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: 6269830
• 负责人: MARK SMULSON
• 金额: $ 20.32万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6269830
• 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）仅在以下情况下具有催化活性： 与DNA链断裂或末端结合，如由化学或 电离辐射这种酶催化聚乙烯的共价连接， （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， 响应于大量DNA支持聚（ADP-核糖基化）反应 损坏或退化。某些PARP底物的酶活性 蛋白质，如核酸内切酶和拓扑异构酶，在细胞中被抑制， 聚（ADP-核糖基化）状态。 在细胞凋亡过程中切割PARP的半胱氨酸蛋白酶已经被发现。 鉴定我们最近提纯并克隆的人类酶， 被称为“脱辅基蛋白酶”，其本身在细胞凋亡早期从细胞凋亡中产生。 前体CPP-32，其与C. Elegans蛋白CED-3和 冰脱辅基蛋白酶在DNA结合结构域（Asp 216-Gly 217）中切割PARP， 从而使酶失活。我们开发了一种有效的肽 脱辅基蛋白酶的醛抑制剂，其也抑制细胞凋亡。 该提案的主要目标是确定PARP为什么 在凋亡早期失活。一个可检验的假设是，PARP是 失活以防止聚（ADP-核糖基）作用诱导的对 DNA降解所需的核酶， 细胞凋亡，并且还避免NAD和ATP的无效降解。 具体目的I是表征核酸内切酶的调节， 拓扑异构酶I和II通过聚（ADP-核糖基化）在分子和 细胞水平以及它们在凋亡中的作用。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Ca 2 + Mg 2+依赖性核酸内切酶，以及 产生各种靶酶的抗体和抑制剂。 具体目标II是开发和表征凋亡事件， 用编码以下任一种的构建体稳定转染的细胞中的NAD/ATP： 对脱辅基蛋白酶切割不敏感的PARP突变体或（I） 脱辅基蛋白反义RNA。也为了阐明这些条件的影响 其他apopain clevage蛋白。因此，骨肉瘤细胞 被操纵以表达抗脱辅基蛋白酶的PARP突变体， 裂缝将构成一个可行的和潜在的信息 实验模型，以测试有关的各种假设 脱辅基蛋白酶破坏PARP的生物学原理以及 细胞凋亡中的ATP和NAD。