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。细胞生命与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看门人。细胞应激激活后，BAX 从无活性的胞质单体转变为有毒孔，破坏线粒体膜并启动细胞凋亡途径。 Walensky 实验室此前在 BAX 的 N 端表面发现了一个触发位点或“开关”。 BCL-2 蛋白的仅 BH3 亚组的特定成员与该相互作用位点的接合启动了 BAX 激活，包括其从细胞质易位到线粒体膜。今年，我们确定 BAX 的 C 端凹槽也与仅 BH3 的蛋白质相互作用兼容。我们相信这个新发现的 C 端触发位点促进了 BAX 在线粒体膜上的激活和寡聚化。在这里，我建议开发新型探针来剖析 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如何在功能上影响 BAX 激活途径。具体来说，我的目标是 (1)：使用 BCL-2 结构域的稳定 Alpha 螺旋 (SAHB) 定义 BAX N 端和 C 端触发位点选择性接合的结合决定簇，以及 (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。通过应用涵盖化学、细胞凋亡生物学和发育性癌症治疗的多学科方法，我希望加深我们对驱动 BAX 介导的细胞凋亡的生化机制的理解，并为开发通过直接 BAX 激活重新激活癌症细胞凋亡的新策略提供信息。

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.