thr785 790 795 800Pro Lys Ser Gly Val Lys Ile His Val Ser Asp Gln Glu Leu Gln Ser 805 810 815Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Glu Glu Leu Lys Ala Thr 820 825 830Leu Pro Ser Pro Asp Lys Leu Pro Gly Phe Lys Met Tyr Pro Ile Asp 835 840 845Phe Glu Lys Asp Asp Asp Ser Asn Phe His Met Asp Phe Ile Val Ala 850 855 860Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Ser Pro Ala Asp Arg865 870 875 880His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala Thr 885 890 895Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys Val 900 905 910Val Gln Gly His Gln Gln Leu Asp Ser Tyr Lys Asn Gly Phe Leu Asn 915 920 925Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro Arg 930 935 940His Gln Tyr Tyr Asn Gln Glu Trp Thr Leu Trp Asp Arg Phe Glu Val945 950 955 960Gln Gly Leu Gln Pro Asn Gly Glu Glu Met Thr Leu Lys Gln Phe Leu 965 970 975Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Ile Thr Met Leu Ser Gln 980 985 990Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu Lys 995 1000 1005Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Val Ser Arg Val Ser 1010 1015 1020Lys Arg Lys Leu Gly Arg His Val Arg Ala Leu Val Leu Glu Leu 1025 1030 1035Cys Cys Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pro Tyr Val 1040 1045 1050Arg Tyr Thr Ile Arg 105517148PRTSaccharomyces cerevisiae 17Met Ser Ser Ser Lys Arg Ile Ala Lys Glu Leu Ser Asp Leu Gly Arg1 5 10 15Asp Pro Pro Ala Ser Cys Ser Ala Gly Pro Val Gly Asp Asp Leu Tyr 20 25 30His Trp Gln Ala Ser Ile Met Gly Pro Ser Asp Ser Pro Tyr Ala Gly 35 40 45Gly Val Phe Phe Leu Ser Ile His Phe Pro Thr Asp Tyr Pro Phe Lys 50 55 60Pro Pro Lys Val Asn Phe Thr Thr Lys Ile Tyr His Pro Asn Ile Asn65 70 75 80Ser Ser Gly Asn Ile Cys Leu Asp Ile Leu Lys Asp Gln Trp Ser Pro 85 90 95Ala Leu Thr Leu Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Thr 100 105 110Asp Ala Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala Gln Ile Tyr 115 120 125Lys Thr Asp Lys Ala Lys Tyr Glu Ala Thr Ala Lys Glu Trp Thr Lys 130 135 140Lys Tyr Ala Val14518185PRTArabidopsis lyrata 18Met Ser Ser Pro Ser Lys Arg Arg Glu Met Asp Leu Met Lys Leu Met1 5 10 15Met Ser Asp Tyr Lys Val Glu Met Ile Asn Asp Gly Met Gln Glu Phe 20 25 30Tyr Val Glu Phe Asn Gly Pro Lys Asp Ser Ile Tyr Glu Gly Gly Val 35 40 45Trp Lys Ile Arg Val Glu Leu Pro Asp Ala Tyr Pro Tyr Lys Ser Pro 50 55 60Ser Val Gly Phe Ile Thr Lys Ile Tyr His Pro Asn Val Asp Glu Met65 70 75 80Ser Gly Ser Val Cys Leu Asp Val Ile Asn Gln Thr Trp Ser Pro Met 85 90 95Phe Asp Leu Val Asn Val Phe Glu Thr Phe Leu Pro Gln Leu Leu Leu 100 105 110Tyr Pro Asn Pro Ser Asp Pro Leu Asn Gly Glu Ala Ala Ala Leu Met 115 120 125Met Arg Asp Arg Pro Thr Tyr Glu Gln Arg Val Lys Glu Tyr Cys Glu 130 135 140Lys Tyr Ala Lys Pro Arg Ala Asp Thr Glu Glu Met Ser Ser Asp Asp145 150 155 160Glu Met Ser Glu Asp Glu Tyr Ala Ser Asp Cys Asp Asp Glu Asp Asp 165 170 175Val Ala Ile Ala Gly Lys Leu Asp Pro 180 18519148PRTSaccharomyces cerevisiae 19Met Ser Ser Ser Lys Arg Ile Ala Lys Glu Leu Ser Asp Leu Glu Arg1 5 10 15Asp Pro Pro Thr Ser Cys Ser Ala Gly Pro Val Gly Asp Asp Leu Tyr 20 25 30His Trp Gln Ala Ser Ile Met Gly Pro Ala Asp Ser Pro Tyr Ala Gly 35 40 45Gly Val Phe Phe Leu Ser Ile His Phe Pro Thr Asp Tyr Pro Phe Lys 50 55 60Pro Pro Lys Ile Ser Phe Thr Thr Lys Ile Tyr His Pro Asn Ile Asn65 70 75 80Ala Asn Gly Asn Ile Cys Leu Asp Ile Leu Lys Asp Gln Trp Ser Pro 85 90 95Ala Leu Thr Leu Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Thr 100 105 110Asp Ala Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala His Ile Tyr 115 120 125Lys Thr Asp Arg Pro Lys Tyr Glu Ala Thr Ala Arg Glu Trp Thr Lys 130 135 140Lys Tyr Ala Val14520170PRTArabidopsis thaliana 20Met Met Ser Asp Tyr Lys Val Glu Met Ile Asn Asp Gly Met Gln Glu1 5 10 15Phe Phe Val Glu Phe Ser Gly Pro Lys Asp Ser Ile Tyr Glu Gly Gly 20 25 30Val Trp Lys Ile Arg Val Glu Leu Pro Asp Ala Tyr Pro Tyr Lys Ser 35 40 45Pro Ser Val Gly Phe Ile Thr Lys Ile Tyr His Pro Asn Val Asp Glu 50 55 60Met Ser Gly Ser Val Cys Leu Asp Val Ile Asn Gln Thr Trp Ser Pro65 70 75 80Met Phe Asp Leu Val Asn Val Phe Glu Thr Phe Leu Pro Gln Leu Leu 85 90 95Leu Tyr Pro Asn Pro Ser Asp Pro Leu Asn Gly Glu Ala Ala Ala Leu 100 105 110Met Met Arg Asp Arg Pro Thr Tyr Glu Gln Arg Val Lys Glu Tyr Cys 115 120 125Glu Lys Tyr Ala Lys Pro Arg Ala Asp Thr Glu Glu Met Ser Ser Asp 130 135 140Asp Glu Met Ser Glu Asp Glu Tyr Ala Ser Asp Gly Asp Asp Glu Asp145 150 155 160Asp Val Ala Ile Ala Gly Lys Leu Asp Pro 165 17021236PRTHomo sapiens 21Met Ala Arg Pro Leu Val Pro Ser Ser Gln Lys Ala Leu Leu Leu Glu1 5 10 15Leu Lys Gly Leu Gln Glu Glu Pro Val Glu Gly Phe Arg Val Thr Leu 20 25 30Val Asp Glu Gly Asp Leu Tyr Asn Trp Glu Val Ala Ile Phe Gly Pro 35 40 45Pro Asn Thr Tyr Tyr Glu Gly Gly Tyr Phe Lys Ala Arg Leu Lys Phe 50 55 60Pro Ile Asp Tyr Pro Tyr Ser Pro Pro Ala Phe Arg Phe Leu Thr Lys65 70 75 80Met Trp His Pro Asn Ile Tyr Glu Thr Gly Asp Val Cys Ile Ser Ile 85 90 95Leu His Pro Pro Val Asp Asp Pro Gln Ser Gly Glu Leu Pro Ser Glu 100 105 110Arg Trp Asn Pro Thr Gln Asn Val Arg Thr Ile Leu Leu Ser Val Ile 115 120 125Ser Leu Leu Asn Glu Pro Asn Thr Phe Ser Pro Ala Asn Val Asp Ala 130 135 140Ser Val Met Tyr Arg Lys Trp Lys Glu Ser Lys Gly Lys Asp Arg Glu145 150 155 160Tyr Thr Asp Ile Ile Arg Lys Gln Val Leu Gly Thr Lys Val Asp Ala 165 170 175Glu Arg Asp Gly Val Lys Val Pro Thr Thr Leu Ala Glu Tyr Cys Val 180 185 190Lys Thr Lys Ala Pro Ala Pro Asp Glu Gly Ser Asp Leu Phe Tyr Asp 195 200 205Asp Tyr Tyr Glu Asp Gly Glu Val Glu Glu Glu Ala Asp Ser Cys Phe 210 215 220Gly Asp Asp Glu Asp Asp Ser Gly Thr Glu Glu Ser225 230 23522147PRTHomo sapiens

22Met Ala Leu Lys Arg Ile His Lys Glu Leu Asn Asp Leu Ala Arg Asp1 5 10 15Pro Pro Ala Gln Cys Ser Ala Gly Pro Val Gly Asp Asp Met Phe His 20 25 30Trp Gln Ala Thr Ile Met Gly Pro Asn Asp Ser Pro Tyr Gln Gly Gly 35 40 45Val Phe Phe Leu Thr Ile His Phe Pro Thr Asp Tyr Pro Phe Lys Pro 50 55 60Pro Lys Val Ala Phe Thr Thr Arg Ile Tyr His Pro Asn Ile Asn Ser65 70 75 80Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg Ser Gln Trp Ser Pro Ala 85 90 95Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Cys Asp 100 105 110Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala Arg Ile Tyr Lys 115 120 125Thr Asp Arg Glu Lys Tyr Asn Arg Ile Ala Arg Glu Trp Thr Gln Lys 130 135 140Tyr Ala Met14523147PRTHomo sapiens 23Met Ala Leu Lys Arg Ile Asn Lys Glu Leu Ser Asp Leu Ala Arg Asp1 5 10 15Pro Pro Ala Gln Cys Ser Ala Gly Pro Val Gly Asp Asp Met Phe His 20 25 30Trp Gln Ala Thr Ile Met Gly Pro Asn Asp Ser Pro Tyr Gln Gly Gly 35 40 45Val Phe Phe Leu Thr Ile His Phe Pro Thr Asp Tyr Pro Phe Lys Pro 50 55 60Pro Lys Val Ala Phe Thr Thr Arg Ile Tyr His Pro Asn Ile Asn Ser65 70 75 80Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg Ser Gln Trp Ser Pro Ala 85 90 95Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Cys Asp 100 105 110Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala Arg Ile Tyr Lys 115 120 125Thr Asp Arg Asp Lys Tyr Asn Arg Ile Ser Arg Glu Trp Thr Gln Lys 130 135 140Tyr Ala Met14524200PRTHomo sapiens 24Met Ala Asn Ile Ala Val Gln Arg Ile Lys Arg Glu Phe Lys Glu Val1 5 10 15Leu Lys Ser Glu Glu Thr Ser Lys Asn Gln Ile Lys Val Asp Leu Val 20 25 30Asp Glu Asn Phe Thr Glu Leu Arg Gly Glu Ile Ala Gly Pro Pro Asp 35 40 45Thr Pro Tyr Glu Gly Gly Arg Tyr Gln Leu Glu Ile Lys Ile Pro Glu 50 55 60Thr Tyr Pro Phe Asn Pro Pro Lys Val Arg Phe Ile Thr Lys Ile Trp65 70 75 80His Pro Asn Ile Ser Ser Val Thr Gly Ala Ile Cys Leu Asp Ile Leu 85 90 95Lys Asp Gln Trp Ala Ala Ala Met Thr Leu Arg Thr Val Leu Leu Ser 100 105 110Leu Gln Ala Leu Leu Ala Ala Ala Glu Pro Asp Asp Pro Gln Asp Ala 115 120 125Val Val Ala Asn Gln Tyr Lys Gln Asn Pro Glu Met Phe Lys Gln Thr 130 135 140Ala Arg Leu Trp Ala His Val Tyr Ala Gly Ala Pro Val Ser Ser Pro145 150 155 160Glu Tyr Thr Lys Lys Ile Glu Asn Leu Cys Ala Met Gly Phe Asp Arg 165 170 175Asn Ala Val Ile Val Ala Leu Ser Ser Lys Ser Trp Asp Val Glu Thr 180 185 190Ala Thr Glu Leu Leu Leu Ser Asn 195 20025428PRTArabidopsis thaliana 25Met Ala Thr Ala Phe Ala Pro Thr Lys Leu Thr Ala Thr Val Pro Leu1 5 10 15His Gly Ser His Glu Asn Arg Leu Leu Leu Pro Ile Arg Leu Ala Pro 20 25 30Pro Ser Ser Phe Leu Gly Ser Thr Arg Ser Leu Ser Leu Arg Arg Leu 35 40 45Asn His Ser Asn Ala Thr Arg Arg Ser Pro Val Val Ser Val Gln Glu 50 55 60Val Val Lys Glu Lys Gln Ser Thr Asn Asn Thr Ser Leu Leu Ile Thr65 70 75 80Lys Glu Glu Gly Leu Glu Leu Tyr Glu Asp Met Ile Leu Gly Arg Ser 85 90 95Phe Glu Asp Met Cys Ala Gln Met Tyr Tyr Arg Gly Lys Met Phe Gly 100 105 110Phe Val His Leu Tyr Asn Gly Gln Glu Ala Val Ser Thr Gly Phe Ile 115 120 125Lys Leu Leu Thr Lys Ser Asp Ser Val Val Ser Thr Tyr Arg Asp His 130 135 140Val His Ala Leu Ser Lys Gly Val Ser Ala Arg Ala Val Met Ser Glu145 150 155 160Leu Phe Gly Lys Val Thr Gly Cys Cys Arg Gly Gln Gly Gly Ser Met 165 170 175His Met Phe Ser Lys Glu His Asn Met Leu Gly Gly Phe Ala Phe Ile 180 185 190Gly Glu Gly Ile Pro Val Ala Thr Gly Ala Ala Phe Ser Ser Lys Tyr 195 200 205Arg Arg Glu Val Leu Lys Gln Asp Cys Asp Asp Val Thr Val Ala Phe 210 215 220Phe Gly Asp Gly Thr Cys Asn Asn Gly Gln Phe Phe Glu Cys Leu Asn225 230 235 240Met Ala Ala Leu Tyr Lys Leu Pro Ile Ile Phe Val Val Glu Asn Asn 245 250 255Leu Trp Ala Ile Gly Met Ser His Leu Arg Ala Thr Ser Asp Pro Glu 260 265 270Ile Trp Lys Lys Gly Pro Ala Phe Gly Met Pro Gly Val His Val Asp 275 280 285Gly Met Asp Val Leu Lys Val Arg Glu Val Ala Lys Glu Ala Val Thr 290 295 300Arg Ala Arg Arg Gly Glu Gly Pro Thr Leu Val Glu Cys Glu Thr Tyr305 310 315 320Arg Phe Arg Gly His Ser Leu Ala Asp Pro Asp Glu Leu Arg Asp Ala 325 330 335Ala Glu Lys Ala Lys Tyr Ala Ala Arg Asp Pro Ile Ala Ala Leu Lys 340 345 350Lys Tyr Leu Ile Glu Asn Lys Leu Ala Lys Glu Ala Glu Leu Lys Ser 355 360 365Ile Glu Lys Lys Ile Asp Glu Leu Val Glu Glu Ala Val Glu Phe Ala 370 375 380Asp Ala Ser Pro Gln Pro Gly Arg Ser Gln Leu Leu Glu Asn Val Phe385 390 395 400Ala Asp Pro Lys Gly Phe Gly Ile Gly Pro Asp Gly Arg Tyr Arg Cys 405 410 415Glu Asp Pro Lys Phe Thr Glu Gly Thr Ala Gln Val 420 42526235PRTMus musculus 26Met Ala Arg Pro Leu Val Pro Ser Ser Gln Lys Ala Leu Leu Leu Glu1 5 10 15Leu Lys Gly Leu Gln Glu Glu Pro Val Glu Gly Phe Arg Val Thr Leu 20 25 30Val Asp Glu Gly Asp Leu Tyr Asn Trp Glu Val Ala Ile Phe Gly Pro 35 40 45Pro Asn Thr Tyr Tyr Glu Gly Gly Tyr Phe Lys Ala Arg Leu Lys Phe 50 55 60Pro Ile Asp Tyr Pro Tyr Ser Pro Pro Ala Phe Arg Phe Leu Thr Lys65 70 75 80Met Trp His Pro Asn Ile Tyr Glu Thr Gly Asp Val Cys Ile Ser Ile 85 90 95Leu His Pro Pro Val Asp Asp Pro Gln Ser Gly Glu Leu Pro Ser Glu 100 105 110Arg Trp Asn Pro Thr Gln Asn Val Arg Thr Ile Leu Leu Ser Val Ile 115 120 125Ser Leu Leu Asn Glu Pro Asn Thr Phe Ser Pro Ala Asn Val Asp Ala 130 135 140Ser Val Met Tyr Arg Lys Trp Lys Glu Ser Lys Gly Lys Asp Arg Glu145 150 155 160Tyr Thr Asp Ile Ile Arg Lys Gln Val Leu Gly Thr Lys Val Asp Ala 165 170 175Glu Arg Asp Gly Val Lys Val Pro Thr Thr Leu Ala Glu Tyr Cys Val 180 185 190Lys Thr Lys Ala Pro Ala Pro Asp Glu Gly Ser Asp Leu Phe Tyr Asp 195 200 205Asp Tyr Tyr Glu Asp Gly Glu Val Glu Glu Ala Asp Ser Cys Phe Gly 210 215 220Asp Glu Glu Asp Asp Ser Gly Thr Glu Glu Ser225 230 23527172PRTSaccharomyces cerevisiae 27Met Ser Thr Pro Ala Arg Arg Arg Leu Met Arg Asp Phe Lys Arg Met1 5 10 15Lys Glu Asp Ala Pro Pro Gly Val Ser Ala Ser Pro Leu Pro Asp Asn 20 25 30Val Met Val Trp Asn Ala Met Ile Ile Gly Pro Ala Asp Thr Pro Tyr 35 40 45Glu Asp Gly Thr Phe Arg Leu Leu Leu Glu Phe Asp Glu Glu Tyr Pro 50 55 60Asn Lys Pro Pro His Val Lys Phe Leu Ser Glu Met Phe His Pro Asn65 70 75 80Val Tyr Ala Asn Gly Glu Ile Cys Leu Asp Ile Leu Gln Asn Arg Trp 85 90 95Thr Pro Thr Tyr Asp Val Ala Ser Ile Leu Thr Ser Ile Gln Ser Leu 100 105 110Phe Asn Asp Pro Asn Pro Ala Ser Pro Ala Asn Val Glu Ala Ala Thr 115 120 125Leu Phe Lys Asp His Lys Ser Gln Tyr Val Lys Arg Val Lys Glu Thr 130 135 140Val Glu Lys Ser Trp Glu Asp Asp Met Asp Asp Met Asp Asp Asp Asp145 150 155 160Asp Asp Asp Asp Asp Asp Asp Asp Asp Glu Ala Asp 165 17028200PRTMus musculus 28Met Ala Asn Ile Ala Val Gln Arg Ile Lys Arg Glu Phe Lys Glu Val1 5 10 15Leu Lys Ser Glu Glu Thr Ser Lys Asn Gln Ile Lys Val Asp Leu Val 20 25 30Asp Glu Asn Phe Thr Glu Leu Arg Gly Glu Ile Ala Gly Pro Pro Asp 35 40 45Thr Pro Tyr Glu Gly Gly Arg Tyr Gln Leu Glu Ile Lys Ile Pro Glu 50 55 60Thr Tyr Pro Phe Asn Pro Pro Lys Val Arg Phe Ile Thr Lys Ile Trp65 70 75 80His Pro Asn Ile Ser Ser Val Thr Gly Ala Ile Cys Leu Asp Ile Leu 85 90 95Lys Asp Gln Trp Ala Ala Ala Met Thr Leu Arg Thr Val Leu Leu Ser 100 105 110Leu Gln Ala Leu Leu Ala Ala Ala Glu Pro Asp Asp Pro Gln Asp Ala 115 120 125Val Val Ala Asn Gln Tyr Lys Gln Asn Pro Glu Met Phe Lys Gln Thr 130 135 140Ala Arg Leu Trp Ala His Val Tyr Ala Gly Ala Pro Val Ser Ser Pro145 150 155 160Glu Tyr Thr Lys Lys Ile Glu Asn Leu Cys Ala Met Gly Phe Asp Arg 165 170 175Asn Ala Val Ile Val Ala Leu Ser Ser Lys Ser Trp Asp Val Glu Thr 180 185 190Ala Thr Glu Leu Leu Leu Ser Asn 195 20029153PRTArabidopsis thaliana 29Met Ala Asn Ser Asn Leu Pro Arg Arg Ile Ile Lys Glu Thr Gln Arg1 5 10 15Leu Leu Ser Glu Pro Ala Pro Gly Ile Ser Ala Ser Pro Ser Glu Asp 20 25 30Asn Met Arg Tyr Phe Asn Val Met Ile Leu Gly Pro Thr Gln Ser Pro 35 40 45Tyr Glu Gly Gly Val Phe Lys Leu Glu Leu Phe Leu Pro Glu Glu Tyr 50 55 60Pro Met Ala Ala Pro Lys Val Arg Phe Leu Thr Lys Ile Tyr His Pro65 70 75 80Asn Ile Asp Lys Leu Gly Arg Ile Cys Leu Asp Ile Leu Lys Asp Lys 85 90 95Trp Ser Pro Ala Leu Gln Ile Arg Thr Val Leu Leu Ser Ile Gln Ala 100 105 110Leu Leu Ser Ala Pro Asn Pro Asp Asp Pro Leu Ser Glu Asn Ile Ala 115 120 125Lys His Trp Lys Ser Asn Glu Ala Glu Ala Val Asp Thr Ala Lys Glu 130 135 140Trp Thr Arg Leu Tyr Ala Ser Gly Ala145 15030295PRTSaccharomyces cerevisiae 30Met Ser Ser Arg Lys Ser Thr Ala Ser Ser Leu Leu Leu Arg Gln Tyr1 5 10 15Arg Glu Leu Thr Asp Pro Lys Lys Ala Ile Pro Ser Phe His Ile Glu 20 25 30Leu Glu Asp Asp Ser Asn Ile Phe Thr Trp Asn Ile Gly Val Met Val 35 40 45Leu Asn Glu Asp Ser Ile Tyr His Gly Gly Phe Phe Lys Ala Gln Met 50 55 60Arg Phe Pro Glu Asp Phe Pro Phe Ser Pro Pro Gln Phe Arg Phe Thr65 70 75 80Pro Ala Ile Tyr His Pro Asn Val Tyr Arg Asp Gly Arg Leu Cys Ile 85 90 95Ser Ile Leu His Gln Ser Gly Asp Pro Met Thr Asp Glu Pro Asp Ala 100 105 110Glu Thr Trp Ser Pro Val Gln Thr Val Glu Ser Val Leu Ile Ser Ile 115 120 125Val Ser Leu Leu Glu Asp Pro Asn Ile Asn Ser Pro Ala Asn Val Asp 130 135 140Ala Ala Val Asp Tyr Arg Lys Asn Pro Glu Gln Tyr Lys Gln Arg Val145 150 155 160Lys Met Glu Val Glu Arg Ser Lys Gln Asp Ile Pro Lys Gly Phe Ile 165 170 175Met Pro Thr Ser Glu Ser Ala Tyr Ile Ser Gln Ser Lys Leu Asp Glu 180 185 190Pro Glu Ser Asn Lys Asp Met Ala Asp Asn Phe Trp Tyr Asp Ser Asp 195 200 205Leu Asp Asp Asp Glu Asn Gly Ser Val Ile Leu Gln Asp Asp Asp Tyr 210 215 220Asp Asp Gly Asn Asn His Ile Pro Phe Glu Asp Asp Asp Val Tyr Asn225 230 235 240Tyr Asn Asp Asn Asp Asp Asp Asp Glu Arg Ile Glu Phe Glu Asp Asp 245 250 255Asp Asp Asp Asp Asp Asp Ser Ile Asp Asn Asp Ser Val Met Asp Arg 260 265 270Lys Gln Pro His Lys Ala Glu Asp Glu Ser Glu Asp Val Glu Asp Val 275 280 285Glu Arg Val Ser Lys Lys Ile 290 29531145PRTHomo sapiens 31Met Ala Val Ser Thr Gly Val Lys Val Pro Arg Asn Phe Arg Leu Leu1 5 10 15Glu Glu Leu Glu Glu Gly Gln Lys Gly Val Gly Asp Gly Thr Val Ser 20 25 30Trp Gly Leu Glu Asp Asp Glu Asp Met Thr Leu Thr Arg Trp Thr Gly 35 40 45Met Ile Ile Gly Pro Pro Arg Thr Asn Tyr Glu Asn Arg Ile Tyr Ser 50 55 60Leu Lys Val Glu Cys Gly Pro Lys Tyr Pro Glu Ala Pro Pro Ser Val65 70 75 80Arg Phe Val Thr Lys Ile Asn Met Asn Gly Ile Asn Asn Ser Ser Gly 85 90 95Met Val Asp Ala Arg Ser Ile Pro Val Leu Ala Lys Trp Gln Asn Ser 100 105 110Tyr Ser Ile Lys Val Val Leu Gln Glu Leu Arg Arg Leu Met Met Ser 115 120 125Lys Glu Asn Met Lys Leu Pro Gln Pro Pro Glu Gly Gln Thr Tyr Asn 130 135 140Asn14532215PRTSaccharomyces cerevisiae 32Met Ser Arg Ala Lys Arg Ile Met Lys Glu Ile Gln Ala Val Lys Asp1 5 10 15Asp Pro Ala Ala His Ile Thr Leu Glu Phe Val Ser Glu Ser Asp Ile 20 25 30His His Leu Lys Gly Thr Phe Leu Gly Pro Pro Gly Thr Pro Tyr Glu 35 40 45Gly Gly Lys Phe Val Val Asp Ile Glu Val Pro Met Glu Tyr Pro Phe 50 55 60Lys Pro Pro Lys Met Gln Phe Asp Thr Lys Val Tyr His Pro Asn Ile65 70 75 80Ser Ser Val Thr Gly Ala Ile Cys Leu Asp Ile Leu Lys Asn Ala Trp 85 90 95Ser Pro Val Ile Thr Leu Lys Ser Ala Leu Ile Ser Leu Gln Ala Leu 100 105 110Leu Gln Ser Pro Glu Pro Asn Asp Pro Gln Asp Ala Glu Val Ala Gln 115 120 125His Tyr Leu Arg Asp Arg Glu Ser Phe Asn Lys Thr Ala Ala Leu Trp 130 135 140Thr Arg Leu Tyr Ala Ser Glu Thr Ser Asn Gly Gln Lys Gly Asn Val145 150 155 160Glu Glu Ser Asp Leu Tyr Gly Ile Asp His Asp Leu Ile Asp Glu Phe 165 170 175Glu Ser Gln Gly Phe Glu Lys Asp Lys Ile Val Glu Val Leu Arg Arg 180 185 190Leu Gly Val Lys Ser Leu Asp Pro Asn Asp Asn Asn Thr Ala Asn Arg 195 200 205Ile Ile Glu Glu Leu Leu Lys 210 21533250PRTSaccharomyces cerevisiae 33Met Ala Thr Lys Gln Ala His Lys Arg Leu Thr Lys Glu Tyr Lys Leu1 5 10 15Met Val Glu Asn Pro Pro Pro Tyr Ile Leu Ala Arg Pro Asn Glu Asp 20 25 30Asn Ile Leu Glu Trp His Tyr Ile Ile Thr Gly Pro Ala Asp Thr Pro 35 40 45Tyr Lys Gly Gly Gln Tyr His Gly Thr Leu Thr Phe Pro Ser Asp Tyr 50 55 60Pro Tyr Lys Pro Pro Ala Ile Arg Met Ile Thr Pro Asn Gly Arg Phe65 70 75 80Lys Pro Asn Thr Arg Leu Cys Leu Ser Met Ser Asp Tyr His Pro Asp 85 90 95Thr Trp Asn Pro Gly Trp Ser Val Ser Thr Ile Leu Asn Gly Leu Leu 100

