Dataset Information

PVHL mediates K63-linked ubiquitination of nCLU.

ABSTRACT: pVHL, product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase that targets proteins for ubiquitination and proteasomal degradation. Hypoxia-inducible factor ? (HIF?) is the well-known substrate of pVHL. Besides HIF?, pVHL also binds to many other proteins and has multiple functions. In this manuscript, we report that the nuclear clusterin (nCLU) is a target of pVHL. We found that pVHL had a direct interaction with nCLU. nCLU bound to pVHL at pVHL's ? domain, the site for recognition of substrate, indicating that nCLU might be a substrate of pVHL. Interestingly, pVHL bound to nCLU but did not lead to nCLU destruction. Further studies indicated that pVHL mediated K63-linked ubiquitination of nCLU and promoted nCLU nuclear translocation. In summary, our results disclose a novel function of pVHL that mediates K63-linked ubiquitination and identify nCLU as a new target of pVHL.

SUBMITTER: Xue J

PROVIDER: S-EPMC3332038 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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pVHL mediates K63-linked ubiquitination of nCLU.

Xue Jing J Lv Dan-Dan DD Jiao Shi S Zhao Wenting W Li Xuebing X Sun Heng H Yan Bing B Fan Li L Hu Rong-Gui RG Fang Jing J

PloS one 20120420 4

pVHL, product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase that targets proteins for ubiquitination and proteasomal degradation. Hypoxia-inducible factor α (HIFα) is the well-known substrate of pVHL. Besides HIFα, pVHL also binds to many other proteins and has multiple functions. In this manuscript, we report that the nuclear clusterin (nCLU) is a target of pVHL. We found that pVHL had a direct interaction with nCLU. ...[more]

PMID: 22532874

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Project description:Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent worldwide, manipulates the host's innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-?B activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses interleukin-1? (IL-1?)-stimulated NF-?B activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with I?B kinase, which regulates the activation of NF-?B. HBeAg suppressed the IL-1?-induced tumor necrosis factor (TNF)-associated factor 6 (TRAF6)-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-?B activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-?B signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells, and clinical liver samples. Our study reveals a molecular mechanism whereby HBeAg suppresses IL-1?-induced NF-?B activation by decreasing the TRAF6-dependent K63-linked ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-?B pathway are controversial. In this study, we showed that HBeAg could suppress both Toll-like receptor 2 (TLR2)- and IL-1?-induced activation of NF-?B in cells and clinical samples, and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for I?B kinase (IKK) that controls the NF-?B signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in downregulation of NF-?B activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher levels of NF-?B activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections. Our findings represent a conceptual advance in HBV-related suppression of NF-?B signaling.

Project description:Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFN?) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFN?-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFN?-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFN?-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.

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PVHL mediates K63-linked ubiquitination of nCLU.

Publications

pVHL mediates K63-linked ubiquitination of nCLU.

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