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Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies.

ABSTRACT: Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4. Mutations in either Doa4 or Bro1 that disrupt catalytic activation of Doa4 impair deubiquitination and sorting of MVB cargo proteins and lead to the formation of lumenal MVB vesicles that are predominantly small compared with the vesicles seen in wild-type cells. Thus, by recruiting Doa4 to late endosomes and stimulating its catalytic activity, Bro1 fulfills a novel dual role in coordinating deubiquitination in the MVB pathway.

SUBMITTER: Richter C

PROVIDER: S-EPMC1868904 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies.

Richter Caleb C West Matthew M Odorizzi Greg G

The EMBO journal 20070419 10

Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a Y ...[more]

PMID: 17446860

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Project description:UnlabelledCoxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to αVβ3 and αVβ6 integrins on the cell surface and utilizes β2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection, as judged by the strong inhibitory effect of the wild-type form of Hrs and dominant negative form of VPS4 in CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection, whereas Rac1 is especially important between 1 and 3 h p.i., when the virus is in endosomes. Several lines of evidence implicate that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have a neutral pH. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection.ImportanceWe demonstrate here that the enterovirus coxsackievirus A9 (CVA9) uses a nonclathrin and nonacidic pathway to infect cells. CVA9 does not accumulate in conventional late endosomes or lysosomes. We found that inhibitors of phospholipase C (PLC), Rac1, and the Na(+)/H(+) exchanger decreased CVA9 infection. The PLC inhibitor acts on early entry, the Rac1 inhibitor acts between 1 and 3 h, when the virus is in endosomes, and the Na(+)/H(+) exchange inhibitor acts during various steps during the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which accumulate CVA9 during the first hours of entry. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger formation of neutral MVBs. The data show that these enteroviruses favor nonacidic conditions and complex MVBs to promote virus infection.

Project description:BackgroundAzithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression.MethodsVA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present.ResultsBioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives.ConclusionsTaken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.

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Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies.

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Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies.

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