Ubiquitin ligase assay

Abstract

The invention relates to assays for measuring ubiquitin ligase activity and for identifying modulators of ubiquitin ligase enzymes.

1 . A method of assaying ubiquitin ligase activity comprising:
 a) combining:
 i) tag1-ubiquitin, wherein the ubiquitin has at least 95% sequence identity to an amino acid sequence depicted in an accession number selected from the group consisting of P02248, AAA36788 and AAB06013 or is encoded by a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence depicted in an accession number selected from the group consisting of M26880, AB003730 and U49869; 
 ii) ubiquitin activating enzyme (E1); 
 iii) ubiquitin conjugating enzyme (E2); 
 iv) tag2-ubiquitin ligase (E3); 
   b) measuring the amount of said tag1-ubiquitin bound to said ubiquitin ligase (E3), whereby the amount of said tag1-ubiquitin bound to said ubiquitin ligase (E3) indicates said ubiquitin ligase activity.   
     
     
         2 . The method of  claim 1 , further comprising:
 a) combining:
 v) a candidate ubiquitin ligase modulator. 
   
     
     
         3 . The method of  claim 1 , wherein the ubiquitin activating enzyme (E1) has at least 95% sequence identity to an amino acid sequence depicted in an accession number selected from the group consisting of A38564, S23770, AAA61246, P22314, CAA40296 and BAA33144. 
     
     
         4 . The method of  claim 1 , wherein the ubiquitin activating enzyme (E1) is encoded by a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence depicted in an accession number selected from the group consisting of M58028, X56976 and AB012190. 
     
     
         5 . The method of  claim 1 , wherein ubiquitin conjugating enzyme (E2) is selected from the group consisting of Ubiquitin conjugating enzyme 5 (Ubc5), Ubiquitin conjugating enzyme 3 (Ubc3), Ubiquitin conjugating enzyme 4 (Ubc4), and Ubiquitin conjugating enzyme X (UbcX). 
     
     
         6 . The method of  claim 1 , wherein ubiquitin conjugating enzyme (E2) has at least 95% sequence identity to an amino acid sequence depicted in an accession number selected from the group consisting of AAC37534, P49427, CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461, CAA63538, AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141, CAA04156, BAA11675, Q16781, NP — 003333, BAB18652, AAH00468, CAC16955, CAB76865, CAB76864, O00762, XP — 009804, XP — 009488, XP — 006823, XP — 006343, XP — 005934, XP — 002869, XP — 003400, XP — 009365, XP — 010361, XP — 004699, XP — 004019, O14933, P50550, P52485, P51668, P51669, P49459, P37286, P23567, P56554, CAB45853, NP — 003331, NP — 003330, NP — 003329, AAB53362 and NP — 008950. 
     
     
         7 . The method of  claim 1 , wherein the ubiquitin conjugating enzyme (E2) is encoded by a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence depicted in an accession number selected from the group consisting of Z29328, L40146, U39317, U39318, X92962, U58522, S81003, AF031141, AF075599, AJ000519, XM — 009488, NM — 007019, U73379 and D83004. 
     
     
         8 . The method of  claim 1 , wherein tag2-ubiquitin ligase (E3) comprises a RING finger protein. 
     
     
         9 . The method of  claim 8 , wherein said RING finger protein is selected from the group consisting of Regulator of cullins 1 (ROC1), Regulator of cullins 2 (ROC2), and Anaphase Promoting complex 11 (APC11). 
     
     
         10 . The method of  claim 8 , wherein said RING finger protein has at least 95% sequence identity to an amino acid sequence depicted in an accession number selected from the group consisting of AAD30147, AAD30146, and 6320196. 
     
     
         11 . The method of  claim 8 , wherein said RING finger protein is encoded by a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence depicted in an accession number selected from the group consisting of AF142059, AF142060 and nucleic acids 433493 to 433990 of NC — 001136. 
     
     
         12 . The method of  claim 1 , wherein tag2-ubiquitin ligase (E3) comprises a Cullin. 
     
     
         13 . The method of  claim 12 , wherein said Cullin is selected from the group consisting of Cullin 1 (CUL1), Cullin 2 (CUL2), Cullin 3 (CUL3), Cullin 4A (CUL4A), Cullin 4B (CUL4B), Cullin 5 (CUL5), and Anaphase Promoting complex 2 (APC2). 
     
     
         14 . The method of  claim 12 , wherein said Cullin has at least 95% sequence identity to an amino acid sequence depicted in an accession number selected from the group consisting of 4503161, AAC50544, AAC36681, 4503163, AAC51190, AAD23581, 4503165, AAC36304, AAC36682, AAD45191, AAC50548, Q13620, 4503167 and AAF05751. 
     
     
         15 . The method of  claim 12 , wherein said Cullin is encoded by a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence depicted in an accession number selected from the group consisting of NM — 003592, U58087, AF062536, AF126404, NM — 003591, U83410, NM — 003590, AF062537, AF064087, AF077188, U58091, NM — 003478, X81882 and AF191337. 
     
     
         16 . The method of  claim 1 , wherein tag2-ubiquitin ligase (E3) comprises a RING finger protein/Cullin combination. 
     
     
         17 . The method of  claim 16 , wherein said RING finger protein/Cullin combination is selected from the group consisting of Anaphase promoting complex 2 (APC2)/Anaphase Promoting complex 11 (APC11), Regulator of cullins 1 (ROC1)/Cullin 1 (CUL1), Regulator of cullins 1 (ROC1)/Cullin 2 (CUL2), and Regulator of cullins 2(ROC2)/Cullin 5 (CUL5). 
     
     
         18 . The method of  claim 1 , wherein said tag1 is a label or a partner of a binding pair. 
     
     
         19 . The method of  claim 18 , wherein said label is a fluorescent label. 
     
     
         20 . The method of  claim 19 , wherein said measuring is by measuring luminescence. 
     
     
         21 . The method of  claim 18 , wherein said partner of a binding pair is selected from the group consisting of an antigen, biotin, and calmodalin binding protein (CBP). 
     
     
         22 . The method of  claim 21 , wherein said partner of a binding pair is labeled by indirect labeling. 
     
     
         23 . The method of  claim 22 , wherein said indirect labeling is with a fluorescent label or a label enzyme. 
     
     
         24 . The method of  claim 23 , wherein said measuring is by measuring luminescence. 
     
     
         25 . The method of  claim 23 , wherein said label enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase and glucose oxidase. 
     
     
         26 . The method of  claim 25 , wherein said label enzyme is reacted with a label enzyme substrate which produces a fluorescent product. 
     
     
         27 . The method of  claim 26 , wherein said measuring is by measuring luminescence. 
     
     
         28 . The method of  claim 27 , wherein said tag1 is FLAG (DYKDDDDK; SEQ ID NO: 16). 
     
     
         29 . The method of  claim 23 , wherein said partner of a binding pair is FLAG and said indirect labeling is via anti-FLAG (DYKDDDDK; SEQ ID NO: 16). 
     
     
         30 . The method of  claim 22 , wherein said partner of a binding pair is FLAG (DYKDDDDK; SEQ ID NO: 16). 
     
     
         31 . The method of  claim 21 , wherein said antigen is FLAG (DYKDDDDK; SEQ ID NO: 16). 
     
     
         32 . The method of  claim 18 , wherein said tag2 is a surface substrate binding molecule. 
     
     
         33 . The method of  claim 32 , wherein said surface substrate binding molecule is a polyhistidine structure (His-tag). 
     
     
         34 . The method of  claim 33 , wherein said assaying is performed in a multi-well plate comprising a surface substrate comprising nickel.