APOPAIN AND PARP IN RADIATION INDUCED APOPTOSIS

APOPAIN 和 PARP 在辐射诱导细胞凋亡中的作用

• 批准号: 6443859
• 负责人: MARK SMULSON
• 金额: $ 34.88万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-02 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6443859
• 关键词: ADP ribosylation; DNA binding protein; DNA damage; DNA topoisomerases; NAD nucleosidase; adenosine triphosphate; apoptosis; cell line; cellular pathology; chemical cleavage; endonuclease; enzyme activity; enzyme inhibitors; immunoprecipitation; molecular cloning; molecular pathology; neoplastic cell culture for noncancer research; neoplastic growth; nucleic acid sequence; protein degradation; transfection; western blottings

Poly (ADP-ribose) polymerase (PARP) is catalytically active only when bound to DNA strand breaks or ends, as induced by either chemical or ionizing radiation. The enzyme catalyzes the covalent attachment of poly (ADP-ribose) chains, derived from NAD (and hense ATP, to a variety of nuclear proteins. Large amounts of ATP can thus be consumed by cells to support poly (ADP-ribosylation) reactions in response to substantial DNA damage or degradation. The enzyme activities of certain PARP substrate proteins, such as endonucleases and topoisomerases, are inhibited in the poly (adp-ribosylated) state. A cysteine protease that cleaves PARP during apoptosis has been identified. The human enzyme, which we have recently purified and cloned, and termed "apopain", is itself generated early in apoptosis from a precursor, CPP-32, that is related to the C. Elegans protein CED-3 and to ICE. Apopain cleaves PARP in the DNA binding domain (Asp 216-Gly 217), thereby inactivating the enzyme. We have developed a potent peptide aldehyde inhibitor of apopain which also inhibits apoptosis. The major objective of this proposal is to determine why PARP is inactivated early in apoptosis. A testable hypothesis is that PARP is inactivated to prevent poly(ADP-ribosyl)action-unduced inhibition of nuclear enzymes required for the DNA degradation that occurs during apoptosis, and also to spare futile degradation of NAD and ATP. Specific Aim I is to characterize the regulation of endonucleases and topoisomerases I and II by poly (ADP-ribosy)lation at the molecular and cellular levels as well as their roles in apoptosis. This aim will be accomplished using the Merck Gene Index and requires the cloning of a Ca2+Mg2+ -dependent endonuclease that is modified by PARP, and the generation of antibodies to, and inhibitors of the various target enzymes. Specific Aim II is to develop and characterize apoptotic events involving NAD/ATP in cells stably transfected with constructs encoding either (I) PARP mutants that are not susceptible to cleavage by apopain or (I) apopain antisense RNA. Also to elucidate the effects of these conditions upon other apopain-clevage proteins. Accordingly, osteosarcoma cells manipulated to express PARP mutants that are resistant to apopain cleveage would constitute a feasible and potentially informative experimental model with which to test various hypotheses concerning the biological rationale for PARP's destruction by apopain and the roles for ATP and NAD during apoptosis.

聚(ADP-核糖)聚合酶(PARP)只有在以下条件下才具有催化活性 脱氧核糖核糖核酸链断裂或末端的，如由化学物质或 电离辐射。该酶催化多聚物的共价连接 (ADP-核糖)链，来源于NAD(和Hense ATP)，到各种 核蛋白。因此，细胞可以消耗大量的ATP来 支持对大量DNA的聚合(ADP-核糖化)反应 损坏或降级。某些PARP底物的酶活性 蛋白质，如核酸内切酶和拓扑异构酶，在 多(ADP-核糖化)状态。 一种在细胞凋亡过程中裂解PARP的半胱氨酸蛋白酶已经被发现 确认身份。我们最近提纯和克隆的人类酶， 被称为“脱氧核糖核酸”，本身就是在细胞凋亡早期产生的 前体CPP-32，与线虫蛋白CED-3和 冰。Apopain在DNA结合域(Asp 216-Gly 217)中切割PARP， 从而使酶失活。我们已经开发出一种有效的多肽 脱氧核糖核酸的醛抑制剂，它也能抑制细胞凋亡。 该提案的主要目标是确定为什么PARP 在细胞凋亡早期失活。一个可测试的假设是，PARP是 灭活以防止聚(ADP-核糖)作用-诱导抑制 DNA降解过程中所需的核酶 细胞凋亡，也避免了NAD和ATP的无效降解。 具体目的一是描述核酸内切酶的调控和 拓扑异构酶I和II通过多聚ADP-核糖在分子和 细胞水平及其在细胞凋亡中的作用。这一目标将是 使用默克基因索引完成，需要克隆一个 由PARP修饰的依赖于钙镁离子的内切酶，以及 产生各种靶标酶的抗体和抑制物。 具体目标二是开发和表征涉及以下因素的凋亡事件 稳定表达NAD/ATP基因的细胞(I) 不易被apopain或(I)切割的PARP突变体 脱氧核糖核酸反义。也是为了阐明这些条件的影响 在其他脱氧核糖核酸的蛋白质上。相应地，骨肉瘤细胞 被操纵以表达对凋亡素具有抗性的PARP突变体 Cleeage将构成一个可行的和潜在的信息量 一种实验模型，用来检验与此有关的各种假设 脱氧核糖核酸酶破坏PARP的生物学基础及其作用 细胞凋亡过程中的ATP和NAD。