| 2021 |
The Cdc48/p97 ATPase complex with its cofactor Ufd1/Npl4 (UN) recognizes polyubiquitin chains (rather than the substrate directly), cooperatively binds the chain, unfolds one ubiquitin molecule (initiator), then pulls all ubiquitins linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding; when the ATPase reaches the isopeptide bond, it translocates and unfolds both N- and C-terminal substrate segments, while ubiquitins linked to the branchpoint dissociate from UN and move outside the pore, releasing unfolded polyubiquitinated substrate. |
In vitro reconstitution with purified yeast components, hydrogen–deuterium exchange MS, biochemical translocation assays, mutagenesis |
Molecular cell |
High |
34951965
|
| 2024 |
Bidirectional substrate shuttling between the 26S proteasome and the Cdc48-Ufd1/Npl4 ATPase complex promotes degradation of well-folded substrates. A minimal reconstituted system requires the 26S proteasome, Cdc48-UN complex, proteasome cofactor Rad23, and Cdc48 cofactors Ubx5 and Shp1: Rad23 and Ubx5 stimulate polyubiquitin binding to the proteasome and Cdc48-UN respectively, allowing competition for substrates; Shp1 stimulates protein unfolding by Cdc48-UN rather than substrate recruitment. |
In vitro reconstitution with purified yeast components, biochemical degradation assays, yeast genetics confirmation |
Molecular cell |
High |
38401542
|
| 2022 |
SUMO modification enhances substrate unfolding by the Ufd1/Npl4/Cdc48 complex: interactions between Ufd1 and SUMO accelerate unfolding of substrates modified by SUMO-polyubiquitin hybrid chains compared to polyubiquitin alone. Cryo-EM structures of the complex with a SUMO-polyubiquitin hybrid-chain substrate reveal features of Ufd1/Npl4/Cdc48 interactions with ubiquitin prior to and during unfolding. |
In vitro unfolding assays with purified yeast proteins, single-particle cryo-EM structural analysis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
36574706
|
| 2009 |
VCP/p97 is required for autophagosome maturation: loss of VCP activity (by RNAi knockdown or dominant-negative overexpression) results in accumulation of immature autophagosomes under basal conditions; these autophagosomes fail to mature into autolysosomes and degrade LC3. Disease-causing IBMPFD mutants (R155H, A232E) cause the same autophagy defect. VCP is selectively required for autophagic degradation of ubiquitinated substrates but not for starvation-induced autophagy. |
RNAi knockdown, dominant-negative overexpression, stable dual-tagged LC3 reporter (mCherry-EGFP-LC3), patient-derived myoblasts |
The Journal of cell biology |
High |
20008565 20104022
|
| 2021 |
VCP/p97 regulates autophagy initiation in two Beclin-1-dependent ways: (1) VCP stabilizes Beclin-1 protein levels by promoting the deubiquitinase activity of ataxin-3 (ATXN3) towards Beclin-1; (2) VCP interacts with and promotes assembly and kinase activity of the Beclin-1-containing PI3K complex I, regulating PI(3)P production. Inhibition of VCP ATPase activity impairs starvation-induced PI(3)P production and limits downstream recruitment of WIPI2, ATG16L, and LC3, decreasing autophagosome formation. |
Small-molecule VCP ATPase inhibitors, PI(3)P lipid assays, co-immunoprecipitation, deubiquitinase activity assays, WIPI2/ATG16L/LC3 recruitment assays |
Nature chemical biology |
High |
33510452 33719912
|
| 2011 |
Cdc48/p97, together with cofactors Ufd1-Npl4, Ubx4, and Ubx5, mediates UV-dependent turnover of RNA Pol II largest subunit Rpb1: ubiquitinated Rpb1, proteasomes, and Cdc48 accumulate on chromatin in UV-treated cells; in cdc48 mutants, Ub conjugates accumulate on the proteasome, indicating Cdc48 acts to facilitate processive degradation at sites of stalled transcription rather than solely upstream of the proteasome. |
Proteasome isolation followed by mass spectrometry, chromatin fractionation, genetic mutant analysis in yeast |
Molecular cell |
High |
21211725
|
| 2005 |
IBMPFD disease-causing VCP/p97 mutant R155H has normal ATPase activity and hexameric structure, yet impairs ERAD: it increases overall ubiquitin-conjugated proteins and specifically blocks degradation of the ERAD substrate ΔF508-CFTR, similar to ATPase-deficient VCP. IBMPFD mutants also promote formation of aggregates containing VCP, ubiquitin conjugates, and ER-resident proteins. |
