Dissecting the biochemical role of BOK in regulating cellular life and death

描述(申请人提供)：细胞凋亡是细胞程序性死亡的一种形式，是一种高度协调的机制，用于在发育期间和整个生命过程中维持新细胞和死亡细胞之间的关键平衡。这种平衡的破坏可能导致不必要的细胞死亡，如神经变性、中风、心脏病发作和细胞减少症，或病理性细胞存活，如癌症和自身免疫性疾病。Bcl-2家族由守护蛋白和刽子手蛋白组成，它们形成了一个复杂的相互作用网络，调节细胞凋亡。已经出现了几种以bcl2家族蛋白为靶点的小分子和基于多肽的方法，并成功地对它们进行了调节，以获得潜在的治疗益处。基于我们在Bax和BAK上的离散触发位点的确定，这两个重要的促凋亡bcl2家族蛋白的直接调节可能提供了在适当的疾病背景下激活或抑制细胞死亡的机会。这项提案的目的是确定难以捉摸的促凋亡蛋白BCL-2家族蛋白BOK的生化、结构和功能。确定BOK的上下文相关角色将促进我们对细胞寿命控制点的理解，并为开发新的药理学策略提供信息，以针对它们的临床益处。我提出了一种多学科的方法，利用合成化学、结构生物学以及生化和细胞研究来研究BOK在维持组织动态平衡方面的功能作用。我的目标是：(1)定义并从结构上表征促凋亡BOK与BH3结构域螺旋多样性之间的相互作用网络；以及(2)确定BOK在体外和细胞内参与BH3的功能后果。首先，我将在最近成功克服以单体形式产生全长重组BOK这一长期挑战的基础上再接再厉。为了探索BOK结合活性及其对功能激活的影响，我将产生并应用BCL-2结构域的稳定化α螺旋(SAHBs)，这是一种独特的研究工具，概括了天然BH3死亡配体的自然结构和特异性。直接与BOK结合的SAHBs将作为分子探针和原型疗法，在体外和细胞内研究和调节BOK的生物学活性。基于对BOK活性的这种机制解剖，我希望发现新的药理学方法来调节不想要的细胞死亡或病理性细胞存活的疾病中的细胞死亡。我致力于科学生涯，专注于解剖细胞死亡途径的蛋白质相互作用动力学，并利用结构、生化和细胞洞察力来推进新的治疗策略，以优化衰老期间谎言的数量和质量。为此，我制定了一项综合培训计划，在动态的生物医学环境中融合化学、结构生物学、细胞凋亡生物学和发育治疗学，以最大限度地发展我作为一名在化学生物学和细胞寿命研究方面具有独特专业知识的独立研究员。

DESCRIPTION (provided by applicant): Apoptosis, a form of programmed cell death, is a highly orchestrated mechanism for maintaining the critical balance between new and dying cells during development and throughout life. Disruption of this balance can lead to unwanted cell death, such as in neurodegeneration, stroke, heart attack and cytopenias, or pathologic cell survival, such as in cancer and autoimmune disease. The BCL-2 family is composed of guardian and executioner proteins that form a complex interaction network that regulates apoptosis. Several small molecule and peptide-based approaches have emerged that target BCL-2 family proteins and successfully modulate them for potential therapeutic benefit. Based on our identification of discrete trigger sites on BAX and BAK, two of the essential pro-apoptotic BCL-2 family proteins, their direct modulation may present an opportunity to activate or inhibit cell death in the appropriate disease context. The goal of this proposal is to define the biochemistry, structure, and function of the elusive pro-apoptotic BCL-2 family protein BOK. Determining the context-dependent roles of BOK will advance our understanding of cellular lifespan control points and inform the development of novel pharmacologic strategies to target them for clinical benefit. I propose a multidisciplinary approach that employs synthetic chemistry, structural biology, and biochemical and cellular studies to investigate the functional role of BOK in maintaining tissue homeostasis. I aim to: (1) Define and structurally characterize the network of interactions between pro-apoptotic BOK and the diversity of BH3 domain helices; and (2) Determine the functional consequences of BH3 engagement of BOK in vitro and in cells. First, I will build upon my recent success in overcoming the longstanding challenge of generating full-length, recombinant BOK in monomeric form. To probe BOK binding activity and its impact on functional activation, I will generate and apply Stabilized Alpha-Helices of BCL-2 domains (SAHBs), unique research tools that recapitulate the natural structure and specificity of native BH3 death ligands. SAHBs that directly bind to BOK will serve as molecular probes and prototype therapeutics to investigate and modulate the biological activity of BOK in vitro and in cells. Based on this mechanistic dissection of BOK activity, I hope to uncover new pharmacologic approaches to regulate cell death in diseases of unwanted cell death or pathologic cell survival. I am committed to a scientific career focused on dissecting the protein interaction dynamics of the cell death pathway and harnessing the structural, biochemical, and cellular insights to advance novel therapeutic strategies for optimizing quantity and quality of lie during aging. To that end, I have developed a comprehensive training program that blends chemistry, structural biology, apoptosis biology, and developmental therapeutics, in the context of a dynamic biomedical environment, to maximize my development as an independent investigator with unique expertise at the interface of chemical biology and cellular longevity research.