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

INSTRUMENT(S): Bruker Daltonics timsTOF series

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Osteosarcoma

SUBMITTER: Andreas Mund

LAB HEAD: Andreas Mund

PROVIDER: PXD043565 | Pride | 2024-03-14

REPOSITORIES: Pride

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			Action	DRS
	FAM104a_AP-MS_raw.rar	Other
	MQsearch.zip	Other
	UP000005640_9606_human_additional.fasta	Fasta
	checksum.txt	Txt

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

2024-03-14 | PXD043563 | Pride

A Non-symmetrical p97 Conformation Favors Ufd1/Npl4 Recruitment

Project description:p97/Cdc48 is a hexameric AAA ATPase in which each monomer consists of an N-terminal domain (NTD) and two ATPase domains (D1 and D2), which cooperate in substrate recruitment and processing. In vitro experiments and cryo-EM structures of p97 and its cofactor, Ufd1/Npl4 (UN), revealed mechanistic insights regarding substrate processing. Yet, dynamics and the intermediate structures of the related ATPase cycle upon UN binding remain unresolved. Using novel mutations, we captured two discrete functional conformations associated with specific nucleotide states: a functional conformation in which D1 protomers are ATP bound, while the D2 subunits are in the ADP state. A configuration that is presumably required for the initial substrate engagement with the D2 pore. We also report on a heterologous nucleotide state within the D1 ring in which only 2NTDs are in the “up” ATP state. Based on the elevated affinity and structural insights, we propose that this conformation favors UN binding. Further analysis suggests that p97-UN complex formation entails a two-state mechanism. Initially, UN binds p97's non-symmetrical conformation (2NTDs in an "up" state), this association promotes a structural transition upon which five NTDs shift to an "up" state and are poised to bind ATP. The UBXL domain of Npl4 selectively binds p97's downed NTD demonstrating a new conformation that may introduce directionality to incoming substrate.

2024-05-16 | PXD045179 | 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

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.

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

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