线粒体蛋白ISCA2突变通过[4Fe-4S]合成缺陷介导脑病发生的机制研究

Iron-sulfur clusters are involved in many important physiological processes ranging from electron transport to enzyme reaction catalyzation. Mitochondrial iron-sulfur cluster assembly protein ISCA2 G77S mutation would lead to a clinical manifestation which is similar to the mitochondria encephalopathy caused by mutation in complex-Ⅰsubunits which contain [4Fe-4S] cluster. Recently, our group conducted experiments and identified that ISCA2 protein can bind both mononuclear iron and the active [2Fe-2S] cluster. G77S mutation of ISCA2 selectively affects the [2Fe-2S] cluster binding activity of protein. In Hela cells, ISCA2 knockdown led to the decline of oxidative phosphorylation activity and down-regulation of subunits containing [4Fe-4S] cluster in respiratory chain. So we propose a hypothesis that the [2Fe-2S] cluster binding in ISCA2 is involved in the process of [4Fe-4S] cluster assembly in mitochondria. Deficiency of ISCA2 protein may lead to defect of [4Fe-4S] cluster biogenesis, which affect the function of oxidative phosphorylation (OXPHOS) and eventually contribute to encephalopathy. Our work will clarify the function of ISCA2 in the assembly of [4Fe-4S] cluster, and further analyse the importance of iron-sulfur cluster assembly function in mitochondrial oxidative phosphorylation and energy metabolism. At last, we will construct ISCA2 conditional knockout mouse model to observe whether ISCA2 knockout in nervous system would lead to mitochondria encephalopathy through the way of blocking the assembly of [4Fe-4S] cluster in mitochondrial. If the work finished, we may demonstrate the pathogenic mechanism of mitochondrial encephalopathy caused by ISCA2-mutation-mediated defect of [4Fe-4S] cluster biogenesis.

幼儿线粒体铁硫簇组装蛋白ISCA2 G77S突变引起的临床表现同呼吸链复合体中[4Fe-4S]亚基缺陷所致的线粒体脑病相似，但机制不清楚。课题组前期工作显示G77S突变选择性影响蛋白质结合[2Fe-2S]，而敲低Hela细胞ISCA2导致复合体活性降低及含[4Fe-4S]亚基下调，为此假设：①ISCA2中[2Fe-2S]的结合参与线粒体中[4Fe-4S]生成；②ISCA2缺陷能引起[4Fe-4S]合成障碍，进而影响氧化磷酸化系统最终促进脑病发生。本项目计划：①蛋白水平阐述ISCA2蛋白在铁硫簇组装中的功能；②细胞水平阐明ISCA2铁硫簇组装功能及在氧化磷酸化和能量代谢中的地位；③动物水平验证ISCA2条件性敲除是否会通过阻断铁硫簇组装影响线粒体能量代谢导致线粒体脑病。预期阐明ISCA2参与[4Fe-4S]组装及缺陷导致线粒体脑病发生的机制，从而为铁硫簇缺陷引起脑病的精准诊治提供理论基础。

ISCA2 G77S突变是一种常染色体隐性遗传病，它的临床症状与线粒体[4Fe-4S]型蛋白亚基缺陷所引起的线粒体脑病和肌病相似，但具体机制并不清楚。本项目主要从以下三个层面进行了研究：①蛋白水平阐述ISCA2蛋白在铁硫簇组装中的功能以及G77S突变生化功能的改变。在大肠杆菌中表达纯化ISCA2蛋白，通过紫外光谱和EPR分析发现它不仅具有[2Fe-2S]簇结合能力同时还有铁结合能力；通过同样的方式我们分析ISCA2 G77S突变蛋白的金属结合特性及其功能，发现G77S突变蛋白失去了[2Fe-2S]簇的结合能力，此外，我们还通过体外铁硫簇组装和传递实验证明了G77S突变对蛋白[2Fe-2S]簇的组装和传递功能具有抑制作用；②细胞水平阐明了ISCA2线粒体内[4Fe-4S]簇组装功能及在线粒体氧化磷酸化和能量代谢中的地位，同时验证了蛋白G77S位点在细胞功能的重要作用。我们利用基因敲除技术敲除Hela细胞中的ISCA2蛋白，发现ISCA2敲除的细胞线粒体内含[4Fe-4S]簇铁硫蛋白产量减少，线粒体氧化磷酸化能力显著降低。然后，我们在敲除的Hela细胞中回补野生型ISCA2和G77S突变蛋白，发现G77S突变蛋白不能像野生型蛋白一样恢复ISCA2蛋白的正常功能；③动物水平构建了ISCA2蛋白G77S点突变基因敲入小鼠模型，发现G77S突变通过阻断线粒体[4Fe-4S]簇组装影响线粒体能量代谢导致小鼠发生线粒体肌病、脑病等表型。从而阐述了ISCA2缺陷导致线粒体脑病的具体机制，为临床诊断、预防和治疗线粒体脑病提供理论基础和新方向。.本课题累计发表学术论文6篇，其中SCI收录4篇（均为二区TOP期刊），授权专利2项，培养研究生4人（已毕业3人），参加国内外学术交流4次，含国际会议分组报告1次。

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