Ubiquitin variant modulators of hect e3 ligases and their uses

Abstract

The invention provides ubiquitin variants that specifically bind to HECT E3 ligases, and methods of using these variants to modulate the activity of HECT E3 ligases.

What is claimed is: 
     
         1 . A ubiquitin variant (Ubv) polypeptide comprising one or more substitution in one or more region of a ubiquitin polypeptide, wherein the region is selected from the group consisting of:
 (a) region 1 (amino acids 2-14 of SEQ ID NO:1) wherein the polypeptide comprises the structure:   X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14      
       wherein 
       X 2  is selected from the group consisting of A, D, E, H, K, L, P, Q, R, S, T, and Y; 
       X 4  is selected from the group consisting of A, C, F, I, L, R, S, V, and Y; 
       X 6  is selected from the group consisting of E, G, H, I, K, N, Q, R, S, V, W, and Y; 
       X 8  is selected from the group consisting of F, G, H, I, L, M, P, R, V, and Y; 
       X 9  is selected from the group consisting of A, F, G, I, K, L, M, N, P, R, S, and T; 
       X 10  is selected from the group consisting of A, D, G, L, R, V, and W; 
       X 11  is selected from the group consisting of D, E, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; 
       X 12  is selected from the group consisting of D, G, I, L, N, P, S, T, V, and Y; 
       X 14  is selected from the group consisting of A, C, F, I, K, N, P, R, S, and T; and 
       any X not specified optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1;
 (b) region 2 (amino acids 42-49 of SEQ ID NO:1) wherein the polypeptide comprises the structure: 
 X 42 -X 43 -X 44 -X- 45 -X 46 -X 47 -X 48 -X 49    
 
       wherein 
       X 42  is selected from the group consisting of F, G, I, K, L, Q, R, S, T, and V; 
       X 44  is selected from the group consisting of F, I, L, T, and V; 
       X 46  is selected from the group consisting of A, G, H, L, R, S, T, and V; 
       X 47  is selected from the group consisting of A, D, G, R, S, and W; 
       X 48  is selected from the group consisting of K, M, N, Q, R, and T; 
       X 49  is selected from the group consisting of D, E, H, I, K, N, P, Q, R, and S; and 
       any X not specified optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1; and
 (c) region 3 (amino acids 62-78 of SEQ ID NO:1) wherein the polypeptide comprises the structure: 
 X 62 -X 63 -X 64 -X 65 -X 66 -X 67 -X 68 -X 69 -X 70 -X 71 -X 72 -X 73 -X 74 -X 75 -X 76 -X 77 -X 78    
 
       wherein 
       X 62  is selected from the group consisting of C, H, K, L, P, Q, R, and Y; 
       X 63  is selected from the group consisting of D, E, G, H, K, M, N, Q, R, and Y; 
       X 64  is selected from the group consisting of A, D, E, F, G, K, Q, R, T, V, W, and Y; 
       X 66  is selected from the group consisting of A, F, H, I, L, N, P, Q, R, S, T, and W; 
       X 68  is selected from the group consisting of G, F, H, L, N, R, S, T, W, and Y; 
       X 70  is selected from the group consisting of A, I, F, K, L, M, R, V, and W; 
       X 71  is selected from the group consisting of F, G, K, L, M, R, V, and Y; 
       X 72  is selected from the group consisting of G, I, K, P, Q, R, S, and T; 
       X 73  is selected from the group consisting of F, I, L, P, R, and T; 
       X 74  is selected from the group consisting of L, H, I, P, R, and V; 
       X 75  is selected from the group consisting of A, D, G, I, P, R, V, W, and Y; 
       X 76  is selected from the group consisting of A, D, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, and Y; 
       X 77  is selected from the group consisting of A, D, E, F, H, I, K, M, N, P, Q, R, S, T, V, and Y, or is absent; 
       X 78  is selected from the group consisting of A, D, E, F, H, I, K, L, N, P, Q, R, S, T, V, W, and Y, or is absent; and any X not specified optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1; 
       wherein any X specified in said Ubv polypeptide optionally has the amino acid sequence of the corresponding position in SEQ ID NO:1; 
       or 
       a fragment thereof, wherein the sequence of said Ubv polypeptide does not consist of SEQ ID NO:1. 
     
     
         2 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to NEDD4 (N4). 
     
     
         3 . The Ubv polypeptide of  claim 2 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:2-4 or a fragment thereof. 
     
     
         4 . The Ubv polypeptide of  claim 3 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:2-4. 
     
     
         5 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to NEDD4L (NL). 
     
     
         6 . The Ubv polypeptide of  claim 5 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:5-8. 
     
     
         7 . The Ubv polypeptide of  claim 6 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:5-8. 
     
     
         8 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to ITCH (IT). 
     
     
         9 . The Ubv polypeptide of  claim 8 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:9-12. 
     
     
         10 . The Ubv polypeptide of  claim 9 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:9-12. 
     
