Proteomic dissection of the von Hippel-Lindau (VHL) interactome

The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex containing elongin B, elongin C, cullin 2, and Rbx1, which acts as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the alpha subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in the maintenance of primary cilium, extracellular matrix formation, and tumor suppression. We undertook a series of proteomic analyses to gain a comprehensive picture of the VHL-interacting proteins. We found that the ARF tumor suppressor interacts with VHL30, a longer VHL isoform, but not with VHL19, a shorter VHL isoform. ARF was found to release VHL30 from the E3 ligase complex, promoting the binding of VHL30 to a protein arginine methyltransferase, PRMT3. Our analysis of the VHL19 interactome also uncovered that VHL19 displays an affinity to collagens and their biosynthesis enzymes.     

UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover

p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.     

Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here we report on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, we provide evidence that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes.     

Loss of VHL confers hypoxia-inducible factor (HIF)-dependent resistance to vesicular stomatitis virus: role of HIF in antiviral response

Hypoxia-inducible factor (HIF) is a central regulator of cellular responses to hypoxia, and under normal oxygen tension the catalytic alpha subunit of HIF is targeted for ubiquitin-mediated destruction via the VHL-containing E3 ubiquitin ligase complex. Principally known for its association with oncogenesis, HIF has been documented to have a role in the antibacterial response. Interferons, cytokines with antiviral functions, have been shown to upregulate the expression of HIF-1alpha, but the significance of HIF in the antiviral response has not been established. Here, using renal carcinoma cells devoid of VHL or reconstituted with functional wild-type VHL or VHL mutants with various abilities to negatively regulate HIF as an ideal model system of HIF activity, we show that elevated HIF activity confers dramatically enhanced resistance to vesicular stomatitis virus (VSV)-mediated cytotoxicity. Inhibition of HIF activity using a small-molecule inhibitor, chetomin, enhanced cellular sensitivity to VSV, while treatment with hypoxia mimetic CoCl2 promoted resistance. Similarly, targeting HIF-2alpha by RNA interference also enhanced susceptibility to VSV. Expression profiling studies show that upon VSV infection, the induction of genes with known antiviral activity, such as that encoding beta interferon (IFN-beta), is significantly enhanced by HIF. These results reveal a previously unrecognized role of HIF in the antiviral response by promoting the expression of the IFN-beta gene and other genes with antiviral activity upon viral infection.     

Regulation of DNA topoisomerase IIα stability by the ECV ubiquitin ligase complex

In this study, we attempted to elucidate the E3 ubiquitin ligase for topo IIα. When cullins and VHL were ectopically expressed in HT1080 and HEK293T cells, topo IIα was degraded most prominently in cullin 2- and VHL-expressing cells. Cullin 2 and the β domain (aa 114-123) of VHL, a subunit of the ECV (Elongin B/C-cullin 2-VHL protein) complex, specifically interact with the ATPase domain of topo IIα. We identified that topo IIα associated with endogenous Elongin C. In HT1080 cells co-transfected with deletion mutants of topo IIα GRDD (glucose-regulated destruction domain) and VHL, topo IIα was degraded by VHL expression. These results demonstrate that ECV acts as E3 ubiquitin ligase targeting GRDD-independent topo IIα to the ubiquitin-proteasome pathway.