Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10

Zhang, Wei; Sartori, Maria A.; Makhnevych, Taras; Federowicz, Kelly; Dong, Xiaohui; Liu, Li; Nim, Satra; Dong, Aiping; Yang, Jing; Li, Yanjun; Haddad, Dania; Ernst, Andreas; Heerding, Dirk A.; Tong, Yufeng; Moffat, Jason; Sidhu, Sachdev S.

doi:10.1016/j.jmb.2017.05.025

Cited by 46 publications

(51 citation statements)

References 54 publications

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“…We previously designed a UbV library (library 2) in which 29 positions on the Ub surface were subjected to a diversification strategy that favored the wild-type sequence (soft randomization) and four C-terminal positions were diversified in a completely random manner (hard randomization) . Library 2 has been used to develop tight and specific UbV binders for a variety of UPS components, including DUBs Zhang et al, 2017) and E3 ligases (Gabrielsen et al, 2017;Gorelik et al, 2016;Zhang et al, 2016b). We analyzed the sequences of UbVs characterized in these previous studies to aid the design of a new library 4 ( Figure 2A and Table S1).…”

Section: Generation and Optimization Of Ubv Binders For Usp15mentioning

confidence: 99%

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

et al. 2019

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Highlights d Tight and selective UbVs target USP15 catalytic and adaptor domains d UbV inhibitors lock the USP15 active site in an inactive conformation d A strand-swapped UbV dimer binds two DUSP domains simultaneously d Linear UbV dimers are potent and specific USP15 inhibitors in cells SUMMARYThe multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor b pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

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Section: Generation and Optimization Of Ubv Binders For Usp15mentioning

confidence: 99%

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

et al. 2019

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“…UbVs are emerging as powerful tools for probing functions of the Ub system. To date, we and others have selected for UbVs that bind catalytic domains from enzymes (DUBs, E3 ligases, E2 conjugating enzymes), interchangeable cullin-binding subcomplexes (Skp1-F-box proteins), and Ub-binding domains (21,28,(38)(39)(40)(41)(42)(43)(44). These selections have yielded hundreds of tools targeting the known domains by modulating catalytic activity and assembly of E3 ligases, and inhibiting Ub binding by well-recognized interacting domains.…”

Section: Discussionmentioning

confidence: 99%

“…Unlike Ub, which is constrained in sequence and structure by the requirement to bind a massive number of partner proteins, sequence variants have the potential to make distinctive contacts that increase affinity for particular partners. We and others previously performed phage display to select variants binding to a variety of E3 ligases and deubiquitylating enzymes (DUBs), and previously identified Ub-binding domains, to test effects of perturbing Ub interactions with known interaction sites (21,28,(38)(39)(40)(41)(42)(43)(44). Indeed, our UbV R , selected from among 10 10 Ub variants (UbVs) displayed on phage as binding APC11's RING domain, proved useful for uncovering the contribution of APC11's Ub-binding exosite to processive affinity amplification of activity with UBE2C, and enabled obtaining cryo EM data visualizing UBE2S bound to the APC2-APC11 catalytic core of APC/C (28).…”

Section: Introductionmentioning

confidence: 99%

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

Watson

Grace

Zhang

et al. 2019

Preprint

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Ubiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitin's C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at "exosites". However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.

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“…Very recently, a phage‐displayed library of billions of Ub variants (UbVs) has been established which is expected to be able to generate specific UPS inhibitors. Through this platform, specific UbVs have been identified that can inhibit USP7 and USP10 with high affinity . Implementation of fragment‐based methods and surface plasmon resonance (SPR) has led to the generation of new compounds with high inhibition potency .…”

Section: Ubiquitin Specific Protease 7 (Usp7)mentioning

confidence: 99%

“…103 with high affinity. 119 Implementation of fragment-based methods and surface plasmon resonance (SPR) has led to the generation of new compounds with high inhibition potency. 120 triterpenes has been documented as potent USP7 inhibitor.…”

Section: Identified Inhibitors Of Usp7mentioning

confidence: 99%

Targeting ubiquitin specific protease 7 in cancer: A deubiquitinase with great prospects

Khusbu

Chen

2018

Cell Biochemistry & Function

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Deubiquitinase (DUB)-mediated cleavage of ubiquitin chain balances ubiquitination and deubiquitination for determining protein fate. USP7 is one of the best characterized DUBs and functionally important. Numerous proteins have been identified as potential substrates and binding partners of USP7; those play crucial roles in diverse array of cellular and biological processes including tumour suppression, cell cycle, DNA repair, chromatin remodelling, and epigenetic regulation. This review aims at summarizing the current knowledge of this wide association of USP7 with many cellular processes that enlightens the possibility of abnormal USP7 activity in promoting oncogenesis and the importance of identification of specific inhibitors.

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Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10

Cited by 46 publications

References 54 publications

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

Targeting ubiquitin specific protease 7 in cancer: A deubiquitinase with great prospects

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