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

描述（由申请人提供）：程序性细胞死亡或细胞凋亡是正常发育和组织稳态所必需的细胞过程。细胞生命和死亡之间微妙平衡的破坏可能导致许多人类疾病，包括癌症。BCL-2家族蛋白是关键的凋亡调节因子，由抗凋亡和促凋亡成员组成。促凋亡BAX是线粒体凋亡的关键守门人。在细胞应激激活后，BAX从无活性的胞质单体转化为破坏线粒体膜并启动凋亡途径的毒性孔。Walensky实验室先前在BAX的N-末端表面确定了一个触发位点或“开关”。BCL-2蛋白质的仅BH 3亚组的特定成员与该相互作用位点的接合启动BAX活化，包括其从细胞质到线粒体膜的易位。今年，我们确定了BAX的C-末端沟也与BH 3-only蛋白质相互作用相容。我们相信这个新发现的C-末端触发位点推动了BAX在线粒体膜上的激活和寡聚化。在这里，我建议开发新的探针来解剖BAX上的两个BH 3结合位点的结合决定簇，并确定它们的选择性触发功能如何影响BAX激活途径。具体而言，我的目标是（1）：定义的N-和C-末端触发位点的BAX使用BCL-2结构域（SAHB）的稳定的α-螺旋的选择性接合的结合决定簇和（2）解剖的BAX在触发其直接激活的两个BH 3结合位点的功能作用。通过应用跨化学，凋亡生物学和发育癌症治疗学的多学科方法，我希望推进我们对驱动BAX介导的细胞凋亡的生化机制的理解，并通过直接BAX激活来重新激活癌症细胞凋亡的新策略的发展。

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.