Modification of human mitoNEET [2Fe-2S] cluster under nitric oxide stress

描述（由申请人提供）：mitoNEET是II型糖尿病药物吡格列酮的新靶点，并通过N-末端跨膜α-螺旋定位于线粒体外膜。遗传学研究表明，mitoNEET对线粒体中的能量代谢具有至关重要的调节作用，并且不仅与II型糖尿病有关，还与人类乳腺癌增殖和神经退行性疾病有关。可溶性C-末端mitoNEET含有一个[2Fe-2S]簇，具有三个半胱氨酸和一个组氨酸残基的不寻常配体排列。然而，mitoNEET的具体功能在很大程度上仍然难以捉摸。初步研究表明，当在大肠杆菌细胞中表达时，mitoNEET [2Fe-2S]簇被完全还原，并且还原的mitoNEET [2Fe-2S]簇对一氧化氮高度敏感，一氧化氮是一种与包括糖尿病在内的许多人类疾病相关的生理自由基。暴露于一氧化氮后，mitoNEET [2Fe-2S]簇容易被修饰，形成mitoNEET结合的二亚硝酰铁络合物（DNIC）。本申请的目的是检验一种假设，即mitoNEET [2Fe-2S]簇可以作为一种新型的一氧化氮传感器来调节线粒体中的能量代谢。有两个具体目标：目标1是确定mitoNEET [2Fe-2S]簇与一氧化氮的特异性反应性。拟议的研究将确定还原的mitoNEET [2Fe-2S]簇与一氧化氮之间的反应动力学，并研究一氧化氮对培养细胞中mitoNEET [2Fe-2S]簇的修饰。还将探索II型糖尿病药物吡格列酮对一氧化氮介导的mitoNEET [2Fe-2S]簇修饰的潜在调节。目的二是研究一氧化氮修饰的[2Fe-2S]团簇在线粒体NEET中的修复机制。为了修复mitoNEET中的一氧化氮修饰的[2Fe-2S]簇，必须在组装新的[2Fe-2S]簇之前去除蛋白质中修饰的损伤二亚硝酰铁复合物（DNIC）。拟议的实验将集中在如何DNIC在mitoNEET可以通过生物硫醇通过硫醇配体交换去除。生物化学分析将与体内siRNA方法相结合，以研究生物硫醇在介导从mitoNEET去除DNIC中的特异性活性和生理相关性。该研究的成功有望阐明线粒体中NO通过mitoNEET [2Fe-2S]簇的潜在新信号通路，通过mitoNEET [2Fe-2S]簇的修饰建立NO信号与II型糖尿病之间的联系，并为开发II型糖尿病的新治疗方法提供信息。这些结果还将为揭示两种与mitoNEET相关的线粒体蛋白--Wolfram Syndrome 2相关的Miner 1和功能未知的蛋白质Miner 2在人类细胞中的潜在调控作用奠定基础。

DESCRIPTION (provided by applicant): mitoNEET is a novel target of type II diabetes drug pioglitazone, and localizes on mitochondrial outer membrane via the N-terminal transmembrane a-helix. Genetics studies have shown that mitoNEET has a crucial regulatory role for energy metabolism in mitochondria, and has been associated with not only type II diabetes, but also human breast cancer proliferation and neurodegenerative diseases. The soluble C-terminal mitoNEET contains a [2Fe-2S] cluster with an unusual ligand arrangement of three cysteine and one histidine residues. Nevertheless, specific function of mitoNEET remains largely elusive. Preliminary studies indicated that the mitoNEET [2Fe-2S] clusters are fully reduced when expressed in Escherichia coli cells, and that the reduced mitoNEET [2Fe-2S] clusters are highly sensitive to nitric oxide, a physiological free radical that has been associated with a number of human diseases including diabetes. Upon exposure to nitric oxide, the mitoNEET [2Fe-2S] clusters are readily modified forming the mitoNEET-bound dinitrosyl iron complex (DNIC). The goal of this application is to test a hypothesis that the mitoNEET [2Fe-2S] cluster may act as a novel sensor of nitric oxide to modulate energy metabolism in mitochondria. There are two specific aims: Aim 1 is to determine the specific reactivity of the mitoNEET [2Fe-2S] clusters with nitric oxide. The proposed research will determine the kinetics of the reaction between the reduced mitoNEET [2Fe-2S] clusters and nitric oxide and investigate modification of the mitoNEET [2Fe-2S] clusters in cultured cells by nitric oxide. Potential regulation of the nitric oxide-mediated modification of the mitoNEET [2Fe-2S] clusters by the type II diabetes drug pioglitazone will also be explored. Aim 2 is to investigate the repair mechanism for the nitric oxide-modified [2Fe-2S] clusters in mitoNEET. To repair the nitric oxide-modified [2Fe-2S] clusters in mitoNEET, the modified lesion dinitrosyl iron complex (DNIC) in the protein must be removed before a new [2Fe-2S] cluster may be assembled. The proposed experiments will focus on how DNIC in mitoNEET may be removed by biological thiols via thiol ligand exchange. Biochemical analyses will be combined with the in vivo siRNA approaches to investigate the specific activity and physiological relevance of biological thiols in mediating removal of DNIC from mitoNEET. Success of the proposed research is expected to illustrate a potentially novel signal pathway of NO in mitochondria via the mitoNEET [2Fe-2S] clusters, to establish a link between NO signal and type II diabetes via the modification of the mitoNEET [2Fe-2S] clusters, and to provide information for developing new therapeutic treatments for type II diabetes. The results will also lay groundwork to unravel the potential regulatory roles of two mitoNEET-related mitochondrial proteins, the Wolfram Syndrome 2-related Miner1 and the function unknown protein Miner2, in human cells.