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Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

ABSTRACT: UBA1 initiates most cellular ubiquitin signaling by activating and transferring ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause a severe inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked autoinflammatory, somatic) syndrome. Despite extensive investigation into the clinical manifestations of this lethal disease, little is known about the underlying molecular mechanisms. Here, we systematically dissect VEXAS-causing mutations in UBA1 to better understand disease pathogenesis. We find that only UBA1 p.Met41 mutations alter cytoplasmic isoform expression, while the remaining mutations reduce catalytic function of both cytoplasmic and nuclear isoforms by diverse mechanisms, including defective ubiquitin adenylation, reduced thioesterification, and aberrant oxyester formation. Strikingly, most non-p.Met41 mutations prominently affect transthioesterification, revealing ubiquitin conjugation to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Yan Wang

PROVIDER: MSV000092914 | MassIVE |

SECONDARY ACCESSION(S): PXD045533

REPOSITORIES: MassIVE

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Project description:Ubiquitination has crucial roles in many cellular processes and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of E2 ubiquitin-conjugating enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, a very promiscuous E2 enzyme that affects a broad range of processes. Here, we set out to identify in vivo targets of UBE2D3 by using SILAC-based quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent and, interestingly, identified pathways related to mRNA translation as top pathways being affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control (RQC), is dependent on UBE2D3. Moreover, we find that UBE2D3 limits monoubiquitination of histone H2B at lysine 120, mediated by the E3 ligase RNF20. This identifies UBE2D3 as a potential regulator of the diverse and critical cellular processes that are controlled by H2B monoubiquitination. Collectively, our findings show that depletion of an E2 ubiquitin-conjugating enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Together with our identification of RQC factors and H2B as novel in vivo targets of UBE2D3, our work provides an important resource for further studies on the in vivo functions of UBE2D3.

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Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

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