Human Molecular Genetics Advance Access originally published online on April 6, 2009
Human Molecular Genetics 2009 18(13):2344-2358; doi:10.1093/hmg/ddp167
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TRIM32 is an E3 ubiquitin ligase for dysbindin
1 Department of Psychological Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN, UK 2 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
* To whom correspondence should be addressed. Tel: +44 2920687051; Fax: +44 2920687068; Email: blakedj{at}cardiff.ac.uk
Received December 15, 2008; Accepted April 2, 2009
Mutations in the gene encoding tripartite motif protein 32 (TRIM32) cause two seemingly diverse diseases: limb-girdle muscular dystrophy type 2H (LGMD2H) or sarcotubular myopathy (STM) and Bardet–Biedl syndrome type 11(BBS11). Although TRIM32 is involved in protein ubiquitination, its substrates and the molecular consequences of disease-causing mutations are poorly understood. In this paper, we show that TRIM32 is a widely expressed ubiquitin ligase that is localized to the Z-line in skeletal muscle. Using the yeast two-hybrid system, we found that TRIM32 binds and ubiquitinates dysbindin, a protein implicated in the genetic aetiology of schizophrenia, augmenting its degradation. Small-interfering RNA-mediated knock-down of TRIM32 in myoblasts resulted in elevated levels of dysbindin. Importantly, the LGMD2H/STM-associated TRIM32 mutations, D487N and R394H impair ubiquitin ligase activity towards dysbindin and were mislocalized in heterologous cells. These mutants were able to self-associate and also co-immunoprecipitated with wild-type TRIM32 in transfected cells. Furthermore, the D487N mutant could bind to both dysbindin and its E2 enzyme but was defective in monoubiquitination. In contrast, the BBS11 mutant P130S did not show any biochemical differences compared with the wild-type protein. Our data identify TRIM32 as a regulator of dysbindin and demonstrate that the LGMD2H/STM mutations may impair substrate ubiquitination.
Present address: Department of Cardiovascular Medicine and the Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
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