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Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

ABSTRACT: 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.

SUBMITTER: Arumughan A

PROVIDER: S-EPMC5080433 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

Arumughan Anup A Roske Yvette Y Barth Carolin C Forero Laura Lleras LL Bravo-Rodriguez Kenny K Redel Alexandra A Kostova Simona S McShane Erik E Opitz Robert R Faelber Katja K Rau Kirstin K Mielke Thorsten T Daumke Oliver O Selbach Matthias M Sanchez-Garcia Elsa E Rocks Oliver O Panáková Daniela D Heinemann Udo U Wanker Erich E EE

Nature communications 20161020

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 s ...[more]

PMID: 27762274

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Project description:The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384-466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0-α- 1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261-573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261-573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1' intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.

Dataset Information

Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

Publications

Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

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