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

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.

The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases

“…As the K8 residue is located in close proximity to the critical C14 residue of the catalytic Josephin domain, it has also been suggested that enhanced ubiquitination at this site may impact polyQ-expanded ataxin-3's DUB function [ 77 ]. Ubiquitination of ataxin-3 at the K8 residue has also been suggested by a recent study [ 107 ].…”

“…Ataxin-3's UIMs therefore appear to be critical to its DUB function. In support of this, the K85 residue (located between ubiquitin-binding sites 1 and 2) is important for ataxin-3's binding and processing of K48-linked polyubiquitin chains [ 107 ] through the previously described mechanism involving the UIMs and site 2 [ 108 ]. K85 is also important for cytoplasmic localisation of ataxin-3 [ 107 ].…”

“…Compared with its wild-type counterpart, polyQ-expanded ataxin-3 appears to be ubiquitinated to a greater extent at the K8 lysine residue [ 77 ]. The K8 and K85 residues appear to be important for the proteostasis of ataxin-3 as the presence of either residue within ataxin-3 increases the stability of the protein compared with ataxin-3 with all lysine residues mutated to arginine [ 107 ]. As the K8 residue is located in close proximity to the critical C14 residue of the catalytic Josephin domain, it has also been suggested that enhanced ubiquitination at this site may impact polyQ-expanded ataxin-3's DUB function [ 77 ].…”