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The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding., (© 2024. The Author(s).)

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry., Competing Interests: Competing interests statement:T.M.K. is a co-founder of and has an ownership interest in RADD Pharmaceuticals, Inc.

Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined. To shed light on the structural basis of the ZFAND1-p97 interaction, we determined the atomic structures of the individual domains of ZFAND1 by solution-state NMR spectroscopy and X-ray crystallography. We further characterized the interaction between ZFAND1 and p97 by methyl NMR spectroscopy and cryo-EM. 15 N spin relaxation dynamics analysis indicated independent domain motions for ZF1, ZF2, and UBL. The crystal structure and NMR structure of UBL showed a conserved β-grasp fold homologous to ubiquitin and other UBLs. Nevertheless, the UBL of ZFAND1 contains an additional N-terminal helix that adopts different conformations in the crystalline and solution states. ZFAND1 uses the C-terminal UBL to bind to p97, evidenced by the pronounced line-broadening of the UBL domain during the p97 titration monitored by methyl NMR spectroscopy. ZFAND1 binding induces pronounced conformational heterogeneity in the N-terminal domain of p97, leading to a partial loss of the cryo-EM density of the N-terminal domain of p97. In conclusion, this work paved the way for a better understanding of the interplay between p97 and ZFAND1 in the context of SG clearance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

p97/valosin-containing protein is an essential eukaryotic AAA+ ATPase with diverse functions including protein homeostasis, membrane remodeling, and chromatin regulation. Dysregulation of p97 function causes severe neurodegenerative disease and is associated with cancer, making this protein a significant therapeutic target. p97 extracts polypeptide substrates from macromolecular assemblies by hydrolysis-driven translocation through its central pore. Growing evidence indicates that this activity is highly coordinated by "adapter" partner proteins, of which more than 30 have been identified and are commonly described to facilitate translocation through substrate recruitment or modification. In so doing, these adapters enable critical p97-dependent functions such as extraction of misfolded proteins from the endoplasmic reticulum or mitochondria, and are likely the reason for the extreme functional diversity of p97 relative to other AAA+ translocases. Here, we review the known functions of adapter proteins and highlight recent structural and biochemical advances that have begun to reveal the diverse molecular bases for adapter-mediated regulation of p97 function. These studies suggest that the range of mechanisms by which p97 activity is controlled is vastly underexplored with significant advances possible for understanding p97 regulation by the most known adapters., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The AAA + ATPase p97 is an essential unfoldase/segragase involved in a multitude of cellular processes. It functions as a molecular machine critical for protein homeostasis, homotypic membrane fusion events and organelle biogenesis during mitosis in which it acts in concert with cofactors p47 and p37. Cofactors assist p97 in extracting and unfolding protein substrates through ATP hydrolysis. In contrast to other p97's cofactors, p37 uniquely increases the ATPase activity of p97. Disease-causing mutations in p97, including mutations that cause neurodegenerative diseases, increase cofactor association with its N-domain, ATPase activity and improper substrate processing. Upregulation of p97 has also been observed in various cancers. This study aims towards the characterization of the protein-protein interaction between p97 and p37 at the atomic level. We defined the interacting residues in p97 and p37. The knowledge will facilitate the design of unique small molecules inhibiting this interaction with insights into cancer therapy and drug design., Results: The homology model of human p37 UBX domain was built from the X-ray crystal structure of p47 C-terminus from rat (PDB code:1S3S, G) as a template and assessed by model validation analysis. According to the HDOCK, HAWKDOCK, MM-GBSA binding free energy calculations and Arpeggio, we found that there are several hydrophobic and two hydrogen-bonding interactions between p37 UBX and p97 N-D1 domain. Residues of p37 UBX predicted to be involved in the interactions with p97 N-D1 domain interface are highly conserved among UBX cofactors., Conclusion: This study provides a reliable structural insight into the p37-p97 complex binding sites at the atomic level though molecular docking coupled with molecular dynamics simulation. This can guide the rational design of small molecule drugs for inhibiting mutant p97 activity., (© 2022. The Author(s).)