105 110Ser Phe Met Thr Ser Asp Glu Ala Thr Thr Gly Ser Ile Thr Thr Ser 115 120 125Asp His Gln Lys Lys Thr Leu Ala Arg Asn Ser Ile Ser Tyr Asn Thr 130 135 140Phe Gln Asn Val Arg Phe Lys Leu Ile Phe Pro Glu Val Val Gln Glu145 150 155 160Asn Val Glu Thr Leu Glu Lys Arg Lys Leu Asp Glu Gly Asp Ala Ala 165 170 175Asn Thr Gly Asp Glu Thr Glu Asp Pro Phe Thr Lys Ala Ala Lys Glu 180 185 190Lys Val Ile Ser Leu Glu Glu Ile Leu Asp Pro Glu Asp Arg Ile Arg 195 200 205Ala Glu Gln Ala Leu Arg Gln Ser Glu Asn Asn Ser Lys Lys Asp Gly 210 215 220Lys Glu Pro Asn Asp Ser Ser Ser Met Val Tyr Ile Gly Ile Ala Ile225 230 235 240Phe Leu Phe Leu Val Gly Leu Phe Met Lys 245 25034148PRTArabidopsis thaliana 34Met Ala Ser Lys Arg Ile Leu Lys Glu Leu Lys Asp Leu Gln Lys Asp1 5 10 15Pro Pro Thr Ser Cys Ser Ala Gly Pro Val Ala Glu Asp Met Phe His 20 25 30Trp Gln Ala Thr Ile Met Gly Pro Ser Glu Ser Pro Tyr Ala Gly Gly 35 40 45Val Phe Leu Val Thr Ile His Phe Pro Pro Asp Tyr Pro Phe Lys Pro 50 55 60Pro Lys Val Ala Phe Arg Thr Lys Val Phe His Pro Asn Ile Asn Ser65 70 75 80Asn Gly Ser Ile Cys Leu Asp Ile Leu Lys Glu Gln Trp Ser Pro Ala 85 90 95Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Thr Asp 100 105 110Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala His Met Tyr Lys 115 120 125Thr Asp Lys Asn Lys Tyr Glu Ser Thr Ala Arg Ser Trp Thr Gln Lys 130 135 140Tyr Ala Met Gly14535318PRTMus musculus 35Met Glu Thr Arg Tyr Asn Leu Lys Ser Pro Ala Val Lys Arg Leu Met1 5 10 15Lys Glu Ala Ala Glu Leu Lys Asp Pro Thr Asp His Tyr His Ala Gln 20 25 30Pro Leu Glu Asp Asn Leu Phe Glu Trp His Phe Thr Val Arg Gly Pro 35 40 45Pro Asp Ser Asp Phe Asp Gly Gly Val Tyr His Gly Arg Ile Val Leu 50 55 60Pro Pro Glu Tyr Pro Met Lys Pro Pro Ser Ile Ile Leu Leu Thr Ala65 70 75 80Asn Gly Arg Phe Glu Val Gly Lys Lys Ile Cys Leu Ser Ile Ser Gly 85 90 95His His Pro Glu Thr Trp Gln Pro Ser Trp Ser Ile Arg Thr Ala Leu 100 105 110Leu Ala Ile Ile Gly Phe Met Pro Thr Lys Gly Glu Gly Ala Ile Gly 115 120 125Ser Leu Asp Tyr Thr Pro Glu Glu Arg Arg Ala Leu Ala Lys Lys Ser 130 135 140Gln Asp Phe Cys Cys Glu Gly Cys Gly Ser Ala Met Lys Asp Val Leu145 150 155 160Leu Pro Leu Lys Ser Gly Ser Gly Ser Ser Gln Ala Asp Gln Glu Ala 165 170 175Lys Glu Leu Ala Arg Gln Ile Ser Phe Lys Ala Glu Val Asn Ser Ser 180 185 190Gly Lys Thr Ile Ala Glu Ser Asp Leu Asn Gln Cys Phe Ser Leu Asn 195 200 205Asp Ser Gln Asp Asp Leu Pro Thr Thr Phe Gln Gly Ala Thr Ala Ser 210 215 220Thr Ser Tyr Gly Ala Gln Asn Pro Ser Gly Ala Pro Leu Pro Gln Pro225 230 235 240Thr Gln Pro Ala Pro Lys Asn Thr Ser Met Ser Pro Arg Gln Arg Arg 245 250 255Ala Gln Gln Gln Ser Gln Arg Arg Pro Ser Thr Ser Pro Asp Val Leu 260 265 270Gln Gly Gln Pro Pro Arg Ala His His Thr Glu His Gly Gly Ser Ala 275 280 285Met Leu Ile Ile Ile Leu Thr Leu Ala Leu Ala Ala Leu Ile Phe Arg 290 295 300Arg Ile Tyr Leu Ala Asn Glu Tyr Ile Phe Asp Phe Glu Leu305 310 31536148PRTArabidopsis thaliana 36Met Ala Ser Lys Arg Ile Leu Lys Glu Leu Lys Asp Leu Gln Lys Asp1 5 10 15Pro Pro Ser Asn Cys Ser Ala Gly Pro Val Ala Glu Asp Met Phe His 20 25 30Trp Gln Ala Thr Ile Met Gly Pro Pro Glu Ser Pro Tyr Ala Gly Gly 35 40 45Val Phe Leu Val Ser Ile His Phe Pro Pro Asp Tyr Pro Phe Lys Pro 50 55 60Pro Lys Val Ser Phe Lys Thr Lys Val Tyr His Pro Asn Ile Asn Ser65 70 75 80Asn Gly Ser Ile Cys Leu Asp Ile Leu Lys Glu Gln Trp Ser Pro Ala 85 90 95Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Thr Asp 100 105 110Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala His Met Tyr Lys 115 120 125Thr Asp Arg Ser Lys Tyr Glu Ser Thr Ala Arg Ser Trp Thr Gln Lys 130 135 140Tyr Ala Met Gly14537491PRTHomo sapiens 37Met Cys Asn Thr Asn Met Ser Val Pro Thr Asp Gly Ala Val Thr Thr1 5 10 15Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro 20 25 30Leu Leu Leu Lys Leu Leu Lys Ser Val Gly Ala Gln Lys Asp Thr Tyr 35 40 45Thr Met Lys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile Met Thr Lys 50 55 60Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp65 70 75 80Leu Leu Gly Asp Leu Phe Gly Val Pro Ser Phe Ser Val Lys Glu His 85 90 95Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln 100 105 110Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Glu Asn Arg Cys His 115 120 125Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu Val Gln Glu Leu Gln Glu 130 135 140Glu Lys Pro Ser Ser Ser His Leu Val Ser Arg Pro Ser Thr Ser Ser145 150 155 160Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu Asn Ser Asp Glu Leu Ser 165 170 175Gly Glu Arg Gln Arg Lys Arg His Lys Ser Asp Ser Ile Ser Leu Ser 180 185 190Phe Asp Glu Ser Leu Ala Leu Cys Val Ile Arg Glu Ile Cys Cys Glu 195 200 205Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu 210 215 220Asp Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser225 230 235 240Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser 245 250 255Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu Asp 260 265 270Asp Glu Val Tyr Gln Val Thr Val Tyr Gln Ala Gly Glu Ser Asp Thr 275 280 285Asp Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys 290 295 300Cys Thr Ser Cys Asn Glu Met Asn Pro Pro Leu Pro Ser His Cys Asn305 310 315 320Arg Cys Trp Ala Leu Arg Glu Asn Trp Leu Pro Glu Asp Lys Gly Lys 325 330 335Asp Lys Gly Glu Ile Ser Glu Lys Ala Lys Leu Glu Asn Ser Thr Gln 340 345 350Ala Glu Glu Gly Phe Asp Val Pro Asp Cys Lys Lys Thr Ile Val Asn 355 360 365Asp Ser Arg Glu Ser Cys Val Glu Glu Asn Asp Asp Lys Ile Thr Gln 370 375 380Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser385 390 395 400Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp Val Lys Glu Phe Glu Arg 405 410 415Glu Glu Thr Gln Asp Lys Glu Glu Ser Val Glu Ser Ser Leu Pro Leu 420 425 430Asn Ala Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro Lys Asn Gly 435 440 445Cys Ile Val His Gly Lys Thr Gly His Leu Met Ala Cys Phe Thr Cys 450 455 460Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro Cys Pro Val Cys Arg Gln465 470 475 480Pro Ile Gln Met Ile Val Leu Thr Tyr Phe Pro 485 49038465PRTHomo sapiens 38Met Ile Val Phe Val Arg Phe Asn Ser Ser His Gly Phe Pro Val Glu1 5 10 15Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys 20 25 30Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys 35 40 45Glu Leu Arg Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln 50 55 60Ser Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met65 70 75 80Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu 85 90 95Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu 100 105 110Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg 115 120 125Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn 130 135 140Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly145 150 155 160Lys Leu Arg Val Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu 165 170 175Thr Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met 180 185 190Ser Gly Glu Cys Gln Ser Pro His Cys Pro Gly Thr Ser Ala Glu Phe 195 200 205Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Glu Thr Ser Val 210 215 220Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr225 230 235 240Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg 245 250 255His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu 260 265 270Asn Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly Tyr Ser Leu Pro 275 280 285Cys Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe 290 295 300Arg Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala305 310 315 320Glu Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly 325 330 335Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys 340 345 350Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys 355 360 365Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser 370 375 380Gly Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala Ala Glu Gln385 390 395 400Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys 405 410 415Pro Cys Pro Arg Cys His Val Pro Val Glu Lys Asn Gly Gly Cys Met 420 425 430His Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn 435 440 445Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp 450 455 460Val465391863PRTHomo sapiens 39Met Asp Leu Ser Ala Leu Arg Val Glu Glu Val Gln Asn Val Ile Asn1 5 10 15Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cys Leu Glu Leu Ile Lys 20 25 30Glu Pro Val Ser Thr Lys Cys Asp His Ile Phe Cys Lys Phe Cys Met 35 40 45Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Ser Gln Cys Pro Leu Cys 50 55 60Lys Asn Asp Ile Thr Lys Arg Ser Leu Gln Glu Ser Thr Arg Phe Ser65 70 75 80Gln Leu Val Glu Glu Leu Leu Lys Ile Ile Cys Ala Phe Gln Leu Asp 85 90 95Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Phe Ala Lys Lys Glu Asn 100 105 110Asn Ser Pro Glu His Leu Lys Asp Glu Val Ser Ile Ile Gln Ser Met 115 120 125Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gln Ser Glu Pro Glu Asn 130 135 140Pro Ser Leu Gln Glu Thr Ser Leu Ser Val Gln Leu Ser Asn Leu Gly145 150 155 160Thr Val Arg Thr Leu Arg Thr Lys Gln Arg Ile Gln Pro Gln Lys Thr 165 170 175Ser Val Tyr Ile Glu Leu Gly Ser Asp Ser Ser Glu Asp Thr Val Asn 180 185 190Lys Ala Thr Tyr Cys Ser Val Gly Asp Gln Glu Leu Leu Gln Ile Thr 195 200 205Pro Gln Gly Thr Arg Asp Glu Ile Ser Leu Asp Ser Ala Lys Lys Ala 210 215 220Ala Cys Glu Phe Ser Glu Thr Asp Val Thr Asn Thr Glu His His Gln225 230 235 240Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Lys Arg Ala Ala Glu Arg 245 250 255His Pro Glu Lys Tyr Gln Gly Ser Ser Val Ser Asn Leu His Val Glu 260 265 270Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gln His Glu Asn Ser 275 280 285Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Val Glu Lys Ala Glu Phe 290 295 300Cys Asn Lys Ser Lys Gln Pro Gly Leu Ala Arg Ser Gln His Asn Arg305 310 315 320Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Arg Arg Thr Pro Ser Thr 325 330 335Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Glu Arg Lys Glu 340 345 350Trp Asn Lys Gln Lys Leu Pro Cys Ser Glu Asn Pro Arg Asp Thr Glu 355 360 365Asp Val Pro Trp Ile Thr Leu Asn Ser Ser Ile Gln Lys Val Asn Glu 370 375 380Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Ser Asp Asp Ser His Asp385 390 395 400Gly Glu Ser Glu Ser Asn Ala Lys Val Ala Asp Val Leu Asp Val Leu 405 410 415Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Glu Lys Ile Asp Leu Leu 420 425 430Ala Ser Asp Pro His Glu Ala Leu Ile Cys Lys Ser Glu Arg Val His 435 440 445Ser Lys Ser Val Glu Ser Asn Ile Glu Asp Lys Ile Phe Gly Lys Thr 450 455 460Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Ser His Val Thr Glu Asn465 470 475 480Leu Ile Ile Gly Ala Phe Val Thr Glu Pro Gln Ile Ile Gln Glu Arg 485 490 495Pro Leu Thr Asn Lys Leu Lys Arg Lys Arg Arg Pro Thr Ser Gly Leu 500 505 510His Pro Glu Asp Phe Ile Lys Lys Ala Asp Leu Ala Val Gln Lys Thr 515 520 525Pro Glu Met Ile Asn Gln Gly Thr Asn Gln Thr Glu Gln Asn Gly Gln 530 535 540Val Met Asn Ile Thr Asn Ser Gly His Glu Asn Lys Thr Lys Gly Asp545 550 555 560Ser Ile Gln Asn Glu Lys Asn Pro Asn Pro Ile Glu Ser Leu Glu Lys 565 570 575Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro Ile Ser Ser Ser Ile Ser 580 585 590Asn Met Glu Leu Glu Leu Asn Ile His Asn Ser Lys Ala Pro Lys Lys 595 600 605Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg His Ile His Ala Leu Glu 610 615 620Leu Val Val Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Glu Leu Gln625 630 635 640Ile Asp Ser Cys Ser Ser Ser Glu Glu Ile Lys Lys Lys Lys Tyr Asn 645 650 655Gln Met Pro Val Arg His Ser Arg Asn Leu Gln Leu Met Glu Gly Lys 660 665 670Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Glu Gln Thr 675 680 685Ser Lys Arg His Asp Ser Asp Thr Phe Pro Glu Leu Lys Leu Thr Asn 690 695 700Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Thr Ser Glu Leu Lys Glu705 710 715 720Phe Val Asn Pro Ser Leu Pro Arg Glu Glu Lys Glu Glu Lys Leu Glu 725 730 735Thr Val Lys Val Ser Asn Asn Ala Glu Asp Pro Lys Asp Leu Met Leu 740 745

