A strategy for modulation of enzymes in the ubiquitin system.
Andreas Ernst,George V. Avvakumov,Jiefei Tong,Yihui Fan,Yanling Zhao,Philipp Alberts,Avinash Persaud,John R. Walker,Ana-Mirela Neculai,Dante Neculai,Andrew Vorobyov,Pankaj Garg,Linda G. Beatty,Pak-Kei Chan,Yu Chi Juang,Marie-Claude Landry,Christina Yeh,Christina Yeh,Elton Zeqiraj,Konstantina Karamboulas,Abdellah Allali-Hassani,Masoud Vedadi,Mike Tyers,Mike Tyers,Jason Moffat,Frank Sicheri,Frank Sicheri,Laurence Pelletier,Laurence Pelletier,Daniel Durocher,Daniel Durocher,Brian Raught,Daniela Rotin,Jianhua Yang,Michael Moran,Sirano Dhe-Paganon,Sirano Dhe-Paganon,Sachdev S. Sidhu +37 more
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TLDR
This work used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography to report a method to target the myriad enzymes that govern ubiquitination of protein substrates.Abstract:
The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.read more
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Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes.
TL;DR: This work focuses on the use of UBDs to directly purify or detect (poly)ubiquitin‐modified proteins and more broadly for the targeted manipulation of ubiquitIn‐mediated processes, highlighting insights into ubiquitin signalling that have been provided.
Journal ArticleDOI
Affinity- and Specificity-Enhancing Mutations Are Frequent in Multispecific Interactions between TIMP2 and MMPs
TL;DR: It is demonstrated that the TIMP2 sequence lies far from the fitness maximum when interacting with its target enzymes, suggesting that this non-optimality of the binding interface and high potential for improvement might characterize all proteins evolved for binding to multiple targets.
Journal ArticleDOI
Ubiquitin-specific protease 8 (USP8/UBPy): a prototypic multidomain deubiquitinating enzyme with pleiotropic functions
TL;DR: An overview of the versatile molecular, cellular and pathology associated function and regulation of USP8 which appears to depend on specific protein binding partners, substrates and the cellular context is provided.
Journal ArticleDOI
Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10.
Wei Zhang,Maria A. Sartori,Taras Makhnevych,Kelly Federowicz,Xiaohui Dong,Li Liu,Satra Nim,Aiping Dong,Jingsong Yang,Yanjun Li,Dania Haddad,Andreas Ernst,Dirk A. Heerding,Yufeng Tong,Jason Moffat,Sachdev S. Sidhu +15 more
TL;DR: A phage-displayed ubiquitin variant (UbV) library is employed to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2, and cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin.
Journal ArticleDOI
Differential Oligomerization of the Deubiquitinases USP25 and USP28 Regulates Their Activities.
Florian Sauer,Theresa Klemm,Ravi B. Kollampally,Ingrid Tessmer,Radhika K. Nair,Nikita Popov,Caroline Kisker +6 more
TL;DR: This work solved the structures of the catalytic domains of both proteins and established substantial differences in their activities, leading to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.
References
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TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
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TL;DR: The content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories.
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Airlie J. McCoy,Ralf W. Grosse-Kunstleve,Paul D. Adams,Martyn Winn,Laurent C. Storoni,Randy J. Read +5 more
TL;DR: A description is given of Phaser-2.1: software for phasing macromolecular crystal structures by molecular replacement and single-wavelength anomalous dispersion phasing.
Journal ArticleDOI
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TL;DR: The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties and the results derived are consistently better than those obtained from least-squares refinement.