Quantitative Proteomics Identifies the Myb-Binding Protein p160 as a Novel Target of the von Hippel-Lindau Tumor Suppressor

Background: The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the a subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression. Methodology/Principal Findings: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL. Conclusions/Significance: A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases

Microtubular Stability Affects pVHL-Mediated Regulation of HIF-1alpha via the p38/MAPK Pathway in Hypoxic Cardiomyocytes

BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes

Low VHL mRNA Expression is Associated with More Aggressive Tumor Features of Papillary Thyroid Carcinoma

Alterations of the von Hippel-Lindau (VHL) tumor suppressor gene can cause different hereditary tumors associated with VHL syndrome, but the potential role of the VHL gene in papillary thyroid carcinoma (PTC) has not been characterized. This study set out to investigate the relationship of VHL expression level with clinicopathological features of PTC in an ethnically and geographically homogenous group of 264 patients from Serbia, for the first time. Multivariate logistic regression analysis showed a strong correlation between low level of VHL expression and advanced clinical stage (OR55.78, 95% CI 3.17-10.53, P<0.0001), classical papillary morphology of the tumor (OR52.92, 95% CI 1.33-6.44, P=50.008) and multifocality (OR51.96, 95% CI 1.06-3.62, P=50.031). In disease-free survival analysis, low VHL expression had marginal significance (P=50.0502 by the log-rank test) but did not appear to be an independent predictor of the risk for chance of faster recurrence in a proportion hazards model. No somatic mutations or evidence of VHL downregulation via promoter hypermethylation in PTC were found. The results indicate that the decrease of VHL expression associates with tumor progression but the mechanism of downregulation remains to be elucidated