750Ser Gly Glu Arg Val Leu Gln Thr Glu Arg Ser Val Glu Ser Ser Ser 755 760 765Ile Ser Leu Val Pro Gly Thr Asp Tyr Gly Thr Gln Glu Ser Ile Ser 770 775 780Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Lys Thr Glu Pro Asn Lys785 790 795 800Cys Val Ser Gln Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu Ile His 805 810 815Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Glu Gly Phe Lys Tyr Pro 820 825 830Leu Gly His Glu Val Asn His Ser Arg Glu Thr Ser Ile Glu Met Glu 835 840 845Glu Ser Glu Leu Asp Ala Gln Tyr Leu Gln Asn Thr Phe Lys Val Ser 850 855 860Lys Arg Gln Ser Phe Ala Pro Phe Ser Asn Pro Gly Asn Ala Glu Glu865 870 875 880Glu Cys Ala Thr Phe Ser Ala His Ser Gly Ser Leu Lys Lys Gln Ser 885 890 895Pro Lys Val Thr Phe Glu Cys Glu Gln Lys Glu Glu Asn Gln Gly Lys 900 905 910Asn Glu Ser Asn Ile Lys Pro Val Gln Thr Val Asn Ile Thr Ala Gly 915 920 925Phe Pro Val Val Gly Gln Lys Asp Lys Pro Val Asp Asn Ala Lys Cys 930 935 940Ser Ile Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser Gln Phe Arg Gly945 950 955 960Asn Glu Thr Gly Leu Ile Thr Pro Asn Lys His Gly Leu Leu Gln Asn 965 970 975Pro Tyr Arg Ile Pro Pro Leu Phe Pro Ile Lys Ser Phe Val Lys Thr 980 985 990Lys Cys Lys Lys Asn Leu Leu Glu Glu Asn Phe Glu Glu His Ser Met 995 1000 1005Ser Pro Glu Arg Glu Met Gly Asn Glu Asn Ile Pro Ser Thr Val 1010 1015 1020Ser Thr Ile Ser Arg Asn Asn Ile Arg Glu Asn Val Phe Lys Glu 1025 1030 1035Ala Ser Ser Ser Asn Ile Asn Glu Val Gly Ser Ser Thr Asn Glu 1040 1045 1050Val Gly Ser Ser Ile Asn Glu Ile Gly Ser Ser Asp Glu Asn Ile 1055 1060 1065Gln Ala Glu Leu Gly Arg Asn Arg Gly Pro Lys Leu Asn Ala Met 1070 1075 1080Leu Arg Leu Gly Val Leu Gln Pro Glu Val Tyr Lys Gln Ser Leu 1085 1090 1095Pro Gly Ser Asn Cys Lys His Pro Glu Ile Lys Lys Gln Glu Tyr 1100 1105 1110Glu Glu Val Val Gln Thr Val Asn Thr Asp Phe Ser Pro Tyr Leu 1115 1120 1125Ile Ser Asp Asn Leu Glu Gln Pro Met Gly Ser Ser His Ala Ser 1130 1135 1140Gln Val Cys Ser Glu Thr Pro Asp Asp Leu Leu Asp Asp Gly Glu 1145 1150 1155Ile Lys Glu Asp Thr Ser Phe Ala Glu Asn Asp Ile Lys Glu Ser 1160 1165 1170Ser Ala Val Phe Ser Lys Ser Val Gln Lys Gly Glu Leu Ser Arg 1175 1180 1185Ser Pro Ser Pro Phe Thr His Thr His Leu Ala Gln Gly Tyr Arg 1190 1195 1200Arg Gly Ala Lys Lys Leu Glu Ser Ser Glu Glu Asn Leu Ser Ser 1205 1210 1215Glu Asp Glu Glu Leu Pro Cys Phe Gln His Leu Leu Phe Gly Lys 1220 1225 1230Val Asn Asn Ile Pro Ser Gln Ser Thr Arg His Ser Thr Val Ala 1235 1240 1245Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu Asn Leu Leu Ser Leu 1250 1255 1260Lys Asn Ser Leu Asn Asp Cys Ser Asn Gln Val Ile Leu Ala Lys 1265 1270 1275Ala Ser Gln Glu His His Leu Ser Glu Glu Thr Lys Cys Ser Ala 1280 1285 1290Ser Leu Phe Ser Ser Gln Cys Ser Glu Leu Glu Asp Leu Thr Ala 1295 1300 1305Asn Thr Asn Thr Gln Asp Pro Phe Leu Ile Gly Ser Ser Lys Gln 1310 1315 1320Met Arg His Gln Ser Glu Ser Gln Gly Val Gly Leu Ser Asp Lys 1325 1330 1335Glu Leu Val Ser Asp Asp Glu Glu Arg Gly Thr Gly Leu Glu Glu 1340 1345 1350Asn Asn Gln Glu Glu Gln Ser Met Asp Ser Asn Leu Gly Glu Ala 1355 1360 1365Ala Ser Gly Cys Glu Ser Glu Thr Ser Val Ser Glu Asp Cys Ser 1370 1375 1380Gly Leu Ser Ser Gln Ser Asp Ile Leu Thr Thr Gln Gln Arg Asp 1385 1390 1395Thr Met Gln His Asn Leu Ile Lys Leu Gln Gln Glu Met Ala Glu 1400 1405 1410Leu Glu Ala Val Leu Glu Gln His Gly Ser Gln Pro Ser Asn Ser 1415 1420 1425Tyr Pro Ser Ile Ile Ser Asp Ser Ser Ala Leu Glu Asp Leu Arg 1430 1435 1440Asn Pro Glu Gln Ser Thr Ser Glu Lys Ala Val Leu Thr Ser Gln 1445 1450 1455Lys Ser Ser Glu Tyr Pro Ile Ser Gln Asn Pro Glu Gly Leu Ser 1460 1465 1470Ala Asp Lys Phe Glu Val Ser Ala Asp Ser Ser Thr Ser Lys Asn 1475 1480 1485Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Ser Lys Cys Pro Ser 1490 1495 1500Leu Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gly Ser Leu Gln 1505 1510 1515Asn Arg Asn Tyr Pro Ser Gln Glu Glu Leu Ile Lys Val Val Asp 1520 1525 1530Val Glu Glu Gln Gln Leu Glu Glu Ser Gly Pro His Asp Leu Thr 1535 1540 1545Glu Thr Ser Tyr Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Tyr 1550 1555 1560Leu Glu Ser Gly Ile Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp 1565 1570 1575Pro Ser Glu Asp Arg Ala Pro Glu Ser Ala Arg Val Gly Asn Ile 1580 1585 1590Pro Ser Ser Thr Ser Ala Leu Lys Val Pro Gln Leu Lys Val Ala 1595 1600 1605Glu Ser Ala Gln Ser Pro Ala Ala Ala His Thr Thr Asp Thr Ala 1610 1615 1620Gly Tyr Asn Ala Met Glu Glu Ser Val Ser Arg Glu Lys Pro Glu 1625 1630 1635Leu Thr Ala Ser Thr Glu Arg Val Asn Lys Arg Met Ser Met Val 1640 1645 1650Val Ser Gly Leu Thr Pro Glu Glu Phe Met Leu Val Tyr Lys Phe 1655 1660 1665Ala Arg Lys His His Ile Thr Leu Thr Asn Leu Ile Thr Glu Glu 1670 1675 1680Thr Thr His Val Val Met Lys Thr Asp Ala Glu Phe Val Cys Glu 1685 1690 1695Arg Thr Leu Lys Tyr Phe Leu Gly Ile Ala Gly Gly Lys Trp Val 1700 1705 1710Val Ser Tyr Phe Trp Val Thr Gln Ser Ile Lys Glu Arg Lys Met 1715 1720 1725Leu Asn Glu His Asp Phe Glu Val Arg Gly Asp Val Val Asn Gly 1730 1735 1740Arg Asn His Gln Gly Pro Lys Arg Ala Arg Glu Ser Gln Asp Arg 1745 1750 1755Lys Ile Phe Arg Gly Leu Glu Ile Cys Cys Tyr Gly Pro Phe Thr 1760 1765 1770Asn Met Pro Thr Asp Gln Leu Glu Trp Met Val Gln Leu Cys Gly 1775 1780 1785Ala Ser Val Val Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gly 1790 1795 1800Val His Pro Ile Val Val Val Gln Pro Asp Ala Trp Thr Glu Asp 1805 1810 1815Asn Gly Phe His Ala Ile Gly Gln Met Cys Glu Ala Pro Val Val 1820 1825 1830Thr Arg Glu Trp Val Leu Asp Ser Val Ala Leu Tyr Gln Cys Gln 1835 1840 1845Glu Leu Asp Thr Tyr Leu Ile Pro Gln Ile Pro His Ser His Tyr 1850 1855 186040809PRTSaccharomyces cerevisiae 40Met Pro Ser Ser Ile Ser Val Lys Leu Val Ala Ala Glu Ser Leu Tyr1 5 10 15Lys Arg Asp Val Phe Arg Ser Pro Asp Pro Phe Ala Val Leu Thr Ile 20 25 30Asp Gly Tyr Gln Thr Lys Ser Thr Ser Ala Ala Lys Lys Thr Leu Asn 35 40 45Pro Tyr Trp Asn Glu Thr Phe Lys Phe Asp Asp Ile Asn Glu Asn Ser 50 55 60Ile Leu Thr Ile Gln Val Phe Asp Gln Lys Lys Phe Lys Lys Lys Asp65 70 75 80Gln Gly Phe Leu Gly Val Val Asn Val Arg Val Gly Asp Val Leu Gly 85 90 95His Leu Asp Glu Asp Thr Ala Thr Ser Ser Gly Arg Pro Arg Glu Glu 100 105 110Thr Ile Thr Arg Asp Leu Lys Lys Ser Asn Asp Gly Met Ala Val Ser 115 120 125Gly Arg Leu Ile Val Val Leu Ser Lys Leu Pro Ser Ser Ser Pro His 130 135 140Ser Gln Ala Pro Ser Gly His Thr Ala Ser Ser Ser Thr Asn Thr Ser145 150 155 160Ser Thr Thr Arg Thr Asn Gly His Ser Thr Ser Ser Thr Arg Asn His 165 170 175Ser Thr Ser His Pro Ser Arg Gly Thr Ala Gln Ala Val Glu Ser Thr 180 185 190Leu Gln Ser Gly Thr Thr Ala Ala Thr Asn Thr Ala Thr Thr Ser His 195 200 205Arg Ser Thr Asn Ser Thr Ser Ser Ala Thr Arg Gln Tyr Ser Ser Phe 210 215 220Glu Asp Gln Tyr Gly Arg Leu Pro Pro Gly Trp Glu Arg Arg Thr Asp225 230 235 240Asn Phe Gly Arg Thr Tyr Tyr Val Asp His Asn Thr Arg Thr Thr Thr 245 250 255Trp Lys Arg Pro Thr Leu Asp Gln Thr Glu Ala Glu Arg Gly Asn Gln 260 265 270Leu Asn Ala Asn Thr Glu Leu Glu Arg Arg Gln His Arg Gly Arg Thr 275 280 285Leu Pro Gly Gly Ser Ser Asp Asn Ser Ser Val Thr Val Gln Val Gly 290 295 300Gly Gly Ser Asn Ile Pro Pro Val Asn Gly Ala Ala Ala Ala Ala Phe305 310 315 320Ala Ala Thr Gly Gly Thr Thr Ser Gly Leu Gly Glu Leu Pro Ser Gly 325 330 335Trp Glu Gln Arg Phe Thr Pro Glu Gly Arg Ala Tyr Phe Val Asp His 340 345 350Asn Thr Arg Thr Thr Thr Trp Val Asp Pro Arg Arg Gln Gln Tyr Ile 355 360 365Arg Thr Tyr Gly Pro Thr Asn Thr Thr Ile Gln Gln Gln Pro Val Ser 370 375 380Gln Leu Gly Pro Leu Pro Ser Gly Trp Glu Met Arg Leu Thr Asn Thr385 390 395 400Ala Arg Val Tyr Phe Val Asp His Asn Thr Lys Thr Thr Thr Trp Asp 405 410 415Asp Pro Arg Leu Pro Ser Ser Leu Asp Gln Asn Val Pro Gln Tyr Lys 420 425 430Arg Asp Phe Arg Arg Lys Val Ile Tyr Phe Arg Ser Gln Pro Ala Leu 435 440 445Arg Ile Leu Pro Gly Gln Cys His Ile Lys Val Arg Arg Lys Asn Ile 450 455 460Phe Glu Asp Ala Tyr Gln Glu Ile Met Arg Gln Thr Pro Glu Asp Leu465 470 475 480Lys Lys Arg Leu Met Ile Lys Phe Asp Gly Glu Glu Gly Leu Asp Tyr 485 490 495Gly Gly Val Ser Arg Glu Phe Phe Phe Leu Leu Ser His Glu Met Phe 500 505 510Asn Pro Phe Tyr Cys Leu Phe Glu Tyr Ser Ala Tyr Asp Asn Tyr Thr 515 520 525Ile Gln Ile Asn Pro Asn Ser Gly Ile Asn Pro Glu His Leu Asn Tyr 530 535 540Phe Lys Phe Ile Gly Arg Val Val Gly Leu Gly Val Phe His Arg Arg545 550 555 560Phe Leu Asp Ala Phe Phe Val Gly Ala Leu Tyr Lys Met Met Leu Arg 565 570 575Lys Lys Val Val Leu Gln Asp Met Glu Gly Val Asp Ala Glu Val Tyr 580 585 590Asn Ser Leu Asn Trp Met Leu Glu Asn Ser Ile Asp Gly Val Leu Asp 595 600 605Leu Thr Phe Ser Ala Asp Asp Glu Arg Phe Gly Glu Val Val Thr Val 610 615 620Asp Leu Lys Pro Asp Gly Arg Asn Ile Glu Val Thr Asp Gly Asn Lys625 630 635 640Lys Glu Tyr Val Glu Leu Tyr Thr Gln Trp Arg Ile Val Asp Arg Val 645 650 655Gln Glu Gln Phe Lys Ala Phe Met Asp Gly Phe Asn Glu Leu Ile Pro 660 665 670Glu Asp Leu Val Thr Val Phe Asp Glu Arg Glu Leu Glu Leu Leu Ile 675 680 685Gly Gly Ile Ala Glu Ile Asp Ile Glu Asp Trp Lys Lys His Thr Asp 690 695 700Tyr Arg Gly Tyr Gln Glu Ser Asp Glu Val Ile Gln Trp Phe Trp Lys705 710 715 720Cys Val Ser Glu Trp Asp Asn Glu Gln Arg Ala Arg Leu Leu Gln Phe 725 730 735Thr Thr Gly Thr Ser Arg Ile Pro Val Asn Gly Phe Lys Asp Leu Gln 740 745 750Gly Ser Asp Gly Pro Arg Arg Phe Thr Ile Glu Lys Ala Gly Glu Val 755 760 765Gln Gln Leu Pro Lys Ser His Thr Cys Phe Asn Arg Val Asp Leu Pro 770 775 780Gln Tyr Val Asp Tyr Asp Ser Met Lys Gln Lys Leu Thr Leu Ala Val785 790 795 800Glu Glu Thr Ile Gly Phe Gly Gln Glu 80541501PRTHomo sapiens 41Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln1 5 10 15Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr 20 25 30Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln 35 40 45Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser 50 55 60Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu65 70 75 80Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala 85 90 95Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys 100 105 110Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg 115 120 125Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg 130 135 140Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser145 150 155 160Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys 165 170 175Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys 180 185 190Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr 195 200 205Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu 210 215 220Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu225 230 235 240Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro 245 250 255Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys 260 265 270Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val 275 280 285Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys Ser 290 295 300Arg Gln His Arg Leu Asp Gln Asp Lys Ile Glu Ala Leu Ser Ser Lys305 310 315 320Val Gln Gln Leu Glu Arg Ser Ile Gly Leu Lys Asp Leu Ala Met Ala 325 330 335Asp Leu Glu Gln Lys Val Leu Glu Met Glu Ala Ser Thr Tyr Asp Gly 340 345 350Val Phe Ile Trp Lys Ile Ser Asp Phe Ala Arg Lys Arg Gln Glu Ala 355 360 365Val Ala Gly Arg Ile Pro Ala Ile Phe Ser Pro Ala Phe Tyr Thr Ser 370 375 380Arg Tyr Gly Tyr Lys Met Cys Leu Arg Ile Tyr Leu Asn Gly Asp Gly385 390 395 400Thr Gly Arg Gly Thr His Leu Ser Leu Phe Phe Val Val Met Lys Gly 405 410 415Pro Asn Asp Ala Leu Leu Arg Trp Pro Phe Asn Gln Lys Val Thr Leu 420 425 430Met Leu Leu Asp Gln Asn Asn Arg Glu His Val Ile Asp Ala Phe Arg 435 440 445Pro Asp Val Thr Ser Ser Ser Phe Gln Arg Pro Val Asn Asp Met Asn 450 455 460Ile Ala Ser Gly Cys Pro Leu Phe Cys Pro Val Ser Lys Met Glu Ala465 470 475 480Lys Asn Ser Tyr Val Arg Asp Asp Ala Ile Phe Ile Lys Ala Ile Val 485 490 495Asp Leu Thr Gly Leu 50042282PRTHomo sapiens 42Met Ser Arg Gln Thr Ala Thr Ala Leu Pro Thr Gly Thr Ser Lys Cys1 5 10 15Pro Pro Ser Gln Arg Val Pro Ala Leu Thr Gly Thr Thr Ala Ser Asn 20

