Dataset Information

Nuclear versus cytoplasmic VHL interactomes

ABSTRACT: Protein misfolding is linked to a wide variety of human disease. Protective cellular protein quality control (PQC) mechanisms evolved to selectively recognize misfolded proteins and limit their toxic effects. Using yeast as a model system, we previously discovered that the misfolded protein VHL interacts with various PQC machines on its way to being cleared by the ubiquitin-proteasome system. However, the clearance of nuclear and cytoplasmic VHL has difference PQC requirements. In this study, we performed SILAC-based AP-MS of nuclear (NLS) versus cytoplasmic (NES) VHL to identify, in an unbiased manner, the similarities and differences between nuclear and cytoplasmic PQC. We found that 4 molecular chaperones of the Hsp70 family, and multiple subunits of the proteasome, were significantly enriched with both nuclear and cytoplasmic VHL. By contrast, the chaperone Hsp90 and Sis1 were only associated with cytoplasmic VHL, whereas 6 of the 8 TRiC/chaperonin subunits were specifically enriched with nuclear VHL. Our study highlights unexpected differences between nuclear and cytoplasmic PQC, with important implications for our understanding of a wide range of protein misfolding diseases.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Rahul Samant

LAB HEAD: Judith Frydman

PROVIDER: PXD010660 | Pride | 2018-11-04

REPOSITORIES: Pride

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Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control.

Samant Rahul S RS Livingston Christine M CM Sontag Emily M EM Frydman Judith J

Nature 20181031 7731

Protein misfolding is linked to a wide array of human disorders, including Alzheimer's disease, Parkinson's disease and type II diabetes<sup>1,2</sup>. Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects<sup>3-9</sup>, thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin-proteasome system cooperate to recognize and promote the ...[more]

PMID: 30429547

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Project description:Gene expression data from VHL teratomas comparing genes differentially expressed based on apoptotic response to tumor microenvironment. Abstract BACKGROUND: The risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations. METHODS: VHL deficient (Vhl -/- ) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided. RESULTS: The P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation-induced apoptosis. CONCLUSIONS: The TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response. Three genotypes of VHL teratomas were examined: Three replicate wildtype VHL, Three replicate R167Q VHL, and Four replicate P81S VHL (representing two replicates of two different P81S clones)

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