BCL 2 FUNCTION IN ENDOPLASMIC RETICULUM

BCL 2 在内质网中的功能

• 批准号: 6089857
• 负责人: CLARK W DISTELHORST
• 金额: $ 23.81万
• 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/6089857
• 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：（改编自研究者摘要）Bcl-2蛋白 家族通过广泛的凋亡信号调节细胞死亡诱导， 从而在癌症的发病机制中起重要作用。虽然 抗凋亡蛋白Bcl-2和Bcl-xL定位于线粒体， 内质网（ER）最近的研究几乎只集中在 在线粒体中的作用。因此，本建议的目的是 研究Bcl-2/Bxl-xL在ER中的功能。基于中心作用， ER在细胞内钙稳态和信号传导中起作用，该提议将 测试Bcl-2/Bcl-xL作为离子通道或 离子通道的调节剂以保持ER腔内的钙稳态， 从而防止触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作为钙传感器调节离子电导率 Bcl-2/Bcl-xL对ER内钙浓度变化的响应 管腔，从而形成一个反馈回路，维持钙稳态， 急诊室目的2将调查的假设，即BH 4结构域的 Bcl-2/Bcl-xL调节Bcl-2/Bcl-xL的离子电导率，以响应 管腔内钙浓度的变化。目标1和2将采用平面 脂质双层技术来测量ER靶向细胞中的离子通道活性 荧光蛋白，骆驼，监测Bcl-2/Bcl-xL的影响， 以单细胞为基础的管腔内钙浓度。目标3将 研究Bcl-2/Bcl-xL通过以下途径调节钙外流的假设： 肌醇1.4.5-三磷酸受体，一种位于内质网的钙通道 膜的目的4将检验凋亡信号干扰 钙依赖性ER功能，如通过钙依赖性处理测量的 的溶酶体天冬氨酸蛋白酶组织蛋白酶D，Bcl-2/Bcl-xL保存， 钙依赖性蛋白质在内质网中的加工，从而抑制细胞器 功能障碍和保持细胞活力。后两项研究将采用 脉冲追踪标记技术和绿色荧光显微成像 蛋白-组织蛋白酶D融合蛋白以确定凋亡信号的作用 和Bcl-2/Bcl-xL对ER内蛋白质加工的影响。总的来说， 该提案研究了关于以下机制的新概念： Bcl-2/Bcl-xL在ER水平发挥作用。