Nitrosylation of Cytochrome C during Apoptosis

描述(申请人提供)：细胞凋亡是一种受到严格控制的细胞死亡形式，它将多余或不需要的细胞从生物体中移除。细胞色素c在许多细胞凋亡级联反应中起关键作用。当线粒体收到凋亡信号时，细胞色素c从线粒体膜间间隙释放到细胞质中。胞质细胞色素c与Apaf-1和Caspase-9形成一个复合体，导致下游caspase的激活和随后的细胞凋亡。细胞色素c在细胞凋亡过程中的调节机制还不是很清楚。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是一种内源性气体，通过与过渡金属或蛋白质上的半胱氨酸残基结合来调节蛋白质的功能，这一过程被称为亚硝化。我们的初步数据表明，在Fas诱导的细胞凋亡过程中，细胞色素c被内源性亚硝化。我们的研究还表明，细胞色素c亚硝化增加了caspase的活性。这是细胞色素c在细胞凋亡过程中内源性翻译后修饰的首次证明。初步研究结果表明，细胞色素c亚硝化可能是一种新的细胞凋亡调控机制。这一假设将在拟议的研究中得到检验。在特定的目标1中，我们将确定细胞色素c是在线粒体中还是在细胞质中被亚硝化。在特定的目标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被亚硝化。在特定的目标3，我们将分析硝化细胞色素c在细胞凋亡中的作用。在特定的目标4中，我们将确定细胞色素c亚硝化是否是调节线粒体依赖性形式的细胞凋亡的一种普遍机制。这些研究的结果将确定细胞色素c亚硝化是否是一种调节细胞凋亡信号的新机制。最终，这些发现可能会导致开发合理的基于NO的疗法，用于治疗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退行性变。

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.