25 30Asn Asp Leu Ala Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val 35 40 45Leu Pro Pro Ile Leu Gln Cys Gln Ser Gly His Leu Val Cys Ser Asn 50 55 60Cys Arg Pro Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Gly65 70 75 80Ser Ile Arg Asn Leu Ala Met Glu Lys Val Ala Asn Ser Val Leu Phe 85 90 95Pro Cys Lys Tyr Ala Ser Ser Gly Cys Glu Ile Thr Leu Pro His Thr 100 105 110Glu Lys Ala Asp His Glu Glu Leu Cys Glu Phe Arg Pro Tyr Ser Cys 115 120 125Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Ser Leu Asp Ala Val 130 135 140Met Pro His Leu Met His Gln His Lys Ser Ile Thr Thr Leu Gln Gly145 150 155 160Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val 165 170 175Asp Trp Val Met Met Gln Ser Cys Phe Gly Phe His Phe Met Leu Val 180 185 190Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile 195 200 205Val Gln Leu Ile Gly Thr Arg Lys Gln Ala Glu Asn Phe Ala Tyr Arg 210 215 220Leu Glu Leu Asn Gly His Arg Arg Arg Leu Thr Trp Glu Ala Thr Pro225 230 235 240Arg Ser Ile His Glu Gly Ile Ala Thr Ala Ile Met Asn Ser Asp Cys 245 250 255Leu Val Phe Asp Thr Ser Ile Ala Gln Leu Phe Ala Glu Asn Gly Asn 260 265 270Leu Gly Ile Asn Val Thr Ile Ser Met Cys 275 28043464PRTMus musculus 43Met Ile Val Phe Val Arg Phe Asn Ser Ser Tyr Gly Phe Pro Val Glu1 5 10 15Val Asp Ser Asp Thr Ser Ile Leu Gln Leu Lys Glu Val Val Ala Lys 20 25 30Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys 35 40 45Glu Leu Pro Asn His Leu Thr Val Gln Asn Cys Asp Leu Glu Gln Gln 50 55 60Ser Ile Val His Ile Val Gln Arg Pro Arg Arg Arg Ser His Glu Thr65 70 75 80Asn Ala Ser Gly Gly Asp Glu Pro Gln Ser Thr Ser Glu Gly Ser Ile 85 90 95Trp Glu Ser Arg Ser Leu Thr Arg Val Asp Leu Ser Ser His Thr Leu 100 105 110Pro Val Asp Ser Val Gly Leu Ala Val Ile Leu Asp Thr Asp Ser Lys 115 120 125Arg Asp Ser Glu Ala Ala Arg Gly Pro Val Lys Pro Thr Tyr Asn Ser 130 135 140Phe Phe Ile Tyr Cys Lys Gly Pro Cys His Lys Val Gln Pro Gly Lys145 150 155 160Leu Arg Val Gln Cys Gly Thr Cys Lys Gln Ala Thr Leu Thr Leu Ala 165 170 175Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met Ser 180 185 190Gly Glu Cys Gln Ser Pro Asp Cys Pro Gly Thr Arg Ala Glu Phe Phe 195 200 205Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Asp Thr Ser Val Ala 210 215 220Leu Asn Leu Ile Thr Ser Asn Arg Arg Ser Ile Pro Cys Ile Ala Cys225 230 235 240Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn His Arg His 245 250 255Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu Asn 260 265 270Asp Arg Gln Phe Val His Asp Ala Gln Leu Gly Tyr Ser Leu Pro Cys 275 280 285Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe Arg 290 295 300Ile Leu Gly Glu Glu Gln Tyr Thr Arg Tyr Gln Gln Tyr Gly Ala Glu305 310 315 320Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly Cys 325 330 335Gly Ala Gly Leu Leu Pro Glu Gln Gly Gln Arg Lys Val Thr Cys Glu 340 345 350Gly Gly Asn Gly Leu Gly Cys Gly Phe Val Phe Cys Arg Asp Cys Lys 355 360 365Glu Ala Tyr His Glu Gly Asp Cys Asp Ser Leu Leu Glu Pro Ser Gly 370 375 380Ala Thr Ser Gln Ala Tyr Arg Val Asp Lys Arg Ala Ala Glu Gln Ala385 390 395 400Arg Trp Glu Glu Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys Pro 405 410 415Cys Pro Arg Cys Asn Val Pro Ile Glu Lys Asn Gly Gly Cys Met His 420 425 430Met Lys Cys Pro Gln Pro Gln Cys Lys Leu Glu Trp Cys Trp Asn Cys 435 440 445Gly Cys Glu Trp Asn Arg Ala Cys Met Gly Asp His Trp Phe Asp Val 450 455 46044303PRTHomo sapiens 44Met Lys Gly Lys Glu Glu Lys Glu Gly Gly Ala Arg Leu Gly Ala Gly1 5 10 15Gly Gly Ser Pro Glu Lys Ser Pro Ser Ala Gln Glu Leu Lys Glu Gln 20 25 30Gly Asn Arg Leu Phe Val Gly Arg Lys Tyr Pro Glu Ala Ala Ala Cys 35 40 45Tyr Gly Arg Ala Ile Thr Arg Asn Pro Leu Val Ala Val Tyr Tyr Thr 50 55 60Asn Arg Ala Leu Cys Tyr Leu Lys Met Gln Gln His Glu Gln Ala Leu65 70 75 80Ala Asp Cys Arg Arg Ala Leu Glu Leu Asp Gly Gln Ser Val Lys Ala 85 90 95His Phe Phe Leu Gly Gln Cys Gln Leu Glu Met Glu Ser Tyr Asp Glu 100 105 110Ala Ile Ala Asn Leu Gln Arg Ala Tyr Ser Leu Ala Lys Glu Gln Arg 115 120 125Leu Asn Phe Gly Asp Asp Ile Pro Ser Ala Leu Arg Ile Ala Lys Lys 130 135 140Lys Arg Trp Asn Ser Ile Glu Glu Arg Arg Ile His Gln Glu Ser Glu145 150 155 160Leu His Ser Tyr Leu Ser Arg Leu Ile Ala Ala Glu Arg Glu Arg Glu 165 170 175Leu Glu Glu Cys Gln Arg Asn His Glu Gly Asp Glu Asp Asp Ser His 180 185 190Val Arg Ala Gln Gln Ala Cys Ile Glu Ala Lys His Asp Lys Tyr Met 195 200 205Ala Asp Met Asp Glu Leu Phe Ser Gln Val Asp Glu Lys Arg Lys Lys 210 215 220Arg Asp Ile Pro Asp Tyr Leu Cys Gly Lys Ile Ser Phe Glu Leu Met225 230 235 240Arg Glu Pro Cys Ile Thr Pro Ser Gly Ile Thr Tyr Asp Arg Lys Asp 245 250 255Ile Glu Glu His Leu Gln Arg Val Gly His Phe Asp Pro Val Thr Arg 260 265 270Ser Pro Leu Thr Gln Glu Gln Leu Ile Pro Asn Leu Ala Met Lys Glu 275 280 285Val Ile Asp Ala Phe Ile Ser Glu Asn Gly Trp Val Glu Asp Tyr 290 295 300451030PRTDanio rerio 45Met Thr Thr Gly Arg Asn Asn Arg Val Met Met Glu Gly Val Gly Ala1 5 10 15Arg Val Ile Arg Gly Pro Asp Trp Lys Trp Gly Lys Gln Asp Gly Gly 20 25 30Glu Gly His Val Gly Thr Val Arg Ser Phe Glu Ser Pro Glu Glu Val 35 40 45Val Val Val Trp Asp Asn Gly Thr Ala Ala Asn Tyr Arg Cys Ser Gly 50 55 60Ala Tyr Asp Val Arg Ile Leu Asp Ser Ala Pro Thr Gly Ile Lys His65 70 75 80Asp Gly Thr Met Cys Asp Thr Cys Arg Gln Gln Pro Ile Ile Gly Ile 85 90 95Arg Trp Lys Cys Ala Glu Cys Thr Asn Tyr Asp Leu Cys Thr Thr Cys 100 105 110Tyr His Gly Asp Lys His His Leu Arg His Arg Phe Tyr Arg Ile Thr 115 120 125Thr Pro Gly Ser Glu Arg Val Leu Leu Glu Ser Arg Arg Lys Ser Lys 130 135 140Lys Ile Thr Ala Arg Gly Ile Phe Ala Gly Gly Arg Val Val Arg Gly145 150 155 160Val Asp Trp Gln Trp Glu Asp Gln Asp Gly Gly Asn Gly Arg Arg Gly 165 170 175Lys Val Thr Glu Ile Gln Asp Trp Ser Ala Ala Ser Pro His Ser Ala 180 185 190Ala Tyr Val Leu Trp Asp Asn Gly Ala Lys Asn Leu Tyr Arg Val Gly 195 200 205Phe Glu Gly Met Ser Asp Leu Lys Cys Val Gln Asp Ala Lys Gly Gly 210 215 220Thr Phe Tyr Arg Asp His Cys Pro Val Leu Gly Glu Gln Asn Gly Asn225 230 235 240Arg Asn Pro Gly Gly Leu Gln Ile Gly Asp Leu Val Asn Ile Asp Leu 245 250 255Asp Leu Glu Ile Val Gln Ser Leu Gln His Gly His Gly Gly Trp Thr 260 265 270Asp Gly Met Phe Glu Thr Leu Thr Thr Thr Gly Thr Val Cys Gly Ile 275 280 285Asp Glu Asp His Asp Ile Val Val Gln Tyr Pro Ser Gly Asn Arg Trp 290 295 300Thr Phe Asn Pro Ala Val Leu Thr Lys Ala Asn Val Val Arg Ser Gly305 310 315 320Glu Val Ala Ala Gly Ala Glu Gly Gly Ser Ser Gln Phe Met Val Gly 325 330 335Asp Leu Val Gln Ile Cys Tyr Asp Ile Asp Arg Ile Lys Leu Leu Gln 340 345 350Arg Gly His Gly Glu Trp Ala Glu Ala Met Leu Pro Thr Leu Gly Lys 355 360 365Val Gly Arg Val Gln Gln Ile Tyr Ser Asp Ser Asp Leu Lys Val Glu 370 375 380Val Cys Gly Thr Ser Trp Thr Tyr Asn Pro Ala Ala Val Thr Lys Val385 390 395 400Ala Pro Ala Gly Ser Ala Val Thr Asn Ala Ser Gly Glu Arg Leu Ser 405 410 415Gln Leu Leu Lys Lys Leu Phe Glu Thr Gln Glu Ser Gly Asp Ile Asn 420 425 430Glu Glu Leu Val Lys Ala Ala Ala Asn Gly Asp Leu Ala Lys Val Glu 435 440 445Asp Ile Leu Lys Arg Pro Asp Val Asp Val Asn Gly Gln Cys Ala Gly 450 455 460His Thr Ala Met Gln Ala Ala Ser Gln Asn Gly His Val Asp Val Leu465 470 475 480Lys Leu Leu Leu Lys His Ser Val Asp Leu Glu Ala Glu Asp Lys Asp 485 490 495Gly Asp Arg Ala Val His His Ala Ser Phe Gly Asp Glu Gly Ser Val 500 505 510Ile Glu Val Leu His Arg Gly Gly Ala Asp Leu Asn Ala Arg Asn Lys 515 520 525Arg Arg Gln Thr Pro Leu His Ile Ala Val Asn Lys Gly His Leu Gln 530 535 540Val Val Lys Thr Leu Leu Asp Phe Gly Cys His Pro Ser Leu Gln Asp545 550 555 560Ser Glu Gly Asp Thr Pro Leu His Asp Ala Ile Ser Lys Lys Arg Asp 565 570 575Asp Met Leu Ser Val Leu Leu Glu Ala Gly Ala Asp Val Thr Ile Thr 580 585 590Asn Asn Asn Gly Phe Asn Ala Leu His His Ala Ala Leu Arg Gly Asn 595 600 605Pro Ser Ala Met Arg Val Leu Leu Ser Lys Leu Pro Arg Pro Trp Ile 610 615 620Val Asp Glu Lys Lys Asp Asp Gly Tyr Thr Ala Leu His Leu Ala Ala625 630 635 640Leu Asn Asn His Val Glu Val Ala Glu Leu Leu Val His Gln Gly Asn 645 650 655Ala Asn Leu Asp Val Gln Asn Val Asn Gln Gln Thr Ala Leu His Leu 660 665 670Ala Val Glu Arg Gln His Thr Gln Ile Val Arg Leu Leu Val Arg Ala 675 680 685Glu Ala Lys Leu Asp Val Gln Asp Lys Asp Gly Asp Thr Pro Leu His 690 695 700Glu Ala Leu Arg His His Thr Leu Ser Gln Leu Arg Gln Leu Gln Asp705 710 715 720Met Gln Asp Val Ser Lys Val Glu Pro Trp Glu Pro Ser Lys Asn Thr 725 730 735Leu Ile Met Gly Leu Gly Thr Gln Gly Ala Glu Lys Lys Ser Ala Ala 740 745 750Ser Ile Ala Cys Phe Leu Ala Ala Asn Gly Ala Asp Leu Thr Ile Arg 755 760 765Asn Lys Lys Gly Gln Ser Pro Leu Asp Leu Cys Pro Asp Pro Ser Leu 770 775 780Cys Lys Ala Leu Ala Lys Cys His Lys Glu Lys Thr Ser Gly Gln Val785 790 795 800Gly Ser Arg Ser Pro Ser Leu Asn Ser Asn Asn Glu Thr Leu Glu Glu 805 810 815Cys Met Val Cys Ser Asp Met Lys Arg Asp Thr Leu Phe Gly Pro Cys 820 825 830Gly His Ile Ala Thr Cys Ser Leu Cys Ser Pro Arg Val Lys Lys Cys 835 840 845Leu Ile Cys Lys Glu Gln Val Gln Ser Arg Thr Lys Ile Glu Glu Cys 850 855 860Val Val Cys Ser Asp Lys Lys Ala Ala Val Leu Phe Gln Pro Cys Gly865 870 875 880His Met Cys Ala Cys Glu Asn Cys Ala Ser Leu Met Lys Lys Cys Val 885 890 895Gln Cys Arg Ala Val Val Glu Arg Arg Thr Pro Phe Val Leu Cys Cys 900 905 910Gly Gly Lys Gly Met Glu Asp Ala Thr Asp Asp Glu Asp Leu Thr Gly 915 920 925Gly Ser Asn Ser Met Ala Gly Gly Ser Gln Asp Leu Leu Gln Pro Asn 930 935 940Asn Leu Ala Leu Ser Trp Ser Ser Gly Asn Ile Pro Ala Leu Gln Arg945 950 955 960Asp Lys Asp Asn Thr Asn Val Asn Ala Asp Val Gln Lys Leu Gln Gln 965 970 975Gln Leu Gln Asp Ile Lys Glu Gln Thr Met Cys Pro Val Cys Leu Asp 980 985 990Arg Leu Lys Asn Met Ile Phe Met Cys Gly His Gly Thr Cys Gln Leu 995 1000 1005Cys Gly Asp Arg Met Ser Glu Cys Pro Ile Cys Arg Lys Ala Ile 1010 1015 1020Glu Arg Arg Ile Leu Leu Tyr1025 103046757PRTHomo sapiens 46Met Ser Asn Pro Gly Thr Arg Arg Asn Gly Ser Ser Ile Lys Ile Arg1 5 10 15Leu Thr Val Leu Cys Ala Lys Asn Leu Ala Lys Lys Asp Phe Phe Arg 20 25 30Leu Pro Asp Pro Phe Ala Lys Ile Val Val Asp Gly Ser Gly Gln Cys 35 40 45His Ser Thr Asp Thr Val Lys Asn Thr Leu Asp Pro Lys Trp Asn Gln 50 55 60His Tyr Asp Leu Tyr Val Gly Lys Thr Asp Ser Ile Thr Ile Ser Val65 70 75 80Trp Asn His Lys Lys Ile His Lys Lys Gln Gly Ala Gly Phe Leu Gly 85 90 95Cys Val Arg Leu Leu Ser Asn Ala Ile Ser Arg Leu Lys Asp Thr Gly 100 105 110Tyr Gln Arg Leu Asp Leu Cys Lys Leu Asn Pro Ser Asp Thr Asp Ala 115 120 125Val Arg Gly Gln Ile Val Val Ser Leu Gln Thr Arg Asp Arg Ile Gly 130 135 140Thr Gly Gly Ser Val Val Asp Cys Arg Gly Leu Leu Glu Asn Glu Gly145 150 155 160Thr Val Tyr Glu Asp Ser Gly Pro Gly Arg Pro Leu Ser Cys Phe Met 165 170 175Glu Glu Pro Ala Pro Tyr Thr Asp Ser Thr Gly Ala Ala Ala Gly Gly 180 185 190Gly Asn Cys Arg Phe Val Glu Ser Pro Ser Gln Asp Gln Arg Leu Gln 195 200 205Ala Gln Arg Leu Arg Asn Pro Asp Val Arg Gly Ser Leu Gln Thr Pro 210 215 220Gln Asn Arg Pro His Gly His Gln Ser Pro Glu Leu Pro Glu Gly Tyr225 230 235 240Glu Gln Arg Thr Thr Val Gln Gly Gln Val Tyr Phe Leu His Thr Gln 245 250 255Thr Gly Val Ser Thr Trp His Asp Pro Arg Ile Pro Ser Pro Ser Gly 260 265 270Thr Ile Pro Gly Gly Asp Ala Ala Phe Leu Tyr Glu Phe Leu Leu Gln 275 280 285Gly His Thr Ser Glu Pro Arg Asp Leu Asn Ser Val Asn Cys Asp Glu 290 295 300Leu Gly Pro Leu Pro Pro Gly Trp Glu Val Arg Ser Thr Val Ser Gly305 310 315 320Arg Ile Tyr Phe Val Asp His Asn Asn Arg Thr Thr Gln Phe Thr Asp 325 330 335Pro Arg Leu His His Ile Met Asn His Gln Cys Gln Leu Lys Glu Pro 340 345 350Ser Gln Pro Leu Pro Leu Pro Ser Glu Gly Ser Leu Glu Asp Glu Glu 355 360 365Leu Pro Ala Gln Arg Tyr Glu Arg Asp Leu Val Gln Lys Leu Lys Val 370 375 380Leu Arg His Glu Leu Ser Leu Gln Gln Pro Gln Ala Gly His Cys Arg385 390 395 400Ile Glu Val Ser Arg Glu Glu Ile Phe Glu Glu Ser Tyr Arg Gln Ile

