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bioRxiv · 10.1101/2024.09.08.611915

Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases

Vostal, L. E.·Dahan, N. E.·Zhang, W.·Reynolds, M. J.·Chait, B.·Kapoor, T. M.
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Abstract

Errors in proteostasis, which requires regulated degradation and recycling of diverse proteins, are linked to aging, cancer and neurodegenerative disease (1). In particular, recycling proteins from multiprotein complexes, organelles and membranes is initiated by ubiquitylation, extraction and unfolding by the essential mechanoenzyme VCP (2-4), and ubiquitin removal by deubiquitinases (DUBs), a class of [~]100 ubiquitin-specific proteases in humans (5, 6). As VCPs substrate recognition requires ubiquitylation, the removal of ubiquitins from substrates for recycling must follow extraction and unfolding. How the activities of VCP and different DUBs are coordinated for protein recycling or other fates is unclear. Here, we employ a photochemistry-based approach to profile proteome-wide domain-specific VCP interactions in living cells (7). We identify DUBs that bind near the entry, exit, or both sites of VCPs central pore, the channel for ATP-dependent substrate translocation (8-10). From this set of DUBs, we focus on VCPIP1, required for organelle assembly and DNA repair (11-13), that our chemical proteomics workflow indicates binds the central pores entry and exit sites. We determine a [~]3[A] cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer. VCPIP1s UBX-L domain binds VCPs N-domain in a down conformation, linked to VCPs ADP-bound state (2, 14), and the deubiquitinase domain is positioned at the central pores exit site, poised to remove ubiquitin following substrate unfolding. We find that VCP stimulates VCPIP1s DUB activity and use mutagenesis and single-molecule mass photometry assays to test the structural model. Together, our data suggest that DUBs bind VCP at distinct sites and reveal how the two enzyme activities can be coordinated to achieve specific downstream outcomes for ubiquitylated proteins.

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Vostal, L. E., Dahan, N. E., Zhang, W., Reynolds, M. J., Chait, B., Kapoor, T. M.. 2024-09-09. Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases. https://doi.org/10.1101/2024.09.08.611915

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