Bcl-2 family proteins in the ER-mediated apoptosis

描述(申请人提供)：本项目重点研究促凋亡的Bcl2家族蛋白Bax和Bak如何在内质网应激反应中调节细胞凋亡，并扩大研究范围以确定如何使用Bax/Bak双缺陷模型系统来调节兴奋性神经细胞死亡。内质网应激触发未折叠蛋白反应(UPR)，最终导致细胞凋亡。目前还不清楚内质网应激信号是如何传导到启动细胞凋亡的。BAX和BAK在启动细胞凋亡中起着重要作用。初步研究表明，Bax和Bak除了存在于线粒体外膜外，还定位于内质网并启动细胞凋亡。将从以下方面研究Bax和Bak从内质网启动细胞凋亡的机制：(A)抗凋亡和促凋亡多域Bcl2家族蛋白的转录和翻译后调节，以及仅BH3蛋白的修饰，例如，响应内质网应激，定位、磷酸化、蛋白酶切割和转录调节的改变，(B)参与细胞内钙离子和可能介导细胞死亡的蛋白应激，(C)通过观察ER腔蛋白释放到胞浆中来确定Bax/Bak是否可以诱导内质网泄漏。第二个目的是利用Bax/Bak双缺陷细胞来表征兴奋性毒性神经细胞的死亡。激毒性细胞死亡与人类神经退行性疾病和脑瘤侵袭有关。细胞死亡既有凋亡性的，也有坏死性的，这使得对其机制的研究变得复杂。Bax和Bak的缺陷阻断了线粒体的凋亡途径，但从Bax/Bak缺陷的小鼠分离的神经前体细胞对NMDA和淀粉样蛋白B(AB)诱导的细胞死亡很敏感。因此，可以在Bax/Bak缺陷细胞中研究参与兴奋性毒性细胞死亡的机制，而不需要复杂的线粒体死亡放大效应。由于兴奋性细胞死亡与内质网介导的细胞死亡具有相同的特征，如细胞内钙稳态的扰动，我计划通过以下方法来研究兴奋性细胞死亡：(A)分离和培养小脑颗粒细胞并建立永生化鼻咽癌细胞系；(B)鉴定NMDA和AP诱导的Bax/Bak缺陷细胞的细胞死亡，以确定这些细胞是否具有典型的凋亡或坏死特征；(C)利用Bax/Bak缺陷细胞，确定酸敏感离子通道(ASIC)或聚(ADP-核糖)聚合酶(PARP)在兴奋性细胞死亡中的参与，以及研究可能参与Bax/Bak非依赖性细胞死亡的天冬氨酸蛋白酶和钙蛋白酶，(D)研究细胞内钙离子在兴奋性毒性神经细胞死亡中的作用。

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.