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

剖析 BOK 在调节细胞生死中的生化作用

• 批准号: 8649696
• 负责人: Rebecca I Goldstein
• 金额: $ 4.92万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649696
• 关键词: Activities of Daily Living; Affinity; Aging; Apoptosis; Apoptotic; Autoimmune Diseases; BAX gene; BCL2 gene; BH3 Domain; BH3 peptide; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biology; Cell Death; Cell Survival; Cells; Cessation of life; Chemicals; Chemistry; Clinical; Commit; Complex; Development; Developmental Therapeutics Program; Disease; Dissection; Environment; Epitopes; Equilibrium; Family; Goals; Hand; Homeostasis; Homologous Gene; Hydrocarbons; In Vitro; Induction of Apoptosis; Laboratories; Lead; Length; Libraries; Life; Ligands; Link; Liposomes; Longevity; Malignant Neoplasms; Measures; Methods; Mitochondria; Molecular Probes; Mutagenesis; Myocardial Infarction; Nerve Degeneration; Pathologic; Pathway interactions; Peptides; Process; Production; Protein Family; Proteins; Protocols documentation; Recombinant Proteins; Recombinants; Regulation; Research; Research Personnel; Role; Site; Specificity; Stress; Stroke; Structure; Structure-Activity Relationship; Synthesis Chemistry; System; Therapeutic; Time; Tissues; Training; Training Programs; X-Ray Crystallography; alpha helix; base; career; cellular longevity; cytopenia; cytotoxic; design; insight; interdisciplinary approach; member; monomer; novel; novel therapeutics; prototype; public health relevance; response; small molecule; structural biology; success; tool

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.

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，在发育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。这种平衡的破坏会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，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，形成一个复杂的相互作用网络，调节细胞凋亡。一些基于小分子和多肽的方法已经出现，靶向BCL-2家族蛋白，并成功地调节它们以获得潜在的治疗效果。基于我们对BAX和BAK（两种重要的促凋亡BCL-2家族蛋白）离散触发位点的鉴定，它们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。本研究的目的是确定难以捉摸的促凋亡BCL-2家族蛋白BOK的生化、结构和功能。确定BOK的环境依赖性作用将促进我们对细胞寿命控制点的理解，并为开发针对它们的新药理策略提供信息，以获得临床益处。我提出了一种多学科方法，采用合成化学、结构生物学、生化和细胞研究来研究BOK在维持组织稳态中的功能作用。我的目标是：(1)定义和结构表征促凋亡BOK与BH3结构域螺旋多样性之间的相互作用网络；(2)在体外和细胞中确定BOK与BH3结合的功能后果。首先，我将以我最近的成功为基础，克服长期存在的挑战，以单体形式生成全长重组BOK。为了探究BOK结合活性及其对功能激活的影响，我将生成并应用BCL-2结构域的稳定α -螺旋（SAHBs），这是一种独特的研究工具，可以概括BH3天然死亡配体的自然结构和特异性。直接与BOK结合的sahb将作为分子探针和原型治疗手段，在体外和细胞中研究和调节BOK的生物活性。基于对BOK活性的机制解剖，我希望发现新的药理学方法来调节非期望细胞死亡或病理性细胞存活疾病中的细胞死亡。我致力于研究细胞死亡途径的蛋白质相互作用动力学，并利用结构，生化和细胞的见解来推进新的治疗策略，以优化衰老过程中睡眠的数量和质量。为此，我制定了一个综合的培训计划，在一个动态的生物医学环境中，将化学、结构生物学、细胞凋亡生物学和发育治疗学融合在一起，最大限度地提高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者的发展。