铁硫簇组装蛋白hIscA参与Wilson病中铜的细胞毒性机制研究

Affected Wilson's disease patients suffer from hepatic disorders and severe neurological defects. Wilson's disease is an autosomal recessive disorder in which the liver does not properly release copper into bile, resulting in prominent copper accumulation in various tissues. But the mechanism of toxicity of copper is not clear, the paper published in MOL MICROBIOL by our team recently claimed that the main toxicity of copper in E.coli is mediated by iron-sulfur assemble protein IscA. Copper can compete with iron for the metal binding site in IscA and effectively inhibit the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. Preliminary experiments indicate that human IscA(hIscA) which is homology with IscA in E.coli has a strong and specific binding activity for Cu in vitro. And the HepG2 cells with copper overload fail to assemble [4Fe-4S] cluster in proteins such as aconitase leading to a decreasing of the activity of [4Fe-4S] type iron-sulfur proteins, but it doesn’t affect the activity of [2Fe-2S] type iron-sulfur protein. what’s more ,the activity of respiratory chain complexes I-III which contain iron-sulfur protein subunits also decreased in high copper HepG2 cells. All this changes in the copper overload cells is similar to the phenotype of hIscA RNA interference cells. So we raise the hypothesis that the molecular mechanism of copper overload toxicity like Wilson disease is similar to our findings in E.coli. To investigate the hypothesis, we conduct a systematic project using the successful established methods in analyzing the assemble of iron-sulfur clusters, explaining how the overloading copper affects the assemble of iron-sulfur clusters. This will not only provide a new theoretical basis for the pathogenesis of Wilson disease at the molecular level, but also offer significant clues and information in treating the disease and developing new drugs.

Wilson病因铜代谢异常而主要累积于肝脏和豆状核引起变性，其中铜介导细胞毒性的具体分子机制还不完全清楚。申请者新近发表在MOL MICROBIOL上的论文阐述了铜与大肠杆菌铁硫簇组装蛋白IscA结合而引起其介导的[4Fe-4S]簇组装障碍的铜杀菌机制。预实验结果显示序列、结构和功能均与大肠杆菌IscA高度保守的人同源蛋白hIscA同样具有铜的结合能力，且铜过载的HepG2细胞内出现aconitase酶中[4Fe-4S]簇组装障碍和含[4Fe-4S]簇的复合体活性下降等类似于低表hIscA的表型。因此假设Wilson病中过多的铜导致的细胞毒性的分子机制可能与在大肠杆菌中的发现相似，本研究拟利用本实验室已成功建立的细胞和线虫铁硫簇组装研究手段系统研究验证假设，预期从蛋白-细胞-线虫-临床等不同水平系统阐明人线粒体蛋白hIscA介导的Wilson 病中铜累积导致的细胞毒性的具体分子机制。

铁硫簇组装蛋白hIscA参与Wilson病中铜的细胞毒性机制研究，本课题运用生物信息学、蛋白质生物化学、细胞生物学等方法与技术，对铜参与破坏铁硫簇组装的分子机制进行了初步研究。我们通过体外纯化真核细胞铁硫簇组装蛋白，进行体外实验表明：体外条件下过高的铜离子会结合在铁硫簇组装蛋白上影响铁硫簇合成。另外我们得到ATP7A突变的Menkes病人淋巴细胞，在培养基中加入少量铜离子即会影响其铁硫簇组装，在构建的高铜肝细胞HepG2细胞模型中我们也得到了相同的实验结果。在动物水平上，我们实验结果表明：在Wilson 疾病模型小鼠中，铜离子累积在小鼠肝脏中，高铜通过影响铁硫簇组装影响相关铁硫蛋白表达，最后影响线粒体功能导致肝细胞损伤。.我们从蛋白-细胞-动物三个不同水平系统阐明人铁硫簇组装蛋白ISCU、ISCA1及ISCA2介导的铜毒性的分子机制。这将为Wilson 病等人类铜代谢紊乱性疾病的致病机制从分子水平提供崭新的理论基础并有望找到新的药物治疗靶点。.在项目资助下，发表期刊论文5篇（含接收一篇），会议论文3篇，其中SCI收录TOP期刊2篇，关于铜离子影响铁硫簇组装成果部分论文还在撰写中；申请国家发明专利5项，其中1项已授权；培养硕士生3名。