Nitrosylation of Cytochrome C during Apoptosis

描述（由申请人提供）：细胞凋亡是一种严格调控的细胞死亡形式，可从生物体中清除多余或不需要的细胞。细胞色素c在许多凋亡级联反应中起关键作用。当线粒体接收到凋亡信号时，细胞色素c从线粒体膜间隙释放到细胞质中。细胞质细胞色素c与Apaf-1和半胱天冬酶-9形成复合物，导致下游半胱天冬酶的激活和随后的凋亡性细胞死亡。细胞凋亡过程中调节细胞色素c功能的机制知之甚少。在我们的初步研究中，我们研究了一氧化氮（NO）在细胞色素c调节中的作用。NO是一种内源性产生的气体，通过与蛋白质上的过渡金属或半胱氨酸残基结合来调节蛋白质功能，这一过程称为亚硝基化。我们的初步数据表明，细胞色素c是内源性亚硝基化Fas诱导的细胞凋亡。我们的研究还表明，细胞色素c亚硝基化增加半胱天冬酶的激活。这是第一次证明细胞色素c在细胞凋亡过程中的内源性翻译后修饰。初步研究结果提出了细胞色素c亚硝基化是一种新的细胞凋亡调控机制的可能性。这一假设将在拟定的研究中进行检验。在具体目标1中，我们将确定细胞色素c是否在线粒体或细胞质中被亚硝基化。在具体目标2中，我们将确定细胞色素c是否在血红素或半胱氨酸残基上亚硝基化。在具体目标3中，我们将分析亚硝基化细胞色素c在细胞凋亡过程中的功能。在具体目标4中，我们将确定细胞色素c亚硝基化是否是一种调节细胞凋亡依赖性形式的普遍机制。这些研究的结果将确定细胞色素c亚硝基化是否是一种调节凋亡信号的新机制。最终，这些发现可能导致合理的基于NO的治疗方法的发展，这些治疗方法用于与细胞凋亡失调相关的疾病，包括癌症，自身免疫性疾病和神经变性。

DESCRIPTION (provided by applicant): Apoptosis is a tightly regulated form of cell death that removes excess or unwanted cells from organisms. Cytochrome c plays a critical role in many apoptotic cascades. When mitochondria receive an apoptotic signal, cytochrome c is released from the mitochondrial intermembrane space into the cytoplasm. Cytoplasmic cytochrome c forms a complex with Apaf-1 and caspase-9 leading to the activation of downstream caspases and subsequent apoptotic cell death. The mechanisms regulating cytochrome c function during apoptosis are poorly understood. In our preliminary studies we investigated the role of nitric oxide (NO) in cytochrome c regulation. NO is an endogenously produced gas that regulates protein function by binding to transition metals or cysteine residues on proteins, a process called nitrosylation. Our preliminary data suggests that cytochrome c is endogenously nitrosylated during Fas-induced apoptosis. Our studies also suggest that cytochrome c nitrosylation increases caspase activation. This is the first demonstration of an endogenous posttranslational modification of cytochrome c during apoptosis. The preliminary findings raise the possibility that cytochrome c nitrosylation is a novel mechanism of apoptosis regulation. This hypothesis will be tested in the proposed studies. In Specific Aim 1 we will determine if cytochrome c is nitrosylated in mitochondria or in the cytoplasm. In Specific Aim 2 we will determine if cytochrome c is nitrosylated on a heme or a cysteine residue. In Specific Aim 3 we will analyze the function of nitrosylated cytochrome c during apoptosis. In Specific Aim 4 we will determine if cytochrome c nitrosylation is a generalized mechanism regulating mitochondria-dependent forms of apoptosis. The results of these studies will determine if cytochrome c nitrosylation is a novel mechanism regulating apoptotic signaling. Ultimately the findings may lead to the development of rational NO-based therapies for diseases associated with dysregulated apoptosis including cancer, autoimmune disease and neurodegeneration.