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）：该项目的重点是研究促凋亡的Bcl-2家族蛋白Bax和Bak如何响应ER应力调节凋亡，并扩展研究以确定兴奋性神经元细胞死亡如何使用Bax/Bak双重缺陷模型系统调节兴奋性神经元细胞死亡。 ER应力触发展开的蛋白质反应（UPR），最终导致凋亡。目前尚不清楚如何转导来自ER应力的信号引发凋亡。 Bax和Bak在启动凋亡中起着基本作用。初步研究表明，除了它们在线粒体外膜的存在外，Bax和Bak还位于ER并启动凋亡。 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对ER应激的响应，（b）细胞内Ca2+的参与以及可能响应ER应激而介导细胞死亡的蛋白酶，（c）确定Bax/Bak是否可以通过查看ER腔蛋白释放到细胞质中来诱导ER泄漏。第二个目的是使用Bax/Bak双重缺乏细胞来表征兴奋性神经元细胞死亡。兴奋性细胞死亡与人神经退行性疾病和脑肿瘤侵袭有关。细胞死亡的凋亡和坏死形式的存在使研究所涉及的机制变得复杂。 Bax和Bak的缺乏均可阻止线粒体凋亡途径，但是从BAX/BAK缺陷型小鼠中分离出的神经祖细胞对NMDAAND淀粉样蛋白B（AB）诱导的细胞死亡敏感。因此，可以在Bax/Bak缺陷型细胞中研究涉及兴奋性细胞死亡的机制，而不会导致线粒体的死亡扩增效应产生的复杂性。由于兴奋性细胞死亡与ER介导的细胞死亡具有特征，例如细胞内Ca2+稳态的扰动，我计划通过以下方式研究兴奋性细胞死亡：凋亡或坏死特征，（c）确定酸敏感的离子通道（ASICS）或聚（ADP-核糖）聚合酶（PARP）在兴奋性细胞中使用Bax/Bax/Bak缺陷细胞的涉及兴奋性细胞死亡，并研究了在bax/bak-nimpert的ca均与bax-bax-nimpertent（bax-bax-nimpertent）中的研究（diepterent+ ca intippertion），兴奋性神经元细胞死亡。