Poly-ubiquitin binding by the polyglutamine disease protein ataxin-3 links its normal function to protein surveillance pathways.
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.
Chai
Yaohui
Y
Department of Neurology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1101, USA.
Berke
Sarah Shoesmith
SS
Cohen
Robert E
RE
Paulson
Henry L
HL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000067699:Ataxin-3; D015374:Biosensing Techniques; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D012097:Repressor Proteins; D025801:Ubiquitin; C097188:polyglutamine; D005982:Glutathione Transferase; C092341:ATXN3 protein, human; D000067699:Ataxin-3