Redox environment modulates aggregation of ataxin‐3 <i>in vitro</i> — Implications for drug screening of cysteine‐rich proteins

Spinocerebellar ataxia type 3 is a debilitating neurodegenerative disorder driven by the pathological aggregation of ataxin-3 (Atx3), a deubiquitinating enzyme with a cysteine-rich catalytic domain and an expandable polyglutamine (polyQ) tract. While the role of polyQ expansion in Atx3 aggregation is well documented, the influence of redox conditions on its self-assembly remains underexplored. Here, we demonstrate that reducing agents and a redox environment critically modulate Atx3 aggregation by regulating disulfide bond formation within the Josephin domain. We demonstrate that dithiothreitol (DTT), through progressive oxidation, promotes the formation of non-native and disulfide-linked conformers, which may serve as nucleation centers for fibril formation. In contrast, tris(2-carboxyethyl)phosphine preserves cysteine residues of Atx3 in the reduced state and inhibits aggregation, but concomitantly promotes cleavage of full-length Atx3. Furthermore, we identify a previously underappreciated role for 4,5-dihydroxy-1,2-dithiane, the DTT oxidation product, in directly triggering Atx3 aggregation. We also demonstrate that running aggregation assays at 50 °C circumvents the redox dependency of Atx3 aggregation, thereby streamlining the aggregation process and enabling the use of a simplified, robust platform for medium- to high-throughput screening of aggregation modulators. These findings provide new insights into the redox-dependent modulation of Atx3 aggregation and highlight critical considerations for in vitro aggregation assays of cysteine-rich proteins, with broad implications for therapeutic strategies targeting cysteine-rich, aggregation-prone proteins in neurodegenerative diseases. Although our study focuses on in vitro investigation, it suggests that redox dysregulation in cells could promote pathogenic aggregation of Atx3, reinforcing the link between cellular redox balance and polyglutamine disease progression.