Cell-based ERAD substrate degradation assays, ATPase activity measurement, native gel electrophoresis, immunofluorescence/co-localization in cultured cells |
Human molecular genetics |
High |
16321991
|
| 2011 |
Cdc48/p97-Ufd1-Npl4 complex removes Aurora B from chromatin during mitotic exit in Xenopus egg extracts; in HeLa cells, Ufd1-Npl4 antagonizes Aurora B on chromosomes from prometaphase onwards. Depletion of Ufd1-Npl4 by siRNA causes increased Aurora B levels and activity on chromosomes, chromosome alignment and anaphase defects, and multi-lobed nuclei; low-dose Aurora B inhibitor partially rescues these defects. |
siRNA depletion, quantitative immunofluorescence, Aurora B kinase activity assay (CENP-A phosphorylation), genetic epistasis with hesperadin inhibitor in HeLa cells |
Journal of cell science |
High |
20130676 21486945
|
| 2010 |
In budding yeast, Cdc48-Shp1 complex promotes chromosome bi-orientation by facilitating nuclear localization of the phosphatase Glc7/PP1, which counteracts Ipl1/Aurora B kinase activity at kinetochores. Temperature-sensitive cdc48-3 and Shp1 depletion cause metaphase arrest due to defective bipolar kinetochore attachment and spindle checkpoint activation. |
Yeast temperature-sensitive mutants, co-immunoprecipitation, nuclear localization imaging, genetic epistasis with ipl1 mutants and kinase inhibitors |
Journal of cell science |
High |
20483956
|
| 2018 |
Cdc48/VCP promotes chromosome condensation by releasing condensin from chromatin entrapment via ubiquitin-dependent extraction. Condensin traps itself in its own reaction product during chromatin compaction; Cdc48 acts as the central segregase that promotes ubiquitin-dependent cycling of condensin on mitotic chromatin. |
Yeast genetics, in vivo chromatin binding assays, ubiquitin mutant epistasis, condensin-chromatin dynamics assays |
Molecular cell |
Medium |
29452641
|
| 2013 |
In yeast, stress granules can be targeted for autophagic degradation (granulophagy) in a process requiring CDC48/VCP. Genetic screen identified CDC48 alleles as affecting stress granule dynamics; in mammalian cells, depletion or pathogenic mutations in VCP reduce stress granule clearance, an effect also seen with autophagy inhibition. |
Yeast genetic screen (125 genes), fluorescence microscopy of stress granule dynamics, RNAi depletion, patient mutation expression in mammalian cells |
Cell |
High |
23791177
|
| 2019 |
ULK1 and ULK2 kinases phosphorylate VCP/p97, thereby increasing VCP's ATPase activity and its ability to disassemble stress granules. ULK1/2 localize to stress granules; disrupted ULK1/2 expression in mice causes a vacuolar myopathy similar to VCP disease with TDP-43-positive inclusions. |
Kinase phosphorylation assays, stress granule disassembly assays, VCP ATPase activity measurement, mouse knockout models, ULK1/2 agonist pharmacology |
Molecular cell |
High |
30979586
|
| 2020 |
A p.Asp395Gly mutation in VCP is associated with dementia characterized by neurofibrillary tangles. Wild-type VCP exhibits tau disaggregase activity in vitro, which is impaired by the p.Asp395Gly mutation. Intracerebral microinjection of pathologic tau into p.Asp395Gly VCP knock-in mice leads to increased tau aggregates compared to wild-type mice. |
In vitro tau disaggregation assay with purified VCP, VCP knock-in mouse model with intracerebral tau microinjection, neuropathological analysis |
Science (New York, N.Y.) |
High |
33004675
|
| 2023 |
In cell culture and neurons, VCP is recruited to ubiquitylated Tau fibrils and efficiently disaggregates them; aggregate clearance depends on functional cooperation of VCP with Hsp70 and the ubiquitin-proteasome machinery. Inhibition of VCP stabilizes large Tau aggregates, whereas VCP-mediated disaggregation generates seeding-active Tau species as a byproduct. |
Cell culture Tau aggregate clearance assays, VCP inhibitors, VCP depletion, live-cell imaging, seeding activity assays in neurons |
Nature communications |
High |
36732333
|
| 2017 |
Endogenous VCP negatively regulates Mitofusin, which is required for outer mitochondrial membrane fusion. IBMPFD disease mutants of VCP act as hyperactive alleles with respect to Mitofusin regulation; VCP inhibitors suppress mitochondrial fusion and respiratory defects in IBMPFD patient fibroblasts and in a Drosophila IBMPFD muscle model. |
