Implications of specific lysine residues within ataxin-3 for the molecular pathogenesis of Machado-Joseph disease

Priscila Pereira Sena; Jonasz Jeremiasz Weber; Sercan Bayezit; Rafael Saup; Rana Dilara Incebacak Eltemur; Xiaoling Li; Ana Velic; Jaqueline Jung; Boris Macek; Huu Phuc Nguyen; Olaf Riess; Thorsten Schmidt

doi:10.3389/fnmol.2023.1133271

Implications of specific lysine residues within ataxin-3 for the molecular pathogenesis of Machado-Joseph disease

Front Mol Neurosci. 2023 May 19:16:1133271. doi: 10.3389/fnmol.2023.1133271. eCollection 2023.

Authors

Affiliations

¹ Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.
² Department of Human Genetics, Ruhr University Bochum, Bochum, Germany.
³ Proteome Center Tübingen, University of Tübingen, Tübingen, Germany.

Abstract

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein.

Keywords: Polyglutamine diseases; ataxin-3; deubiquitinase; posttranslational modification; proteasomal degradation; protein aggregation; spinocerebellar ataxia type 3; ubiquitination.