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J Biol Chem. 2004 Jan 30;279(5):3605-11. Epub 2003 Nov 5.

Poly-ubiquitin binding by the polyglutamine disease protein ataxin-3 links its normal function to protein surveillance pathways.

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Department of Neurology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1101, USA.


In at least nine inherited diseases polyglutamine expansions cause neurodegeneration associated with protein misfolding and the formation of ubiquitin-conjugated aggregates. Although expanded polyglutamine triggers disease, functional properties of host polyglutamine proteins also must influence pathogenesis. Using complementary in vitro and cell-based approaches we establish that the polyglutamine disease protein, ataxin-3, is a poly-ubiquitin-binding protein. In stably transfected neural cell lines, normal and expanded ataxin-3 both co-precipitate with poly-ubiquitinated proteins that accumulate when the proteasome is inhibited. In vitro pull-down assays show that this reflects direct interactions between ataxin-3 and higher order ubiquitin conjugates; ataxin-3 binds K48-linked tetraubiquitin but not di-ubiquitin or mono-ubiquitin. Further studies with domain-deleted and site-directed mutants map tetra-ubiquitin binding to ubiquitin interaction motifs situated near the polyglutamine domain. In surface plasmon resonance binding analyses, normal and expanded ataxin-3 display similar submicromolar dissociation constants for tetra-ubiquitin. Binding kinetics, however, are markedly influenced by the surrounding protein context; ataxin-3 that lacks the highly conserved, amino-terminal josephin domain shows significantly faster association and dissociation rates for tetra-ubiquitin binding. Our results establish ataxin-3 as a poly-ubiquitin-binding protein, thereby linking its normal function to protein surveillance pathways already implicated in polyglutamine pathogenesis.

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