MECHANISM OF ANTI-APOPTOTIC FUNCTION OF GRP78/BiP

描述（由申请人提供）：虽然已经确定内质网（ER）在细胞稳态中起关键作用，但其对细胞凋亡的重要作用现在才变得明显。本研究旨在探讨内质网应激诱导细胞凋亡的分子机制及内质网伴侣蛋白GRP 78/BJP的抗凋亡作用。我们假设ER应激诱导的细胞凋亡起始于ER，但通过线粒体经由多个途径被放大，并且GRP 78的一种抗细胞凋亡功能依赖于其对启动来自ER的细胞死亡程序的促细胞凋亡组分的活化的预防。我们的假设是基于最近的发现，ER是一个网站的收敛和调节的亲和抗凋亡的组件，GRP 78，一个关键的调节器的未折叠的蛋白质反应，可以保护细胞免受细胞凋亡诱导的ER应力以及DNA损伤拓扑异构酶抑制剂。此外，GRP 78可以作为跨膜蛋白存在，并与ER应激或依托泊苷诱导的半胱天冬酶相互作用，并阻断其活化。为了了解潜在的体内分子机制，我们有三个具体的目标。在目的1中，通过使用Apaf-1缺陷和BAX/巴克双敲MEFs，我们将评估线粒体分支对ER启动的凋亡途径的需求，以及已知的ER凋亡途径和ER巴克/BAX激活之间的关系。我们将进一步研究内质网应激与线粒体凋亡通路激活的相关分子及其诱导机制。在目标2中，我们将确定ER应激和依托泊苷诱导的细胞凋亡途径的步骤，该途径被GRP 78抑制，并测试GRP 78是否是BIK的新型抑制剂，BIK是BAX的上游调节剂。在目标3中，我们将确定ER应激诱导的凋亡途径是否在癌症中改变，以及GRP 78在成神经细胞瘤和肿瘤内内皮细胞中淀粉样蛋白β毒性中的细胞保护功能。这项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性癌症和神经变性方面具有临床意义。

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.