BCL 2 FUNCTION IN ENDOPLASMIC RETICULUM

BCL 2 在内质网中的功能

• 批准号: 6700231
• 负责人: CLARK W DISTELHORST
• 金额: $ 24.1万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-03 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6700231
• 关键词: BCL2 gene /protein; MCF7 cell; apoptosis; calcium channel; calcium flux; carbohydrate receptor; cathepsin D; electrophysiology; endoplasmic reticulum; green fluorescent proteins; inositol phosphates; intracellular transport; protein transport; receptor expression; tissue /cell culture

DESCRIPTION: (Adapted from the investigator's abstract) The Bcl-2 protein family regulates cell death induction by a wide range of apoptotic signals and thereby plays an important role in the pathogenesis of cancer. Although the antiapoptotic proteins Bcl-2 and Bcl-xL are localized to both mitochondria and the endoplasmic reticulum (ER) recent studies have focused almost exclusively on their role in mitochondria. Therefore, the purpose of this proposal is to investigate how Bcl-2/Bxl-xL function in the ER. Based on the central role the ER plays in intracellular calcium homeostasis and signaling, this proposal will test the overall hypothesis that Bcl-2/Bcl-xL work either as ion channels or regulators of ion channels to preserve calcium homeostasis within the ER lumen, thereby preventing organelle dysfunction that triggers apoptosis. Aim 1 will investigate the hypothesis that the transmembrane alpha 5, 6 helices of Bcl-2/Bcl-xL function as a calcium sensor that regulates the ion conductivity of Bcl-2/Bcl-xL in response to changes in calcium concentration within the ER lumen, thereby forming a feedback loop that maintains calcium homeostasis in the ER. Aim 2 will investigate the hypothesis that the BH4 domain of the Bcl-2/Bcl-xL regulates the ion conductivity of Bcl-2/Bcl-xL in response to changes in intraluminal calcium concentration. Aims 1 & 2 will employ planar lipid bilayer techniques to measure ion channel activity in ER-targeted fluorescent proteins, cameleons, to monitor effects of Bcl-2/Bcl-xL on intraluminal calcium concentration on a single cell basis. Aim 3 will investigate the hypothesis that Bcl-2/Bcl-xL regulate calcium efflux through the inositol 1.4.5-triphosphate receptor, a calcium channel located in the ER membrane. Aim 4 will test the hypothesis that apoptotic signals disrupt calcium-dependent ER function, as measured by the calcium-dependent processing of the lysosomal aspartic protease cathepsin D, and that Bcl-2/Bcl-xL preserve calcium-dependent protein processing in the ER, thereby inhibiting organelle dysfunction and preserving cell viability. The latter studies will employ both pulse-chase labeling techniques and microscopic imaging of green fluorescent protein-cathepsin D fusion proteins to determine effects of apoptotic signals and Bcl-2/Bcl-xL on protein processing within the ER. Collectively, the aims of this proposal investigate novel concepts regarding the mechanism of Bcl-2/Bcl-xL function at the level of the ER.

描述：（改编自研究者的摘要）Bcl-2 蛋白 家族通过广泛的细胞凋亡信号调节细胞死亡诱导 因此在癌症的发病机制中发挥着重要作用。虽然 抗凋亡蛋白 Bcl-2 和 Bcl-xL 定位于线粒体和 最近的研究几乎完全集中于内质网（ER） 关于它们在线粒体中的作用。因此，本提案的目的是 研究 Bcl-2/Bxl-xL 在 ER 中的功能。以核心角色为基础 ER 在细胞内钙稳态和信号传导中发挥作用，该提案将 检验 Bcl-2/Bcl-xL 作为离子通道或 离子通道调节剂以保持 ER 腔内的钙稳态， 从而防止引发细胞凋亡的细胞器功能障碍。目标1将 研究跨膜 α 5、6 螺旋的假设 Bcl-2/Bcl-xL 用作调节离子电导率的钙传感器 Bcl-2/Bcl-xL 响应 ER 内钙浓度的变化 管腔，从而形成反馈回路，维持钙稳态 急诊室。目标 2 将研究以下假设： Bcl-2/Bcl-xL 调节 Bcl-2/Bcl-xL 的离子电导率 腔内钙浓度的变化。目标 1 和 2 将采用平面 脂质双层技术测量 ER 靶向离子通道活性 荧光蛋白、cameleons，用于监测 Bcl-2/Bcl-xL 对 以单细胞为基础的管腔内钙浓度。目标3将 研究 Bcl-2/Bcl-xL 通过调节钙流出的假设 肌醇 1.4.5-三磷酸受体，位于 ER 的钙通道 膜。目标 4 将检验细胞凋亡信号破坏的假设 钙依赖性 ER 功能，通过钙依赖性加工测量 溶酶体天冬氨酸蛋白酶组织蛋白酶 D，并且 Bcl-2/Bcl-xL 保留 内质网中钙依赖性蛋白质加工，从而抑制细胞器 功能障碍并保持细胞活力。后面的研究将同时采用 绿色荧光的脉冲追踪标记技术和显微成像 蛋白质-组织蛋白酶 D 融合蛋白用于确定细胞凋亡信号的影响 和 Bcl-2/Bcl-xL 对 ER 内蛋白质加工的影响。总体而言，目标 该提案研究了有关机制的新概念 Bcl-2/Bcl-xL 在 ER 水平发挥作用。