The protein unfoldase Cdc48/p97 targets a wide variety of cellular substrates, but the molecular basis of substrate turnover remains incompletely understood. Two recent reports, by Ji et al. and van den Boom et al., provide detailed insights into the unfolding process and reveal pronounced flexibility of substrate handling by Cdc48/p97., Competing Interests: Declaration of interests None declared by author., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise β-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97 R155H , p97 R191Q , p97 A232E , p97 D592N , as well as p97 E470D , implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97 R155H and p97 R191Q ), (2) the interprotomer interactions (p97 A232E ), or (3) the coupling between D1 and D2 nucleotide domains (p97 D592N , p97 E470D ). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function., Competing Interests: Conflict of interest S. S. is Founder and CEO of Gandeeva Therapeutics Inc, a drug discovery company based in Vancouver. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Viruses are obligate intracellular parasites that are dependent on host factors for their replication. One such host protein, p97 or the valosin-containing protein (VCP), is a highly conserved AAA ATPase that facilitates replication of diverse RNA- and DNA-containing viruses. The wide range of cellular functions attributed to this ATPase is consistent with its participation in multiple steps of the virus life cycle from entry and uncoating to viral egress. Studies of VCP/p97 interactions with viruses will provide important information about host processes and cell biology, but also viral strategies that take advantage of these host functions. The critical role of p97 in viral replication might be exploited as a target for development of pan-antiviral drugs that exceed the capability of virus-specific vaccines or therapeutics.

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Å away., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCF Dia2 . This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48., Competing Interests: TD, PM, RF, CP, KL No competing interests declared, (© 2020, Deegan et al.)

Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.

The AAA+ ATPase, p97, also referred to as VCP, plays an essential role in cellular homeostasis by regulating endoplasmic reticulum-associated degradation (ERAD), mitochondrial-associated degradation (MAD), chromatin-associated degradation, autophagy, and endosomal trafficking. Mutations in p97 have been linked to a number of neurodegenerative diseases, and overexpression of wild type p97 is observed in numerous cancers. Furthermore, p97 activity has been shown to be essential for the replication of certain viruses, including poliovirus, herpes simplex virus (HSV), cytomegalovirus (CMV), and influenza. Taken together, these observations highlight the potential for targeting p97 as a therapeutic approach in neurodegeneration, cancer, and certain infectious diseases. This Perspective reviews recent advances in the discovery of small molecule inhibitors of p97, their optimization and characterization, and therapeutic potential.

Several pathologies have been associated with the AAA+ ATPase p97, an enzyme essential to protein homeostasis. Heterozygous polymorphisms in p97 have been shown to cause neurological disease, while elevated proteotoxic stress in tumours has made p97 an attractive cancer chemotherapy target. The cellular processes reliant on p97 are well described. High-resolution structural models of its catalytic D2 domain, however, have proved elusive, as has the mechanism by which p97 converts the energy from ATP hydrolysis into mechanical force to unfold protein substrates. Here, we describe the high-resolution structure of the p97 D2 ATPase domain. This crystal system constitutes a valuable tool for p97 inhibitor development and identifies a potentially druggable pocket in the D2 domain. In addition, its P6 1 symmetry suggests a mechanism for substrate unfolding by p97. DATABASE: The atomic coordinates and structure factors have been deposited in the PDB database under the accession numbers 6G2V, 6G2W, 6G2X, 6G2Y, 6G2Z and 6G30., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide: N -glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing.

Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. Despite the identification of numerous pathways that are perturbed in MSP, the molecular-level defects of these p97 mutants are not completely understood. Here, we use biochemistry and cryoelectron microscopy to explore the effects of MSP mutations on the unfoldase activity of p97 in complex with its substrate adaptor NPLOC4⋅UFD1L (UN). We show that all seven analyzed MSP mutants unfold substrates faster. Mutant homo- and heterohexamers exhibit tighter UN binding and faster substrate processing. Our structural studies suggest that the increased UN affinity originates from a decoupling of p97's nucleotide state and the positioning of its N-terminal domains. Together, our data support a gain-of-function model for p97-UN-dependent processes in MSP and underscore the importance of N-terminal domain movements for adaptor recruitment and substrate processing by p97., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mitofusins are dynamin-related GTPases that drive mitochondrial fusion by sequential events of oligomerization and GTP hydrolysis, followed by their ubiquitylation. Here, we show that fusion requires a trilateral salt bridge at a hinge point of the yeast mitofusin Fzo1, alternatingly forming before and after GTP hydrolysis. Mutations causative of Charcot-Marie-Tooth disease massively map to this hinge point site, underlining the disease relevance of the trilateral salt bridge. A triple charge swap rescues the activity of Fzo1, emphasizing the close coordination of the hinge residues with GTP hydrolysis. Subsequently, ubiquitylation of Fzo1 allows the AAA-ATPase ubiquitin-chaperone Cdc48 to resolve Fzo1 clusters, releasing the dynamin for the next fusion round. Furthermore, cross-complementation within the oligomer unexpectedly revealed ubiquitylated but fusion-incompetent Fzo1 intermediates. However, Cdc48 did not affect the ubiquitylated but fusion-incompetent variants, indicating that Fzo1 ubiquitylation is only controlled after membrane merging. Together, we present an integrated model on how mitochondrial outer membranes fuse, a critical process for their respiratory function but also putatively relevant for therapeutic interventions., (© 2019 Anton et al.)