405 410 415Met Lys Met Arg Pro Lys Asp Leu Lys Lys Arg Leu Met Val Lys Phe 420 425 430Arg Gly Glu Glu Gly Leu Asp Tyr Gly Gly Val Ala Arg Glu Trp Leu 435 440 445Tyr Leu Leu Cys His Glu Met Leu Asn Pro Tyr Tyr Gly Leu Phe Gln 450 455 460Tyr Ser Thr Asp Asn Ile Tyr Met Leu Gln Ile Asn Pro Asp Ser Ser465 470 475 480Ile Asn Pro Asp His Leu Ser Tyr Phe His Phe Val Gly Arg Ile Met 485 490 495Gly Leu Ala Val Phe His Gly His Tyr Ile Asn Gly Gly Phe Thr Val 500 505 510Pro Phe Tyr Lys Gln Leu Leu Gly Lys Pro Ile Gln Leu Ser Asp Leu 515 520 525Glu Ser Val Asp Pro Glu Leu His Lys Ser Leu Val Trp Ile Leu Glu 530 535 540Asn Asp Ile Thr Pro Val Leu Asp His Thr Phe Cys Val Glu His Asn545 550 555 560Ala Phe Gly Arg Ile Leu Gln His Glu Leu Lys Pro Asn Gly Arg Asn 565 570 575Val Pro Val Thr Glu Glu Asn Lys Lys Glu Tyr Val Arg Leu Tyr Val 580 585 590Asn Trp Arg Phe Met Arg Gly Ile Glu Ala Gln Phe Leu Ala Leu Gln 595 600 605Lys Gly Phe Asn Glu Leu Ile Pro Gln His Leu Leu Lys Pro Phe Asp 610 615 620Gln Lys Glu Leu Glu Leu Ile Ile Gly Gly Leu Asp Lys Ile Asp Leu625 630 635 640Asn Asp Trp Lys Ser Asn Thr Arg Leu Lys His Cys Val Ala Asp Ser 645 650 655Asn Ile Val Arg Trp Phe Trp Gln Ala Val Glu Thr Phe Asp Glu Glu 660 665 670Arg Arg Ala Arg Leu Leu Gln Phe Val Thr Gly Ser Thr Arg Val Pro 675 680 685Leu Gln Gly Phe Lys Ala Leu Gln Gly Ser Thr Gly Ala Ala Gly Pro 690 695 700Arg Leu Phe Thr Ile His Leu Ile Asp Ala Asn Thr Asp Asn Leu Pro705 710 715 720Lys Ala His Thr Cys Phe Asn Arg Ile Asp Ile Pro Pro Tyr Glu Ser 725 730 735Tyr Glu Lys Leu Tyr Glu Lys Leu Leu Thr Ala Val Glu Glu Thr Cys 740 745 750Gly Phe Ala Val Glu 755471319PRTHomo sapiens 47Met Ala Gln Ser Leu Arg Leu His Phe Ala Ala Arg Arg Ser Asn Thr1 5 10 15Tyr Pro Leu Ser Glu Thr Ser Gly Asp Asp Leu Asp Ser His Val His 20 25 30Met Cys Phe Lys Arg Pro Thr Arg Ile Ser Thr Ser Asn Val Val Gln 35 40 45Met Lys Leu Thr Pro Arg Gln Thr Ala Leu Ala Pro Leu Ile Lys Glu 50 55 60Asn Val Gln Ser Gln Glu Arg Ser Ser Val Pro Ser Ser Glu Asn Val65 70 75 80Asn Lys Lys Ser Ser Cys Leu Gln Ile Ser Leu Gln Pro Thr Arg Tyr 85 90 95Ser Gly Tyr Leu Gln Ser Ser Asn Val Leu Ala Asp Ser Asp Asp Ala 100 105 110Ser Phe Thr Cys Ile Leu Lys Asp Gly Ile Tyr Ser Ser Ala Val Val 115 120 125Asp Asn Glu Leu Asn Ala Val Asn Asp Gly His Leu Val Ser Ser Pro 130 135 140Ala Ile Cys Ser Gly Ser Leu Ser Asn Phe Ser Thr Ser Asp Asn Gly145 150 155 160Ser Tyr Ser Ser Asn Gly Ser Asp Phe Gly Ser Cys Ala Ser Ile Thr 165 170 175Ser Gly Gly Ser Tyr Thr Asn Ser Val Ile Ser Asp Ser Ser Ser Tyr 180 185 190Thr Phe Pro Pro Ser Asp Asp Thr Phe Leu Gly Gly Asn Leu Pro Ser 195 200 205Asp Ser Thr Ser Asn Arg Ser Val Pro Asn Arg Asn Thr Thr Pro Cys 210 215 220Glu Ile Phe Ser Arg Ser Thr Ser Thr Asp Pro Phe Val Gln Asp Asp225 230 235 240Leu Glu His Gly Leu Glu Ile Met Lys Leu Pro Val Ser Arg Asn Thr 245 250 255Lys Ile Pro Leu Lys Arg Tyr Ser Ser Leu Val Ile Phe Pro Arg Ser 260 265 270Pro Ser Thr Thr Arg Pro Thr Ser Pro Thr Ser Leu Cys Thr Leu Leu 275 280 285Ser Lys Gly Ser Tyr Gln Thr Ser His Gln Phe Ile Ile Ser Pro Ser 290 295 300Glu Ile Ala His Asn Glu Asp Gly Thr Ser Ala Lys Gly Phe Leu Ser305 310 315 320Thr Ala Val Asn Gly Leu Arg Leu Ser Lys Thr Ile Cys Thr Pro Gly 325 330 335Glu Val Arg Asp Ile Arg Pro Leu His Arg Lys Gly Ser Leu Gln Lys 340 345 350Lys Ile Val Leu Ser Asn Asn Thr Pro Arg Gln Thr Val Cys Glu Lys 355 360 365Ser Ser Glu Gly Tyr Ser Cys Val Ser Val His Phe Thr Gln Arg Lys 370 375 380Ala Ala Thr Leu Asp Cys Glu Thr Thr Asn Gly Asp Cys Lys Pro Glu385 390 395 400Met Ser Glu Ile Lys Leu Asn Ser Asp Ser Glu Tyr Ile Lys Leu Met 405 410 415His Arg Thr Ser Ala Cys Leu Pro Ser Ser Gln Asn Val Asp Cys Gln 420 425 430Ile Asn Ile Asn Gly Glu Leu Glu Arg Pro His Ser Gln Met Asn Lys 435 440 445Asn His Gly Ile Leu Arg Arg Ser Ile Ser Leu Gly Gly Ala Tyr Pro 450 455 460Asn Ile Ser Cys Leu Ser Ser Leu Lys His Asn Cys Ser Lys Gly Gly465 470 475 480Pro Ser Gln Leu Leu Ile Lys Phe Ala Ser Gly Asn Glu Gly Lys Val 485 490 495Asp Asn Leu Ser Arg Asp Ser Asn Arg Asp Cys Thr Asn Glu Leu Ser 500 505 510Asn Ser Cys Lys Thr Arg Asp Asp Phe Leu Gly Gln Val Asp Val Pro 515 520 525Leu Tyr Pro Leu Pro Thr Glu Asn Pro Arg Leu Glu Arg Pro Tyr Thr 530 535 540Phe Lys Asp Phe Val Leu His Pro Arg Ser His Lys Ser Arg Val Lys545 550 555 560Gly Tyr Leu Arg Leu Lys Met Thr Tyr Leu Pro Lys Thr Ser Gly Ser 565 570 575Glu Asp Asp Asn Ala Glu Gln Ala Glu Glu Leu Glu Pro Gly Trp Val 580 585 590Val Leu Asp Gln Pro Asp Ala Ala Cys His Leu Gln Gln Gln Gln Glu 595 600 605Pro Ser Pro Leu Pro Pro Gly Trp Glu Glu Arg Gln Asp Ile Leu Gly 610 615 620Arg Thr Tyr Tyr Val Asn His Glu Ser Arg Arg Thr Gln Trp Lys Arg625 630 635 640Pro Thr Pro Gln Asp Asn Leu Thr Asp Ala Glu Asn Gly Asn Ile Gln 645 650 655Leu Gln Ala Gln Arg Ala Phe Thr Thr Arg Arg Gln Ile Ser Glu Glu 660 665 670Thr Glu Ser Val Asp Asn Arg Glu Ser Ser Glu Asn Trp Glu Ile Ile 675 680 685Arg Glu Asp Glu Ala Thr Met Tyr Ser Asn Gln Ala Phe Pro Ser Pro 690 695 700Pro Pro Ser Ser Asn Leu Asp Val Pro Thr His Leu Ala Glu Glu Leu705 710 715 720Asn Ala Arg Leu Thr Ile Phe Gly Asn Ser Ala Val Ser Gln Pro Ala 725 730 735Ser Ser Ser Asn His Ser Ser Arg Arg Gly Ser Leu Gln Ala Tyr Thr 740 745 750Phe Glu Glu Gln Pro Thr Leu Pro Val Leu Leu Pro Thr Ser Ser Gly 755 760 765Leu Pro Pro Gly Trp Glu Glu Lys Gln Asp Glu Arg Gly Arg Ser Tyr 770 775 780Tyr Val Asp His Asn Ser Arg Thr Thr Thr Trp Thr Lys Pro Thr Val785 790 795 800Gln Ala Thr Val Glu Thr Ser Gln Leu Thr Ser Ser Gln Ser Ser Ala 805 810 815Gly Pro Gln Ser Gln Ala Ser Thr Ser Asp Ser Gly Gln Gln Val Thr 820 825 830Gln Pro Ser Glu Ile Glu Gln Gly Phe Leu Pro Lys Gly Trp Glu Val 835 840 845Arg His Ala Pro Asn Gly Arg Pro Phe Phe Ile Asp His Asn Thr Lys 850 855 860Thr Thr Thr Trp Glu Asp Pro Arg Leu Lys Ile Pro Ala His Leu Arg865 870 875 880Gly Lys Thr Ser Leu Asp Thr Ser Asn Asp Leu Gly Pro Leu Pro Pro 885 890 895Gly Trp Glu Glu Arg Thr His Thr Asp Gly Arg Ile Phe Tyr Ile Asn 900 905 910His Asn Ile Lys Arg Thr Gln Trp Glu Asp Pro Arg Leu Glu Asn Val 915 920 925Ala Ile Thr Gly Pro Ala Val Pro Tyr Ser Arg Asp Tyr Lys Arg Lys 930 935 940Tyr Glu Phe Phe Arg Arg Lys Leu Lys Lys Gln Asn Asp Ile Pro Asn945 950 955 960Lys Phe Glu Met Lys Leu Arg Arg Ala Thr Val Leu Glu Asp Ser Tyr 965 970 975Arg Arg Ile Met Gly Val Lys Arg Ala Asp Phe Leu Lys Ala Arg Leu 980 985 990Trp Ile Glu Phe Asp Gly Glu Lys Gly Leu Asp Tyr Gly Gly Val Ala 995 1000 1005Arg Glu Trp Phe Phe Leu Ile Ser Lys Glu Met Phe Asn Pro Tyr 1010 1015 1020Tyr Gly Leu Phe Glu Tyr Ser Ala Thr Asp Asn Tyr Thr Leu Gln 1025 1030 1035Ile Asn Pro Asn Ser Gly Leu Cys Asn Glu Asp His Leu Ser Tyr 1040 1045 1050Phe Lys Phe Ile Gly Arg Val Ala Gly Met Ala Val Tyr His Gly 1055 1060 1065Lys Leu Leu Asp Gly Phe Phe Ile Arg Pro Phe Tyr Lys Met Met 1070 1075 1080Leu His Lys Pro Ile Thr Leu His Asp Met Glu Ser Val Asp Ser 1085 1090 1095Glu Tyr Tyr Asn Ser Leu Arg Trp Ile Leu Glu Asn Asp Pro Thr 1100 1105 1110Glu Leu Asp Leu Arg Phe Ile Ile Asp Glu Glu Leu Phe Gly Gln 1115 1120 1125Thr His Gln His Glu Leu Lys Asn Gly Gly Ser Glu Ile Val Val 1130 1135 1140Thr Asn Lys Asn Lys Lys Glu Tyr Ile Tyr Leu Val Ile Gln Trp 1145 1150 1155Arg Phe Val Asn Arg Ile Gln Lys Gln Met Ala Ala Phe Lys Glu 1160 1165 1170Gly Phe Phe Glu Leu Ile Pro Gln Asp Leu Ile Lys Ile Phe Asp 1175 1180 1185Glu Asn Glu Leu Glu Leu Leu Met Cys Gly Leu Gly Asp Val Asp 1190 1195 1200Val Asn Asp Trp Arg Glu His Thr Lys Tyr Lys Asn Gly Tyr Ser 1205 1210 1215Ala Asn His Gln Val Ile Gln Trp Phe Trp Lys Ala Val Leu Met 1220 1225 1230Met Asp Ser Glu Lys Arg Ile Arg Leu Leu Gln Phe Val Thr Gly 1235 1240 1245Thr Ser Arg Val Pro Met Asn Gly Phe Ala Glu Leu Tyr Gly Ser 1250 1255 1260Asn Gly Pro Gln Ser Phe Thr Val Glu Gln Trp Gly Thr Pro Glu 1265 1270 1275Lys Leu Pro Arg Ala His Thr Cys Phe Asn Arg Leu Asp Leu Pro 1280 1285 1290Pro Tyr Glu Ser Phe Glu Glu Leu Trp Asp Lys Leu Gln Met Ala 1295 1300 1305Ile Glu Asn Thr Gln Gly Phe Asp Gly Val Asp 1310 131548887PRTMus musculus 48Met Ser Ser Asp Met Ala Ala Asp Glu Ser Glu Ala Pro Val Leu Ser1 5 10 15Glu Asp Glu Val Trp Glu Phe Cys Leu Asp Lys Thr Glu Asp Gly Gly 20 25 30Gly Ser Pro Gly Ser Asp Val Thr Asp Thr Cys Glu Pro Pro Cys Gly 35 40 45Cys Trp Glu Leu Asn Pro Asn Ser Leu Glu Glu Glu His Val Leu Phe 50 55 60Thr Ala Asp Pro Tyr Leu Glu Leu His Asn Asp Asp Thr Arg Val Val65 70 75 80Arg Val Lys Val Ile Ala Gly Ile Gly Leu Ala Lys Lys Asp Ile Leu 85 90 95Gly Ala Ser Asp Pro Tyr Val Arg Val Thr Leu Tyr Asp Pro Met Ser 100 105 110Gly Ile Leu Thr Ser Val Gln Thr Lys Thr Ile Lys Lys Ser Leu Asn 115 120 125Pro Lys Trp Asn Glu Glu Ile Leu Phe Arg Val Leu Pro Gln Arg His 130 135 140Arg Ile Leu Phe Glu Val Phe Asp Glu Asn Arg Leu Thr Arg Asp Asp145 150 155 160Phe Leu Gly Gln Val Asp Val Pro Leu Tyr Pro Leu Pro Thr Glu Asn 165 170 175Pro Arg Met Glu Arg Pro Tyr Thr Phe Lys Asp Phe Val Leu His Pro 180 185 190Arg Ser His Lys Ser Arg Val Lys Gly Tyr Leu Arg Leu Lys Met Thr 195 200 205Tyr Leu Pro Lys Asn Gly Ser Glu Asp Glu Asn Ala Asp Gln Ala Glu 210 215 220Glu Leu Glu Pro Gly Trp Val Val Leu Asp Gln Pro Asp Ala Ala Thr225 230 235 240His Leu Pro His Pro Pro Glu Pro Ser Pro Leu Pro Pro Gly Trp Glu 245 250 255Glu Arg Gln Asp Val Leu Gly Arg Thr Tyr Tyr Val Asn His Glu Ser 260 265 270Arg Arg Thr Gln Trp Lys Arg Pro Ser Pro Asp Asp Asp Leu Thr Asp 275 280 285Glu Asp Asn Asp Asp Met Gln Leu Gln Ala Gln Arg Ala Phe Thr Thr 290 295 300Arg Arg Gln Ile Ser Glu Asp Val Asp Gly Pro Asp Asn Arg Glu Ser305 310 315 320Pro Glu Asn Trp Glu Ile Val Arg Glu Asp Glu Asn Thr Glu Tyr Ser 325 330 335Gly Gln Ala Val Gln Ser Pro Pro Ser Gly His Ile Asp Val Gln Thr 340 345 350His Leu Ala Glu Glu Phe Asn Thr Arg Leu Ala Val Cys Gly Asn Pro 355 360 365Ala Thr Ser Gln Pro Val Thr Ser Ser Asn His Ser Ser Arg Gly Gly 370 375 380Ser Leu Gln Thr Cys Ile Phe Glu Glu Gln Pro Thr Leu Pro Val Leu385 390 395 400Leu Pro Thr Ser Ser Gly Leu Pro Pro Gly Trp Glu Glu Lys Gln Asp 405 410 415Asp Arg Gly Arg Ser Tyr Tyr Val Asp His Asn Ser Lys Thr Thr Thr 420 425 430Trp Ser Lys Pro Thr Met Gln Asp Asp Pro Arg Ser Lys Ile Pro Ala 435 440 445His Leu Arg Gly Lys Thr Asp Ser Asn Asp Leu Gly Pro Leu Pro Pro 450 455 460Gly Trp Glu Glu Arg Thr His Thr Asp Gly Arg Val Phe Phe Ile Asn465 470 475 480His Asn Ile Lys Lys Thr Gln Trp Glu Asp Pro Arg Leu Gln Asn Val 485 490 495Ala Ile Thr Gly Pro Ala Val Pro Tyr Ser Arg Asp Tyr Lys Arg Lys 500 505 510Tyr Glu Phe Phe Arg Arg Lys Leu Lys Lys Gln Thr Asp Ile Pro Asn 515 520 525Lys Phe Glu Met Lys Leu Arg Arg Ala Asn Ile Leu Glu Asp Ser Tyr 530 535 540Arg Arg Ile Met Gly Val Lys Arg Ala Asp Leu Leu Lys Ala Arg Leu545 550 555 560Trp Ile Glu Phe Asp Gly Glu Lys Gly Leu Asp Tyr Gly Gly Val Ala 565 570 575Arg Glu Trp Phe Phe Leu Ile Ser Lys Glu Met Phe Asn Pro Tyr Tyr 580 585 590Gly Leu Phe Glu Tyr Ser Ala Thr Asp Asn Tyr Thr Leu Gln Ile Asn 595 600 605Pro Asn Ser Gly Leu Cys Asn Glu Asp His Leu Ser Tyr Phe Lys Phe 610 615 620Ile Gly Arg Val Ala Gly Met Ala Val Tyr His Gly Lys Leu Leu Asp625 630 635 640Gly Phe Phe Ile Arg Pro Phe Tyr Lys Met Met Leu Gln Lys Leu Ile 645 650 655Thr Leu His Asp Met Glu Ser Val Asp Ser Glu Tyr Tyr Ser Ser Leu 660 665 670Arg Trp Ile Leu Glu Asn Asp Pro Thr Glu Leu Asp Leu Arg Phe Ile 675 680 685Ile Asp Glu Glu Leu Phe Gly Gln Thr His Gln His Glu Leu Lys Thr 690 695 700Gly Gly Ser Glu Ile Val Val Thr Asn Lys Asn Lys Lys Glu Tyr Ile705 710 715 720Tyr Leu Val Ile Gln Trp Arg Phe Val Asn Arg Ile Gln Lys Gln Met 725 730 735Ala Ala Phe Lys Glu Gly Phe Phe Glu Leu Ile Pro Gln Asp Leu Ile 740 745 750Lys Ile Phe Asp Glu Asn Glu Leu Glu Leu Leu Met Cys Gly Leu Gly 755 760 765Asp Val Asp Val Asn Asp Trp Arg Glu His Thr Lys Tyr Lys Asn Gly 770 775 780Tyr Ser Met Asn His Gln Val Ile His Trp Phe Trp Lys Ala Val Trp785 790 795 800Met Met

