Understanding the role of the Josephin domain in the PolyUb binding and cleavage properties of ataxin-3

• Published in: PloS one 2010-08, Vol.5 (8), p.e12430-e12430
• Main Authors: Nicastro, Giuseppe, Todi, Sokol V, Karaca, Ezgi, Bonvin, Alexandre M J J, Paulson, Henry L, Pastore, Annalisa
• Format: Article
• Language: English
• Subjects: Ataxia; Ataxin; Ataxin-3; Binding Sites; Biochemistry/Biocatalysis; Biophysics/Biocatalysis; Biophysics/Experimental Biophysical Methods; C-Terminus; Catalysis; Cell cycle; Chains; Cleavage; Cysteine; Cysteine proteinase; Deoxyribonucleic acid; DNA; DNA polymerase; Drosophila; Humans; Huntingtons disease; Insects; Linkages; Machado-Joseph disease; Machado-Joseph Disease - metabolism; Medical research; Molecular Conformation; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Nervous system diseases; Neurodegenerative diseases; NMR; Nuclear magnetic resonance; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Polyubiquitin - chemistry; Polyubiquitin - genetics; Polyubiquitin - metabolism; Properties (attributes); Proteases; Proteasomes; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteins; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Topology; Transgenic animals; Ubiquitin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0012430

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology.