Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ben Collins

LAB HEAD: Ben Collins

PROVIDER: PXD007876 | Pride | 2018-04-04

REPOSITORIES: Pride

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Publications

AP-SWATH Reveals Direct Involvement of VCP/p97 in Integrated Stress Response Signaling Through Facilitating CReP/PPP1R15B Degradation.

Hülsmann Julia J Kravic Bojana B Weith Matthias M Gstaiger Matthias M Aebersold Ruedi R Collins Ben C BC Meyer Hemmo H

Molecular & cellular proteomics : MCP 20180329 7

The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of damaged or misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of its interactions with partner and substrate proteins and understanding its links to stress signaling is therefore a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with the substrate-trapping mutant, p97-E578Q. AP-SWATH identified differen ...[more]

PMID: 29599191

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Project description:Werner syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS is believed to be involved in different aspects of transcription, replication, and/or DNA repair. We generated a mouse model with a deletion in the helicase domain of the murine WRN homologue that recapitulates most of the WS phenotypes including an abnormal hyaluronic acid excretion, higher reactive oxygen species (ROS) levels, increased genomic instability and cancer incidence resulting in a 10-15% decreased life span expectancy. In addition, WS patients and Wrn mutant mice show hallmarks of a metabolic syndrome including premature visceral obesity, hypertriglyceridemia, insulin-resistant diabetes type 2 and associated cardiovascular diseases. In this study, we compared the expression profile of liver tissues from 9 months old Wrn mutant and wild type animals. Gene set enrichment analysis of the microarray data indicated that Wrn mutant mice exhibited down-regulation of genes normally decreased in several transgenic mouse models of hepatoma and during caloric restriction. Wrn mutant mice also altered the expression of genes involved in inflammation as well as in glutathione and xenobiotic metabolisms. These results indicate that Wrn mutant mice respond to the observed oxidative stress by altering the necessary pathways to survive. Vitamin C supplementation rescued the life span expectancy of Wrn mutant mice and reversed several age-related abnormalities in adipose, cardiac, and liver tissues, genomic integrity and inflammatory status. Finally, gene set enrichment analyses revealed that vitamin C decreased genes normally up regulated in human WS fibroblasts and cancers and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Experiment Overall Design: Microarray analyses were performed on the liver tissues of 9 months old mice. Four independent biological replicates of this experiment (wild type vs Wrn mutant or wild type vs vitamin C treated mutant mice) were carried out with a dye swap on two replicates of each genotype.

Dataset Information

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

Publications

AP-SWATH Reveals Direct Involvement of VCP/p97 in Integrated Stress Response Signaling Through Facilitating CReP/PPP1R15B Degradation.

Similar Datasets

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