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：（改编自调查员的摘要）Bcl-2蛋白 家庭通过广泛的凋亡信号调节细胞死亡诱导和 因此，在癌症的发病机理中起着重要作用。虽然 抗凋亡蛋白Bcl-2和Bcl-XL位于线粒体和 内质网（ER）最近的研究几乎完全关注 关于它们在线粒体中的作用。因此，该提议的目的是 研究BCl-2/BXL-XL在ER中的功能。基于中心角色 ER在细胞内钙稳态和信号传导中发挥作用，该建议将 检验Bcl-2/bcl-XL作为离子通道或 离子通道的调节剂，以保留ER管腔内钙稳态的调节器， 从而防止触发细胞凋亡的细胞器功能障碍。目标1意志 研究跨膜alpha 5，6螺旋的假设 Bcl-2/bcl-XL充当调节离子电导率的钙传感器 Bcl-2/bcl-XL的响应于ER内钙浓度的变化 管腔，从而形成一个保持钙稳态的反馈回路 急诊室。 AIM 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 脂质双层技术测量靶向ER的离子通道活性 荧光蛋白，Cameleons，以监测Bcl-2/bcl-XL的影响 腔内钙浓度在单个细胞基础上。目标3意志 研究Bcl-2/bcl-XL通过 肌醇1.4.5-三磷酸受体，一种位于ER中的钙通道 膜。 AIM 4将检验凋亡信号破坏的假设 依赖钙的ER功能，通过钙依赖性处理测量 溶酶体天冬氨酸蛋白酶组织蛋白酶D，该蛋白酶和Bcl-2/bcl-XL保存 ER中的钙依赖性蛋白质加工，从而抑制细胞器 功能障碍和保留细胞活力。后者的研究将两者都采用 脉冲马的标记技术和绿色荧光的显微成像 蛋白质 - 触摸蛋白D融合蛋白确定凋亡信号的作用 在ER内的蛋白质加工上，Bcl-2/bcl-XL。共同的目的 该建议研究了有关机理的新颖概念 Bcl-2/bcl-XL功能在ER的水平上。