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Variety in the USP deubiquitinase catalytic mechanism.

ABSTRACT: The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical residue) is directly adjacent to this third critical residue. Although both critical residues accommodate catalysis in USP2, these residues have not been comprehensively investigated in other USPs. Here, we quantitatively investigate their roles in five USPs. Although USP7 relies on the third critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40, and USP48, where the fourth critical residue is vital instead. Furthermore, these residues vary in importance for nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. This unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs.

SUBMITTER: Keijzer N

PROVIDER: S-EPMC10867860 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Variety in the USP deubiquitinase catalytic mechanism.

Keijzer Niels N Priyanka Anu A Stijf-Bultsma Yvette Y Fish Alexander A Gersch Malte M Sixma Titia K TK

Life science alliance 20240214 4

The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical ...[more]

PMID: 38355287

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Project description:AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to the lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery. First, we performed mutational and kinetic analysis of the putative residues involved in diubiquitin recognition and catalysis with a view of better understanding the catalytic mechanism of AMSH. Our mutational and kinetic analysis reveals that recognition of the proximal ubiquitin is imperative for the linkage specificity and catalytic efficiency of the enzyme. The MIC-CAP disease mutation, Thr313Ile, yields a substantial loss of catalytic activity without any significant change in the thermodynamic stability of the protein, indicating that its perturbed catalytic activity is the basis of the disease. The catalytic activity of AMSH is stimulated upon binding to the ESCRT-0 member STAM; however, the precise mechanism and its significance are not known. On the basis of a number of biochemical and biophysical analyses, we are able to propose a model for activation according to which activation of AMSH is allowed by facile, simultaneous binding to two ubiquitin groups in a polyubiquitin substrate, one by the catalytic domain of the DUB (binding to the distal ubiquitin) and the other (the proximal ubiquitin) by the ubiquitin interacting motif (UIM) from STAM. Such a mode of binding would stabilize the ubiquitin chain in a productive orientation, resulting in an enhancement of the activity of the enzyme. These data together provide a mechanism for understanding the recruitment and activation of AMSH at ESCRT-0, providing biochemical and biophysical evidence that supports a role for AMSH when it is recruited to the initial ESCRT complex: it functions to facilitate the transfer of ubiquitinated receptors (cargo) from one ESCRT member to the next by disassembling the polyubiquitin chain while leaving some ubiquitin groups still attached to the cargo.

Dataset Information

Variety in the USP deubiquitinase catalytic mechanism.

Publications

Variety in the USP deubiquitinase catalytic mechanism.

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