Bcl-2 family proteins in the ER-mediated apoptosis

项目描述（申请人提供）：本项目主要研究促凋亡Bcl-2家族蛋白Bax和Bak如何在内质网应激下调节细胞凋亡，并将研究扩展到利用Bax/Bak双缺陷模型系统来确定兴奋性毒性神经元细胞死亡的调控机制。内质网应激触发未折叠蛋白反应（UPR），最终导致细胞凋亡。目前尚不清楚内质网应激信号如何被转导以启动细胞凋亡。Bax和Bak在启动细胞凋亡中起着重要作用。初步研究表明，除了存在于线粒体外膜外，Bax和Bak也定位于内质网并启动细胞凋亡。我们将从以下几个方面研究内质网中Bax和Bak启动细胞凋亡的机制：(a)抗凋亡和促凋亡的多结构域Bcl-2家族蛋白的转录和翻译后调控，以及bh3蛋白的修饰，例如，内质网应激下定位、磷酸化、蛋白酶切割和转录调控的改变；(b)内质网应激下细胞内Ca2+和蛋白酶的参与，可能介导细胞死亡；(c)通过观察内质网管蛋白向细胞质内的释放，确定Bax/Bak是否能诱导内质网渗漏。第二个目的是利用Bax/Bak双缺陷细胞表征兴奋性毒性神经元细胞死亡。兴奋性毒性细胞死亡与人类神经退行性疾病和脑肿瘤侵袭有关。细胞凋亡和坏死两种死亡形式的存在，使其机制的研究变得复杂。Bax和Bak的缺乏阻断了线粒体凋亡途径，但从Bax/Bak缺陷小鼠分离的神经祖细胞对nmda和淀粉样蛋白B （AB）诱导的细胞死亡敏感。因此，可以在Bax/ bak缺陷细胞中研究兴奋性毒性细胞死亡的机制，而不需要线粒体死亡扩增效应带来的复杂性。由于兴奋性毒性细胞死亡与er介导的细胞死亡具有相同的特征，例如细胞内Ca2+稳态的扰动，因此我计划通过：(a)分离培养小脑颗粒细胞并建立永生化NPC细胞系，(b)表征NMDA和Ap在Bax/ bak缺陷细胞中诱导的细胞死亡，以确定这些细胞是否以典型的凋亡或坏死特征死亡，(c)确定酸敏感离子通道（asic）或聚（adp -核糖）聚合酶（PARP）参与使用Bax/ bak缺陷细胞的兴奋性毒性细胞死亡。研究可能参与Bax/ baker不依赖性细胞死亡的天冬氨酸和钙蛋白酶。(d)研究细胞内Ca2+在兴奋性毒性神经元细胞死亡中的作用。

DESCRIPTION (provided by applicant): This project focuses on studying how proapoptotic Bcl-2 family proteins Bax and Bak regulate apoptosis in response to ER stress, and extends the study to determine how excitotoxic neuronal cell death is regulated using Bax/Bak doubly deficient model system. ER stress triggers the unfolded protein response (UPR), which ultimately results in apoptosis. It remains unclear how signals from ER stress is transduced to initiate apoptosis. Bax and Bak play a fundamental role in initiating apoptosis. Preliminary studies have suggested that in addition to their presence at the mitochondrial outer membrane, Bax and Bak also localize to the ER and initiate apoptosis. The following areas will be addressed to study the mechanisms involved in the initiation of apoptosis by Bax and Bak from the ER: (a) Transcriptional and post-translational regulation of both anti-apoptotic and proapoptotic multi-domain Bcl-2 family proteins and, modification of the BH3-only proteins, e.g., change of localization, phosphorylation, protease cleavage, and transcriptional regulation, in response to ER stress, (b) Involvement of intracellular Ca2+ and proteases that may mediate cell death in response to ER stress, (c) Determining whether Bax/Bak can induce ER leakage by looking at the release of ER lumenal proteins into cytosol. The second aim is to characterize excitotoxic neuronal cell death using Bax/Bak doubly deficient cells. Excitotoxic cell death has been implicated in human neurodegenerative diseases and brain tumor invasion. The existence of both apoptotic and necrotic forms of cell death makes it complicated to study the mechanisms involved. Deficiency in both Bax and Bak blocks mitochondrial apoptotic pathways, yet neural progenitor cells isolated from Bax/Bak-deficient mice are sensitive to NMDAand amyloid B (AB)-induced cell death. Thus, mechanisms involved in excitotoxic cell death can be studied in Bax/Bak-deficient cells without the complexity resulting from the death amplification effect of mitochondria. Since excitotoxic cell death shares features with the ER-mediated cell death, such as the perturbation of intracellular Ca2+ homeostasis, I plan to study excitotoxic cell death by: (a) Isolating and culturing cerebellar granule cells and establishing immortalized NPC lines, (b) Characterizing cell death induced by NMDA and Ap in Bax/Bak-deficient cells to determine whether these cells die with characteristic apoptotic or necrotic features, (c) Determining the involvement of the acid-sensitive ionic channels (ASICs) or poly(ADP-ribose) polymerase (PARP) in excitotoxic cell death using Bax/Bak-deficient cells, and study aspartyl and calpain proteases that may be involved in Bax/Bak-independent cell death, (d) Studying the role of intracellular Ca2+ in excitotoxic neuronal cell death.