Bcl-2 family proteins in the ER-mediated apoptosis

Bcl-2 家族蛋白在 ER 介导的细胞凋亡中的作用

• 批准号: 7066569
• 负责人: Wei-Xing Zong
• 金额: $ 13.79万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7066569
• 关键词: BCL2 gene /protein; Bax gene /protein; apoptosis; aspartic endopeptidases; calcium flux; calcium transporting ATPase; cell line; chemical cleavage; cysteine endopeptidases; endoplasmic reticulum; enzyme activity; excitatory aminoacid; genetic transcription; granule cell; laboratory mouse; membrane channels; neural degeneration; northern blottings; pentosyltransferase; phosphorylation; posttranslational modifications; protein localization; transfection /expression vector; western blottings

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.

描述（由申请人提供）：本项目主要研究促凋亡Bcl-2家族蛋白Bax和巴克如何响应ER应激调节细胞凋亡，并将研究扩展到确定如何使用Bax/巴克双缺陷模型系统调节兴奋性毒性神经元细胞死亡。ER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ER应激信号如何被转导以启动细胞凋亡。Bax和巴克在启动细胞凋亡中起重要作用。初步研究表明，Bax和巴克除了存在于线粒体外膜外，还定位于ER并启动细胞凋亡。将致力于以下领域以研究参与由来自ER的Bax和巴克引发细胞凋亡的机制：（a）抗细胞凋亡和促细胞凋亡多结构域Bcl-2家族蛋白的转录和翻译后调节以及仅BH 3蛋白的修饰，例如，响应于ER应激的定位、磷酸化、蛋白酶切割和转录调节的变化，（B）细胞内Ca 2+和蛋白酶的参与，所述细胞内Ca 2+和蛋白酶可介导响应于ER应激的细胞死亡，（c）通过观察ER内腔蛋白释放到胞质溶胶中来确定Bax/巴克是否可诱导ER渗漏。第二个目的是使用Bax/巴克双缺陷细胞表征兴奋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。细胞凋亡和坏死形式的存在使得研究所涉及的机制变得复杂。Bax和巴克的缺乏阻断了线粒体凋亡途径，然而从Bax/Bak缺乏小鼠中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，参与兴奋性毒性细胞死亡的机制可以在Bax/Bak缺陷细胞中进行研究，而不需要线粒体的死亡放大效应所导致的复杂性。由于兴奋性毒性细胞死亡与ER介导的细胞死亡具有共同的特征，例如细胞内Ca 2+稳态的扰动，因此我计划通过以下方式研究兴奋性毒性细胞死亡：（a）分离和培养小脑颗粒细胞并建立永生化NPC系，（B）表征Bax/Bak缺陷细胞中由NMDA和A β诱导的细胞死亡，以确定这些细胞是否以特征性凋亡或坏死特征死亡，（c）使用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）在兴奋性毒性细胞死亡中的参与，并研究可能参与Bax/Bak非依赖性细胞死亡的乙酰胆碱和钙蛋白酶。