Abstract
The ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) is encoded by STUB1 and promotes ubiquitination of misfolded and damaged proteins. CHIP deficiency has been linked to several diseases, and mutations in the human STUB1 gene are associated with recessive and dominant forms of spinocerebellar ataxias (SCAR16/SCA48). Here, we examine the effects of impaired CHIP ubiquitin ligase activity in zebrafish (Danio rerio). We characterized the zebrafish stub1 gene and Chip protein, and generated and characterized a zebrafish mutant causing truncation of the Chip functional U-box domain. Zebrafish stub1 has a high degree of conservation with mammalian orthologs and was detected in a wide range of tissues in adult stages, with highest expression in brain, eggs, and testes. In the brain, stub1 mRNA was predominantly detected in the cerebellum, including the Purkinje cell layer and granular layer. Recombinant wild-type zebrafish Chip showed ubiquitin ligase activity highly comparable to human CHIP, while the mutant Chip protein showed impaired ubiquitination of the Hsc70 substrate and Chip itself. In contrast to SCAR16/SCA48 patients, no gross cerebellar atrophy was evident in mutant fish, however, these fish displayed reduced numbers and sizes of Purkinje cell bodies and abnormal organization of Purkinje cell dendrites. Mutant fish also had decreased total 26S proteasome activity in the brain and showed behavioral changes. In conclusion, truncation of the Chip U-box domain leads to impaired ubiquitin ligase activity and behavioral and anatomical changes in zebrafish, illustrating the potential of zebrafish to study STUB1-mediated diseases.
Highlights
C-terminus of Hsc70-interacting protein (CHIP), encoded by the STUB1 (STIP1 homology and U-box containing protein 1) gene, is a dimeric co-chaperone that negatively regulates the efficiency of Hsp70 and Hsp90 chaperones by interfering with the ATPase cycles of the folding process (Ballinger et al, 1999)
We identified one copy of the stub1 zebrafish gene in the zebrafish genome based on searches in Ensembl7 and Uniprot8 databases, our analysis refers to the gene annotated as “stub1” in the Ensembl genome database (ENSDARG00000045228, Ensembl release 102—November 2020)
The U-box domain seems to play a significant role in the pathogenesis of CHIP, and in the present study, we aimed to characterize the effects of truncation of this domain in zebrafish
Summary
C-terminus of Hsc70-interacting protein (CHIP), encoded by the STUB1 (STIP1 homology and U-box containing protein 1) gene, is a dimeric co-chaperone that negatively regulates the efficiency of Hsp and Hsp chaperones by interfering with the ATPase cycles of the folding process (Ballinger et al, 1999). CHIP was the first ubiquitin ligase to be directly associated with molecular chaperones (McDonough and Patterson, 2003). The N-terminal TPR (tetratricopeptide repeat) domain is responsible for the interaction of CHIP with molecular chaperones, while the C-terminal U-box domain acts as an E3 ubiquitin ligase and facilitates ubiquitination of chaperone substrates for further proteasomal degradation (McDonough and Patterson, 2003). Analysis of CHIP expression and localization in the rodent brain (Anderson et al, 2010; Shi et al, 2013) suggested co-localization of CHIP with the calciumbinding protein calbindin in cerebellar Purkinje cells of the cerebellum as the critical elements of precise motor coordination (Shi et al, 2013)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
