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：(改编自研究人员摘要)Bcl2蛋白 家族通过广泛的凋亡信号调节细胞死亡诱导和 因此在癌症的发病机制中起着重要的作用。尽管 抗凋亡蛋白Bcl2和Bclxl定位于线粒体和 内质网(ER)最近的研究几乎完全集中在 关于它们在线粒体中的作用。因此，这项建议的目的是 探讨Bcl2/Bxl-xl在内质网中的作用。基于中心角色， 内质网在细胞内钙稳态和信号转导中发挥作用，这一提议将 测试总体假设，即Bcl2/Bclxl作为离子通道或作为离子通道工作 离子通道的调节器，以保持内质网管腔内的钙稳态， 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α5，6螺旋的假设 BCL-2/BCL-XL作为钙传感器调节离子电导 内质网钙离子浓度变化对Bcl2/Bclxl表达的影响 管腔，从而形成一个反馈环路，以维持钙稳态 急诊室。目标2将调查假设的BH4结构域的 BCL-2/BCL-xL调节Bc l-2/Bc l-xl离子电导 血管内钙离子浓度的变化。AIMS 1和2将采用平面 脂质双分子膜技术测量内质网靶向离子通道活性 用于监测Bcl2/Bclxl对细胞周期的影响的荧光蛋白 单细胞基础上的管腔内钙浓度。目标3将 Bcl2/Bclxl通过调节钙外流的假说探讨 内质网钙通道--肌醇1.4.5-三磷酸受体 薄膜。目标4将检验这样一种假设：细胞凋亡信号干扰 钙依赖的内质网功能，通过钙依赖的加工来测量 溶酶体天冬氨酸蛋白酶组织蛋白D的表达及Bcl2/Bclxl的保存 内质网中钙依赖蛋白的处理，从而抑制细胞器 功能障碍和维持细胞活力。后一项研究将同时采用这两种方法 脉冲追逐标记技术与绿色荧光的显微成像 蛋白质-组织蛋白酶D融合蛋白对细胞凋亡信号的影响 和Bcl2/Bclxl对内质网内蛋白质加工的影响。总体而言， 这项建议调查了关于 BCL-2/BCL-XL在内质网水平发挥作用。