Nitric Oxide Cytotoxicity and Iron-Sulfur Proteins

描述（由申请人提供）：NO的过量产生与神经退行性疾病、心血管氧化损伤和癌症的发生有关。本研究旨在验证一个假设，即NO对蛋白质中铁硫簇的修饰代表了NO细胞毒性的急性细胞氧化损伤。与血红素中可逆的NO结合不同，NO破坏铁硫簇形成稳定的蛋白质结合二硝基铁络合物（DNIC）。由于铁硫蛋白参与多种生物过程，主要涉及能量转换、DNA修复、氨基酸代谢、血红素和生物素的生物合成以及铁的体内平衡，因此NO修饰铁硫簇可能导致多种细胞功能的失败，最终导致癌症等人类疾病的发生。该提案的总体目标是研究1)NO对铁硫团簇生物修饰的氧化还原反应，以及2)NO修饰铁硫团簇修复的细胞机制。目的1是利用放射性标记的铁硫团簇定量分析铁硫团簇释放的铁和硫化物以及NO对蛋白质结合DNIC的形成。目的2是确定铁硫簇和小分子硫醇在大肠杆菌和培养的人成纤维细胞中与NO的相对反应性，并使用蛋白质组学方法鉴定大肠杆菌细胞中特定的NO修饰铁硫蛋白。目的3是研究l -半胱氨酸介导的蛋白质结合DNIC分解的氧化还原反应，基于初步研究表明l -半胱氨酸可以分解蛋白质结合的DNIC并促进蛋白质中新的铁硫簇的重组。目的4是探索l -半胱氨酸在大肠杆菌细胞中修复蛋白结合DNIC的生理作用，其中细胞内l -半胱氨酸含量将被调节。该研究如果成功，将为开发预防或减轻NO细胞毒性造成的细胞氧化损伤的治疗方法提供基础知识。

DESCRIPTION (provided by applicant): Excessive production of NO has been implicated in causing neurodegenerative diseases, cardiovascular oxidative injuries and cancers. This proposal is aimed to test a hypothesis that modification of iron-sulfur clusters in proteins by NO represents an acute cellular oxidative damage of NO cytotoxicity. Unlike the reversible NO binding in heme, NO disrupts iron-sulfur clusters forming the stable protein-bound dinitrosyl iron complex (DNIC). As iron-sulfur proteins are involved in diverse biological processes, primarily in energy conversion, DNA repair, amino acid metabolism, heme and biotin biosynthesis and iron homeostasis, modification of iron-sulfur clusters by NO could lead to failure of multiple cellular functions and eventually contribute to development of human diseases such as cancer. The overall goals of the proposal are to investigate 1) the redox reaction underlying the biological modification of iron-sulfur clusters by NO, and 2) the cellular mechanism by which the NO-modified iron-sulfur clusters are repaired. Aim 1 is to quantitatively analyze the iron and sulfide released from iron-sulfur clusters and formation of the protein-bound DNIC by NO using the radioactive labeled iron-sulfur clusters. Aim 2 is to determine the relative reactivity of iron sulfur clusters and small molecular thiols with NO in both E. coli and cultured human fibroblast cells and to identify specific NO-modified iron-sulfur proteins in E. coli cells using the proteomic approaches. Aim 3 is to nvestigate the redox reactions of the L-cysteine-mediated decomposition of the protein-bound DNIC, based on preliminary studies showing that L-cysteine can decompose the protein-bound DNIC and facilitate reassembly of new iron-sulfur clusters in the proteins. Aim 4 is to explore the physiological role of L-cysteine in repairing the protein-bound DNIC in the E. coli cells in which the intracellular L-cysteine content will be modulated. The proposed research, if successful, will provide fundamental knowledge for developing therapeutic approaches to prevent or alleviate cellular oxidative damages inflicted by NO cytotoxicity.