Peng, I. | Search bioRxiv

bioRxiv2025

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

AO_SCPLOWBSTRACTC_SCPLOWThe 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 CHIPs 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.

neuroscience↗

bioRxiv2025

Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering

Protein quality control is crucial for cellular homeostasis, involving the heat shock response, the ubiquitin-proteasome system, and the autophagy-lysosome pathway. Central to these systems are the chaperone homologs heat shock protein 70 (HSP70) and heat shock cognate 70 (HSC70), which manage protein folding and degradation. This study investigated the impact of the C-terminal phosphorylation of HSP70 on its interaction with the co-chaperone CHIP (C-terminus of HSC70 interacting protein), an E3 ligase that ubiquitinates protein substrates for degradation. Using both cell-free and cell-based approaches, including X-ray crystallography, biolayer interferometry, and live cell biocomplementation assays, we demonstrate that phosphorylation at HSP70 T636 reduces CHIPs binding affinity, shifting the preference toward other co-chaperones like HOP. Structural analysis reveals that phosphorylation disrupts key hydrogen bonds, altering binding dynamics. We engineered a CHIP variant (CHIP-G132N) to restore binding affinity to phosphorylated HSP70. While CHIP-G132N effectively restored binding without additional functional domains, its effectiveness was diminished in full-length phosphomimetic constructs in cell-free and in-cell assays, suggesting that additional interactions may influence binding. Functional assays indicate that phosphorylation of HSP70 affects its stability and degradation, with implications for diseases such as cancer and neurodegeneration. Our findings highlight the complexity of chaperone-co-chaperone interactions and underscore the importance of post-translational modifications in regulating protein quality control mechanisms. By elucidating the molecular details of HSP70 and CHIP interactions, our study provides a foundation for developing therapeutic interventions for diseases characterized by proteostasis imbalance.

biochemistry↗