Redox environment modulates aggregation of ataxin-3 in vitro Implications for drug screening of cysteine-rich proteins

Spinocerebellar ataxia type 3 is a debilitating neurodegenerative disorderdriven by the pathological aggregation of ataxin-3 (Atx3), a deubiquitinat-ing enzyme with a cysteine-rich catalytic domain and an expandable poly-glutamine (polyQ) tract. While the role of polyQ expansion in Atx3aggregation is well documented, the influence of redox conditions on itsself-assembly remains underexplored. Here, we demonstrate that reducingagents and a redox environment critically modulate Atx3 aggregation byregulating disulfide bond formation within the Josephin domain. We dem-onstrate that dithiothreitol (DTT), through progressive oxidation, pro-motes the formation of non-native and disulfide-linked conformers, whichmay serve as nucleation centers for fibril formation. In contrast, tris(2-car-boxyethyl)phosphine preserves cysteine residues of Atx3 in the reducedstate and inhibits aggregation, but concomitantly promotes cleavage offull-length Atx3. Furthermore, we identify a previously underappreciatedrole for 4,5-dihydroxy-1,2-dithiane, the DTT oxidation product, in directlytriggering Atx3 aggregation. We also demonstrate that running aggregationassays at 50 °C circumvents the redox dependency of Atx3 aggregation,thereby streamlining the aggregation process and enabling the use of a sim-plified, robust platform for medium- to high-throughput screening ofaggregation modulators. These findings provide new insights into theredox-dependent modulation of Atx3 aggregation and highlight criticalconsiderations for in vitro aggregation assays of cysteine-rich proteins, withbroad implications for therapeutic strategies targeting cysteine-rich,aggregation-prone proteins in neurodegenerative diseases. Although ourstudy focuses on in vitro investigation, it suggests that redox dysregulation in cells could promote pathogenic aggregation of Atx3, reinforcing the linkbetween cellular redox balance and polyglutamine disease progression.