Targeting BAX Oligomerization in Hematologic Disease

描述（由申请人提供）：BCL-2家族的蛋白质相互作用调节程序性细胞死亡或凋亡，从而维持细胞生命和死亡之间的关键平衡。促凋亡BAX是一种关键的刽子手蛋白，其在细胞溶质中休眠，直到被细胞应激触发以对线粒体造成不可逆的损伤。由于其作为细胞死亡的看门人的作用，BAX的药理学调节具有在细胞凋亡失调的疾病中交替阻断或重新激活细胞死亡的潜力。BAX活化被认为是一个高度调节的多步骤过程，涉及相互作用触发的构象变化、线粒体易位和同源寡聚化以在外线粒体膜内形成致死孔。使用直接启动BAX介导的线粒体凋亡的BCL-2结构域（SAHB）的稳定化α-抑制剂，我们最近通过NMR分析确定了一个触发其激活的新的BAX相互作用位点。在解决了BAX激活的初始步骤之后，我现在建议确定导致同源寡聚化的BAX自激活的难以捉摸的分子机制，以便可以在治疗上利用凋亡途径的这个关键控制点来抑制血液病中的细胞死亡。具体而言，我的目标是（1）合成结构增强的α-螺旋对应于BAX的BH 3死亡结构域，以确定和表征其与促凋亡BAX的相互作用，（2）确定BAX SAHB-BAX复合物和中间体BAX构象的溶液结构，以及（3）研究BAX传播的机制及其在血液细胞中的药理学抑制的影响。通过在化学，生物学和血液学的界面上操作，我希望为我们对BAX自激活途径的理解提供新的见解，揭示BCL-2家族蛋白相互作用的新位点，并确定如何重新编程以改善血液病患者。 这项建议涉及多学科，需要高级培训和专门知识。在Loren D博士的指导下，Walensky和Alan D 'Andrea博士，我将获得化学生物学，细胞凋亡生物学和血液学方面的新技能和知识，此外还将通过培训，实验室管理，工作申请流程和其他初级教师生存技能为向独立过渡做准备。达纳-法伯癌症研究所和哈佛医学院儿科肿瘤学系内拟议的培训和职业发展计划提供了最先进的资源，世界一流的教师顾问和合作者，以及一个优秀的环境，以促进成功过渡到学术独立。我的职业目标是成为一名独立资助的首席研究员，在一家主要的学术研究中心担任终身职位。我致力于科学事业，专注于调节细胞死亡的蛋白质相互作用的结构和功能，并直接应用于开发治疗血液病的新型药理学策略。 公共卫生相关性：程序性细胞死亡或细胞凋亡调节细胞生命和死亡之间的关键平衡，并且当失调时，有助于以血细胞过多或过少为特征的多种血液病的发病机制。BCL-2家族蛋白调节细胞凋亡，并且对它们的结构和功能的分析有望阐明调节细胞凋亡以获得治疗益处的机会。使用新的化学工具，结构生物学分析和血液细胞实验的组合，我的目标是解剖和抑制一个关键的刽子手蛋白BAX的激活机制，以保护血液细胞免于过早或不必要的细胞死亡。

DESCRIPTION (provided by applicant): The protein interactions of the BCL-2 family regulate programmed cell death or apoptosis, and thereby maintain the critical balance between cellular life and death. Pro-apoptotic BAX is a critical executioner protein that lies dormant in the cytosol until triggered by cellular stress to inflict irreversible damage on the mitochondria. Because of its role as a gatekeeper of cell death, pharmacologic modulation of BAX has the potential to alternatively block or reactivate cell death in diseases of deregulated apoptosis. BAX activation is believed to be a highly regulated, multi-step process involving an interaction-triggered conformational change, mitochondrial translocation, and homo-oligomerization to form a lethal pore within the outer mitochondrial membrane. Using Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis, we recently identified by NMR analysis a novel BAX interaction site that triggers its activation. Having tackled the initial step of BAX activation, I now propose to determine the elusive molecular mechanism of BAX auto-activation that leads to homo-oligomerization, so that this critical control point of the apoptotic pathway can be exploited therapeutically to inhibit cell death in hematologic disease. Specifically, I aim to (1) synthesize structurally-reinforced alpha-helices corresponding to the BH3 death domain of BAX to identify and characterize its interaction(s) with pro-apoptotic BAX, (2) determine the solution structures of the BAX SAHB-BAX complex and an intermediate BAX conformer, and (3) investigate the mechanism of BAX propagation and the impact of its pharmacologic inhibition in hematologic cells. By operating at the interface of chemistry, biology, and hematology, I hope to contribute new insight into our understanding of the BAX auto- activation pathway, revealing new sites of BCL-2 family protein interaction and determining how they can be pharmacologically reprogrammed for the betterment of hematology patients. The multidisciplinary scope of this proposal will require advanced training and expertise. With the mentorship of Dr. Loren D. Walensky and Dr. Alan D'Andrea, I will acquire new skills and knowledge in chemical biology, apoptosis biology, and hematology, in addition to preparing for the transition to independence through training in grantsmanship, laboratory management, the job application process, and other junior faculty survival skills. The proposed training and career development program within the Department of Pediatric Oncology at the Dana-Farber Cancer Institute and Harvard Medical School offers state-of-the-art resources, world class faculty advisors and collaborators, and an outstanding environment to facilitate a successful transition to academic independence. My career goal is to become an independently funded principal investigator with a tenure-track position at a major academic research center. I am committed to a scientific career focused on the structure and function of protein interactions that regulate cell death, with direct application to the development of novel pharmacologic strategies to treat hematologic disease. PUBLIC HEALTH RELEVANCE: Programmed cell death or apoptosis regulates the critical balance between cellular life and death and, when deregulated, contributes to the pathogenesis of a wide variety of hematologic diseases characterized by too many or too few blood cells. BCL-2 family proteins regulate apoptosis and analysis of their structure and function promises to elucidate opportunities for modulating apoptosis for therapeutic benefit. Using a combination of novel chemical tools, structural biology analyses, and hematologic cell experimentation, I aim to dissect and inhibit the activation mechanism of a critical executioner protein called BAX in order to protect hematologic cells from premature or unwanted cell death.