MECHANISM OF ANTI-APOPTOTIC FUNCTION OF GRP78/BiP

描述(申请人提供)：虽然内质网(ER)在细胞内稳态中起着关键作用是众所周知的，但它对细胞凋亡的重大贡献直到现在才变得明显。本研究旨在揭示内质网应激诱导细胞凋亡的分子机制以及内质网伴侣蛋白GRP78/BJP的抗凋亡作用。我们假设，内质网应激诱导的细胞凋亡始于内质网，但通过多条途径通过线粒体放大，GRP78的一个抗凋亡功能依赖于它阻止从内质网启动细胞死亡程序的促凋亡成分的激活。我们的假设是基于最近的发现，即内质网是促凋亡和抗凋亡成分的汇聚和调节部位，而GRP78是未折叠蛋白反应的关键调节因子，可以保护细胞免受内质网应激和DNA损伤拓扑异构酶抑制剂诱导的细胞凋亡。此外，GRP78可以跨膜蛋白的形式存在，并与内质网应激或依托泊苷诱导的半胱氨酸酶相互作用，并阻断其激活。为了了解体内潜在的分子机制，我们有三个具体目标。在目标1中，通过使用APAF-1缺陷和Bax/BAK双敲击MEF，我们将评估线粒体分支对ER启动的凋亡通路的需求，以及已知的ER凋亡通路与ER BAK/Bax激活的关系。我们将进一步确定内质网应激与线粒体凋亡通路激活相关的分子及其诱导机制。在目标2中，我们将识别内质网应激和依托泊苷诱导的被GRP78抑制的凋亡途径的步骤，并测试GRP78是否是BIK的新抑制剂，BIK是Bax的上游调节因子。在目标3中，我们将确定内质网应激诱导的凋亡通路在肿瘤中是否发生改变，以及GRP78在淀粉样β蛋白在神经母细胞瘤和肿瘤内皮细胞中的细胞保护作用。这项拟议的工作不仅将为基础细胞生物学提供新的信息，而且在消除患者的耐药癌症和神经变性方面也具有临床意义。

DESCRIPTION (provided by applicant): While it is well-established that the endoplasmic reticulum (ER) plays a critical role in cellular homeostasis, its significant contribution to apoptosis is only now becoming apparent. This proposal aims at discovering the molecular mechanisms of the ER stress-induced apoptosis and of the antiapoptotic function of ER chaperone protein GRP78/BJP. We hypothesize that the ER stress-induced apoptosis initiates from the ER but is amplified through the mitochondria via multiple pathways and that one anti-apoptotic function of GRP78 relies on its prevention of activation of pro-apoptotic components that initiate the cell death program from the ER. Our hypothesis is based on the recent discovery that the ER is a site of convergence and regulation of both pro- and anti-apoptotic components, and that GRP78, a key regulator of the unfolded protein response, can protect cells against apoptosis induced by ER stress as well as DNA-damaging topoisomerase inhibitors. Further, GRP78 can exist as a transmembrane protein and interact with caspases inducible by ER stress or etoposide and block their activation. Towards understanding the underlying in vivo molecular mechanisms, we have three specific aims. In Aim 1, through the use of Apaf-1 deficient and BAX/BAK double knock MEFs, we will assess the requirement of the mitochondrial branch for ER-initiated apoptotic pathways, the relationship between known ER apoptotic pathways and ER BAK/BAX activation. We will further identify the molecules linking ER stress to activation of mitochondrial apoptotic pathway and their induction mechanism by ER stress. In Aim 2, we will identify steps of the ER-stress and etoposide-induced apoptotic pathway that is suppressed by GRP78 and test whether GRP78 is a novel inhibitor of BIK, an upstream regulator of BAX. In Aim 3, we will determine whether the ER stress-induced apoptotic pathways are altered in cancer, and the cytoprotective function of GRP78 in amyloid-beta toxicity in neuroblastoma and endothelial cells within tumors. The proposed work will not only contribute novel information on basic cell biology but also has clinical relevance in eliminating drug-resistant cancers and neurodegeneration in patients.