     
         11 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to SMURF1 (S1). 
     
     
         12 . The Ubv polypeptide of  claim 11 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:13-17. 
     
     
         13 . The Ubv polypeptide of  claim 12 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:13-17. 
     
     
         14 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to SMURF2 (S2). 
     
     
         15 . The Ubv polypeptide of  claim 14 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:18-22. 
     
     
         16 . The Ubv polypeptide of  claim 15 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:18-22. 
     
     
         17 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to WWP1 (P1). 
     
     
         18 . The Ubv polypeptide of  claim 17 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:23-26. 
     
     
         19 . The Ubv polypeptide of  claim 18 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:23-26. 
     
     
         20 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to WWP2 (P2). 
     
     
         21 . The Ubv polypeptide of  claim 20 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:27-30. 
     
     
         22 . The Ubv polypeptide of  claim 21 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:27-30. 
     
     
         23 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HECW1 (W1). 
     
     
         24 . The Ubv polypeptide of  claim 23 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:31-34. 
     
     
         25 . The Ubv polypeptide of  claim 24 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:31-34. 
     
     
         26 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HECW2 (W2). 
     
     
         27 . The Ubv polypeptide of  claim 26 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:35-38. 
     
     
         28 . The Ubv polypeptide of  claim 27 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:35-38. 
     
     
         29 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to RSP5 (R5). 
     
     
         30 . The Ubv polypeptide of  claim 29 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:39-44. 
     
     
         31 . The Ubv polypeptide of  claim 30 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:39-44. 
     
     
         32 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HERC1 (H1). 
     
     
         33 . The Ubv polypeptide of  claim 32 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:45 and 46. 
     
     
         34 . The Ubv polypeptide of  claim 33 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:45 and 46. 
     
     
         35 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HERC2 (H2). 
     
     
         36 . The Ubv polypeptide of  claim 35 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:47 and 48. 
     
     
         37 . The Ubv polypeptide of  claim 36 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:47 and 48. 
     
     
         38 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HERC4 (H4). 
     
     
         39 . The Ubv polypeptide of  claim 38 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:49-52. 
     
     
         40 . The Ubv polypeptide of  claim 39 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:49-52. 
     
     
         41 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HERC6 (H6). 
     
     
         42 . The Ubv polypeptide of  claim 41 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:53. 
     
     
         43 . The Ubv polypeptide of  claim 42 , wherein the sequence of said Ubv polypeptide comprises SEQ ID NO:53. 
     
     
         44 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HACE1 (HA). 
     
     
         45 . The Ubv polypeptide of  claim 44 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:54-56. 
     
     
         46 . The Ubv polypeptide of  claim 45 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:54-56. 
     
     
         47 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HUWE1 (HU). 
     
     
         48 . The Ubv polypeptide of  claim 47 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:57 and 58. 
     
     
         49 . The Ubv polypeptide of  claim 48 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:57 and 58. 
     
     
         50 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to UBE3C (3C). 
     
     
         51 . The Ubv polypeptide of  claim 50 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:59 and 60. 
     
     
         52 . The Ubv polypeptide of  claim 51 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:59 and 60. 
     
     
         53 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to HECTD1 (D1). 
     
     
         54 . The Ubv polypeptide of  claim 53 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:61-64. 
     
     
         55 . The Ubv polypeptide of  claim 54 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:61-64. 
     
     
         56 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to EDD1 (ED). 
     
     
         57 . The Ubv polypeptide of  claim 56 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:65 and 66. 
     
     
         58 . The Ubv polypeptide of  claim 57 , wherein the sequence of said Ubv polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:65 and 66. 
     
     
         59 . The Ubv polypeptide of  claim 1 , wherein said Ubv polypeptide binds to KIAA0317 (KI). 
     
     
         60 . The Ubv polypeptide of  claim 59 , wherein the sequence of said Ubv polypeptide comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs:67-70. 
     
     
         61 . The Ubv polypeptide of  claim 60 , wherein the sequence of said Ubv polypeptide is selected from the group consisting of SEQ ID NOs:67-70. 
     
     
         62 . The Ubv polypeptide of any one of  claims 3 ,  4 ,  6 ,  7 ,  9 ,  10 ,  12 ,  13 ,  15 ,  16 ,  18 ,  19 ,  21 ,  22 ,  24 ,  25 ,  27 ,  28 ,  30 ,  31 ,  33 ,  34 ,  36 ,  37 ,  39 ,  40 ,  42 ,  43 ,  45 ,  46 ,  48 ,  49 ,  51 ,  52 ,  54 ,  55 ,  57 ,  58 ,  60 , or  61 , wherein said Ubv polypeptide comprises a fragment of a polypeptide comprising the indicated sequence identifier or a sequence at least 90% identical thereto. 
     
     
         63 . A nucleic acid molecule encoding a Ubv polypeptide of any one of  claims 1 - 62 . 
     
     
         64 . A recombinant expression vector comprising the nucleic acid molecule of  claim 63 . 
     
