Study of CED-4 translocation during apoptosis

描述(由申请人提供)： 细胞程序性死亡或凋亡在组织内稳态和生物体的正常发育中起着至关重要的作用。细胞死亡的异常调节可能导致人类疾病的发展，包括癌症、神经退行性疾病和自身免疫性疾病。因此，必须严格控制细胞死亡的调控。在线虫中，内源性CED-4定位于线粒体与CED-9蛋白的复合体中，从而拮抗CED-3和CED-4在非凋亡胚胎中的促死亡活性。然而，在被诱导死亡的胚胎中，CED-4移位到核膜上。虽然CED-4在核膜上的重新定位与细胞程序性死亡相一致，但尚不清楚这一过程是激活CED-3还是执行细胞凋亡所必需的。在这项提案中，我将启动研究，以确定CED-4在细胞凋亡过程中向核膜转运的机制。通过错误定位CED-4：：GFP的筛选，我们分离到了四个小突变体。我将通过遗传、分子和生化分析来确定这些突变体的特征，首先检查它们对核凋亡事件的影响。然后，将通过粘粒救援法克隆MIC-1(sm]58)和Mic-2(Sm160)。最后，我建议确定MIC基因是否与CED-4在复合体中起作用，以及在凋亡过程中，转移到细胞核的其他蛋白质是否受到MIC基因的影响。通过这些研究，我希望了解CED-4移位到细胞核的机制，并确定这一事件在细胞程序性死亡中的作用。

DESCRIPTION (provided by applicant): Programmed cell death or apoptosis plays an essential role in tissue homeostasis and proper development of an organism. The aberrant regulation of cell death may lead to the development of human diseases including cancer, neurodegenerative and autoimmune diseases. Thus, the regulation of cell death must be tightly controlled. In C. elegans, endogenous CED-4 localizes to the mitochondria in a complex with the CED-9 protein, which antagonizes the pro-death activity of CED-3 and CED-4, in non-apoptotic embryos. However, in embryos induced to die, CED-4 translocates to the nuclear envelope. Although the relocalization of CED-4 to the nuclear envelope coincides with programmed cell death it is unclear whether this process is required for the activation of CED-3 or the execution of apoptosis. In this proposal, I will initiate studies to determine the mechanism of CED-4 transport to the nuclear envelope during apoptosis. Using a screen for mislocalized CED-4::GFP we have isolated four mic mutants. I will characterize through genetic, molecular, and biochemical analysis these mutants, first examining their affect on nuclear apoptotic events. Then mic-1(sm] 58) and mic-2 (sm160) will be cloned by cosmid rescue. Finally, I propose to determine whether the mic genes act in a complex with CED-4 and if during apoptosis other proteins that translocate to the nucleus are affected by the mic genes. Through these studies I hope to understand the mechanism underlying CED-4 translocation to the nucleus and determine the role of this event in programmed cell death.