The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo-electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.7-angstrom-resolution structure of Cdc48 in complex with an adaptor protein and a native substrate. Cdc48 engages substrate by adopting a helical configuration of substrate-binding residues that extends through the central pore of both of the ATPase rings. These findings indicate a unified hand-over-hand mechanism of protein translocation by Cdc48 and other AAA+ ATPases., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Carcinoma-associated fibroblasts are fibroblasts activated by surrounding cancer cells. Carcinoma-associated fibroblasts exhibit enhanced cell migration, which plays an important role in cancer metastasis. Previously, we demonstrated enhanced migration of NIH3T3 fibroblasts when they were cultured in the presence of MCF7 breast cancer cells. Human fibroblasts displayed a similar phenomenon even when they were co-cultured with cancer cells other than MCF7 cells. In this study, we screened ∼16,000 compounds from the RIKEN Natural Products Depository chemical library for inhibitors of enhanced NIH3T3 cell migration in the presence of MCF7. We identified NPD8733 as an inhibitor of cancer cell-enhanced fibroblast migration. This inhibition was observed not only in a wound-healing co-culture assay but also in a Transwell migration assay. Using NPD8733 and a structurally similar but inactive derivative, NPD8126, on immobilized beads, we found that NPD8733, but not NPD8126, specifically binds to valosin-containing protein (VCP)/p97, a member of the ATPase-associated with diverse cellular activities (AAA+) protein family. Using VCP truncation variants, we found that NPD8733 binds to the D1 domain of VCP. Because VCP's D1 domain is important for its function, we concluded that NPD8733 may act on VCP by binding to this domain. siRNA-mediated silencing of VCP in NIH3T3 fibroblasts, but not in MCF7 cells, reduced the migration of the co-cultured NIH3T3 fibroblasts. These results indicate that MCF7 activates the migration of NIH3T3 cells through VCP and that NPD8733 binds VCP and thereby inhibits its activity., (© 2019 Suvarna et al.)

Cdc48/p97 is an essential and highly conserved AAA+ ATPase that uses its protein-unfoldase activity to extract ubiquitinated polypeptides from macromolecular complexes and membranes. This motor has also been implicated in protein-degradation pathways, yet its exact role in acting upstream of the 26S proteasome remains elusive. Ubiquitinated proteins destined for degradation by the proteasome require an unstructured initiation region to engage with the proteasomal translocation machinery, and Cdc48 was proposed to generate these unfolded segments, yet direct evidence has been missing. Here, we used an in vitro reconstituted system to demonstrate the collaboration of Cdc48 and the 26S proteasome from S . cerevisiae in degrading ubiquitinated, well-folded proteins that lack unstructured segments. Our data indicate that a critical role for Cdc48 in the ubiquitin-proteasome system is to create flexible initiation regions in compact substrates that otherwise would be refractory to engagement and degradation by the proteasome., Competing Interests: The authors declare no competing interests.

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding., Competing Interests: The authors declare no conflict of interest.

Intracellular protein aggregation is characterized by accumulation of misfolded proteins. Chaperones, degradation machineries, and quality-control mechanisms counteract protein aggregation. In this study, we report that the ATPase valosin-containing protein (VCP/p97) acts as a functional disaggregase that disassembles Huntingtin-exon1 aggregates in vitro and in HeLa cells. The N-terminal part of VCP (Cdc48_N domain) interacts with the N-terminal 17-amino acid region of Huntingtin-exon1. We show that VCP has properties of a disaggregase, since it is capable of reducing preformed protein aggregates and displays increased ATPase activity in the presence of protein aggregates. However, VCP shows high divergence/disparity from other disaggregases. Taken together, our studies show the novel function of VCP/p97 as a disaggregase which detangles protein aggregates to probably channelize their degradation., (© 2018 Federation of European Biochemical Societies.)

Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1-Npl4 heterodimer. Here, we report structures of the Cdc48-Ufd1-Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn 2+ -finger domains to anchor the enzymatically inactive Mpr1-Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48's central pore, a position similar to the MPN domain from deubiquitinase Rpn11 in the proteasome. Our results indicate that Npl4 is unique among Cdc48 cofactors and suggest a mechanism for binding and translocation of polyubiquitinated substrates into the ATPase.

A balance between protein synthesis and degradation is necessary to maintain cellular homeostasis. Failure to triage aberrant proteins may result in their accumulation and aggregation in the cytosol. The valosin-containing protein (VCP)-BCL2-associated athanogene 6 (BAG6) complex facilitates a wide variety of ubiquitin-mediated quality control events at the endoplasmic reticulum (ER), both prior to ER translocation and during ER-associated degradation (ERAD). However, how ubiquitylated clients associated with BAG6 are recognized by VCP for proteasomal degradation is presently unknown. We have identified UBXN1 as the VCP adaptor in BAG6-dependent processes occurring prior to ER insertion but not during ERAD. The loss of VCP-UBXN1 results in the inappropriate stabilization of ubiquitylated BAG6 clients and their accumulation in insoluble aggregates and sensitizes cells to proteotoxic stress. Our results identify how VCP is specifically targeted to ubiquitylated substrates in the BAG6 triage pathway and suggest that the degradation of ubiquitylated clients by the proteasome is reliant on the association of UBXN1 with ubiquitylated substrates and the catalytic activity of VCP., (Copyright © 2018 American Society for Microbiology.)

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments., Competing Interests: The authors declare no conflict of interest.

p97 is an essential ATPase associated with various cellular activities (AAA + ) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum-associated degradation. However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Yeast Cdc48 is a well-conserved, essential chaperone of ATPases associated with diverse cellular activity (AAA) proteins, which recognizes substrate proteins and modulates their conformations to carry out many cellular processes. However, the fundamental mechanisms underlying the diverse pivotal roles of Cdc48 remain unknown. Almost all AAA proteins form a ring-shaped structure with a conserved aromatic amino acid residue that is essential for proper function. The threading mechanism hypothesis suggests that this residue guides the intrusion of substrate proteins into a narrow pore of the AAA ring, thereby becoming unfolded. By contrast, the aromatic residue in one of the two AAA rings of Cdc48 has been eliminated through evolution. Here, we show that artificial retrieval of this aromatic residue in Cdc48 is lethal, and essential features to support the threading mechanism are required to exhibit the lethal phenotype. In particular, genetic and biochemical analyses of the Cdc48 lethal mutant strongly suggested that when in complex with the 20S proteasome, essential proteins are abnormally forced to thread through the Cdc48 pore to become degraded, which was not detected in wild-type Cdc48. Thus, the widely applicable threading model is less effective for wild-type Cdc48; rather, Cdc48 might function predominantly through an as-yet-undetermined mechanism.

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable., Competing Interests: Conflict of interest statement: R.J.D. is a founder and a shareholder of Cleave Biosciences, which is developing CB-5083 for therapy of cancer. The other authors declare that no competing interests exist.

Valosin-containing protein (VCP/p97) ATPase (a.k.a. Cdc48) is a key member of the ER-associated protein degradation (ERAD) pathway. ERAD and VCP/p97 have been implicated in a multitude of human diseases, such as neurodegenerative diseases and cancer. Inhibition of VCP/p97 induces proteotoxic ER stress and cell death in cancer cells, making it an attractive target for cancer treatment. However, no drugs exist against this protein in the market. Repositioning of drugs towards new indications is an attractive alternative to the de novo drug development due to the potential for significantly shorter time to clinical translation. Here, we employed an integrative strategy for the repositioning of drugs as novel inhibitors of the VCP/p97 ATPase. We integrated structure-based virtual screening with the chemical genomics analysis of drug molecular signatures, and identified several candidate inhibitors of VCP/p97 ATPase. Importantly, experimental validation with cell-based and in vitro ATPase assays confirmed three (ebastine, astemizole and clotrimazole) out of seven tested candidates (~40% true hit rate) as direct inhibitors of VCP/p97 and ERAD. This study introduces an effective integrative strategy for drug repositioning, and identified new drugs against the VCP/p97/ERAD pathway in human diseases.

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.