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Science 339 (6119): 590-595

Copyright © 2013 by the American Association for the Advancement of Science

A Strategy for Modulation of Enzymes in the Ubiquitin System

Andreas Ernst1, George Avvakumov2, Jiefei Tong3, Yihui Fan4, Yanling Zhao4, Philipp Alberts3, Avinash Persaud3,5, John R. Walker2, Ana-Mirela Neculai1, Dante Neculai2, Andrew Vorobyov1, Pankaj Garg1, Linda Beatty1, Pak-Kei Chan6, Yu-Chi Juang7, Marie-Claude Landry7, Christina Yeh7,8, Elton Zeqiraj7, Konstantina Karamboulas1, Abdellah Allali-Hassani2, Masoud Vedadi2, Mike Tyers6,7, Jason Moffat1,8,9,10, Frank Sicheri7,8, Laurence Pelletier7,8, Daniel Durocher7,8, Brian Raught10, Daniela Rotin3,5, Jianhua Yang4, Michael F. Moran3,8,9, Sirano Dhe-Paganon2,11, and Sachdev S. Sidhu1,8,9,10,*

1 Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
2 Structural Genomics Consortium, MaRS Centre, 101 College Street, Suite 700, Toronto, Ontario M5G 1L7, Canada.
3 Hospital for Sick Children, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
4 Texas Children's Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
5 Biochemistry Department, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
6 Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada.
7 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
8 Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
9 Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
10 Ontario Cancer Institute and McLaughlin Centre for Molecular Medicine, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
11 Department of Physiology, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.


Figure 1
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Fig. 1. Structural rationale and library design for Ub-based USP inhibitors. (A) Superposition of Ub in complex USP21 (blue; PDB entry 3I3T), USP2a (green; PDB entry 2HD5), USP5 (yellow; PDB entry 3IHP), USP7 (magenta; PDB entry 1NBF), and USP14 (red; PDB entry 2AYO) (22, 45, 46). The superposition was performed with complete coordinates for the Ub:USP complexes, but for clarity, only USP21 is shown (gray). (B) The Ub-binding site of USP21. USP21 is shown as a molecular surface, and residues that form the Ub-binding site are colored red or blue, indicating residues that are <50% or ≥50% conserved, respectively, in the sequences of 48 human USPs (fig. S1). (C) The phage-displayed Ub library design mapped onto the Ub structure (PDB entry 1UBQ). The Ub main chain is shown as a gray tube, and positions that were diversified in the libraries are shown as spheres, colored as follows: region 1, purple; region 2, blue; and region 3, orange. (D) The primary sequence of the regions targeted in the library design. Diversified sequences are shaded and colored as in (C). Two libraries were constructed. Amongst the shaded sequences, library 1 (7 x 1010 distinct variants) did not target six of the positions in region 3 (73 to 78), and library 2 (9 x 109 distinct variants) did not target four of the positions in region 2 (35, 37, 39, and 40).

 

Figure 2
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Fig. 2. Selective binding of Ub variants to DUBs, Ub ligases, and UBDs, and inhibition of DUB function in vitro. (A) Sequence alignment of Ub.wt and representative Ub variants selected for binding to USP8 (8.2), USP21 (21.4), USP2a (2.3), OTUB1 (B1.1), the BRISC protein complex (BR.1), NEDD4 (N.2), ITCH (IT.1), USP37-UBD (37.1), or Cdc34 (R1.2). The alignment shows only those positions that were diversified in the Ub library, and positions that were conserved as the wt sequence are indicated by dashes. A complete list of selected variants is provided in table S2. (B) Ub variants bind selectively to their cognate targets, as shown by phage enzyme-linked immunosorbent assays for binding to the following immobilized proteins (color coded as indicated in the panel): USP2a, USP5, USP7, USP8, USP9x, USP9y, USP10, USP14, USP16, USP21, USP48, OTUB1, BRISC, NEDD4, ITCH, and USP37-UBD. Bound phage were detected spectrophotometrically (optical density at 450 nm), and background binding to neutravidin was subtracted from the signal. (C) Inhibition of USP2a, USP28, or USP21 shown as dose-response curves for Ub variants. The IC50 value was determined as the concentration of Ub variant that reduced USP activity by 50%. (D) The effects of Ubv.B1.1 or Ub.wt on binding of OTUB1 to Ub~UbcH5B measured as relative fluorescence by means of time-resolved Förster-energy transfer. The median effective concentration (EC50) value is defined as the half-maximal effective concentration of OTUB1.

 

