Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights

Abstract

The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology.

Keywords: E1; E3; activity-based probe; activity-based protein profiling; chemoproteomics; deubiquitinase; protease; ubiquitin; ubiquitin-proteasome system.

Figure 1

Ubiquitin conjugation and deconjugation machinery. (A) X‐ray crystal structure of human ubiquitin (PDB: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1UBI). Lysine side chains are shown in magenta, N‐terminal methionine (M1) shown in cyan, and C‐terminal di‐Gly shown in blue; (B) overview of enzymes involved in Ub conjugation and deconjugation, with catalytic Cys residues indicated; (C) the ‘three Rs’ of an activity‐based probe (ABP)—reactive group, recognition element, and reporter tag—and the reaction of an ABP with an enzyme containing an active site Cys residue, such as a Cys protease.

Figure 2

(A) DUB probe designs. Almost all DUB ABPs contain one (designs i and ii) or two (designs iii and iv) full‐length Ub proteins, with an electrophile (E) positioned at the site of the scissile bond. The reporter tag is typically at the N terminus (designs i, iii, and iv) but may be located at the C terminus, attached to the electrophile (design ii); (B) reaction between a DUB active site Cys and probe electrophiles. Mechanistically, the reactions can be classified as direct addition (e.g., Ub‐PA), conjugate addition (e.g., Ub‐VS), or nucleophilic displacement (e.g., Ub‐Br2).

Figure 3

Deubiquitinases specificity. Ub‐binding sites in the DUB can convey specificity for: a particular linkage type such as K63‐ or K48‐linked chains (A); or position within the Ub chain (B) 18.

Figure 4

Activity‐based probes for the Ub conjugation machinery. (A) E1 probes that label the enzyme active site (Ubl~AMP shown for comparison): Ubl‐AVSN resembles Ubl~AMP but reacts with E1 to form a covalent mimic of the E1:Ubl~AMP complex; Ubl‐AMSN is an unreactive Ubl~AMP analog 107, 108; Ub‐Probe3 reacts in a similar way to Ubl‐AVSN, but incorporates an alkyne tag for detection 109; (B) an E1 ABP, ABP1 41, which labels Ub, and the NEDD8 E1 inhibitor MLN4924 (pevonedistat) which shares a similar structure and mechanism; (C) transthiolation probes for the E1‐E2 reaction 114 and the E2‐E3 reaction 115; (D) ‘cascading’ ABP Ub‐Dha, which can label E1, E2, and some E3 enzymes 61.