Abstract
Spinocerebellar ataxia, autosomal recessive 16 (SCAR16) is caused by biallelic mutations in the STIP1 homology and U-box containing protein 1 (STUB1) gene encoding the ubiquitin E3 ligase and dimeric co-chaperone C-terminus of Hsc70-interacting protein (CHIP). It has been proposed that the disease mechanism is related to CHIP’s impaired E3 ubiquitin ligase properties and/or interaction with its chaperones. However, there is limited knowledge on how these mutations affect the stability, folding, and protein structure of CHIP itself. To gain further insight, six previously reported pathogenic STUB1 variants (E28K, N65S, K145Q, M211I, S236T, and T246M) were expressed as recombinant proteins and studied using limited proteolysis, size-exclusion chromatography (SEC), and circular dichroism (CD). Our results reveal that N65S shows increased CHIP dimerization, higher levels of α-helical content, and decreased degradation rate compared with wild-type (WT) CHIP. By contrast, T246M demonstrates a strong tendency for aggregation, a more flexible protein structure, decreased levels of α-helical structures, and increased degradation rate compared with WT CHIP. E28K, K145Q, M211I, and S236T also show defects on structural properties compared with WT CHIP, although less profound than what observed for N65S and T246M. In conclusion, our results illustrate that some STUB1 mutations known to cause recessive SCAR16 have a profound impact on the protein structure, stability, and ability of CHIP to dimerize in vitro. These results add to the growing understanding on the mechanisms behind the disorder.
Highlights
The recessively inherited group of cerebellar ataxias is commonly characterized by early onset and gradual worsening of gait, balance, and coordination over months and years
STUB1 encodes STUB1 (STIP1 homology and U-box containing protein 1), known as C-terminus of Hsc70-interacting protein (CHIP), an evolutionary conserved protein of ∼35 kDa that is highly expressed as a dimeric co-chaperone in tissues that are active in terms of metabolism and protein turnover, such as brain, heart, and skeletal muscle
CHIP participates in the ubiquitination of unfolded or misfolded substrates bound to chaperones via its U-box domain
Summary
The recessively inherited group of cerebellar ataxias (autosomal recessive cerebellar ataxia, ARCA) is commonly characterized by early onset and gradual worsening of gait, balance, and coordination over months and years. STUB1 encodes STUB1 (STIP1 homology and U-box containing protein 1), known as C-terminus of Hsc70-interacting protein (CHIP), an evolutionary conserved protein of ∼35 kDa that is highly expressed as a dimeric co-chaperone in tissues that are active in terms of metabolism and protein turnover, such as brain, heart, and skeletal muscle. Further structural analysis revealed similarities between the C-terminus of CHIP (the U-box) and the E3 ligase component of the ubiquitin–proteasome pathway, suggesting an active role in ubiquitination of chaperone substrates for CHIP [12]. CHIP labels non-native proteins unable to be refolded by chaperones for proteasomal degradation [12,13,14]
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