The UBX domain in UBXD1 organizes ubiquitin binding at the C-terminus of the VCP/p97 AAA-ATPase.

The AAA+ ATPase p97/VCP together with different sets of substrate-delivery adapters and accessory cofactor proteins unfolds ubiquitinated substrates to facilitate degradation by the proteasome. The UBXD1 cofactor is connected to p97-associated multisystem proteinopathy but its biochemical function and structural organization on p97 has remained largely elusive. Using a combination of crosslinking mass spectrometry and biochemical assays, we identify an extended UBX (eUBX) module in UBXD1 related to a lariat in another cofactor, ASPL. Of note, the UBXD1-eUBX intramolecularly associates with the PUB domain in UBXD1 close to the substrate exit pore of p97. The UBXD1 PUB domain can also bind the proteasomal shuttling factor HR23b via its UBL domain. We further show that the eUBX domain has ubiquitin binding activity and that UBXD1 associates with an active p97-adapter complex during substrate unfolding. Our findings suggest that the UBXD1-eUBX module receives unfolded ubiquitinated substrates after they exit the p97 channel and before hand-over to the proteasome. The interplay of full-length UBXD1 and HR23b and their function in the context of an active p97:UBXD1 unfolding complex remains to be studied in future work.

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.

Protein Phosphatase-1 Complex Disassembly by p97 is Initiated through Multivalent Recognition of Catalytic and Regulatory Subunits by the p97 SEP-domain Adapters.

The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.

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.

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.