Drosophila in vivo IBMPFD muscle model, patient fibroblast assays, VCP inhibitor treatment, mitochondrial morphology/respiration assays |
eLife |
Medium |
28322724
|
| 2016 |
VCP, together with cofactor P47 and the ER morphology regulator ATL1, regulates tubular ER formation and controls protein synthesis efficiency to influence dendritic spine formation in neurons. Knockdown or disease mutation knockin of VCP reduces dendritic spine density; leucine supplementation (increasing protein synthesis) rescues spine defects caused by VCP deficiency. |
Knockdown, disease-mutation knockin mice, live imaging of ER morphology, protein synthesis measurement, dendritic spine morphometry in neurons |
Nature communications |
Medium |
26984393
|
| 2009 |
VCP/p97 is highly modified by phosphorylation and acetylation at numerous sites throughout the protein. Amino acid substitutions at Lys696 and Thr761 (in the D2alpha/VAR domain) profoundly affect VCP ATPase activity, identifying this region as an ATPase regulatory domain. Lys251 and Lys524 (Walker A motifs of D1 and D2 domains) are potential acetylation sites. |
Mass spectrometry identification of modification sites, site-directed mutagenesis with ATPase activity measurement |
Genes to cells : devoted to molecular & cellular mechanisms |
Medium |
19335618
|
| 2011 |
The UBX domain protein Ubx4 modulates the Cdc48-Ufd1-Npl4 complex for correct ERAD function. Ubx4 mutants defective in Cdc48 binding lead to defective degradation of ERAD substrates and accumulation of polyubiquitinated proteins bound to Cdc48. Ubx2 and Ubx4 are not found together in one Cdc48 complex, suggesting distinct steps in modulating Cdc48 activity in ERAD. |
Yeast genetics with ERAD substrate degradation assays, co-immunoprecipitation, polyubiquitin accumulation assays |
The Journal of biological chemistry |
Medium |
19359248
|
| 2018 |
In budding yeast, defective RNA polymerase III (Pol III) is negatively regulated by a sequential SUMO–ubiquitin–Cdc48 pathway: Pol III is sumoylated, then ubiquitylated, and subsequently targeted by Cdc48/p97 segregase, leading to proteasomal degradation of Pol III subunits and repression of Pol III transcription. |
Yeast genetics, sumoylation and ubiquitylation assays, Cdc48 mutant analysis, proteasomal degradation assays |
eLife |
Medium |
30192228
|
| 2022 |
WIPI2 binds VCP/p97 and promotes its recruitment to damaged mitochondria during PINK1-PRKN-mediated mitophagy. WIPI2 depletion blunts VCP recruitment to damaged mitochondria, leading to reduced degradation of outer mitochondrial membrane proteins and impaired mitophagy. |
Co-immunoprecipitation, RNAi depletion, mitophagy flux assays, OMM protein degradation assays, cell death assays |
Autophagy |
Medium |
35389758
|
| 2021 |
VCP cofactor UBXN1/SAKS1 translocates to mitochondria upon depolarization in a PRKN-dependent manner and facilitates MFN2 removal from the outer mitochondrial membrane; loss of UBXN1 impairs VCP and PRKN translocation to mitochondria and reduces mitophagic flux. UBXN1 physically interacts with PRKN in a UBX domain-dependent manner. |
Co-immunoprecipitation, siRNA depletion, mitophagy flux assays, immunofluorescence imaging of mitochondrial translocation, domain mapping |
Autophagy |
Medium |
33966597
|
| 2022 |
Polo-like kinase 1 (Plk1) phosphorylates VCP/p97 at Thr76, recruiting VCP to the centrosome from prophase to anaphase and regulating centrosome orientation. Dephosphorylation of Thr76 by PTEN is required for VCP and Eg5 enrichment at the mitotic spindle, ensuring proper spindle architecture and chromosome segregation. Cryo-EM structures of phosphomimetic (T76E) and phospho-deficient (T76A) VCP revealed that Thr76 phosphorylation alters inter-domain/inter-subunit interactions and the nucleotide-binding pocket conformation. |
Kinase assay, phospho-mutant cell biology, siRNA depletion, cryo-EM structural analysis, co-localization imaging, xenograft tumor growth assay |
Cell death and differentiation |
High |
35430615
|
| 2024 |
VCP/p97 is UFMylated at K109 by the E3 ligase UFL1; this modification promotes BECN1 stabilization through ATXN3-mediated deubiquitination and facilitates assembly of the BECN1-containing PI3K complex, thereby promoting autophagy initiation. UFMylation-defective VCP mutant cannot rescue VCP depletion-induced LC3B accumulation. Several pathogenic VCP mutations are associated with reduced UFMylation. |