Figure 3
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Fig. 3. Molecular basis for DUB inhibition by Ub variants. (A) Superposition of USP21 (yellow) in complex with Ubv.21.4 (purple) and USP21 (gray) in complex with Ub.wt (blue). (B) Surface representation of the Ub-binding site of USP21 in complex with Ubv.21.4 (wheat tube). Residues in Ubv.21.4 that are mutated relative to Ub.wt are shown as colored sticks, and residues on the USP21 surface that make contact with these residues are colored red or blue if they are <50% or ≥50% conserved in the sequences of 48 human USPs, respectively. (C) Details of the superposition of USP21 in complex Ubv.21.4 or Ub.wt, showing the changes in molecular interactions caused by the mutations at positions 64 (left), 68 (center), and 70 (right). The Ubv.21.4 side chains are colored as in (B), and Ub.wt side chains are colored blue. Main chains and USP21 side chains are colored as in (A). Residues are numbered according to the PDB files, and asterisks indicate USP residues. (D) Superposition of USP8 (yellow) in complex with Ubv.8.2 (purple) and apo-USP8 (gray). Dashed ovals demarcate structural changes in the zinc finger region (red) or the BL2 loop (black) of USP8. (E) Surface representation of the Ub-binding site of USP8 in complex with Ubv.8.2 (wheat tube). Residues in Ubv.8.2 that are mutated relative to Ub.wt and make contact with USP8 are shown as colored sticks, and residues on the USP8 surface that make contact with these residues are colored red or blue, as in (B). (F) Details of USP8 in complex with Ubv.8.2, showing molecular interactions involving residues in Ubv.8.2 that are mutated relative to Ub.wt and are in contact with USP8. The Ubv.8.2 side chains are colored as in (E). Main chains and USP8 side chains are colored as in (D). (G) Superposition of USP2a (yellow) in complex with Ubv.2.3 (purple) and USP2a (gray) in complex with Ub.wt (blue). (H) Surface representation of the Ub-binding site of USP2a in complex with Ubv.2.3 (wheat tube). Residues in Ubv.2.3 that are mutated relative to Ub.wt are shown as colored sticks, and residues on the USP2a surface that make contact with these residues are colored red or blue, as in (B). (I) Details of the superposition of USP2a in complex with Ubv.2.3 or Ub.wt, showing changes in molecular interactions caused by the mutations at positions 6 (left), 12 (center), and 9 (right). The Ubv.2.3 side chains are colored as in (H). Main chains and USP2a side chains are colored as in (G). (J) Superposition of OTUB1 (yellow) in complex with Ubv.B1.1 (purple) and OTUB1 (gray) in complex with distal Ub.wt (blue) and an E2-Ub covalent conjugate consisting of E2 conjugating enzyme UbcH5b (red) and Ub.wt (green). (K) Surface representation of the Ub-binding site of OTUB1 in complex with Ubv.B1.1 (wheat tube). Residues in Ubv.B1.1 that are mutated relative to Ub.wt are shown as colored sticks, and residues on the OTUB1 surface that are in contact with these residues are colored red. (L) Details of the superposition of OTUB1 in complex with Ubv.B1.1 or Ub.wt, showing changes in molecular interactions caused by the mutations at positions 47 (left), 66 and 68 (center), and 42 and 44 (right). The Ubv.B1.1 side chains are colored as in (K). Main chains and OTUB1 side chains are colored as in (J).

 

Figure 4
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Fig. 4. Modulation of enzyme function in cells by Ub variants. (A) Flag-tagged Ubv.21.4C{Delta}2, but not Ub.wtC{Delta}2, interacts with Myc-tagged USP21, as evidenced by immunoprecipitations with anti-Myc followed by immunoblotting with anti-Flag. (B) Transfection of Ubv.21.4C{Delta}2, but not Ub.wtC{Delta}2, causes increased RIP1 ubiquitination. The blot is overexposed so as to reveal the ubiquitination state of RIP1. (C) NF-{kappa}B–mediated luciferase activity, which is activated by RIP1 transfection and down-regulated by USP21 cotransfection, is restored by cotransfection of Ubv.21.4C{Delta}2 but not by cotransfection of Ub.wtC{Delta}2. (D) Ubv.8.2C{Delta}2, but not Ub.wtC{Delta}2, binds to USP8 in an EGF-stimulation–independent manner, as shown by means of immunoprecipitation of Flag-tagged Ub variants, followed by immunoblotting with antibodies to USP8. Immunoblotting of whole-cell lysates (WCLs) with anti-phosphotyrosine (pY) and antibodies to USP8 indicates that EGFR phosphorylation and endogenous USP8 concentrations are not affected by the expression of Ub.wtC{Delta}2 or Ubv.8.2C{Delta}2. An irrelevant mouse antibody was included as negative control (first two lanes). (E) Transient expression of Ubv.8.2C{Delta}2, but not Ub.wtC{Delta}2, leads to increased ubiquitination of EGFR in response to stimulation with EGF (10 ng/ml, 10 min) in HEK293 cells stably expressing Flag-tagged EGFR, as shown by means of immunoprecipitation with anti-Flag antibody from denatured lysates, followed by immunoblotting with anti-Ub antibody. (F) Expression of Ubv.8.2C{Delta}2, but not Ub.wtC{Delta}2, leads to decreased levels of endogenous EGFR in response to stimulation with EGF, as shown through analysis of total cellular EGFR levels by immunoblotting with antibody to EGFR. Immunoblotting of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) indicates that similar amounts of sample were probed. (G) Quantification of colocalization of EGFR with EEA1, RAB11, or LAMP observed in immunoflourescence microscopy images. The mean colocalization coefficients, averaged from 10 independent single-cell images, represent pixel overlap between EGFR and EEA1, Rab11, or LAMP. The coefficients vary from 0 to 1, with 0 corresponding to nonoverlapping images and 1 corresponding to 100% colocalization. All error bars represent SD *P < 0.001. (H) Auto-ubiquitination of NEDD4 in vitro. Recombinant full-length NEDD4 was incubated for 3 hours with E1, E2 (UbcH7), adenosine 5'-triphosphate (ATP), Ub, and Ub.wtC{Delta}2 or Ubv.N.2, and the reaction mixture was immunoblotted with antibody to NEDD4. Ubv.N.2 is not a substrate for the E1-E2-E3 enzyme ubiquitination cascade because its C terminus does not contain a di-glycine motif (Fig. 2A). (I) Ubiquitination of the NEDD4 substrate YY1 in cells. HEK293T cells were transfected with Flag-tagged YY1, V5-tagged NEDD4 (wt or catalytically inactive CS mutant), and Ub.wtC{Delta}2 or Ubv.N.2. YY1 was immunoprecipitated with antibody to Flag, and gels were immunoblotted for Ub (top) or YY1 (middle). (Bottom) The amount of NEDD4 in the lysates.

 


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