Dysfunction of the protein quality control system in neurodegeneration: A study of the co-chaperone and ubiquitin ligase CHIP in vitro and in zebrafish

Abstract

The C-terminus of Hsc70 interacting protein (CHIP) is a dimeric co-chaperone and E3 ubiquitin ligase encoded by the STIP1 homology and U-box containing protein 1 (STUB1) gene. As a major player in the protein quality control system, CHIP acts to regulate ubiquitination and elimination of chaperone-bound, misfolded and damaged proteins. The primary structure of CHIP consists of an N-terminal tetratricopeptide repeat (TPR) domain which mediates interaction of CHIP with molecular chaperones and a C-terminal U-box domain that functions as E3 ubiquitin ligase and facilitates ubiquitination of substrates. A central coiled coil region is responsible for dimerization and stability of CHIP structure.

Several bi-allelic mutations, scattered over all domains of the STUB1 gene, are reported in patients with the autosomal recessive spinocerebellar ataxia type 16 (SCAR16) disease. SCAR16 is characterized by early-onset and progressive cerebellar atrophy, mainly resulting in gait disturbance and lack of coordination in muscles. We studied the pathogenic effect of six selected STUB1 mutations in a heterologous system and reported profound impacts for some of these mutations on the ubiquitin ligase activity, structural stability and dimerization of CHIP.

Heterozygous mutations in the STUB1 gene have been lately described in association with a dominantly inherited cerebellar ataxia disease called spinocerebellar ataxia type 48 (SCA48). SCA48 patients present adult-onset ataxia phenotypes often associated with cognitive-psychiatric features. We reported novel STUB1 mutations in three families affected with SCA48. In vitro functional analyses of these variants revealed significant biochemical impacts on CHIP structure and function, similar to those previously reported for the recessive STUB1 variants associated with SCAR16. However, the healthy status of SCAR16 carriers is not consistent with the reported pathogenicity of heterozygous STUB1 variants in SCA48 patients, and should be further investigated.

Zebrafish animal models are being increasingly utilized in the genetic studies of neurodegeneration due to the strong similarity of their genome to that of mammalian orthologs, and a simple yet highly conserved structure of their nervous system. There is a high degree of conservation in the amino acid sequence of CHIP as well as its ubiquitin ligase activity and expression pattern between zebrafish and human orthologs. We have generated zebrafish animals with mutated STUB1 gene (stub1), causing a truncation in the U-box domain of Chip. Characterization of the mutant zebrafish indicated development of phenotypes overlapping with those of mammalian models with CHIP deficiency and SCAR16 patients. These phenotypes include significant reduction of body size, altered morphology of Purkinje cells of cerebellum and changes in anxietylike behavior. Our findings provide the first evidence of zebrafish potential in modeling STUB1-mediated diseases, and may improve our knowledge regarding the pathogenic role of an impaired U-box domain in the development of SCAR16/SCA48 diseases.