The deubiquitylase USP9X controls ribosomal stalling

Abstract

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome.It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

ABPP-HT - High-Throughput Activity-Based Profiling of Deubiquitylating Enzyme Inhibitors in a Cellular Context

“…In this case, a panel of 22 DUBs were quantified (Turnbull et al, 2017). The ABPP assay was used also to assess cellular target engagement and DUB selectivity in crude cell extracts for small molecule inhibitors against USP9X (Clancy et al, 2020), and USP28 (Ruiz et al, 2020).…”

Small molecules as tools for functional assessment of deubiquitinating enzyme function

“…Although the state of DUB inhibitor discovery is relatively young, with only one DUB inhibitor having entered clinical trials, a number of potent and selective DUB inhibitors have recently been described and characterized, which we reviewed (Schauer et al, 2020b). Some of the most promising inhibitors include several small-molecule inhibitors of USP7 (XL188, ALM5, FT671) (Di Gavory et al, 2018;Kategaya et al, 2017;Lamberto et al, 2017;O'Dowd et al, 2018;Reverdy et al, 2012;Turnbull et al, 2017), USP28/25 (AZ1) (Wrigley et al, 2017), and USP9X (FT709) (Clancy et al, 2020). Notably, over a decade ago, development began on inhibitors for the SARS-CoV papainlike protease (PLpro), a protease in the SARS genome that has DUB activity, and subsequently second-generation inhibitors have been described (Baez-Santos et al, 2014;Ghosh et al, 2009Ghosh et al, , 2010Ratia et al, 2008).…”

Structure Activity Relationship of USP5 Allosteric Inhibitors

“…The copyright holder for this preprint (which this version posted May 18, 2021. ; https://doi.org/10.1101/2021.05.17.444542 doi: bioRxiv preprint 4 directed mutagenesis experiments show USP5 processes Met1 and Lys48-linked polyUb chains sequentially from the proximal end of Ub chains 2,31 . The USP domain is highly conserved making it difficult to develop selective catalytic inhibitors; to date, out of 56 human USPs, selective inhibitors have been reported for USP1, USP7, USP9x, USP14 and USP30 [32][33][34][35][36][37][38] . The ZnF-UBD domain is present in a limited number of proteins in the human genome: USP3, USP5, USP13, USP16, USP20, USP22, USP33, USP39, USP44, USP45, USP49, USP51, HDAC6, a histone deacetylase protein, and BRAP, a BRCA1associated protein [39][40][41] .…”