肝豆状核变性是以铜代谢障碍为特征的常染色体隐性遗传疾病，ATP7B基因突变导致的蛋白功能缺陷是其最主要的致病因素。COMMD1对ATP7B正常功能的发挥有着重要的调控作用，但具体的分子机制仍不明确。课题组前期发现，ATP7B基因3号外显子可能与COMMD1的调控相关。因此，本项目拟针对影响该外显子的剪接突变c.1286-2A>G、c.1543+1G>C，以体外转录实验确认其造成的ATP7B基因剪接改变，同时在体内及体外模型中观察突变造成的蛋白功能变化。以Co-IP检测不同铜离子浓度下野生型及突变型ATP7B与COMMD1之间相互作用的差异，并鉴定ATP7B的泛素化位点。利用质谱搜索并验证特异的泛素连接酶及其它潜在的相互作用蛋白。本研究旨在揭示3号外显子的剪接突变通过影响COMMD1诱导的ATP7B泛素化而导致肝豆状核变性的分子机制，为临床疾病诊断及判断病情严重程度提供有价值的参考信息。

Hepatolenticular degeneration, an autosomal recessive genetic disease characterized by copper metabolism disorder, mainly caused by the dysfunction of mutant ATP7B variants. COMMD1 plays an important role in the regulation of ATP7B function, but the exact molecular mechanism is still unknown. Our previous studies indicated that the third exon of ATP7B gene might involved in the regulation rules of COMMD1. Therefore, this project attempts to focus on the splicing mutations c.1286-2A>G and c.1543+1G>C, which both affect the alternative splicing of ATP7B exon 3. We use in vitro transcription assay to confirm the novel ATP7B variants that generated by these splicing mutations, followed by the exploration of the mutant variants’ function in both cell line and animal models. Co-IP will be performed to detect the protein-protein interactions between wild-type/mutant ATP7B and COMMD1 in HepG2 cells co-cultured with different concentrations of CuCl2 solution, and the ubiquitination site of ATP7B will be detected. Besides, Co-IP-MS will be used to search for the specific E3 ubiquitin ligases and other potential interacting proteins of ATP7B. This project will reveal the molecular mechanisms of hepatolenticular degeneration that caused by ATP7B splicing mutations, mainly through regulating the COMMD1-mediated ubiquitination of ATP7B, and provide valuable information for the diagnosis and prognosis.