Ataxin-3 is subject to autolytic cleavage.
The protein ataxin-3 is responsible for spinocerebellar ataxia type 3, a neurodegenerative disease triggered when the length of a stretch of consecutive glutamines exceeds a critical threshold. Different physiologic roles have been suggested for this protein. More specifically, recent papers have shown that the highly conserved N-terminal Josephin domain of ataxin-3 binds ubiquitin and has ubiquitin hydrolase activity, thanks to a catalytic device specific to cysteine proteases. This article shows that the protein also has autoproteolytic activity, sustained by the same residues responsible for the ubiquitin hydrolase activity. The autolytic activity was abolished when these residues, i.e. Cys14 and His119, were replaced by noncatalytic ones. Furthermore, we found that pretreatment of the protein with tosyl l-phenylalanine chloromethyl ketone also abolished this activity, and that this site-specific reagent covalently bound His119, findings supported by MS experiments. MS also allowed us to establish that the attack was aspecific, as cleavage sites were observed at the carboxyl side of apolar, acidic and polar uncharged residues, clustered in the C-terminal, unstructured domain of the protein. In contrast, the Josephin domain was preserved from attack. We propose that the autolytic activity reported here may play a role in pathogenesis, as fragments carrying expanded polyglutamines are thought to be significantly more toxic than the whole protein.
Mauri
Pier Luigi
PL
Istituto di Tecnologie Biomediche, CNR, Milano, Italy.
Riva
Matteo
M
Ambu
Daniela
D
De Palma
Antonella
A
Secundo
Francesco
F
Benazzi
Louise
L
Valtorta
Marco
M
Tortora
Paolo
P
Fusi
Paola
P
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000067699:Ataxin-3; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D005973:Glutamine / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D000096926:Benzenesulfonamides
D000590:Amino Acid Chloromethyl Ketones; D000067699:Ataxin-3; D005973:Glutamine; D006867:Hydrolases; D009687:Nuclear Proteins; D011480:Protease Inhibitors; D014157:Transcription Factors; D025801:Ubiquitin; C009508:tosylglycine chloromethyl ketone; D000096926:Benzenesulfonamides; C511594:Atxn3 protein, mouse