Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN.

The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy.

Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly.

The AAA-ATPase VCP/p97/Cdc48 unfolds proteins by threading them through its central pore, but how substrates are recognized and inserted into the pore in diverse pathways has remained controversial. Here, we show that p97, with its adapter p37, binds an internal recognition site (IRS) within inhibitor-3 (I3) and then threads a peptide loop into its channel to strip I3 off protein phosphatase-1 (PP1). Of note, the IRS is adjacent to the prime interaction site of I3 to PP1, and IRS mutations block I3 processing both in vitro and in cells. In contrast, amino- and carboxy-terminal regions of I3 are not required, and even circularization of I3 does not prevent I3 processing. This was confirmed by an in vitro Förster resonance energy transfer assay that allowed kinetic analysis of the reaction. Thus, our data uncover how PP1 is released from its inhibitory partner for activation and demonstrate a remarkable plasticity in substrate threading by p97.

Insights into ubiquitin chain architecture using Ub-clipping.

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies.

In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.