Kelsall, I. R. | bioRxiv Science

bioRxiv2026

FAF1 and FAF2 enhance human p97-UFD1-NPL4 complex unfoldase activity enabling rational design of p97 activators

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), binds and unfolds ubiquitylated substrates to maintain cellular homeostasis. The human p97-UN complex associates with additional cofactors, but how these cofactors modulate p97-UN activity is not fully understood. Here, we screen for cofactors that enhance p97-UN activity and identify FAF2 as the strongest activator. Using biochemical and structural approaches, we show how FAF2 engages p97-UN and polyubiquitin to promote unfolding. We define a conserved activation motif in FAF2 that contacts both UFD1 and the ubiquitin proximal to the initiator, stabilizing and initiating unfolding in a UFD1-dependent manner. We leverage the features of FAF2 AM to engineer de novo proteins that potently enhance unfolding, providing a rational strategy to boost p97 activity. Our findings reveal how cofactors can provide additional adaptive control, fine-tuning human p97 activity to unfold challenging substrates and those modified with short ubiquitin chains.

biochemistry↗

bioRxiv2021

HOIL-1-catalysed ubiquitylation of unbranched glucosaccharides and its activation by ubiquitin oligomers

HOIL-1, a component of the Linear Ubiquitin Assembly Complex (LUBAC), ubiquitylates serine and threonine residues in proteins, forming ester bonds (Kelsall et al, 2019, PNAS 116, 13293-13298). Here we report that mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, cardiac muscle and other organs, indicating that HOIL-1s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and 1:4-linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The HOIL-1-catalysed monoubiquitylation of maltoheptaose was accelerated >100-fold by Met1-linked or Lys63-linked ubiquitin oligomers, which interact with the catalytic RBR domain of HOIL-1. HOIL-1 also transferred preformed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to amylose resin in vitro, suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal by glycophagy to prevent precipitation as polyglucosan.

biochemistry↗