Domain-dependent uncoupling of the co-chaperone and E3 ubiquitin ligase CHIP underlies heterogeneity in spinocerebellar ataxia 48.

The carboxyl terminus of Hsp70-interacting protein (CHIP, encoded by STUB1) combines co-chaperone and E3 ubiquitin ligase activities to regulate protein quality control. Heterozygous mutations in STUB1 cause spinocerebellar ataxia type 48 (SCA48), a progressive cerebellar ataxia with variable extrapyramidal and cognitive features. To understand the molecular basis of this variability, we systematically analyzed 13 SCA48-associated variants spanning the TPR and U-box domains through recombinant protein biochemistry and cellular models. TPR variants retained intrinsic ligase activity but showed significantly reduced HSP70 binding, impaired substrate ubiquitination, and decreased stability. Conversely, U-box variants abolished ligase function, promoted the formation of high-molecular-weight oligomers, and often increased CHIP levels while only partially impairing co-chaperone activity. Many mutants displayed temperature-sensitive defects and defective stress-induced nuclear translocation. Principal component analysis revealed distinct biochemical clustering specific to each domain. RNA-seq following STUB1 knockdown modeled CHIP insufficiency and showed preserved HSF1-dependent transactivation, but loss of CHIP's capacity to amplify ubiquitination, chaperone function, and stress-related transcriptional programs. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical signatures to upper motor neuron involvement and U-box-like profiles to prominent dysarthria. Overall, these data indicate that SCA48 results from domain-specific disruption of CHIP's dual functions, producing varying degrees of CHIP insufficiency and/or gain-of-toxic effects that together contribute to the phenotypic diversity observed across patients. This work refines the mechanistic framework for SCA48 pathogenesis and highlights strategies for therapeutic modulation of residual CHIP activity.