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

描述（由申请人提供）：程序性细胞死亡或凋亡是正常发育和组织稳态所必需的细胞过程。破坏细胞生与死之间的微妙平衡可能导致包括癌症在内的许多人类疾病。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。在细胞应激的激活下，BAX从一个无活性的细胞质单体转化为一个有毒的孔，破坏线粒体膜并启动凋亡途径。Walensky实验室之前在BAX的n端表面发现了一个触发位点或“开关”。BCL-2蛋白bh3亚群的特定成员参与这个相互作用位点，启动BAX激活，包括其从细胞质溶胶到线粒体膜的易位。今年，我们确定BAX的c端凹槽也与BH3-only蛋白相互作用兼容。我们认为这个新发现的c端触发位点促进了线粒体膜上BAX的激活和低聚化。在这里，我建议开发新的探针来解剖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α -螺旋（SAHBs）定义BAX的N端和c端触发位点选择性结合的结合决定因素；(2)剖析BAX上两个bh3结合位点在触发其直接激活方面的功能作用。通过应用跨化学、细胞凋亡生物学和发育性癌症治疗的多学科方法，我希望推进我们对驱动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来重新激活癌症细胞凋亡的新策略的发展提供信息。

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.