Proximity proteomics reveals OTUD6B regulation of stress granule dynamics through coalescence with VCP/p97

Stress granules (SGs) are membrane-less organelles formed through liquid-liquid phase separation of proteins and RNAs, serving as temporary repositories for biomacromolecules to protect cells under stress conditions. Impaired SG disassembly is closely implicated in neurodegenerative diseases and aging, yet the mechanisms regulating SG dynamics are incompletely investigated. The constituents of heterogenous SGs are complicated and broadly categorized as core and shell components. In contrary to the relatively stable core components, our understanding of the diverse SG shell is deficient. By combining interactomic and proximity proteomic approaches, we reveal that the deubiquitinating enzyme OTUD6B is associated with SG-related functions. Immunofluorescence assays showed that OTUD6B localized to SGs, as well as regulated their early assembly and clearance, partially dependent on its enzymatic activity. Further proximity proteomics and interactomics results uncover the ATPase VCP/p97, a key SG disassembly factor, as an OTUD6B-associated protein. OTUD6B and VCP association is governed through their disordered regions normally participated in biomolecular condensation. VCP knockdown or pharmacological inhibition phenocopied OTUD6B silencing by leading to defects in SG dynamics. Mechanistically, SG coalescence of VCP incurred by OTUD6B in a partially enzymatic activity-dependent manner functions to accelerate not only the early assembly, but also SG clearance following stress removal. Therefore, our findings establish OTUD6B as a critical modulator of SG dynamics, linking its function to stress responses and potential disease mechanisms.