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VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates

ABSTRACT: The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a novel p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved novel -helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ivo Hendriks

LAB HEAD: Michael Lund Nielsen

PROVIDER: PXD043563 | Pride | 2024-03-14

REPOSITORIES: Pride

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VCF1 is a p97 cofactor promoting recruitment of p97-UFD1-NPL4 to ubiquitylated substrates

Project description:The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discovered that FAM104A, a protein of unknown function that we named VCF1, acts as a novel p97 cofactor in human cells. Detailed structure-function studies revealed that VCF1 directly binds p97 via a conserved novel alpha-helical motif that recognizes the p97 N-domain with unusually high affinity exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 stimulates the affinity of the p97-UFD1-NPL4 complex for ubiquitin conjugates indirectly via its binding to p97 but does not itself interact with ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

2024-03-14 | PXD043565 | Pride

Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

Project description:The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules 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 of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

2022-01-19 | PXD027639 | Pride

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation

Project description:Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex, rather than substrate recruitment. In vivo experiments confirm bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase and identify proteins that require both machines for their degradation.

2024-02-23 | PXD042878 | Pride

Ubiquitin-independent disassembly by a p97 AAA-ATPase complex drives PP1 holoenzyme formation

Project description:Protein phosphatase-1 (PP1) dephosphorylates a broad spectrum of substrates to regulate a myriad of cellular processes. This functional diversity relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held in an inactive complex by its partners SDS22 and inhibitor-3 (I3) that 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 of 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.

2018-10-23 | PXD010750 | Pride

Functional characterization of the p97/valosin-containing protein (VCP) interacting network in Leishmania Functional characterization of the p97/valosin-containing protein (VCP) interacting network in Leishmania

Project description:Valosin‐containing protein (VCP)/p97/Cdc48 is an essential AAA+ ATPase associated with many ubiquitin-dependent cellular pathways that plays a central role in protein quality control in all eukaryotes. Its functional diversity relies on its ability to cooperate with a large number of protein cofactors that provide pathway selectivity and fine-tune substrate processing. Here, we identified the main partners of the recently characterized Leishmania infantum VCP homolog (LiVCP) and we provide the first insights into the biology of the LiVCP network in Trypanosomatidae. Among the LiVCP core partners detected by immunoprecipitation (IP) and LC-MS/MS mass spectra acquisition, we determined p47, FAF1, UFD1 and PUB1 as the major cofactors of the Leishmania VCP. Furthermore, we provide in silico analyses demonstrating the conserved regions of these cofactors that potentially interact with LiVCP. We employed ‘‘network proteomics’’ using IP and LC-MS/MS studies for each cofactor to confirm their close partnership with LiVCP and to identify the cofactor-specific interacting partners as well as the partners that are shared among multiple cofactors and LiVCP complexes, such as the important heterotrimer VCP-NPL4-UFD1 complex in Leishmania. Our results suggest a glycosome/peroxisome association of LiFAF1. Gene Ontology analysis of each proteome coupled with digitonin fractionation and immunofluorescence studies indicated nuclear association for Lip47, cytoplasmic and vacuolar association for LiUFD1, glycosome association for LiFAF1, and endoplasmic reticulum interaction for LiPUB1 protein. All together, these data provide the first VCP protein network in Trypanosomatidae. Further functional and structural analysis of the essential LiVCP quality control protein network may lead to the development of new anti-parasitic drugs.

2020-08-06 | PXD013731 | Pride

Targeting of SUMO substrates to a Cdc48-Ufd1-Npl4 segregase and STUbL pathway in fission yeast

Project description:In eukaryotes, the conjugation of proteins to the small ubiquitin-like modifier SUMO regulates numerous cellular functions. A proportion of SUMO conjugates are targeted for degradation by SUMO-targeted ubiquitin ligases (STUbLs) and it has been proposed that the ubiquitin-selective chaperone Cdc48/p97-Ufd1-Npl4 facilitates this process. However, the extent to which the two pathways overlap, and how the substrates are selected, remains unknown. Here, we address these questions in fission yeast through proteome-wide analyses of SUMO modification sites. We identify over a thousand sumoylated lysines in a total of 468 proteins and quantify changes occurring in the SUMO modification status when the STUbL or Ufd1 pathways are compromised by mutations. The data suggest the coordinated processing of several classes of SUMO conjugates, many dynamically associated with centromeres or telomeres. They provide new insights into subnuclear organization and chromosome biology, and, altogether, constitute an extensive resource for the molecular characterization of SUMO function and dynamics.

2015-10-30 | PXD002972 | Pride

AP-SWATH analysis reveals a direct involvement of VCP/p97 in integrated stress response signalling through CReP/PPP1R15B degradation

Project description:The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of dynamic interactions with hosts of accessory proteins and substrates has therefore been a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with a p97 substrate-trapping mutant, p97-E578Q. AP-SWATH identified differential interactions over a large detection range from abundant cofactors to pathway-specific partners and individual ligases such as RNF185 and MUL1 that were trapped in p97-E578Q complexes. In addition, we identified substrate candidates including the PP1 regulator CReP/PPP1R15B that dephosphorylates eIF2α and thus counteracts attenuation of translation by stress-kinases. We demonstrate here that p97 with its Ufd1-Npl4 adapter ensures rapid turnover and balanced levels of CReP in unperturbed cells. Moreover, we show that p97 is essential for the quantitative stress-induced degradation of CReP and, consequently, robust eIF2α phosphorylation to enforce the stress response. Thus, p97 not only facilitates bulk degradation of misfolded proteins upon stress, but also has an unanticipated function in directly modulating the integrated stress response at the level of signalling.

2018-04-04 | PXD007876 | Pride

Immunoprecipitation of p97 from X.laevis S-phase chromatin

Project description:Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process emerged: the replicative helicase at the heart of the replisome is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. To understand how p97 is targeted to the terminated replisome, we immunoprecipitated p97 from chromatin replicating in Xenopus laevis egg extract. We have found a p97 cofactor, Ubxn7, which can interact with active Cul2Lrr1 and with p97, facilitating efficient recognition and processing of terminated helicases ubiquitylated by Cul2.

2022-10-14 | PXD029705 | Pride

VCP-dependent CNN2 proximity proteomics

Project description:Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and eliminated by VCP/p97 and proteasome for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in extraction of CNN2, and show that other membrane regulators including SNAREs, PICALM, AGFG1 and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.

2022-06-23 | PXD032903 | Pride

Human P47 interactome for comparison against CryoEM data

Project description:p97 is an essential and abundant AAA+ ATPase that unfolds ubiquitylated substrates and is a central regulator of protein homeostasis. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a “hand-over-hand” mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification

2022-05-16 | PXD033451 | Pride

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