Profiling DUBs and Ubl-specific proteases with activity-based probes

Abstract

Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse Ub signaling by disassembly of one or multiple poly-Ub chain types and their malfunction is often associated with human disease. The Ub system displays significant crosstalk with structurally homologous ubiquitin-like proteins (Ubls), including SUMO, Nedd8, and ISG15. This can be seen with Methods in Enzymology, Volume 618

Mechanism and inhibition of the papain‐like protease, PLpro, of SARS‐CoV‐2

“…Ub‐MesNa (2‐mercaptoethanesulfonate as a sodium salt) and subsequently Ub‐PA were prepared as described previously (Wilkinson et al , ; Ekkebus et al , ; Gersch et al , ). Human ISG15 CTD (79–156)‐MesNa (ISG15 CTD ‐MesNa) and the ISG15 CTD ‐PA suicide probe were prepared as described previously(Geurink et al , ).…”

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity

“…We next used activity-based probes, namely a highly reactive propargylamide (Prg) ‘warhead’, which forms a covalent bond with catalytic cysteines, and a 7-amido-4-methylcoumarine (AMC) probe, which emits fluorescence upon cleavage, enabling the monitoring of the kinetics of the protease activity 9 – 13 . SCoV2-PLpro preferentially reacted with the ISG15–Prg probe, but showed weak activity towards K48-linked di-ubiquitin (K48-Ub 2 ) and Nedd8, and no activity towards SUMO-based Prg probes ( Fig.…”

Detecting Active Deconjugating Enzymes with Genetically Encoded Activity-Based Ubiquitin and Ubiquitin-like Protein Probes

“…20 Similarly, Nedd8 and ISG15 probes were designed to cross-link Nedd8 or ISG15 deconjugating enzymes, respectively. 21 Genetically encoded unnatural amino acids (UAAs) carrying reactive moieties have been extensively applied to the study of protein−protein interactions. 22−25 UAAs have been also incorporated into Ubs to identify DUBs or Ub-binding proteins in vitro.…”

“…A UFM1 probe has been applied to profiling UFM1 conjugating and deconjugating enzymes in vitro and in vivo . Similarly, Nedd8 and ISG15 probes were designed to cross-link Nedd8 or ISG15 deconjugating enzymes, respectively …”