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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Publications

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:Altered metabolism is an important part of malignant transformation of tumor cells. Oncogenic transformation may reprogram tumor metabolism and render tumor cells addicted to extracellular nutrients. Such nutrient addictions associated with oncogenic mutations may offer therapeutic opportunities; however, it remains difficult to predict these nutrient addictions. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear-cell renal cancer cells (ccRCC) with or without VHL upon the deprivation of individual amino acids. We identified that cystine deprivation triggered rapid programmed necrosis in VHL-deficient RCC, but not in their VHL-restored counterparts. Similar cystine addiction was also observed in VHL-deficient primary RCC tumors cells. Blockage of cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status. Therefore, metabolic differences due to cystine deprivation are not different enough to readily explain the distinct fate of life vs. death in VHL-deficient and restored cell.. Instead, we found that increased levels of TNFÎ± associated with VHL loss in the VHL-deficient RCC force them to rely on intact RIPK1 to inhibit apoptosis. However, this pre-existing elevated TNFÎ± in the VHL-deficient ccRCC renders these cells susceptible to the necrosis signaling triggered by cystine deprivation. In addition, we identified that cystine-deprived necrosis in VHL-deficient RCC depends on reciprocal amplification of the Src-p38-Noxa signaling and TNFÎ±-RIP1/3-MLKL necrosis pathways that culminate in MLKL oligomerization and programmed necrosis. Together, our data reveal that the contextual cystine-addictions in VHL-deficient ccRCC is dependent on activating pre-existing oncogenic pathways to trigger programmed necrosis. RNA was extracted by RNAeasy kits (Qiagen) from the RCC4 Vec and VHL-reconstituted cells which were exposed to the control full DMEM or cystine deprived DMEM media for 6 hours (three replicates in each condition).

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)

Project description:Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.

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Nuclear versus cytoplasmic VHL interactomes

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

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