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.

泛素连接酶 CHIP（Hsc70 相互作用蛋白的 C 端）由 存根1并促进错误折叠和受损蛋白质的泛素化。CHIP 缺陷与多种疾病和人类基因突变有关存根1基因与隐性和显性形式的脊髓小脑性共济失调 (SCAR16/SCA48) 相关。在这里，我们检查了斑马鱼中 CHIP 泛素连接酶活性受损的影响。达尼奥雷里奥）。我们对斑马鱼进行了表征存根1基因和芯片蛋白，并生成并表征了导致芯片功能 U 盒域截断的斑马鱼突变体。斑马鱼存根1与哺乳动物直向同源物具有高度保守性，并在成年阶段的多种组织中检测到，在脑、卵和睾丸中表达最高。在大脑中，存根1mRNA 主要在小脑中检测到，包括浦肯野细胞层和颗粒层。重组野生型斑马鱼芯片显示出与人类芯片高度可比的泛素连接酶活性，而突变芯片蛋白显示出 Hsc70 底物和芯片本身的泛素化受损。与 SCAR16/SCA48 患者相比，突变鱼没有明显的小脑萎缩，然而，这些鱼表现出浦肯野细胞体的数量和大小减少以及浦肯野细胞树突的异常组织。突变鱼也降低了大脑中的总 26S 蛋白酶体活性，并表现出行为变化。总之，Chip U-box 结构域的截断导致斑马鱼泛素连接酶活性受损以及行为和解剖学变化，说明斑马鱼的研究潜力存根1-介导的疾病。