Chemical and semisynthetic approaches to study and target deubiquitinases

Abstract

Ubiquitination is a key posttranslational modification, which affects numerous biological processes and is reversed by a class of enzymes known as deubiquitinases (DUBs). This family of enzymes cleaves mono-ubiquitin or poly-ubiquitin chains from a target protein through different mechanisms and mode of interactions with their substrates. Studying the role of DUBs in health and diseases has been a major goal for many laboratories both in academia and in industry. However, the field has been challenged by the difficulties in obtaining native substrates and novel reagents using traditional enzymatic and molecular biology approaches. Recent advancements in the synthesis and semisynthesis of proteins made it possible to prepare several unique ubiquitin conjugates to study various aspects of DUBs such as their specificities and structures. Moreover, these approaches enable the preparation of novel activity based probes and assays to monitor DUB activities in vitro and in cellular contexts. Efforts made to bring new chemical entities for the selective inhibition of DUBs based on these tools are also highlighted with selected examples.

Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

“…[47] Therefore, failure in its regulation can lead to several pathologies,f or example,cancer and neurodegenerative disease.While aprotein modified with the specific Ub chain can proceed to degradation, this process is further complicated by the activity of DUBs,which remove Ub units from proteins to affect Ub signaling.T he largest sub family of Cys protease DUBs-Ub specific proteases (USPs) [26,47] is involved in av ariety of regulatory processes and has been implicated in various diseases.D espite progress in studying the function of many USPs,c omprehensive understanding of their structure,s pecificities,a nd cellular function is still lacking for many of its members. [47] Therefore, failure in its regulation can lead to several pathologies,f or example,cancer and neurodegenerative disease.While aprotein modified with the specific Ub chain can proceed to degradation, this process is further complicated by the activity of DUBs,which remove Ub units from proteins to affect Ub signaling.T he largest sub family of Cys protease DUBs-Ub specific proteases (USPs) [26,47] is involved in av ariety of regulatory processes and has been implicated in various diseases.D espite progress in studying the function of many USPs,c omprehensive understanding of their structure,s pecificities,a nd cellular function is still lacking for many of its members.…”

“…[26] Notably,the high similarity of the active sites between many USPs hinders the development of selective inhibitors.T odate [26] Notably,the high similarity of the active sites between many USPs hinders the development of selective inhibitors.T odate …”

Understanding and predicting the potency of ROS-based enzyme inhibitors, exemplified by naphthoquinones and ubiquitin specific protease-2

“…8,9 Recent studies revealed that DUBs are susceptible to hydrogen peroxide, suggesting a potential way of regulating their cellular activity under oxidative stress (Fig. 1).…”

“…13 DUBs are hence emerging as promising drug targets, and their targeting via a novel mechanism of inhibition has become a major goal in academia and in industry. 9 …”

Activity‐Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α‐Globin‐Modulating Deubiquitinase

“…[12] In these examples,anelectrophile was placed between the two Ub units to trap the catalytic Cys residue of the specific DUB,w hereby the linkage type dictates the specificity of the DUB.For example, our group temporally incorporated aC ys residue into as pecific Lyss ide chain in Ub to enable ligation with synthetic Ub(1-75)-thioester,f ollowed by dehydroalanine (DHA) formation to serve as atrap for the DBU catalytic Cys residue. [13] However,d espite aw ide range of applicability of these tools,n ot every protein target is synthetically or even semisynthetically accessible,a nd ag eneral strategy for the preparation of ubiquitinated protein ABPs is highly desirable. Themain obstacle in designing such ap robe,h owever, is the nature of the ubiquitinated protein substrate.Chemical protein synthesis is am ajor tool for the preparation of both ubiquitinated proteins and diUb probes with ad efined linkage type.…”