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

Abstract

Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now, no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo. Using these Ub probes combined with mass spectrometry, we have successfully identified endogenous deconjugating enzymes in live cells. We believe that these genetically encoded Ub/Ubl probes are valuable for investigating biological functions of deconjugating enzymes in physiological environments.

Biospecific Chemistry for Covalent Linking of Biomacromolecules

“…The compatibility of GECX with pre-prepared samples can be valuable for studying cells or tissues isolated from animals or patients. In a different study, BprY was incorporated into ubiquitin and ubiquitin-like proteins, which function as activity-based protein probes and cross-link with deconjugating enzymes both in vitro and in vivo . Additionally, upon expressing BprY-modified ubiquitin in HEK293T cells, quantitative MS analysis identified 57 deubiquitinating enzymes and a substantial array of ubiquitin-interacting proteins.…”

Genetically encoded crosslinkers to address protein–protein interactions

“…Besides proof‐of‐principle examples of application, ncAAs for GECX have potential to facilitate studies in different research fields, from the study of protein–protein, protein–RNA, and protein–glycan interactions to the development of covalent protein drugs. Applications of ncAA for GECX include the generation of intermolecular bridges in proteins, either enhancing protein stability (Coin, 2018 ; Xu et al, 2022 ) or allowing conformational control (Coin, 2018 ), as well as the stabilization of protein–protein interactions for structural (Cigler et al, 2017 ; Du et al, 2021 ) or functional studies (Cigler et al, 2017 ; Du et al, 2021 ; Shu et al, 2023 ). For instance, small G proteins of the Rab family were equipped with BrC6K ( 12a ) to capture different domains of the enzyme DrrA from the pathogen Legionella pneumophila .…”

“…For instance, BrEtY replacing different phosphorylated Tyr residues of HER2 captured protein tyrosine phosphatases responsible for the dephosphorylation of each Tyr residue, some of which were previously unknown (Figure 4c ; Tang et al, 2018 ). In a recent work, BrPrY ( 11a ) was incorporated into ubiquitin and ubiquitin‐like proteins to generate molecular probes able to capture deubiquitinating and deconjugating enzymes both in vitro and in vivo (Shu et al, 2023 ).…”