Cell Death Regulation by Pro-Apoptotic BOK During Hematopoiesis

描述(由申请人提供)：程序性细胞死亡是发展和维持组织内稳态所必需的基本生物学过程。放松对细胞凋亡的管制可能会导致细胞过度死亡，如细胞减少症(如血小板减少症)或病理性细胞存活，如血液病(如滤泡性淋巴瘤)。细胞的生命和死亡由一个庞大的bcl2蛋白家族调控，该家族组成了一个复杂的守护者和刽子手蛋白网络。多结构域促凋亡成员BAX和BAK是细胞死亡的终极守门人，一旦被激活，就不可逆转地诱导细胞死亡。尽管Bax和BAK在细胞凋亡中起着重要作用，但双基因敲除小鼠发育出正常的胸部和腹部器官，造血细胞适度过剩积累。这些数据表明，在这样的组织中，更多的促凋亡多结构域蛋白可能取代了Bax和BAK的功能。在线粒体凋亡的执行过程中发挥核心作用的候选基因中，首当其冲的是BOK，它是一种广泛表达的BAX和BAK的密切同源物。我提出了一种多学科的方法，利用合成化学技术、生化和细胞实验以及小鼠模型来研究BOK在维持组织动态平衡方面的生理学作用，重点是造血。我的目标是：(1)设计、合成和纯化重组BOK蛋白和装订多肽调节剂，以研究BH3触发的BOK激活；(2)确定配体诱导的BOK寡聚的功能活性；(3)确定BOK介导的细胞凋亡在造血系统发育和动态平衡中的生理学作用。稳定的BCL-2结构域的α-螺旋(SAHBs)保留了天然BH3死亡配体的特异性并直接激活BOK，将作为独特的分子探针和原型疗法来研究和调节bcl2家族在体内外的相互作用。因此，提出的对BOK活性的机制剖析将推进我们的目标，即开发新的药理学方法来调节BOK，以利于治疗病理性细胞存活或细胞过早死亡的血液疾病。研究计划将在全面的职业发展计划的背景下执行，该计划包括课程、会议、研讨会、丰富的学术环境、具有深厚专业知识的科学顾问委员会以及一位经验丰富的多学科内科科学家导师。这些组件将推动该项目的成功完成，并推动研究人员作为一名专门研究造血细胞凋亡生物学及其临床应用的血液病理学家的独立职业生涯。 与公共健康相关：在决定生死的细胞信号网络中嵌入了一种名为BOK的难以捉摸的刽子手蛋白，它独特地有可能引发细胞死亡。解开BOK活动的奥秘将影响治疗方法的发展，以阻止不必要的细胞死亡，并在细胞过度存活的情况下重新激活细胞死亡。我建议建立基于多肽的化合物和新的小鼠模型，以探索蛋白质相互作用如何触发BOK来调节血细胞发育过程中的细胞死亡这一关键问题，并最终将这些见解应用于产生BOK介导的细胞死亡的药物调节剂，从而达到治疗的目的。

DESCRIPTION (provided by applicant): Programmed cell death is a fundamental biological process required for the development and maintenance of tissue homeostasis. Deregulation of apoptosis can result in excessive cellular demise such as in cytopenias (e.g. thrombocytopenia) or pathologic cellular survival as seen in hematologic cancers (e.g. follicular lymphoma). Cellular life and death is regulated by an expansive family of BCL-2 proteins, which comprise an intricate network of guardians and executioner proteins. The multi-domain pro-apoptotic members BAX and BAK serve as the ultimate gatekeepers of cell death and, once activated, irreversibly induces cellular demise. Despite BAX and BAK's essential role in apoptosis, double knockout mice develop normal thoracic and abdominal organs with moderate excess accumulations of hematopoietic cells. These data suggest that additional pro-apoptotic multidomain proteins may subserve the functions of BAX and BAK in such tissues. Chief among the candidates for a central role in the execution of mitochondrial apoptosis is BOK, a widely- expressed close homologue of BAX and BAK. I propose a multidisciplinary approach that employs synthetic chemistry techniques, biochemical and cellular experiments, and mouse modeling to investigate the physiologic role of BOK in maintaining tissue homeostasis, with a focus on hematopoiesis. I aim to: (1) Design, synthesize, and purify recombinant BOK protein and stapled peptide modulators to investigate BH3- triggered BOK activation; (2) Define the functional activity of ligand-induced BOK oligomerization; and (3) Determine the physiologic role of BOK-mediated apoptosis in the development and homeostasis of the hematopoietic system. Stabilized Alpha-Helices of BCL-2 domains (SAHBs) that retain the specificity of native BH3 death ligands and directly activate BOK will serve as unique molecular probes and prototype therapeutics to investigate and modulate BCL-2 family interactions in vitro and in vivo. Thus, the proposed mechanistic dissection of BOK activity will advance our goal of developing novel pharmacologic approaches to regulate BOK for therapeutic benefit in blood diseases of pathologic cell survival or premature cell death. The research plan will be executed in the context of a comprehensive career development plan that includes courses, meetings, workshops, a rich academic environment, a scientific advisory board with deep expertise, and an accomplished multidisciplinary physician scientist mentor. These components will propel the project to successful completion and advance the investigator's independent career as a hematopathologist specializing in hematopoietic apoptosis biology and its clinical applications. PUBLIC HEALTH RELEVANCE: Embedded within the cellular signaling network that dictates life and death lay an elusive executioner protein named BOK, which is uniquely poised to trigger cellular demise. Unlocking the mysteries of BOK activity will impact the development of therapeutics to thwart unwanted cell death and reactivate cell death in conditions of excessive cellular survival. I propose to generate peptide-based compounds and novel mouse models to probe the critical question of exactly how protein interaction triggers BOK to regulate cell death during the development of blood cells and ultimately apply these insights to generate pharmacologic modulators of BOK-mediated cell death for therapeutic benefit.