Chromatin Degradation During Apoptosis

描述(申请人提供)：染色体DNA片段化是细胞凋亡的关键步骤，它阻止细胞转录和复制其基因，从而促进细胞杀伤过程。这一过程中的缺陷会导致各种病理情况，包括自身免疫性疾病和癌症。在线虫中，我们已经鉴定了10种参与调节和执行细胞凋亡染色体碎裂的核酸酶和非核酸酶因子。它们以顺序和CED-3 caspase依赖的方式起作用，促进染色体的逐步碎裂和降解。这一过程是由一种新型的CED-3介导的二聚体核糖核酸酶(RNase)转化为脱氧核糖核酸酶(DNase)启动的，脱氧核糖核酸酶是对染色体的第一次切割。与此同时，线粒体核酸酶cps-6及其激活剂wah-1被从线粒体释放出来并转移到细胞核，在那里它们与其他细胞死亡核酸酶相互作用和合作，从而改变了由DICER进行的初始切割。 变成双链DNA断裂，导致染色体断裂和退化。在这项拟议的工作中，我们将进行分子遗传学、生化、细胞生物学和结构分析，以了解这两个关键的凋亡DNA降解事件。在目标1中，我们将研究CED-3介导的DICER RNase向DNase转化的分子和结构基础。在目标2中，我们将剖析控制细胞内钙离子增加和线粒体凋亡基因因子在细胞凋亡过程中释放的新的信号通路。在目标3中，我们将对两个新基因cps-13和cps-14进行分子遗传学和功能鉴定，它们调节和协调两个关键的细胞死亡执行事件，染色体断裂和磷脂酰丝氨酸(PS)外化。这些研究将揭示控制DICER核酸酶的特异性和功能切换的新机制，以及控制线粒体促凋亡因子释放的新的信号机制和角色。

DESCRIPTION (provided by applicant): Fragmentation of chromosomal DNA is a critical step in apoptosis that prevents a cell from transcribing and replicating its genes and thus facilitates the cell killing process. Defects in this process can cause various pathological conditions, including autoimmune disorders and cancer. We have identified ten apoptotic nucleases and several non-nuclease factors involved in regulating and executing apoptotic chromosome fragmentation in C. elegans. They act in a sequential and CED-3 caspase-dependent manner to promote stepwise fragmentation and degradation of chromosomes. The process is initiated by a novel CED-3-mediated conversion of the dicer ribonuclease (RNase) into a deoxyribonuclease (DNase), which makes the first cuts on chromosomes. In parallel, a mitochondrial nuclease CPS-6 and its activator WAH-1 are released from mitochondria and translocated to the nucleus, where they interact and cooperate with other cell death nucleases to turn the initial cuts by dicer into double-strand DNA breaks, leading to fragmentation and degradation of chromosomes. In this proposed work, we will carry out molecular genetic, biochemical, cell biological, and structural analyses to understand these two critical events of apoptotic DNA degradation. In Aim 1, we will investigate the molecular and structural basis underlying CED-3-mediated conversion of the dicer RNase into a DNase. In Aim 2, we will dissect the new signaling pathway that controls cytosolic calcium increase and release of the mitochondrial apoptogeneic factors during apoptosis. In Aim 3, we will perform molecular genetic and functional characterization of two new genes, cps-13 and cps-14, that regulate and coordinate two key cell death execution events, chromosome fragmentation and phosphatidylserine (PS) externalization. These studies should reveal the novel mechanism that controls the specificity and function switch of the dicer nuclease and new signaling mechanisms and players that control the release of the mitochondrial apoptogenic factors.