Domain-specific decoupling of co-chaperone and ligase functions in STUB1 underlies biochemical and clinical heterogeneity in SCA48

The carboxyl terminus of HSC70-interacting protein (CHIP, encoded by STUB1 ) integrates co-chaperone and E3 ubiquitin ligase activities to maintain proteostasis. Heterozygous STUB1 mutations cause the dominant cerebellar ataxia SCA48 through incompletely understood mechanisms. We characterized 13 SCA48-associated variants in the TPR and U-box domains using recombinant protein and cellular assays. TPR mutations retained intrinsic ligase activity but lost HSC70 binding, substrate ubiquitination efficiency, and protein stability. Conversely, U-box mutations abolished ligase function, induced aberrant high-molecular-weight oligomerization, and frequently elevated steady-state CHIP levels while only partially impairing co-chaperone activity. Many variants exhibited temperature-sensitive defects and stress-induced nuclear mislocalization. Principal component analysis revealed robust domain-specific biochemical clustering. RNA-seq following STUB1 knockdown demonstrated preserved HSF1-dependent transactivation but a loss of CHIP-dependent amplification of ubiquitination, chaperone, and transcriptional pathways under heat stress. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical profiles to upper motor neuron involvement and U-box profiles to prominent dysarthria. Collectively, SCA48 mutations decouple CHIP’s dual functions in a domain-dependent manner, exerting dominant-negative or gain-of-toxic effects that drive the observed clinical heterogeneity. These findings establish a direct biochemical–clinical correlation in SCA48 and provide a framework for domain-targeted therapeutic strategies exploiting residual ligase or chaperone activity.