Asp Ser Glu Lys Arg Ile Arg Leu Leu Gln Phe Val Thr Gly 805 810 815Thr Ser Arg Val Pro Met Asn Gly Phe Ala Glu Leu Tyr Gly Ser Asn 820 825 830Gly Pro Gln Ser Phe Thr Val Glu Gln Trp Gly Thr Pro Asp Lys Leu 835 840 845Pro Arg Ala His Thr Cys Phe Asn Arg Leu Asp Leu Pro Pro Tyr Glu 850 855 860Ser Phe Asp Glu Leu Trp Asp Lys Leu Gln Met Ala Ile Glu Asn Thr865 870 875 880Gln Gly Phe Asp Gly Val Asp 88549324PRTHomo sapiens 49Met Ser Arg Pro Ser Ser Thr Gly Pro Ser Ala Asn Lys Pro Cys Ser1 5 10 15Lys Gln Pro Pro Pro Gln Pro Gln His Thr Pro Ser Pro Ala Ala Pro 20 25 30Pro Ala Ala Ala Thr Ile Ser Ala Ala Gly Pro Gly Ser Ser Ala Val 35 40 45Pro Ala Ala Ala Ala Val Ile Ser Gly Pro Gly Gly Gly Gly Gly Ala 50 55 60Gly Pro Val Ser Pro Gln His His Glu Leu Thr Ser Leu Phe Glu Cys65 70 75 80Pro Val Cys Phe Asp Tyr Val Leu Pro Pro Ile Leu Gln Cys Gln Ala 85 90 95Gly His Leu Val Cys Asn Gln Cys Arg Gln Lys Leu Ser Cys Cys Pro 100 105 110Thr Cys Arg Gly Ala Leu Thr Pro Ser Ile Arg Asn Leu Ala Met Glu 115 120 125Lys Val Ala Ser Ala Val Leu Phe Pro Cys Lys Tyr Ala Thr Thr Gly 130 135 140Cys Ser Leu Thr Leu His His Thr Glu Lys Pro Glu His Glu Asp Ile145 150 155 160Cys Glu Tyr Arg Pro Tyr Ser Cys Pro Cys Pro Gly Ala Ser Cys Lys 165 170 175Trp Gln Gly Ser Leu Glu Ala Val Met Ser His Leu Met His Ala His 180 185 190Lys Ser Ile Thr Thr Leu Gln Gly Glu Asp Ile Val Phe Leu Ala Thr 195 200 205Asp Ile Asn Leu Pro Gly Ala Val Asp Trp Val Met Met Gln Ser Cys 210 215 220Phe Gly His His Phe Met Leu Val Leu Glu Lys Gln Glu Lys Tyr Glu225 230 235 240Gly His Gln Gln Phe Phe Ala Ile Val Leu Leu Ile Gly Thr Arg Lys 245 250 255Gln Ala Glu Asn Phe Ala Tyr Arg Leu Glu Leu Asn Gly Asn Arg Arg 260 265 270Arg Leu Thr Trp Glu Ala Thr Pro Arg Ser Ile His Asp Gly Val Ala 275 280 285Ala Ala Ile Met Asn Ser Asp Cys Leu Val Phe Asp Thr Ala Ile Ala 290 295 300His Leu Phe Ala Asp Asn Gly Asn Leu Gly Ile Asn Val Thr Ile Ser305 310 315 320Thr Cys Cys Pro50301PRTArabidopsis thaliana 50Met Ala Phe Asn His Arg Lys Met Leu Leu Ser Cys Leu Gln Phe Lys1 5 10 15Asp Leu Arg Phe Cys Phe Arg Gln Tyr Pro Pro Pro Pro Pro Pro Pro 20 25 30Pro Pro Pro Arg Glu Leu Ser Leu Leu Leu Pro Thr Ser Ile Cys Val 35 40 45Val Gly Ser Ile Ile Leu Phe Leu Phe Leu Val Phe Phe Leu Tyr Leu 50 55 60His Ile Thr Gln Gln Arg Arg Ile Ser Ala Ala Ser Val Thr Pro Gly65 70 75 80Asp Thr Asn Gln Gln Glu Asp Glu Asp Glu Thr Glu Glu Arg Asp Phe 85 90 95Ser Asp Phe His His Val Trp Gln Ile Pro Thr Val Gly Leu His Arg 100 105 110Ser Ala Ile Asn Ser Ile Thr Val Val Gly Phe Lys Lys Gly Glu Gly 115 120 125Ile Ile Asp Gly Thr Glu Cys Ser Val Cys Leu Asn Glu Phe Glu Glu 130 135 140Asp Glu Ser Leu Arg Leu Leu Pro Lys Cys Ser His Ala Phe His Leu145 150 155 160Asn Cys Ile Asp Thr Trp Leu Leu Ser His Lys Asn Cys Pro Leu Cys 165 170 175Arg Ala Pro Val Leu Leu Ile Thr Glu Pro Pro His Gln Glu Thr Glu 180 185 190Thr Asn His Gln Pro Asp Ser Glu Ser Ser Asn Asp Leu Arg Gly Arg 195 200 205Gln Asp Ser Ser Arg Ser Arg Arg Asn His Asn Ile Phe Leu Pro Arg 210 215 220Ala Gln Ser Asp Leu Ala Asn Tyr Cys Gly Ser Gly Arg Val Glu Asn225 230 235 240Val Arg Arg Ser Phe Ser Ile Gly Gly Ser Leu Ser Leu Cys Asp Gly 245 250 255Ile Asn Asn Ala Thr Arg Ser Gly Arg Gln Phe Tyr Thr Ser Phe Ser 260 265 270Ala Asn Leu Phe Ser Ser Ser Arg Arg Val Arg Asn Glu Gln Pro Ile 275 280 285Pro Gln Asn Gln Met Pro Ser Val Thr Gly Asn Thr Ser 290 295 300511030PRTDanio rerio 51Met Thr Thr Gly Arg Asn Asn Arg Val Met Met Glu Gly Val Gly Ala1 5 10 15Arg Val Ile Arg Gly Pro Asp Trp Lys Trp Gly Lys Gln Asp Gly Gly 20 25 30Glu Gly His Val Gly Thr Val Arg Ser Phe Glu Ser Pro Glu Glu Val 35 40 45Val Val Val Trp Asp Asn Gly Thr Ala Ala Asn Tyr Arg Cys Ser Gly 50 55 60Ala Tyr Asp Val Arg Ile Leu Asp Ser Ala Pro Thr Gly Ile Lys His65 70 75 80Asp Gly Thr Met Cys Asp Thr Cys Arg Gln Gln Pro Ile Ile Gly Ile 85 90 95Arg Trp Lys Cys Ala Glu Cys Thr Asn Tyr Asp Leu Cys Thr Thr Cys 100 105 110Tyr His Gly Asp Lys His His Leu Arg His Arg Phe Tyr Arg Ile Thr 115 120 125Thr Pro Gly Ser Glu Arg Val Leu Leu Glu Ser Arg Arg Lys Ser Lys 130 135 140Lys Ile Thr Ala Arg Gly Ile Phe Ala Gly Gly Arg Val Val Arg Gly145 150 155 160Val Asp Trp Gln Trp Glu Asp Gln Asp Gly Gly Asn Gly Arg Arg Gly 165 170 175Lys Val Thr Glu Ile Gln Asp Trp Ser Ala Ala Ser Pro His Ser Ala 180 185 190Ala Tyr Val Leu Trp Asp Asn Gly Ala Lys Asn Leu Tyr Arg Val Gly 195 200 205Phe Glu Gly Met Ser Asp Leu Lys Cys Val Gln Asp Ala Lys Gly Gly 210 215 220Thr Phe Tyr Arg Asp His Cys Pro Val Leu Gly Glu Gln Asn Gly Asn225 230 235 240Arg Asn Pro Gly Gly Leu Gln Ile Gly Asp Leu Val Asn Ile Asp Leu 245 250 255Asp Leu Glu Ile Val Gln Ser Leu Gln His Gly His Gly Gly Trp Thr 260 265 270Asp Gly Met Phe Glu Thr Leu Thr Thr Thr Gly Thr Val Cys Gly Ile 275 280 285Asp Glu Asp His Asp Ile Val Val Gln Tyr Pro Ser Gly Asn Arg Trp 290 295 300Thr Phe Asn Pro Ala Val Leu Thr Lys Ala Asn Val Val Arg Ser Gly305 310 315 320Glu Val Ala Ala Gly Ala Glu Gly Gly Ser Ser Gln Phe Met Val Gly 325 330 335Asp Leu Val Gln Ile Cys Tyr Asp Ile Asp Arg Ile Lys Leu Leu Gln 340 345 350Arg Gly His Gly Glu Trp Ala Glu Ala Met Leu Pro Thr Leu Gly Lys 355 360 365Val Gly Arg Val Gln Gln Ile Tyr Ser Asp Ser Asp Leu Lys Val Glu 370 375 380Val Cys Gly Thr Ser Trp Thr Tyr Asn Pro Ala Ala Val Thr Lys Val385 390 395 400Ala Pro Ala Gly Ser Ala Val Thr Asn Ala Ser Gly Glu Arg Leu Ser 405 410 415Gln Leu Leu Lys Lys Leu Phe Glu Thr Gln Glu Ser Gly Asp Ile Asn 420 425 430Glu Glu Leu Val Lys Ala Ala Ala Asn Gly Asp Leu Ala Lys Val Glu 435 440 445Asp Ile Leu Lys Arg Pro Asp Val Asp Val Asn Gly Gln Cys Ala Gly 450 455 460His Thr Ala Met Gln Ala Ala Ser Gln Asn Gly His Val Asp Val Leu465 470 475 480Lys Leu Leu Leu Lys His Ser Val Asp Leu Glu Ala Glu Asp Lys Asp 485 490 495Gly Asp Arg Ala Val His His Ala Ser Phe Gly Asp Glu Gly Ser Val 500 505 510Ile Glu Val Leu His Arg Gly Gly Ala Asp Leu Asn Ala Arg Asn Lys 515 520 525Arg Arg Gln Thr Pro Leu His Ile Ala Val Asn Lys Gly His Leu Gln 530 535 540Val Val Lys Thr Leu Leu Asp Phe Gly Cys His Pro Ser Leu Gln Asp545 550 555 560Ser Glu Gly Asp Thr Pro Leu His Asp Ala Ile Ser Lys Lys Arg Asp 565 570 575Asp Met Leu Ser Val Leu Leu Glu Ala Gly Ala Asp Val Thr Ile Thr 580 585 590Asn Asn Asn Gly Phe Asn Ala Leu His His Ala Ala Leu Arg Gly Asn 595 600 605Pro Ser Ala Met Arg Val Leu Leu Ser Lys Leu Pro Arg Pro Trp Ile 610 615 620Val Asp Glu Lys Lys Asp Asp Gly Tyr Thr Ala Leu His Leu Ala Ala625 630 635 640Leu Asn Asn His Val Glu Val Ala Glu Leu Leu Val His Gln Gly Asn 645 650 655Ala Asn Leu Asp Val Gln Asn Val Asn Gln Gln Thr Ala Leu His Leu 660 665 670Ala Val Glu Arg Gln His Thr Gln Ile Val Arg Leu Leu Val Arg Ala 675 680 685Glu Ala Lys Leu Asp Val Gln Asp Lys Asp Gly Asp Thr Pro Leu His 690 695 700Glu Ala Leu Arg His His Thr Leu Ser Gln Leu Arg Gln Leu Gln Asp705 710 715 720Met Gln Asp Val Ser Lys Val Glu Pro Trp Glu Pro Ser Lys Asn Thr 725 730 735Leu Ile Met Gly Leu Gly Thr Gln Gly Ala Glu Lys Lys Ser Ala Ala 740 745 750Ser Ile Ala Cys Phe Leu Ala Ala Asn Gly Ala Asp Leu Thr Ile Arg 755 760 765Asn Lys Lys Gly Gln Ser Pro Leu Asp Leu Cys Pro Asp Pro Ser Leu 770 775 780Cys Lys Ala Leu Ala Lys Cys His Lys Glu Lys Thr Ser Gly Gln Val785 790 795 800Gly Ser Arg Ser Pro Ser Leu Asn Ser Asn Asn Glu Thr Leu Glu Glu 805 810 815Cys Met Val Cys Ser Asp Met Lys Arg Asp Thr Leu Phe Gly Pro Cys 820 825 830Gly His Ile Ala Thr Cys Ser Leu Cys Ser Pro Arg Val Lys Lys Cys 835 840 845Leu Ile Cys Lys Glu Gln Val Gln Ser Arg Thr Lys Ile Glu Glu Cys 850 855 860Val Val Cys Ser Asp Lys Lys Ala Ala Val Leu Phe Gln Pro Cys Gly865 870 875 880His Met Cys Ala Cys Glu Asn Cys Ala Ser Leu Met Lys Lys Cys Val 885 890 895Gln Cys Arg Ala Val Val Glu Arg Arg Thr Pro Phe Val Leu Cys Cys 900 905 910Gly Gly Lys Gly Met Glu Asp Ala Thr Asp Asp Glu Asp Leu Thr Gly 915 920 925Gly Ser Asn Ser Met Ala Gly Gly Ser Gln Asp Leu Leu Gln Pro Asn 930 935 940Asn Leu Ala Leu Ser Trp Ser Ser Gly Asn Ile Pro Ala Leu Gln Arg945 950 955 960Asp Lys Asp Asn Thr Asn Val Asn Ala Asp Val Gln Lys Leu Gln Gln 965 970 975Gln Leu Gln Asp Ile Lys Glu Gln Thr Met Cys Pro Val Cys Leu Asp 980 985 990Arg Leu Lys Asn Met Ile Phe Met Cys Gly His Gly Thr Cys Gln Leu 995 1000 1005Cys Gly Asp Arg Met Ser Glu Cys Pro Ile Cys Arg Lys Ala Ile 1010 1015 1020Glu Arg Arg Ile Leu Leu Tyr1025 103052522PRTArabidopsis thaliana 52Met Ser Val Lys Asn Asn Ser Phe Ser Ser Ala Glu Ile Pro Asp Val1 5 10 15Ala Asp Gln Pro Arg Asp Arg Phe Asn Pro Glu Ala Thr Gln Asp Leu 20 25 30Gln Glu Lys Asp Glu Thr Lys Glu Glu Lys Glu Gly Asp Glu Glu Val 35 40 45Lys His Asp Glu Ala Glu Glu Asp Gln Glu Val Val Lys Pro Asn Asp 50 55 60Ala Glu Glu Asp Asp Asp Gly Asp Asp Ala Glu Glu Asp Glu Glu Glu65 70 75 80Glu Val Glu Ala Glu Glu Asp Glu Glu Ala Glu Glu Glu Glu Glu Glu 85 90 95Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Ser Lys Glu Arg Ser Pro 100 105 110Ser Ser Ile Ser Gly Asp Gln Ser Glu Phe Met Glu Ile Asp Leu Gly 115 120 125Glu Ile Arg Lys Asp Val Gln Cys Pro Ile Cys Leu Gly Ile Ile Lys 130 135 140Lys Thr Arg Thr Val Met Glu Cys Leu His Arg Phe Cys Arg Glu Cys145 150 155 160Ile Asp Lys Ser Met Arg Leu Gly Asn Asn Glu Cys Pro Ala Cys Arg 165 170 175Lys His Cys Ala Ser Arg Arg Ser Leu Arg Asp Asp Pro Lys Phe Asp 180 185 190Ala Leu Ile Ala Ala Leu Phe Thr Asn Ile Asp Ser Tyr Glu Glu Glu 195 200 205Glu Leu Ala Phe His Glu Asp Glu Met Ala Arg Asn Lys Gln Ile Gln 210 215 220Ala Ser Ile Ala Gln Ile Ser Gln Arg Gln Ser Glu Ala Leu Val Lys225 230 235 240Arg Arg Ser Leu Gly Lys Glu Ala Ala Val Leu Met Arg Ser Pro Arg 245 250 255Ile Ala Ser Gly Ser Arg Arg Arg Arg Asn Ser Arg Asn Met Glu Gln 260 265 270Gln Asn Ala Ser Glu Ala His Glu Asp Asp Asp Asn Asp Asp Asn Asn 275 280 285Asn Arg Gly Arg Asp Lys Asp Ser Ser Ser Asp Glu Arg Gly Thr Glu 290 295 300Val Arg Gln Lys Lys Arg Arg Lys Arg Ser Thr Ser Arg Ser Thr Gln305 310 315 320His Pro Ser Ser Ser Gly Ala Asn Lys Asn Asn Gly Asn Cys Ala Asp 325 330 335Asn Asp Thr Glu Val Tyr Arg Asp Thr Lys Gly Ile Ser Pro Gly Leu 340 345 350Val Trp Asn Pro Glu Ile Leu Ala Trp Gly Arg Gly Gly Thr Arg Ser 355 360 365Asn Thr Arg His Gly Asn Asn Thr Ser Gly Gly Ser Ser Lys Ser Val 370 375 380Arg Asn Ala Arg Val Asn Lys Leu Val Glu Tyr Leu Arg Ser Ser Val385 390 395 400Asp Gly Ser Ser Val Glu Leu Asp Ile His Val Lys Leu Val Ser Leu 405 410 415Asp Thr Lys Cys Ile Pro Asp Leu Pro Gln Pro Tyr Leu Cys Cys Arg 420 425 430Pro Thr Leu Leu Val Lys Gln Leu Arg Glu Phe Val Ala Leu Gln Ile 435 440 445His Leu Lys Thr Glu Glu Val Glu Leu Leu Val Thr Arg Arg Arg Val 450 455 460Gly Glu Asp Ala Ala Ile Glu Asn Leu Pro Ala Val Thr Pro Ala Ser465 470 475 480Ala Ala Ala Ser Lys Asp Glu Met Leu Ser Leu Glu Asp Asn Glu Thr 485 490 495Leu Ser Arg Leu Lys Ile Asp Phe Ile Ser Ser His Glu Gln His Leu 500 505 510Ile Ile Ala Tyr Arg Lys Lys Gln Thr Glu 515 52053574PRTHomo sapiens 53Met Ala Thr Leu Val Val Asn Lys Leu Gly Ala Gly Val Asp Ser Gly1 5 10 15Arg Gln Gly Ser Arg Gly Thr Ala Val Val Lys Val Leu Glu Cys Gly 20 25 30Val Cys Glu Asp Val Phe Ser Leu Gln Gly Asp Lys Val Pro Arg Leu 35 40 45Leu Leu Cys Gly His Thr Val Cys His Asp Cys Leu Thr Arg Leu Pro 50 55 60Leu His Gly Arg Ala Ile Arg Cys Pro Phe Asp Arg Gln Val Thr Asp65 70 75 80Leu Gly Asp Ser Gly Val Trp Gly Leu Lys Lys Asn Phe Ala Leu Leu 85 90 95Glu Leu Leu Glu Arg Leu Gln Asn Gly Pro Ile Gly Gln Tyr Gly Ala 100 105 110Ala Glu Glu Ser Ile Gly Ile Ser Gly Glu Ser Ile Ile Arg Cys Asp 115 120 125Glu Asp Glu Ala His Leu Ala Ser Val Tyr Cys Thr Val Cys Ala Thr 130 135 140His Leu Cys Ser Glu Cys Ser Gln Val Thr His Ser Thr Lys Thr Leu145 150 155 160Ala Lys His Arg Arg Val Pro Leu Ala Asp Lys Pro His Glu Lys Thr 165 170 175Met Cys Ser Gln His Gln Val His Ala Ile Glu Phe Val Cys Leu Glu 180 185 190Glu Gly Cys Gln Thr Ser Pro Leu Met Cys Cys

