Apoptotic Signaling Pathways

细胞凋亡信号通路

• 批准号: 6879358
• 负责人: Sally A Kornbluth
• 金额: $ 34.65万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879358
• 关键词: Drosophilidae; Xenopus; Xenopus oocyte; apoptosis; binding proteins; biological signal transduction; cysteine endopeptidases; cytochrome c; laboratory rabbit; mitochondria; molecular chaperones; protein degradation; protein localization; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): Apoptosis is a program of cellular suicide wherein individual cells are removed from the midst of a living tissue without destroying overall tissue architecture. Although the convergence of multiple signaling pathways determines whether a cell will live or die, once the decision to die is made, most cells activate a family of lethal proteases called caspases. By cleaving key intracellular substrates, these enzymes package cellular contents for their orderly elimination by phagocytosis. Caspases can be inhibited by the binding of a class of proteins known as lAPs (inhibitors of apoptosis). In turn, the lAPs can be antagonized by pro-apoptotic IBM (IAP binding motif) proteins that displace lAPs from their caspase binding sites. One such IBM protein, isolated from Drosophila Melanogaster, is the pro-apoptotic protein, Reaper. Reaper not only releases lAPs from caspases, but also promotes IAP ubquitination and degradation, at least in part, through activation of an lAP-intrinsic E3 ubiquitin ligase activity. Although the IBM motif, present at the extreme Nterminus of Reaper, is required for induction of IAP auto-ubiquitination, an additional domain in Reaper, (the GH3 domain) is required for both Reaper-stimulated IAP degradation and localization of Reaper to mitochondria. In a Reaper mutant lacking the GH3 domain, both mitochondria! localization and the ability to induce IAP degradation can be rectified by appending a heterologous mitochondrial targeting sequence. These observations suggest that mitochondrial localization of Reaper and its ability to induce IAP degradation are in some way linked. Upon expression in vertebrate cells, Reaper binds not only lAPs, but also a 150kD protein called Scythe, which participates in Reaper-induced release of mitochondrial cytochrome c to the cytoplasm. The Scythe/Reaper interaction and consequent cytochrome c release is critical for Reaper-mediated caspase activation in vertebrate systems, though its role in Drosophila cell death has not yet been examined. The goals of this proposal are to understand how Reaper functions at mitochondria to promote IAP degradation in both fly and vertebrate cells, to understand how Scythe participates in Reaper-mediated alterations of mitochondria in vertebrate cells, to identify a vertebrate modulator of Scythe function and to determine whether Scythe is also important for fly cell apoptosis. In aggregate, these experiments should not only help to reconcile the effects of Reaper in fly and vertebrate systems, but should also lead to a detailed picture of how the various functions of Reaper contribute to the induction of full-blown apoptosis. These studies will employ both fly and vertebrate systems, including a powerful in vitro reconstitution system derived from Xenopus eggs that allows the recapitulation of apoptotic signaling in vitro.

描述（由申请人提供）：细胞凋亡是一种细胞自杀程序，其中单个细胞从活组织中被移除，而不破坏整体组织结构。尽管多种信号通路的汇聚决定了细胞的生存还是死亡，但一旦做出死亡的决定，大多数细胞都会激活称为半胱天冬酶的致命蛋白酶家族。通过裂解关键的细胞内底物，这些酶包装细胞内容物，以便通过吞噬作用有序消除。 Caspases 可以通过一类称为 lAP（细胞凋亡抑制剂）的蛋白质的结合来抑制。反过来，IAP 可以被促凋亡 IBM（IAP 结合基序）蛋白拮抗，从而将 IAP 从其 caspase 结合位点取代。从果蝇中分离出的一种 IBM 蛋白是促凋亡蛋白 Reaper。 Reaper 不仅从半胱天冬酶中释放 lAP，而且至少部分地通过激活 lAP 固有的 E3 泛素连接酶活性来促进 IAP 泛素化和降解。尽管存在于 Reaper 末端 N 末端的 IBM 基序是诱导 IAP 自动泛素化所必需的，但 Reaper 中的一个附加结构域（GH3 结构域）对于 Reaper 刺激的 IAP 降解和 Reaper 定位至线粒体是必需的。在缺乏 GH3 结构域的 Reaper 突变体中，两个线粒体！定位和诱导 IAP 降解的能力可以通过附加异源线粒体靶向序列来纠正。这些观察结果表明，Reaper 的线粒体定位与其诱导 IAP 降解的能力以某种方式相关。 在脊椎动物细胞中表达后，Reaper 不仅结合 lAP，还结合一种称为 Scythe 的 150kD 蛋白质，该蛋白质参与 Reaper 诱导的线粒体细胞色素 c 向细胞质的释放。 Scythe/Reaper 相互作用和随后的细胞色素 c 释放对于脊椎动物系统中 Reaper 介导的 caspase 激活至关重要，尽管其在果蝇细胞死亡中的作用尚未得到研究。该提案的目标是了解 Reaper 如何在线粒体上发挥作用，以促进果蝇和脊椎动物细胞中的 IAP 降解，了解 Scythe 如何参与脊椎动物细胞中 Reaper 介导的线粒体改变，识别 Scythe 功能的脊椎动物调节剂，并确定 Scythe 是否对果蝇细胞凋亡也很重要。总的来说，这些实验不仅应该有助于协调 Reaper 在果蝇和脊椎动物系统中的作用，而且还应该详细了解 Reaper 的各种功能如何有助于诱导全面的细胞凋亡。这些研究将采用果蝇和脊椎动物系统，包括源自非洲爪蟾卵的强大体外重建系统，该系统可以在体外重现细胞凋亡信号。