Dissecting the Functional Roles of Two BH3-Binding Sites on Pro-Apoptotic BAX

描述(由申请人提供)：程序性细胞死亡，或称细胞凋亡，是正常发育和组织动态平衡所必需的细胞过程。破坏细胞生存和死亡之间的微妙平衡可能会导致包括癌症在内的一系列人类疾病。BCL-2家族蛋白是关键的细胞凋亡调节因子，由抗凋亡和促凋亡两种成员组成。促凋亡基因Bax是线粒体凋亡的关键守门人。当被细胞应激激活时，Bax从一个不活跃的胞浆单体转变为一个有毒的孔洞，破坏线粒体膜并启动凋亡途径。沃伦斯基实验室此前在BAX的N端表面发现了一个触发点或“开关”。BCL-2蛋白的BH3-Only亚群中的特定成员与该相互作用位点结合可启动Bax的激活，包括其从胞浆到线粒体膜的转位。今年，我们确定Bax的C-端槽也与BH3-Only蛋白质相互作用兼容。我们认为，这个新发现的C末端触发位点推动了线粒体膜上Bax的激活和寡聚化。在这里，我建议开发新的探针来剖析Bax上两个BH3结合位点的结合决定因素，并确定它们的选择性触发功能如何影响Bax激活途径。具体地说，我的目标是：(1)利用BCL-2结构域的稳定的α-螺旋(SAHBs)来确定选择性结合Bax的N-端和C-端触发位点的结合决定因素；(2)剖析Bax上两个BH3结合位点在触发其直接激活中的功能作用。我希望通过应用跨越化学、凋亡生物学和发育癌症治疗学的多学科方法，促进我们对驱动Bax介导的细胞凋亡的生化机制的理解，并为通过直接激活Bax来重新激活癌症中的细胞凋亡的新策略的开发提供信息。

DESCRIPTION (provided by applicant): Programmed cell death, or apoptosis, is a cellular process essential for normal development and tissue homeostasis. Disruption of the delicate balance between cellular life and death can lead to a host of human diseases, including cancer. BCL-2 family proteins are key apoptotic regulators and are composed of anti- and pro-apoptotic members. Pro-apoptotic BAX is a critical gatekeeper of mitochondrial apoptosis. Upon activation by cellular stress, BAX transforms from an inactive cytosolic monomer into a toxic pore that disrupts the mitochondrial membrane and initiates the apoptotic pathway. The Walensky laboratory previously identified a trigger site or "on switch" at BAX's N-terminal surface. Engagement of this interaction site by specific members of the BH3-only subgroup of BCL-2 proteins initiates BAX activation, including its translocation from the cytosol to the mitochondria membrane. This year, we determined that the C-terminal groove of BAX is also compatible with BH3-only protein interaction. We believe this newly identified C-terminal trigger site propels the activation and oligomerization of BAX at the mitochondrial membrane. Here, I propose to develop novel probes to dissect the binding determinants of the two BH3-binding sites on BAX and determine how their selective triggering functionally impacts the BAX activation pathway. Specifically, I aim to (1) : Define the binding determinants for selective engagement of the N- and C-terminal trigger sites of BAX using Stabilized Alpha-Helices of BCL-2 domains (SAHBs) and (2) Dissect the functional roles of the two BH3-binding sites on BAX in triggering its direct activation. By applying multidisciplinary approaches that span chemistry, apoptosis biology, and developmental cancer therapeutics, I hope to advance our understanding of the biochemical mechanism that drives BAX-mediated apoptosis and inform the development of a new strategy to reactivate apoptosis in cancer through direct BAX activation.