Mass spectrometry identification of UFMylation site, site-directed mutagenesis, co-immunoprecipitation, BECN1 stability assays, LC3B reporter assays |
Autophagy |
Medium |
38762759
|
| 2023 |
DUSP1 phosphatase interacts with VCP on mitochondria and dephosphorylates VCP at Ser784. LPS-induced endotoxemia promotes VCP Ser784 phosphorylation; DUSP1 overexpression prevents this, preserving mitochondrial quality control. A phosphomimetic VCP S784E mutant abolishes DUSP1's protective effects on mitochondrial dynamics, mitophagy, and cardiomyocyte contractility. |
Co-immunoprecipitation, phospho-specific antibodies, site-directed mutagenesis (phosphomimetic), DUSP1 transgenic mice and HL-1 cell transfection, mitochondrial function assays |
Cellular and molecular life sciences : CMLS |
Medium |
37464072
|
| 2022 |
PTP4A2 phosphatase dephosphorylates VCP/p97 at Tyr805; this dephosphorylation enables VCP to associate with its C-terminal cofactors UBXN6/UBXD1 and PLAA, which are components of the ELDR complex responsible for lysophagy (autophagic clearance of damaged lysosomes). Loss of PTP4A2 compromises recovery from acute kidney injury due to impaired lysophagy. |
Substrate trapping and mass spectrometry, biochemical dephosphorylation assays, co-immunoprecipitation, lysophagy assays, Ptp4a2 knockout mice |
Autophagy |
High |
36300783
|
| 2021 |
VCP's cofactor FAF1 facilitates VCP-dependent extraction of SUMOylated and ubiquitylated proteins accumulating on chromatin after DNA replication blocks; this VCP(FAF1) activity cooperates with the deubiquitylase USP7 (which maintains a SUMO-high/ubiquitin-low environment at active replication forks). Inactivation of USP7 and FAF1 is synthetically lethal in C. elegans and mammalian cells. |
Co-immunoprecipitation, chromatin fractionation, C. elegans and mammalian cell genetics, synthetic lethality assays, VCP and USP7 inhibitor synergy |
Cell reports |
Medium |
34644576
|
| 2023 |
The p97/VCP UFD1-NPLOC4 segregase complex is required for arsenic-induced degradation of PML and PML-RARA. p97/VCP localizes to PML bodies after arsenic treatment; pharmacological p97 inhibition or siRNA depletion of UFD1/NPLOC4 blocks arsenic-induced degradation of PML-RARA, accumulates SUMO- and ubiquitin-modified PML, and alters PML body number, morphology, and size. |
Proteomic analysis of PML bodies, pharmacological p97 inhibition, siRNA depletion, immunofluorescence, biochemical PML modification analysis |
The Journal of cell biology |
Medium |
36880596
|
| 2024 |
Inhibited WRN helicase is trapped on chromatin and requires p97/VCP for extraction and proteasomal degradation in microsatellite-instability-high (MSI-H) cancer cells. The PIAS4-RNF4 SUMO-ubiquitin axis is responsible for generating the SUMOylated/ubiquitylated WRN signal recognized by p97/VCP for chromatin extraction. |
Single-molecule tracking in living cells, pharmacological WRN inhibition, p97 inhibition, PIAS4 and RNF4 genetic epistasis, chromatin fractionation |
Nature communications |
Medium |
39025847
|
| 2023 |
Ubx5-Cdc48 assists the protease Wss1 in clearing DNA-protein crosslinks (DPCs): Ubx5 accumulates at persistent DPC lesions in the absence of Wss1; abolishing Cdc48 binding by Ubx5 or complete Ubx5 loss suppresses wss1Δ sensitivity to DPC-inducing agents by favoring alternate repair pathways. Ubx5-Cdc48 and Wss1 cooperate in genotoxin-induced degradation of RNA Pol II at DPC lesions. |
Yeast genetics, inducible site-specific crosslink assay, chromatin immunoprecipitation, genetic epistasis, RNAPII degradation assays |
The EMBO journal |
Medium |
37144685
|
| 1993 |
VCP forms a stoichiometric complex with clathrin and Hsc70 in mammalian cell lysates within 15 min of synthesis, identifying VCP as a ubiquitous clathrin-binding protein and suggesting a role in modulating protein-protein interactions in membrane transport processes. |
Biochemical co-purification, co-immunoprecipitation, stoichiometric complex analysis from mammalian cell lysates |
Nature |
Medium |
8413590
|
| 2012 |
Both Cdc48 and its cofactor Vms1 (but not Ufd1 or Ufd2) are required for degradation of the telomere regulator Cdc13; this degradation involves both autophagy and the proteasome under non-stress conditions. |
