Valosin-containing protein (VCP/p97) is capable of unfolding polyubiquitinated proteins through its ATPase domains

Abstract

Valosin-containing protein (VCP or p97) is required for the proteasomal degradation of polyubiquitinated proteins. However, the molecular mechanism for VCP to process the polyubiquitinated proteins remains unclear. Here, we show that VCP can unfold polyubiquitinated proteins. It preferably unfolds the pentaubiquitin- over monoubiquin-conjugated dihydrofolate reductase (Ub5-DHFR or Ub-DHFR) in a dose dependent manner. In addition, the unfolding activity of VCP does not depend on its ATPase activity, on the contrary, ATP and its non-hydrolysable analogs suppress the unfolding of Ub5-DHFR. The structural and functional analysis showed that either D1 or D2 domain of VCP is sufficient to carry out this unfolding activity. The structure of the substrates also affects its unfolding by VCP. VCP is unable to unfold Ub5-DHFR in a tight structure when it binds with methotrexate, a folate analog with high affinity to DHFR. Thus, these results support that VCP is capable of unfolding polyubiquitinated proteins and suggest that VCP may facilitate the proteasomal degradation of polyubiquitinated proteins through its unfolding activity.

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L1 is enhanced by a mutation that causes multisystem proteinopathy

“…The final requirement for unfolding of GFP is ATP hydrolysis by the D2 ATPase of p97. Our results are in direct contrast with a previous study which showed that p97-dependent unfolding is inhibited by ATP (94), but are consistent with overwhelming in vitro and in vivo data (1, 23, 25, 34). Our experiments with p97 mutants and the D2-specific ATP-competitive inhibitor CB-5083 demonstrate the importance of D2 ATPase activity over that of D1, which confirms prior observations (19–25).…”

Pharmacologically targeting a novel pathway of sodium iodide symporter trafficking to enhance radioiodine uptake

“…Additionally, ATP-competitive VCP inhibitors N2,N4-dibenzylquinazoline-2,4diamine (DBeQ) (51,52) and sorafenib, which inhibits VCP activity at the PM by blocking its phosphorylation (53), failed to increase radioiodine uptake in NIS+ve cells ( Figure S7, C and D). Collectively, these results suggest that VCP functions in an ATPase-independent manner to affect NIS function, a facet which has been reported in VCP's ability to unfold proteins for subsequent proteasomal degradation (54).…”

p97/VCP promotes Cullin‐RING‐ubiquitin‐ligase/proteasome‐dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα

“…DNA or protein complexes) or membranes , its unfoldase/remodelling and Ub‐chain editing activities could be involved as well, both of which might facilitate capture and uptake of IκBα‐Ub by the proteasome . p97/VCP unfoldase/remodelling activity might depend or not on ATP hydrolysis and be driven by either the chaperone alone or in synergy with associated 26S proteasome . Furthermore, p97/VCP might shuttle IκBα‐Ub to either downstream Ub‐binding proteins or the proteasome .…”

“…B), and that NMS‐873 supposedly inhibits (potentially/presumably) p97/VCP ATPase‐independent activities ( e.g . unfoldase and Ub‐chain editing activities or mechanical coupling to the proteasome ) besides (ongoing) ATP hydrolysis alike , suggests a combination of activities to be at play in the turnover of IκBα‐Ub. A contributive role of p97/VCP ATPase activity has, however, been demonstrated through overexpression of an ATPase‐deficient mutant .…”