Abstract
Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.
Highlights
Protein ubiquitination is a reversible post-translational modification that regulates several crucial intracellular events ranging from signaling pathways to cell cycle regulation and DNA repair (Hershko and Ciechanover, 1998)
Our results provide a structural explanation on how ubiquitination can directly regulate the deubiquitinating enzymes (DUB) activity of ataxin-3 and enable us to propose a general regulatory mechanism that can modulate the activity of other such enzymes
We capitalized on the ability to produce large quantities of mono-ubiquitinated JosK117-only using a protocol previously described (Faggiano et al, 2013)
Summary
Protein ubiquitination is a reversible post-translational modification that regulates several crucial intracellular events ranging from signaling pathways to cell cycle regulation and DNA repair (Hershko and Ciechanover, 1998). Despite the typical cysteine protease fold, it contains an unusually long helical hairpin not observed in other cysteine proteases (such as papain) or in other DUBs (for example YUH1 or UCH-L3) (Nicastro et al, 2005) (Scheme 1 in Supplementary Material) It contains at least two binding sites for ubiquitin in addition to the multiple UIMs in the C-terminus of ataxin-3. Despite increasing interest in the role of ataxin-3 ubiquitination and in how ubiquitination may influence DUB activity, little is known about the mechanism by which this modification leads to enzyme activation Knowing this mechanism would increase our understanding of the ubiquitin pathways and provide new information on polyglutamine expansion diseases that may be translated into specific treatments. Our results provide a structural explanation on how ubiquitination can directly regulate the DUB activity of ataxin-3 and enable us to propose a general regulatory mechanism that can modulate the activity of other such enzymes
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