Val Cys Lys Glu Tyr 195 200 205Gly Lys His Gln Gly His Lys His Ser Val Leu Glu Pro Glu Ala Asn 210 215 220Gln Ile Arg Ala Ser Ile Leu Asp Met Ala His Cys Ile Arg Thr Phe225 230 235 240Thr Glu Glu Ile Ser Asp Tyr Ser Arg Lys Leu Val Gly Ile Val Gln 245 250 255His Ile Glu Gly Gly Glu Gln Ile Val Glu Asp Gly Ile Gly Met Ala 260 265 270His Thr Glu His Val Pro Gly Thr Ala Glu Asn Ala Arg Ser Cys Ile 275 280 285Arg Ala Tyr Phe Tyr Asp Leu His Glu Thr Leu Cys Arg Gln Glu Glu 290 295 300Met Ala Leu Ser Val Val Asp Ala His Val Arg Glu Lys Leu Ile Trp305 310 315 320Leu Arg Gln Gln Gln Glu Asp Met Thr Ile Leu Leu Ser Glu Val Ser 325 330 335Ala Ala Cys Leu His Cys Glu Lys Thr Leu Gln Gln Asp Asp Cys Arg 340 345 350Val Val Leu Ala Lys Gln Glu Ile Thr Arg Leu Leu Glu Thr Leu Gln 355 360 365Lys Gln Gln Gln Gln Phe Thr Glu Val Ala Asp His Ile Gln Leu Asp 370 375 380Ala Ser Ile Pro Val Thr Phe Thr Lys Asp Asn Arg Val His Ile Gly385 390 395 400Pro Lys Met Glu Ile Arg Val Val Thr Leu Gly Leu Asp Gly Ala Gly 405 410 415Lys Thr Thr Ile Leu Phe Lys Leu Lys Gln Asp Glu Phe Met Gln Pro 420 425 430Ile Pro Thr Ile Gly Phe Asn Val Glu Thr Val Glu Tyr Lys Asn Leu 435 440 445Lys Phe Thr Ile Trp Asp Val Gly Gly Lys His Lys Leu Arg Pro Leu 450 455 460Trp Lys His Tyr Tyr Leu Asn Thr Gln Ala Val Val Phe Val Val Asp465 470 475 480Ser Ser His Arg Asp Arg Ile Ser Glu Ala His Ser Glu Leu Ala Lys 485 490 495Leu Leu Thr Glu Lys Glu Leu Arg Asp Ala Leu Leu Leu Ile Phe Ala 500 505 510Asn Lys Gln Asp Val Ala Gly Ala Leu Ser Val Glu Glu Ile Thr Glu 515 520 525Leu Leu Ser Leu His Lys Leu Cys Cys Gly Arg Ser Trp Tyr Ile Gln 530 535 540Gly Cys Asp Ala Arg Ser Gly Met Gly Leu Tyr Glu Gly Leu Asp Trp545 550 555 560Leu Ser Arg Gln Leu Val Ala Ala Gly Val Leu Asp Val Ala 565 57054864PRTMus musculus 54Met Ser Asp Ser Gly Pro Gln Leu Asp Ser Met Gly Ser Leu Thr Met1 5 10 15Lys Ser Gln Leu Gln Ile Thr Val Ile Ser Ala Lys Leu Lys Glu Asn 20 25 30Lys Lys Asn Trp Phe Gly Pro Ser Pro Tyr Val Glu Val Thr Val Asp 35 40 45Gly Gln Ser Lys Lys Thr Glu Lys Cys Asn Asn Thr Asn Ser Pro Lys 50 55 60Trp Lys Gln Pro Leu Thr Val Ile Val Thr Pro Thr Ser Lys Leu Cys65 70 75 80Phe Arg Val Trp Ser His Gln Thr Leu Lys Ser Asp Val Leu Leu Gly 85 90 95Thr Ala Gly Leu Asp Ile Tyr Glu Thr Leu Lys Ser Asn Asn Met Lys 100 105 110Leu Glu Glu Val Val Met Thr Leu Gln Leu Val Gly Asp Lys Glu Pro 115 120 125Thr Glu Thr Met Gly Asp Leu Ser Val Cys Leu Asp Gly Leu Gln Val 130 135 140Glu Ala Glu Val Val Thr Asn Gly Glu Thr Ser Cys Ser Glu Ser Thr145 150 155 160Thr Gln Asn Asp Asp Gly Cys Arg Thr Arg Asp Asp Thr Arg Val Ser 165 170 175Thr Asn Gly Ser Glu Asp Pro Glu Val Ala Ala Ser Gly Glu Asn Lys 180 185 190Arg Ala Asn Gly Asn Asn Ser Pro Ser Leu Ser Asn Gly Gly Phe Lys 195 200 205Pro Ser Arg Pro Pro Arg Pro Ser Arg Pro Pro Pro Pro Thr Pro Arg 210 215 220Arg Pro Ala Ser Val Asn Gly Ser Pro Ser Thr Asn Ser Asp Ser Asp225 230 235 240Gly Ser Ser Thr Gly Ser Leu Pro Pro Thr Asn Thr Asn Val Asn Thr 245 250 255Ser Thr Ser Glu Gly Ala Thr Ser Gly Leu Ile Ile Pro Leu Thr Ile 260 265 270Ser Gly Gly Ser Gly Pro Arg Pro Leu Asn Thr Val Ser Gln Ala Pro 275 280 285Leu Pro Pro Gly Trp Glu Gln Arg Val Asp Gln His Gly Arg Val Tyr 290 295 300Tyr Val Asp His Val Glu Lys Arg Thr Thr Trp Asp Arg Pro Glu Pro305 310 315 320Leu Pro Pro Gly Trp Glu Arg Arg Val Asp Asn Met Gly Arg Ile Tyr 325 330 335Tyr Val Asp His Phe Thr Arg Thr Thr Thr Trp Gln Arg Pro Thr Leu 340 345 350Glu Ser Val Arg Asn Tyr Glu Gln Trp Gln Leu Gln Arg Ser Gln Leu 355 360 365Gln Gly Ala Met Gln Gln Phe Asn Gln Arg Phe Ile Tyr Gly Asn Gln 370 375 380Asp Leu Phe Ala Thr Ser Gln Asn Lys Glu Phe Asp Pro Leu Gly Pro385 390 395 400Leu Pro Pro Gly Trp Glu Lys Arg Thr Asp Ser Asn Gly Arg Val Tyr 405 410 415Phe Val Asn His Asn Thr Arg Ile Thr Gln Trp Glu Asp Pro Arg Ser 420 425 430Gln Gly Gln Leu Asn Glu Lys Pro Leu Pro Glu Gly Trp Glu Met Arg 435 440 445Phe Thr Val Asp Gly Ile Pro Tyr Phe Val Asp His Asn Arg Arg Ala 450 455 460Thr Thr Tyr Ile Asp Pro Arg Thr Gly Lys Ser Ala Leu Asp Asn Gly465 470 475 480Pro Gln Ile Ala Tyr Val Arg Asp Phe Lys Ala Lys Val Gln Tyr Phe 485 490 495Arg Phe Trp Cys Gln Gln Leu Ala Met Pro Gln His Ile Lys Ile Thr 500 505 510Val Thr Arg Lys Thr Leu Phe Glu Asp Ser Phe Gln Gln Ile Met Ser 515 520 525Phe Ser Pro Gln Asp Leu Arg Arg Arg Leu Trp Val Ile Phe Pro Gly 530 535 540Glu Glu Gly Leu Asp Tyr Gly Gly Val Ala Arg Glu Trp Phe Phe Leu545 550 555 560Leu Ser His Glu Val Leu Asn Pro Met Tyr Cys Leu Phe Glu Tyr Ala 565 570 575Gly Lys Asp Asn Tyr Cys Leu Gln Ile Asn Pro Ala Ser Tyr Ile Asn 580 585 590Pro Asp His Leu Lys Tyr Phe Arg Phe Ile Gly Arg Phe Ile Ala Met 595 600 605Ala Leu Phe His Gly Lys Phe Ile Asp Thr Gly Phe Ser Leu Pro Phe 610 615 620Tyr Lys Arg Ile Leu Asn Lys Pro Val Gly Leu Lys Asp Leu Glu Ser625 630 635 640Ile Asp Pro Glu Phe Tyr Asn Ser Leu Ile Trp Val Lys Glu Asn Asn 645 650 655Ile Glu Glu Cys Gly Leu Glu Met Tyr Phe Ser Val Asp Lys Glu Ile 660 665 670Leu Gly Glu Ile Lys Ser His Asp Leu Lys Pro Asn Gly Gly Asn Ile 675 680 685Leu Val Thr Glu Glu Asn Lys Glu Glu Tyr Ile Arg Met Val Ala Glu 690 695 700Trp Arg Leu Ser Arg Gly Val Glu Glu Gln Thr Gln Ala Phe Phe Glu705 710 715 720Gly Phe Asn Glu Ile Leu Pro Gln Gln Tyr Leu Gln Tyr Phe Asp Ala 725 730 735Lys Glu Leu Glu Val Leu Leu Cys Gly Met Gln Glu Ile Asp Leu Asn 740 745 750Asp Trp Gln Arg His Ala Ile Tyr Arg His Tyr Thr Arg Thr Ser Lys 755 760 765Gln Ile Met Trp Phe Trp Gln Phe Val Lys Glu Ile Asp Asn Glu Lys 770 775 780Arg Met Arg Leu Leu Gln Phe Val Thr Gly Thr Cys Arg Leu Pro Val785 790 795 800Gly Gly Phe Ala Asp Leu Met Gly Ser Asn Gly Pro Gln Lys Phe Cys 805 810 815Ile Glu Lys Val Gly Lys Glu Asn Trp Leu Pro Arg Ser His Thr Cys 820 825 830Phe Asn Arg Leu Asp Leu Pro Pro Tyr Lys Ser Tyr Glu Gln Leu Lys 835 840 845Glu Lys Leu Leu Phe Ala Ile Glu Glu Thr Glu Gly Phe Gly Gln Glu 850 855 86055353PRTHomo sapiens 55Met Asp Tyr Lys Ser Ser Leu Ile Gln Asp Gly Asn Pro Met Glu Asn1 5 10 15Leu Glu Lys Gln Leu Ile Cys Pro Ile Cys Leu Glu Met Phe Thr Lys 20 25 30Pro Val Val Ile Leu Pro Cys Gln His Asn Leu Cys Arg Lys Cys Ala 35 40 45Asn Asp Ile Phe Gln Ala Ala Asn Pro Tyr Trp Thr Ser Arg Gly Ser 50 55 60Ser Val Ser Met Ser Gly Gly Arg Phe Arg Cys Pro Thr Cys Arg His65 70 75 80Glu Val Ile Met Asp Arg His Gly Val Tyr Gly Leu Gln Arg Asn Leu 85 90 95Leu Val Glu Asn Ile Ile Asp Ile Tyr Lys Gln Glu Cys Ser Ser Arg 100 105 110Pro Leu Gln Lys Gly Ser His Pro Met Cys Lys Glu His Glu Asp Glu 115 120 125Lys Ile Asn Ile Tyr Cys Leu Thr Cys Glu Val Pro Thr Cys Ser Met 130 135 140Cys Lys Val Phe Gly Ile His Lys Ala Cys Glu Val Ala Pro Leu Gln145 150 155 160Ser Val Phe Gln Gly Gln Lys Thr Glu Leu Asn Asn Cys Ile Ser Met 165 170 175Leu Val Ala Gly Asn Asp Arg Val Gln Thr Ile Ile Thr Gln Leu Glu 180 185 190Asp Ser Arg Arg Val Thr Lys Glu Asn Ser His Gln Val Lys Glu Glu 195 200 205Leu Ser Gln Lys Phe Asp Thr Leu Tyr Ala Ile Leu Asp Glu Lys Lys 210 215 220Ser Glu Leu Leu Gln Arg Ile Thr Gln Glu Gln Glu Lys Lys Leu Ser225 230 235 240Phe Ile Glu Ala Leu Ile Gln Gln Tyr Gln Glu Gln Leu Asp Lys Ser 245 250 255Thr Lys Leu Val Glu Thr Ala Ile Gln Ser Leu Asp Glu Pro Gly Gly 260 265 270Ala Thr Phe Leu Leu Thr Ala Lys Gln Leu Ile Lys Ser Ile Val Glu 275 280 285Ala Ser Lys Gly Cys Gln Leu Gly Lys Thr Glu Gln Gly Phe Glu Asn 290 295 300Met Asp Phe Phe Thr Leu Asp Leu Glu His Ile Ala Asp Ala Leu Arg305 310 315 320Ala Ile Asp Phe Gly Thr Asp Glu Glu Glu Glu Glu Phe Ile Glu Glu 325 330 335Glu Asp Gln Glu Glu Glu Glu Ser Thr Glu Gly Lys Glu Glu Gly His 340 345 350Gln56870PRTHomo sapiens 56Met Ala Ser Ala Ser Ser Ser Arg Ala Gly Val Ala Leu Pro Phe Glu1 5 10 15Lys Ser Gln Leu Thr Leu Lys Val Val Ser Ala Lys Pro Lys Val His 20 25 30Asn Arg Gln Pro Arg Ile Asn Ser Tyr Val Glu Val Ala Val Asp Gly 35 40 45Leu Pro Ser Glu Thr Lys Lys Thr Gly Lys Arg Ile Gly Ser Ser Glu 50 55 60Leu Leu Trp Asn Glu Ile Ile Ile Leu Asn Val Thr Ala Gln Ser His65 70 75 80Leu Asp Leu Lys Val Trp Ser Cys His Thr Leu Arg Asn Glu Leu Leu 85 90 95Gly Thr Ala Ser Val Asn Leu Ser Asn Val Leu Lys Asn Asn Gly Gly 100 105 110Lys Met Glu Asn Met Gln Leu Thr Leu Asn Leu Gln Thr Glu Asn Lys 115 120 125Gly Ser Val Val Ser Gly Gly Glu Leu Thr Ile Phe Leu Asp Gly Pro 130 135 140Thr Val Asp Leu Gly Asn Val Pro Asn Gly Ser Ala Leu Thr Asp Gly145 150 155 160Ser Gln Leu Pro Ser Arg Asp Ser Ser Gly Thr Ala Val Ala Pro Glu 165 170 175Asn Arg His Gln Pro Pro Ser Thr Asn Cys Phe Gly Gly Arg Ser Arg 180 185 190Thr His Arg His Ser Gly Ala Ser Ala Arg Thr Thr Pro Ala Thr Gly 195 200 205Glu Gln Ser Pro Gly Ala Arg Ser Arg His Arg Gln Pro Val Lys Asn 210 215 220Ser Gly His Ser Gly Leu Ala Asn Gly Thr Val Asn Asp Glu Pro Thr225 230 235 240Thr Ala Thr Asp Pro Glu Glu Pro Ser Val Val Gly Val Thr Ser Pro 245 250 255Pro Ala Ala Pro Leu Ser Val Thr Pro Asn Pro Asn Thr Thr Ser Leu 260 265 270Pro Ala Pro Ala Thr Pro Ala Glu Gly Glu Glu Pro Ser Thr Ser Gly 275 280 285Thr Gln Gln Leu Pro Ala Ala Ala Gln Ala Pro Asp Ala Leu Pro Ala 290 295 300Gly Trp Glu Gln Arg Glu Leu Pro Asn Gly Arg Val Tyr Tyr Val Asp305 310 315 320His Asn Thr Lys Thr Thr Thr Trp Glu Arg Pro Leu Pro Pro Gly Trp 325 330 335Glu Lys Arg Thr Asp Pro Arg Gly Arg Phe Tyr Tyr Val Asp His Asn 340 345 350Thr Arg Thr Thr Thr Trp Gln Arg Pro Thr Ala Glu Tyr Val Arg Asn 355 360 365Tyr Glu Gln Trp Gln Ser Gln Arg Asn Gln Leu Gln Gly Ala Met Gln 370 375 380His Phe Ser Gln Arg Phe Leu Tyr Gln Ser Ser Ser Ala Ser Thr Asp385 390 395 400His Asp Pro Leu Gly Pro Leu Pro Pro Gly Trp Glu Lys Arg Gln Asp 405 410 415Asn Gly Arg Val Tyr Tyr Val Asn His Asn Thr Arg Thr Thr Gln Trp 420 425 430Glu Asp Pro Arg Thr Gln Gly Met Ile Gln Glu Pro Ala Leu Pro Pro 435 440 445Gly Trp Glu Met Lys Tyr Thr Ser Glu Gly Val Arg Tyr Phe Val Asp 450 455 460His Asn Thr Arg Thr Thr Thr Phe Lys Asp Pro Arg Pro Gly Phe Glu465 470 475 480Ser Gly Thr Lys Gln Gly Ser Pro Gly Ala Tyr Asp Arg Ser Phe Arg 485 490 495Trp Lys Tyr His Gln Phe Arg Phe Leu Cys His Ser Asn Ala Leu Pro 500 505 510Ser His Val Lys Ile Ser Val Ser Arg Gln Thr Leu Phe Glu Asp Ser 515 520 525Phe Gln Gln Ile Met Asn Met Lys Pro Tyr Asp Leu Arg Arg Arg Leu 530 535 540Tyr Ile Ile Met Arg Gly Glu Glu Gly Leu Asp Tyr Gly Gly Ile Ala545 550 555 560Arg Glu Trp Phe Phe Leu Leu Ser His Glu Val Leu Asn Pro Met Tyr 565 570 575Cys Leu Phe Glu Tyr Ala Gly Lys Asn Asn Tyr Cys Leu Gln Ile Asn 580 585 590Pro Ala Ser Ser Ile Asn Pro Asp His Leu Thr Tyr Phe Arg Phe Ile 595 600 605Gly Arg Phe Ile Ala Met Ala Leu Tyr His Gly Lys Phe Ile Asp Thr 610 615 620Gly Phe Thr Leu Pro Phe Tyr Lys Arg Met Leu Asn Lys Arg Pro Thr625 630 635 640Leu Lys Asp Leu Glu Ser Ile Asp Pro Glu Phe Tyr Asn Ser Ile Val 645 650 655Trp Ile Lys Glu Asn Asn Leu Glu Glu Cys Gly Leu Glu Leu Tyr Phe 660 665 670Ile Gln Asp Met Glu Ile Leu Gly Lys Val Thr Thr His Glu Leu Lys 675 680 685Glu Gly Gly Glu Ser Ile Arg Val Thr Glu Glu Asn Lys Glu Glu Tyr 690 695 700Ile Met Leu Leu Thr Asp Trp Arg Phe Thr Arg Gly Val Glu Glu Gln705 710 715 720Thr Lys Ala Phe Leu Asp Gly Phe Asn Glu Val Ala Pro Leu Glu Trp 725 730 735Leu Arg Tyr Phe Asp Glu Lys Glu Leu Glu Leu Met Leu Cys Gly Met 740 745 750Gln Glu Ile Asp Met Ser Asp Trp Gln Lys Ser Thr Ile Tyr Arg His 755 760 765Tyr Thr Lys Asn Ser Lys Gln Ile Gln Trp Phe Trp Gln Val Val Lys 770 775 780Glu Met Asp Asn Glu Lys Arg Ile Arg Leu Leu Gln Phe Val Thr Gly785 790 795 800Thr Cys Arg Leu Pro Val Gly Gly Phe Ala Glu Leu Ile Gly Ser Asn 805 810 815Gly Pro Gln Lys Phe Cys Ile Asp Lys Val Gly Lys Glu Thr Trp Leu 820 825 830Pro Arg Ser His Thr Cys Phe Asn Arg Leu Asp Leu Pro Pro Tyr Lys 835 840 845Ser Tyr Glu Gln Leu Arg Glu Lys Leu Leu Tyr Ala Ile Glu Glu Thr 850 855 860Glu Gly Phe Gly Gln Glu865 87057180PRTHomo sapiens 57Met Ala Ala Ala

Glu Glu Glu Asp Gly Gly Pro Glu Gly Pro Asn Arg1 5 10 15Glu Arg Gly Gly Ala Gly Ala Thr Phe Glu Cys Asn Ile Cys Leu Glu 20 25 30Thr Ala Arg Glu Ala Val Val Ser Val Cys Gly His Leu Tyr Cys Trp 35 40 45Pro Cys Leu His Gln Trp Leu Glu Thr Arg Pro Glu Arg Gln Glu Cys 50 55 60Pro Val Cys Lys Ala Gly Ile Ser Arg Glu Lys Val Val Pro Leu Tyr65 70 75 80Gly Arg Gly Ser Gln Lys Pro Gln Asp Pro Arg Leu Lys Thr Pro Pro 85 90 95Arg Pro Gln Gly Gln Arg Pro Ala Pro Glu Ser Arg Gly Gly Phe Gln 100 105 110Pro Phe Gly Asp Thr Gly Gly Phe His Phe Ser Phe Gly Val Gly Ala 115 120 125Phe Pro Phe Gly Phe Phe Thr Thr Val Phe Asn Ala His Glu Pro Phe 130 135 140Arg Arg Gly Thr Gly Val Asp Leu Gly Gln Gly His Pro Ala Ser Ser145 150 155 160Trp Gln Asp Ser Leu Phe Leu Phe Leu Ala Ile Phe Phe Phe Phe Trp 165 170 175Leu Leu Ser Ile 18058875PRTHomo sapiens 58Met Glu Lys Leu His Gln Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser1 5 10 15Asp Asp Ile Glu Ala Ser Arg Met Lys Arg Ala Ala Ala Lys His Leu 20 25 30Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala 35 40 45Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu Arg Met Asp 50 55 60Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala65 70 75 80Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr 85 90 95Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys 100 105 110Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu 115 120 125Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu 130 135 140Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe Ser Ser Ala145 150 155 160Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His Thr Lys Glu 165 170 175Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu 180 185 190Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu Asp Ser Glu 195 200 205Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu 210 215 220Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp Ala Ile Arg225 230 235 240Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe 245 250 255Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys Asp Leu Thr 260 265 270Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile 275 280 285Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr Leu Glu Met 290 295 300Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro Leu Ala Ala305 310 315 320Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile 325 330 335Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile 340 345 350Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp Asp Ala Ile 355 360 365Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala Asn Val Val 370 375 380Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp Glu Glu Pro385 390 395 400Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu 405 410 415Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu Thr Glu Leu 420 425 430Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu 435 440 445Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp Lys Asp Tyr 450 455 460Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe Met Thr Cys465 470 475 480Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp 485 490 495Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser 500 505 510Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg 515 520 525Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala 530 535 540Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu545 550 555 560Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln 565 570 575Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr 580 585 590Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr 595 600 605Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr 610 615 620Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val Tyr Arg Lys625 630 635 640Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro 645 650 655Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val 660 665 670Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe 675 680 685Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro 690 695 700Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr705 710 715 720Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly 725 730 735Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro 740 745 750Glu Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln 755 760 765Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser 770 775 780Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu785 790 795 800Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro 805 810 815Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro 820 825 830Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu 835 840 845Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys 850 855 860Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu865 870 875591068PRTHomo sapiens 59Met Phe Thr Leu Ser Gln Thr Ser Arg Ala Trp Phe Ile Asp Arg Ala1 5 10 15Arg Gln Ala Arg Glu Glu Arg Leu Val Gln Lys Glu Arg Glu Arg Ala 20 25 30Ala Val Val Ile Gln Ala His Val Arg Ser Phe Leu Cys Arg Ser Arg 35 40 45Leu Gln Arg Asp Ile Arg Arg Glu Ile Asp Asp Phe Phe Lys Ala Asp 50 55 60Asp Pro Glu Ser Thr Lys Arg Ser Ala Leu Cys Ile Phe Lys Ile Ala65 70 75 80Arg Lys Leu Leu Phe Leu Phe Arg Ile Lys Glu Asp Asn Glu Arg Phe 85 90 95Glu Lys Leu Cys Arg Ser Ile Leu Ser Ser Met Asp Ala Glu Asn Glu 100 105 110Pro Lys Val Trp Tyr Val Ser Leu Ala Cys Ser Lys Asp Leu Thr Leu 115 120 125Leu Trp Ile Gln Gln Ile Lys Asn Ile Leu Trp Tyr Cys Cys Asp Phe 130 135 140Leu Lys Gln Leu Lys Pro Glu Ile Leu Gln Asp Ser Arg Leu Ile Thr145 150 155 160Leu Tyr Leu Thr Met Leu Val Thr Phe Thr Asp Thr Ser Thr Trp Lys 165 170 175Ile Leu Arg Gly Lys Gly Glu Ser Leu Arg Pro Ala Met Asn His Ile 180 185 190Cys Ala Asn Ile Met Gly His Leu Asn Gln His Gly Phe Tyr Ser Val 195 200 205Leu Gln Ile Leu Leu Thr Arg Gly Leu Ala Arg Pro Arg Pro Cys Leu 210 215 220Ser Lys Gly Thr Leu Thr Ala Ala Phe Ser Leu Ala Leu Arg Pro Val225 230 235 240Ile Ala Ala Gln Phe Ser Asp Asn Leu Ile Arg Pro Phe Leu Ile His 245 250 255Ile Met Ser Val Pro Ala Leu Val Thr His Leu Ser Thr Val Thr Pro 260 265 270Glu Arg Leu Thr Val Leu Glu Ser His Asp Met Leu Arg Lys Phe Ile 275 280 285Ile Phe Leu Arg Asp Gln Asp Arg Cys Arg Asp Val Cys Glu Ser Leu 290 295 300Glu Gly Cys His Thr Leu Cys Leu Met Gly Asn Leu Leu His Leu Gly305 310 315 320Ser Leu Ser Pro Arg Val Leu Glu Glu Glu Thr Asp Gly Phe Val Ser 325 330 335Leu Leu Thr Gln Thr Leu Cys Tyr Cys Arg Lys Tyr Val Ser Gln Lys 340 345 350Lys Ser Asn Leu Thr His Trp His Pro Val Leu Gly Trp Phe Ser Gln 355 360 365Ser Val Asp Tyr Gly Leu Asn Glu Ser Met His Leu Ile Thr Lys Gln 370 375 380Leu Gln Phe Leu Trp Gly Val Pro Leu Ile Arg Ile Phe Phe Cys Asp385 390 395 400Ile Leu Ser Lys Lys Leu Leu Glu Ser Gln Glu Pro Ala His Ala Gln 405 410 415Pro Ala Ser Pro Gln Asn Val Leu Pro Val Lys Ser Leu Leu Lys Arg 420 425 430Ala Phe Gln Lys Ser Ala Ser Val Arg Asn Ile Leu Arg Pro Val Gly 435 440 445Gly Lys Arg Val Asp Ser Ala Glu Val Gln Lys Val Cys Asn Ile Cys 450 455 460Val Leu Tyr Gln Thr Ser Leu Thr Thr Leu Thr Gln Ile Arg Leu Gln465 470 475 480Ile Leu Thr Gly Leu Thr Tyr Leu Asp Asp Leu Leu Pro Lys Leu Trp 485 490 495Ala Phe Ile Cys Glu Leu Gly Pro His Gly Gly Leu Lys Leu Phe Leu 500 505 510Glu Cys Leu Asn Asn Asp Thr Glu Glu Ser Lys Gln Leu Leu Ala Met 515 520 525Leu Met Leu Phe Cys Asp Cys Ser Arg His Leu Ile Thr Ile Leu Asp 530 535 540Asp Ile Glu Val Tyr Glu Glu Gln Ile Ser Phe Lys Leu Glu Glu Leu545 550 555 560Val Thr Ile Ser Ser Phe Leu Asn Ser Phe Val Phe Lys Met Ile Trp 565 570 575Asp Gly Ile Val Glu Asn Ala Lys Gly Glu Thr Leu Glu Leu Phe Gln 580 585 590Ser Val His Gly Trp Leu Met Val Leu Tyr Glu Arg Asp Cys Arg Arg 595 600 605Arg Phe Thr Pro Glu Asp His Trp Leu Arg Lys Asp Leu Lys Pro Ser 610 615 620Val Leu Phe Gln Glu Leu Asp Arg Asp Arg Lys Arg Ala Gln Leu Ile625 630 635 640Leu Gln Tyr Ile Pro His Val Ile Pro His Lys Asn Arg Val Leu Leu 645 650 655Phe Arg Thr Met Val Thr Lys Glu Lys Glu Lys Leu Gly Leu Val Glu 660 665 670Thr Ser Ser Ala Ser Pro His Val Thr His Ile Thr Ile Arg Arg Ser 675 680 685Arg Met Leu Glu Asp Gly Tyr Glu Gln Leu Arg Gln Leu Ser Gln His 690 695 700Ala Met Lys Gly Val Ile Arg Val Lys Phe Val Asn Asp Leu Gly Val705 710 715 720Asp Glu Ala Gly Ile Asp Gln Asp Gly Val Phe Lys Glu Phe Leu Glu 725 730 735Glu Ile Ile Lys Arg Val Phe Asp Pro Ala Leu Asn Leu Phe Lys Thr 740 745 750Thr Ser Gly Asp Glu Arg Leu Tyr Pro Ser Pro Thr Ser Tyr Ile His 755 760 765Glu Asn Tyr Leu Gln Leu Phe Glu Phe Val Gly Lys Met Leu Gly Lys 770 775 780Ala Val Tyr Glu Gly Ile Val Val Asp Val Pro Phe Ala Ser Phe Phe785 790 795 800Leu Ser Gln Leu Leu Gly His His His Ser Val Phe Tyr Ser Ser Val 805 810 815Asp Glu Leu Pro Ser Leu Asp Ser Glu Phe Tyr Lys Asn Leu Thr Ser 820 825 830Ile Lys Arg Tyr Asp Gly Asp Ile Thr Asp Leu Gly Leu Thr Leu Ser 835 840 845Tyr Asp Glu Asp Val Met Gly Gln Leu Val Cys His Glu Leu Ile Pro 850 855 860Gly Gly Lys Thr Ile Pro Val Thr Asn Glu Asn Lys Ile Ser Tyr Ile865 870 875 880His Leu Met Ala His Phe Arg Met His Thr Gln Ile Lys Asn Gln Thr 885 890 895Ala Ala Leu Ile Ser Gly Phe Arg Ser Ile Ile Lys Pro Glu Trp Ile 900 905 910Arg Met Phe Ser Thr Pro Glu Leu Gln Arg Leu Ile Ser Gly Asp Asn 915 920 925Ala Glu Ile Asp Leu Glu Asp Leu Lys Lys His Thr Val Tyr Tyr Gly 930 935 940Gly Phe His Gly Ser His Arg Val Ile Ile Trp Leu Trp Asp Ile Leu945 950 955 960Ala Ser Asp Phe Thr Pro Asp Glu Arg Ala Met Phe Leu Lys Phe Val 965 970 975Thr Ser Cys Ser Arg Pro Pro Leu Leu Gly Phe Ala Tyr Leu Lys Pro 980 985 990Pro Phe Ser Ile Arg Cys Val Glu Val Ser Asp Asp Gln Asp Thr Gly 995 1000 1005Asp Thr Leu Gly Ser Val Leu Arg Gly Phe Phe Thr Ile Arg Lys 1010 1015 1020Arg Glu Pro Gly Gly Arg Leu Pro Thr Ser Ser Thr Cys Phe Asn 1025 1030 1035Leu Leu Lys Leu Pro Asn Tyr Ser Lys Lys Ser Val Leu Arg Glu 1040 1045 1050Lys Leu Arg Tyr Ala Ile Ser Met Asn Thr Gly Phe Glu Leu Ser 1055 1060 106560216PRTEscherichia coli 60Met Pro Phe Ser Phe Asn Leu Ser Ser Gly Asn Tyr Leu Ser Thr Gln1 5 10 15Asp Val Glu Val Leu Gln Arg Ala Thr Arg Asp His Gln Met Glu Arg 20 25 30Leu Thr Ile Gly Glu Arg Ser Phe Ser Val Arg Tyr Gln Ser Ala Met 35 40 45Asp Ala Phe Ile Val Asp Pro Val Gln Gly Glu Leu Tyr Ser Gly Leu 50 55 60Ser His Thr Glu Leu Ala Asp Ile Ile Arg Leu Ala Asp Ser Val Glu65 70 75 80Asn Gln Leu Asn Gly Gly Asn Ser Phe Leu Asp Val Phe Ser Thr Tyr 85 90 95Met Gly Gln Val Ile Ser Glu Phe Met His Ser Asn Asp Asn Arg Ile 100 105 110Glu Leu Leu Gln Arg Arg Leu His Ser Cys Ser Phe Leu Val Asn Ile 115 120 125Glu Glu Met Ser Tyr Ile Asp Glu Ala Leu Gln Cys Pro Ile Thr Leu 130 135 140Ala Ile Pro Gln Arg Gly Val Phe Leu Arg Asn Ala Glu Gly Ser Arg145 150 155 160Val Cys Ser Leu Tyr Asp Glu Met Ala Leu Ser Arg Ile Ile Asn Asp 165 170 175Gly Met His His Pro Leu Ser Arg Glu Pro Ile Thr Leu Ser Met Leu 180 185 190Val Ala Arg Glu Gln Cys Glu Phe Asp Cys Ser Ile Gly His Phe Thr 195 200 205Val Arg Ser Asp Cys Tyr Ser Val 210 21561140PRTEscherichia coli 61Met Glu Arg Arg Ala Val Ala Leu Glu Arg Gln Leu Asn Gly Gly Val1 5 10 15Asp Phe Leu Arg Ser Val Asn Asn Tyr Phe Gln Ser Val Met Ala Glu 20 25 30His Arg Glu Asn Lys Thr Ser Asn Lys Ile Leu Met Glu Lys Ile Asn 35 40 45Ser Cys Val Phe Gly Thr Asp Ser Asn His Phe Ser Cys Pro Glu Ser 50 55 60Phe Leu Thr Cys Pro Ile Thr Leu Asp Thr Pro Ala Asn Gly Val Phe65 70 75 80Met Arg Asn Ser Gln Gly Ala Glu Ile Cys Ser Leu Tyr Asp Lys Asp 85 90 95Thr Leu Val Gln Leu Val Glu Thr Gly Gly Ala His Pro Leu Ser Arg 100 105 110Glu Pro Ile Thr Glu Ser Met Ile Met Arg Lys Asp Glu Cys His Phe

115 120 125Asp Ser Lys Lys Glu Ser Phe Val Ala Ser Asp Ala 130 135 14062123PRTEscherichia coli 62Met Leu Gln Leu Ser Ser Asn Ile Gly Trp Lys Lys Gly Ala Glu Asn1 5 10 15Ala Leu Lys Asn Lys Ile His Ser His Ser Phe Val Val Asn Pro Asp 20 25 30Glu Phe Ser Cys Asp Thr Gln Phe Leu Lys Cys Pro Ile Thr Leu Cys 35 40 45Val Pro Glu Lys Gly Val Phe Val Lys Asn Ala Leu Asn Ser Asn Ile 50 55 60Cys Thr Leu Tyr Asp Lys Ser Ala Phe Met Asn Leu Thr Arg Glu His65 70 75 80Leu Pro His Pro Leu Ser Arg Glu Lys Ile Val Lys Glu Met Ile Ile 85 90 95Glu Arg Asn Met Cys Tyr Phe Asp Thr Ile Ser Gln His Phe Ile Ile 100 105 110Met Asp Ala Asp Gln Gln Lys Gln His Cys Lys 115 12063660DNAArtificial sequenceAn exemplary DNA sequence that encodes full length CAT 63atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 120attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 180cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt 240gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 300acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 360tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 420aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 480gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 540gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtctgtga tggcttccat 600gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 66064219PRTArtificial sequenceamino acid sequence of CAT 64Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gln Trp1 5 10 15His Arg Lys Glu His Phe Glu Ala Phe Gln Ser Val Ala Gln Cys Thr 20 25 30Tyr Asn Gln Thr Val Gln Leu Asp Ile Thr Ala Phe Leu Lys Thr Val 35 40 45Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His Ile Leu Ala 50 55 60Arg Leu Met Asn Ala His Pro Glu Phe Arg Met Ala Met Lys Asp Gly65 70 75 80Glu Leu Val Ile Trp Asp Ser Val His Pro Cys Tyr Thr Val Phe His 85 90 95Glu Gln Thr Glu Thr Phe Ser Ser Leu Trp Ser Glu Tyr His Asp Asp 100 105 110Phe Arg Gln Phe Leu His Ile Tyr Ser Gln Asp Val Ala Cys Tyr Gly 115 120 125Glu Asn Leu Ala Tyr Phe Pro Lys Gly Phe Ile Glu Asn Met Phe Phe 130 135 140Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu Asn Val145 150 155 160Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly Lys Tyr 165 170 175Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gln Val His 180 185 190His Ala Val Cys Asp Gly Phe His Val Gly Arg Met Leu Asn Glu Leu 195 200 205Gln Gln Tyr Cys Asp Glu Trp Gln Gly Gly Ala 210 2156593DNAArtificial sequenceCAT N-terminal fragment nucleic acid sequence 65atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60cattttgagg catttcagtc agttgctcaa taa 9366573DNAArtificial sequenceCAT C-terminal fragment nucleic acid sequence 66atgtgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag 60aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct 120catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac 180ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac 240cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa 300aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc 360tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc 420gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt 480caggttcatc atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa 540cagtactgcg atgagtggca gggcggggcg taa 5736730PRTArtificial sequenceCAT N-terminal fragment amino acid sequence 67Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gln Trp1 5 10 15His Arg Lys Glu His Phe Glu Ala Phe Gln Ser Val Ala Gln 20 25 3068190PRTArtificial sequenceCAT C-terminal fragment amino acid sequence 68Met Cys Thr Tyr Asn Gln Thr Val Gln Leu Asp Ile Thr Ala Phe Leu1 5 10 15Lys Thr Val Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His 20 25 30Ile Leu Ala Arg Leu Met Asn Ala His Pro Glu Phe Arg Met Ala Met 35 40 45Lys Asp Gly Glu Leu Val Ile Trp Asp Ser Val His Pro Cys Tyr Thr 50 55 60Val Phe His Glu Gln Thr Glu Thr Phe Ser Ser Leu Trp Ser Glu Tyr65 70 75 80His Asp Asp Phe Arg Gln Phe Leu His Ile Tyr Ser Gln Asp Val Ala 85 90 95Cys Tyr Gly Glu Asn Leu Ala Tyr Phe Pro Lys Gly Phe Ile Glu Asn 100 105 110Met Phe Phe Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp 115 120 125Leu Asn Val Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met 130 135 140Gly Lys Tyr Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile145 150 155 160Gln Val His His Ala Val Cys Asp Gly Phe His Val Gly Arg Met Leu 165 170 175Asn Glu Leu Gln Gln Tyr Cys Asp Glu Trp Gln Gly Gly Ala 180 185 190

Claims

1. A method of determining whether an enzyme is capable of ubiquitinating a test substrate, the method comprising (a) expressing the enzyme in a bacterial cell; (b) expressing ubiquitin in said bacterial cell, wherein said ubiquitin is attached to a first polypeptide fragment; (c) expressing the test substrate in said bacterial cell, wherein said substrate is attached to a second polypeptide fragment, wherein said first polypeptide fragment associates with said second polypeptide fragment to generate a reporter polypeptide on ubiquitination of the test substrate; and (d) analyzing for the presence of said reporter polypeptide in the bacterial cell, wherein a presence of said reporter polypeptide is indicative that the enzyme is capable of ubiquitinating the test substrate.

2. The method of claim 1, further expressing all the enzymes of the ubiquitinating enzyme cascade of the enzyme.

3. The method of claim 1, wherein said reporter polypeptide is a detectable polypeptide or a selectable polypeptide.

4. The method of claim 1, wherein said enzyme is selected from the group consisting of E3 ligase, ubiquitin E1-activating enzyme and ubiquitin E2 conjugating enzyme.

5. The method of claim 3, wherein said selectable polypeptide is a split antibiotic resistance polypeptide.

6. The method of claim 1, wherein said first polypeptide fragment is attached to said ubiquitin via a linker and/or wherein said second polypeptide fragment is attached to said substrate via a linker.

7. The method of claim 3, wherein said detectable polypeptide is an optically detectable signal.

8. The method of claim 1, wherein said analyzing is effected by bimolecular complementation of an antibiotic resistance protein.

9. A kit comprising: (i) a first polynucleotide which encodes a first polypeptide fragment which is operably linked to a bacterial regulatory sequence, and a cloning site, wherein a position of said cloning site is selected such that upon insertion of a sequence which encodes a test polypeptide into said cloning site, following expression in a bacterial cell, a fusion protein is generated which comprises said test polypeptide in frame with said first polypeptide fragment; and (ii) a second polynucleotide comprising a second nucleic acid sequence encoding a second polypeptide fragment which is attached to ubiquitin, the second nucleic acid sequence being operably linked to a bacterial regulatory sequence, wherein said first polypeptide fragment associates with said second polypeptide fragment to generate a reporter polypeptide dependent on ubiquitination of said test polypeptide.

10. The kit of claim 9, wherein said reporter polypeptide is a selectable polypeptide.

11. The kit of claim 9, further comprising a third polynucleotide which encodes at least one ubiquitinating enzyme.

12. The kit of claim 9, wherein said first polynucleotide and/or said second polynucleotide comprises a sequence which encodes at least one ubiquitinating enzyme.

13. The kit of claim 9, wherein said at least one ubiquitinating enzyme comprises: (a) ubiquitin E1-activating enzyme and ubiquitin E2-conjugating enzyme; or (b) E3 ligase.