Yeast deletion mutant analysis, Cdc13 stability assays, autophagy and proteasome inhibition genetics |
The Journal of biological chemistry |
Medium |
22718752
|
| 2013 |
In IBMPFD, all twelve pathogenic p97/VCP missense mutants cause ERAD substrate accumulation. Most IBMPFD mutants show enhanced binding to cofactors p47 and Ufd1-Npl4. However, the P137L mutant uniquely abolishes interactions with Ufd1, Npl4, and p47 while retaining gp78-VIM binding, and shows a distinct solubility profile and subcellular localization compared to other IBMPFD mutants. |
ERAD substrate degradation assays, in vitro protein-protein binding assays with recombinant proteins, co-immunoprecipitation, subcellular fractionation, immunofluorescence |
The international journal of biochemistry & cell biology |
Medium |
23333620
|
| 2015 |
An ALS-disease-associated aspartate mutation in the pore-2 loop of the D2 ring of Cdc48/p97 impairs 20S proteasome binding and proteolytic communication but does not affect hexamer stability, ATP hydrolysis rate, or protein unfolding activity, identifying the pore-2 loop as a critical element for direct Cdc48–20S proteasome interaction. |
In vitro binding assays between purified archaeal/human Cdc48 variants and 20S proteasome, ATPase activity measurement, protein unfolding assays, hexamer stability analysis, site-directed mutagenesis |
Protein science : a publication of the Protein Society |
Medium |
26134898
|
| 2014 |
Archaeal Cdc48 and 20S proteasome form a stable, enzymatically active coaxial complex stabilized by site-specific cross-linking. The N-terminal domain of Cdc48 packs against the D1 ring in a coplanar fashion in this complex, and the structure demonstrates that coaxial alignment without AAA+ wobbling is sufficient for function. |
Site-specific chemical cross-linking, single-particle electron microscopy structure determination, in vitro proteolytic activity assay |
Proceedings of the National Academy of Sciences of the United States of America |
High |
24711419
|
| 2019 |
VCP inactivation in skeletal muscle of adult mice (conditional knockout) causes lysosomal membrane damage, persistent TFEB nuclear localization, and a necrotic myopathy distinct from ATG5 knockout, identifying VCP as a central mediator of both lysosomal clearance and TFEB-regulated lysosomal biogenesis in differentiated muscle. |
Conditional muscle-specific knockout mice, lysosomal damage markers (LGALS3), TFEB localization by immunofluorescence, comparison with ATG5 knockout mice, chemical lysosomal membrane permeabilization |
Autophagy |
High |
30654731
|
| 2023 |
Nuclear VCP binds to HDAC1 and facilitates its degradation, thereby promoting transcription of FAO genes including CPT1A, upregulating fatty acid oxidation in colorectal cancer cells. |
Co-immunoprecipitation, VCP inhibitor and knockdown experiments, chromatin-based transcription assays, HDAC1 stability assays, cancer cell functional assays |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
37788309
|
| 2025 |
VCP is a top hit in a proximity-labeling screen for proteins that control tau seed amplification within 5 h of seed exposure. VCP knockdown reduces tau seeding; two VCP inhibitors (ML-240 and NMS-873) have opposing effects on seeding, with effects only observed within 8 h of seed exposure. Screening 30 VCP cofactors identified ATXN3, NSFL1C, UBE4B, NGLY1, OTUB1, and NPLOC4 as suppressors of tau seeding, while FAF2 reduction increases seeding. |
Split-APEX2 proximity labeling screen, CRISPR/siRNA cofactor screen, tau seeding biosensor assays in HEK293T cells and human neurons, VCP inhibitor pharmacology |
Molecular neurodegeneration |
Medium |
39773263
|
| 2021 |
Neuronal VCP loss of function (conditional knockout in postnatal forebrain neurons) causes cortical atrophy, neuronal loss, autophagolysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. A single disease-associated VCP-R155C mutation in a VCP null background similarly recapitulates VCP inactivation features, suggesting the R155C MSP mutation is hypomorphic (loss-of-function rather than dominant-negative). |
Conditional neuronal knockout mice, VCP-R155C knock-in in null background, transcriptomic and proteomic analysis, TDP-43 immunohistochemistry |
Cell reports |
Medium |
34289347
|