Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

Structural basis of ubiquitin-independent PP1 complex disassembly by p97.

The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.

Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases.

Protein phosphatase-1 catalytic subunit (PP1) joins diverse targeting subunits to form holophosphatases that regulate many cellular processes. Newly synthesized PP1 is known to be transiently sequestered in an inhibitory complex with Suppressor-of-Dis2-number-2 (SDS22) and Inhibitor-3 (I3), which is disassembled by the ATPases Associated with diverse cellular Activities plus (AAA+) protein p97. Here, we show that the SDS22-PP1-I3 complex also acts as a thermodynamic sink for mature PP1 and that cycles of SDS22-PP1-I3 formation and p97-driven disassembly regulate PP1 function and subunit exchange beyond PP1 biogenesis. Förster Resonance energy transfer (FRET) analysis of labeled proteins in vitro revealed that in the p97-mediated disassembly step, both SDS22 and I3 dissociate concomitantly, releasing PP1. In presence of a targeting subunit, for instance Growth Arrest and DNA Damage-inducible protein 34 (GADD34), liberated PP1 formed an active holophosphatase that dephosphorylated its substrate, eukaryotic translation initiation factor 2 alpha (eIF2α). Inhibition of p97 results in displacement of the GADD34 targeting subunit by rebinding of PP1 to SDS22 and I3 indicating that the SDS22-PP1-I3 complex is thermodynamically favored. Likewise, p97 inhibition in cells causes rapid sequestration of PP1 by free SDS22 and I3 at the expense of other subunits. This suggests that PP1 exists in a steady state maintained by spontaneous SDS22-PP1-I3 formation and adenosine triphosphate (ATP) hydrolysis, p97-driven disassembly that recycles active PP1 between different holophosphatase complexes to warrant a dynamic holophosphatase landscape.

VCP/p97-Mediated Unfolding as a Principle in Protein Homeostasis and Signaling.

The AAA+-type ATPase p97 governs an ever-expanding number of cellular processes reaching from degradation of damaged proteins and organelles to key signaling events and chromatin regulation with thousands of client proteins. With its relevance for cellular homeostasis and genome stability, it is linked to muscular and neuronal degeneration and, conversely, constitutes an attractive anti-cancer drug target. Its molecular function is ATP-driven protein unfolding, which is directed by ubiquitin and assisted by a host of cofactor proteins. This activity underlies p97's diverse ability to pull proteins out of membranes, unfold proteins for proteasomal degradation, or segregate proteins from partners for downstream activity. Recent advances in structural analysis and biochemical reconstitution have underscored this notion, resolved detailed molecular motions within the p97 hexamer, and suggested substrate threading through the central channel of the p97 hexamer as the driving mechanism. We will discuss the mechanisms and open questions in the context of the diverse cellular activities.

Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.

VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair.

During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

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.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.

A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases.

Peptidyl succinimidyl peptides as taspase 1 inhibitors.

Taspase 1 is an N-terminal threonine protease implicated in leukemia and other cancers. Despite intensive efforts in recent years, only a limited number of Taspase 1 inhibitors are currently available, and they lack general applicability. Here we present a novel class of Taspase 1 inhibitors based on a peptidyl succinimidyl peptide motif. These inhibitors were obtained from the substrate cleavage sequence and mechanistic considerations involving the previously proposed asparaginase-type cleavage mechanism. We anticipate that this class of Taspase 1 inhibitor will find wide application in further biochemical and structural studies, for example for better investigating the molecular details of the unusual enzymatic cleavage mechanism of Taspase 1.