Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

Abstract

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing’s disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645–684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing’s disease due to USP8 mutations.

Genetic Basis of ACTH-Secreting Adenomas

“…The USP8 protein is constituted by several domains including the catalytic USP domain, which has deubiquitylating activity and can be activated through cleavage. USP8 also contains a WW-like autoinhibitory domain, which interacts with the catalytic domain, impeding its bonding with ubiquitylated proteins, and avoids the activating cleavage of the protein [ 7 ]. USP8 is modulated through the binding of 14-3-3 proteins with the WW-like domain, which stabilize it in an inactive conformation [ 7 ].…”

“…Most of the reported mutations in ACTH-secreting adenomas alter the amino acid sequence in the 14-3-3 binding site and disrupt the USP8 and 14-3-3 bond, thus un-inhibiting USP8 activity because of increased cleavage of the catalytic domain [ 9 ] and/or increased spontaneous enzymatic activity even without such cleavage [ 7 ]. Other mutations also reduce 14-3-3 binding, not because of altered sequences in the bonding region but because of conformational abnormalities [ 10 ].…”

Molecular mechanisms involved in somatostatin receptor regulation in corticotroph tumors: the role of cytoskeleton and USP8 mutations

“…Gain-of-function somatic mutations in the USP8 gene have been identified in 11–60% of patients with ACTH-secreting PitNETs ( Ma et al 2015 , Perez-Rivas et al 2015 , Reincke et al 2015 , Hayashi et al 2016 , Albani et al 2018 , Losa et al 2019 , Castellnou et al 2020 , Sesta et al 2020 , Treppiedi et al 2021 b ). They are missense variants or small single codon deletions that mostly occur within or in proximity to the consensus 14-3-3 binding motif RSYSSP at amino acid positions 715–720 of USP8 ( Centorrino et al 2018 , Dufner & Knobeloch 2019 ), resulting in compromised 14-3-3 protein binding or more rarely in the autoinhibitory domain of USP8 ( Kakihara et al 2021 , Treppiedi et al 2021 b ), leading in both cases to constitutively enhanced deubiquitinase activity. As a consequence, the EGF receptor (EGFR), that is a well-studied target of USP8, undergoes an increased deubiquitination and recycling on the plasma membrane, and its sustained signaling contributes to cell proliferation, POMC transcription, and ACTH secretion in USP8-mutated tumoral corticotrophs cells ( Ma et al 2015 , Reincke et al 2015 , Treppiedi et al 2021 b ).…”

Ubiquitin-specific protease 8 controls B-cell proteostasis and survival representing a target in multiple myeloma

“…Interactions with components of endosomal sorting complexes required for transport (ESCRT), such as STAM2 and charged multivesicular body (CHMP) proteins, have established the regulation of endosomal sorting and the control of transmembrane protein stability as canonical USP8 functions 3 . The C-terminal part of USP8 harbors the catalytic domain, which is inhibited by phosphorylation-dependent 14-3-3 binding to the N-terminus 4,5 . Somatic mutations in the exon encoding the 14-3-3 binding motif of USP8 are associated with Cushing’s disease (CD) leading to the stabilization of the EGFR in ACTH-secreting pituitary adenomas 6,7 .…”