     
         65 . A host cell comprising the nucleic acid molecule of  claim 63  or the recombinant expression vector of  claim 64 . 
     
     
         66 . A method of obtaining a Ubv polypeptide that modulates an activity or property of a HECT E3 ligase, the method comprising:
 (a) randomizing ubiquitin residues to produce a combinatorial library of Ubv polypeptides, and   (b) selecting Ubv polypeptides that specifically bind to one or more HECT E3 ligase.   
     
     
         67 . The method of  claim 66 , wherein the randomized residues (a) contact the N-lobe exosite of a HECT E3 ligase, (b) contact the E2 site of a HECT E3 ligase, and/or (c) are within region 1 (amino acids 2-14), region 2 (amino acids 42-49), and/or region 3 (amino acids 62-78) of ubiquitin. 
     
     
         68 . The method of  claim 66  or  67 , wherein said HECT E3 ligase is selected from the group consisting of NEDD4 (N4), NEDD4L (NL), ITCH (IT), SMURF1 (S1), SMURF2 (S2), WWP1 (P1), WWP2 (P2), HECW1 (W1), HECW2 (W2), RSP5 (R5), HERC1 (H1), HERC2 (H2), HERC3 (H3), HERC4 (H4), HERC5 (H5), HERC6 (H6), HACE1 (HA), HUWE1 (HU), UBE3A (3A), UBE3B (3B), UBE3C (3C), HECTD1 (D1), EDD1 (ED), KIAA0317 (KI), HECTD1, HECTD2, HECTD3, HECTD4, G2E3, and TRIP12. 
     
     
         69 . The method of any one of  claims 66 - 68 , further comprising assessing a selected Ubv for its effects on an activity of a HECT E3 ligase or on a cellular function or activity modulated by a HECT E3 ligase. 
     
     
         70 . A method of modulating an activity or property of a HECT E3 ligase, the method comprising contacting said HECT E3 ligase with an agent that binds to the N-lobe exosite of said HECT E3 ligase or to the E2 site of said HECT E3 ligase. 
     
     
         71 . The method of  claim 70 , wherein said agent increases the activity of said HECT E3 ligase. 
     
     
         72 . The method of  claim 70 , wherein said agent decreases the activity of said HECT E3 ligase. 
     
     
         73 . The method of any one of  claims 70 - 72 , wherein said agent comprises a Ubv polypeptide, a nucleic acid molecule encoding a Ubv polypeptide, or a fragment thereof. 
     
     
         74 . The method of  claim 73 , wherein said Ubv polypeptide is a Ubv polypeptide of any one of  claims 1  to  62  or a fragment thereof, or said nucleic acid molecule is a nucleic acid molecule of  claim 63  or a fragment thereof. 
     
     
         75 . The method of any one of  claims 70 - 74 , wherein said agent has specificity for a particular HECT E3 ligase. 
     
     
         76 . The method of any one of  claims 70 - 74 , wherein said agent is active against more than one HECT E3 ligase. 
     
     
         77 . The method of any one of  claims 70 - 76 , wherein said HECT E3 ligase is in a cell. 
     
     
         78 . The method of  claim 77 , wherein said cell is comprised within a subject. 
     
     
         79 . The method of  claim 78 , wherein said subject has a disease or condition selected from the group consisting of hypertension, inflammation, and cancer. 
     
     
         80 . A method of treating hypertension, inflammation, or cancer in a subject, the method comprising modulating the activity of a HECT E3 ligase in a cell of the subject according to the method of any one of  claims 70 - 79 . 
     
     
         81 . A method of identifying an agent that modulates the activity of a HECT E3 ligase, the method comprising contacting a complex comprising a HECT E3 ligase and a Ubv polypeptide that binds to said HECT E3 ligase with a candidate agent, and determining whether said agent affects the binding of said Ubv to said HECT E3 ligase or an activity of said complex. 
     
     
         82 . The method of  claim 81 , wherein said candidate agent comprises a small molecule compound. 
     
     
         83 . The method of  claim 81  or  82 , wherein said HECT E3 ligase is selected from the group consisting of NEDD4 (N4), NEDD4L (NL), ITCH (IT), SMURF1 (S1), SMURF2 (S2), WWP1 (P1), WWP2 (P2), HECW1 (W1), HECW2 (W2), RSP5 (R5), HERC1 (H1), HERC2 (H2), HERC3 (H3), HERC4 (H4), HERC5 (H5), HERC6 (H6), HACE1 (HA), HUWE1 (HU), UBE3A (3A), UBE3B (3B), UBE3C (3C), HECTD1 (D1), EDD1 (ED), KIAA0317 (KI), HECTD1, HECTD2, HECTD3, HECTD4, G2E3, and TRIP12. 
     
     
         84 . The method of any one of  claims 81 - 83 , wherein said Ubv polypeptide is selected from a Ubv polypeptide of any one of  claims 1 - 62  or a fragment thereof.