Coordination of Apoptosis and Cell Proliferation in Drosophila

描述(由申请者提供)：我们的长期目标是了解细胞凋亡和细胞增殖在动物发育过程中是如何协调的。增殖的细胞容易发生损伤引起的细胞凋亡，而有丝分裂后的细胞对许多凋亡刺激具有抵抗力。对凋亡的抵抗对于防止无法替代的重要有丝分裂后细胞的丧失非常重要。相反，促进增殖组织中的细胞死亡被认为是应对细胞损伤的有效方法，因为细胞损失可以通过代偿性增殖来弥补。因此，细胞增殖和凋亡的协调对维持动物健康至关重要，而无法协调这些过程会导致疾病，如癌症中的细胞过度增殖，或神经退行性疾病中的过度凋亡。在这个提案中，我们将检验这样的假设，即凋亡体是一个由启动子caspase靶子和接头蛋白Apafl组成的全酶复合体，作为细胞凋亡和细胞增殖的关键协调者。为了测试这一点，我们将利用简单的果蝇细胞死亡范例，其中Diapl(果蝇凋亡蛋白1的抑制物)直接抑制活细胞中的凋亡体，而Diapl拮抗剂Reaper、HID和Grim的表达先于凋亡来解除这种对凋亡体的抑制。一旦凋亡体激活效应半胱氨酸天冬氨酸酶，细胞就会完全参与细胞死亡。我们将追求三个具体目标。首先，我们将研究Diapl是如何抑制细胞凋亡体的。特别是，我们将测试这一假设，即Diapl直接泛素化Apafl以在活细胞中降解，而在注定要死亡的细胞中DIAPL的失活允许稳定的凋亡体形成。其次，我们将研究凋亡细胞在增殖组织中引发代偿性增殖的机制。具体地说，我们将重点研究由活跃的凋亡体启动的有丝分裂信号通路的机制，并确定这是否构成补偿性增殖的基础。最后，我们将探讨有丝分裂后细胞获得抗凋亡的机制。在这里，我们将检验一个假设，即细胞周期退出通过泛素介导的蛋白质降解来增强细胞凋亡体的不稳定性。我们在果蝇组织中的遗传方法将与生化分析相辅相成，以进行进一步的机制研究。这项研究的进展可能有助于设计出对抗癌症和神经退行性疾病的新策略。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how apoptosis and cell proliferation are coordinated during animal development. Proliferating cells readily undergo injury-provoked apoptosis, while postmitotic cells acquire resistance to many apoptotic stimuli. Resistance to apoptosis is important to prevent the loss of vital postmitotic cells that cannot be replaced. Conversely, facilitated cell death in proliferating tissues is thought to be an effective way to cope with cellular injury, as cell loss can be replaced through compensatory proliferation. Accordingly, coordination of cell proliferation and apoptosis is essential for maintaining animal health, and inability to coordinate these processes leads to diseases caused by excess cell proliferation as in cancer, or excess apoptosis as seen in neurodegenerative disorders. In this proposal, we will test the hypothesis that the Apoptosome, a holoenzyme complex that consists of the initiator caspase Drone and the adaptor protein Apafl, serve as a critical coordinator of apoptosis and cell proliferation. To test this, we will exploit the simple Drosophila cell death paradigm, in which Diapl (Drosophila Inhibitor of Apoptosis Protein 1) directly inhibits the Apoptosome in living cells, and the expression of Diapl antagonists Reaper, Hid and Grim precedes apoptosis to relieve this inhibition of the Apoptosome. Once Apoptosomes activate effector caspases, cells become fully committed to cell death. We will pursue three specific aims. First, we will investigate how Diapl inhibits the Apoptosome. In particular, we will test the hypothesis that Diapl directly ubiquitylates Apafl for degradation in living cells, while inactivation of Diapl in cells destined to die allow stable Apoptosome formation. Second, we will investigate the mechanism by which apoptotic cells trigger compensatory proliferation in proliferating tissues. Specifically, we will focus on the mechanism of a mitogenic signaling pathway initiated by active Apoptosomes and determine whether this underlies compensatory proliferation. Finally, we will investigate the mechanism by which postmitotic cells acquire resistance to apoptosis. Here, we will test the hypothesis that cell cycle exit enhances Apoptosome instability through ubiquitin-mediated protein degradation. Our genetic approaches in Drosophila tissues will be complemented with biochemical assays for further mechanistic studies. Progress from this research may help devise new strategies against cancer and neurodegenerative disorders.