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.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Animals, Biosensing Techniques, Cattle, Cell Line, DNA, Complementary, Glutathione Transferase, Humans, Kinetics, Molecular Sequence Data, Nerve Tissue Proteins, Neurons, Nuclear Proteins, Peptides, Plasmids, Precipitin Tests, Protein Binding, Protein Folding, Protein Structure, Tertiary, Repressor Proteins, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Time Factors, Transfection, Tumor Cells, Cultured, Ubiquitin
Amino Acid Motifs, Amino Acid Sequence, Animals, Biosensing Techniques, Cattle, Cell Line, DNA, Complementary, Glutathione Transferase, Humans, Kinetics, Molecular Sequence Data, Nerve Tissue Proteins, Neurons, Nuclear Proteins, Peptides, Plasmids, Precipitin Tests, Protein Binding, Protein Folding, Protein Structure, Tertiary, Repressor Proteins, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Time Factors, Transfection, Tumor Cells, Cultured, Ubiquitin
J. Biol. Chem.
Date: Jan. 30, 2004
PubMed ID: 14602712
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