Cell Death Regulation by Pro-Apoptotic BOK During Hematopoiesis

描述（由申请人提供）：程序性细胞死亡是组织内稳态发展和维持所需的基本生物学过程。细胞凋亡的失调可导致过度的细胞死亡，如细胞减少症（如血小板减少症）或病理性细胞存活，如血液学癌症（如滤泡性淋巴瘤）。细胞的生命和死亡是由一个庞大的BCL-2蛋白家族调控的，该家族由一个复杂的守护者和刽子手蛋白网络组成。多结构域促凋亡成员BAX和BAK作为细胞死亡的最终看门人，一旦被激活，就不可逆地诱导细胞死亡。尽管BAX和BAK在细胞凋亡中起着重要作用，但双敲除小鼠发育出正常的胸腔和腹腔器官，并伴有造血细胞的适度过量积累。这些数据表明，在这些组织中可能有其他促凋亡多结构域蛋白参与BAX和BAK的功能。在线粒体凋亡过程中发挥核心作用的主要候选基因是BOK，它是BAX和BAK广泛表达的密切同源基因。我提出了一种多学科方法，采用合成化学技术，生化和细胞实验，以及小鼠模型来研究BOK在维持组织稳态中的生理作用，重点是造血。我的目标是：(1)设计、合成和纯化重组BOK蛋白和钉接肽调节剂，研究BH3触发的BOK激活；(2)明确配体诱导的BOK寡聚化的功能活性；(3)确定bok介导的细胞凋亡在造血系统发育和稳态中的生理作用。BCL-2结构域（SAHBs）的稳定α -螺旋（SAHBs）保留了BH3天然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治疗方法，用于研究和调节BCL-2家族在体外和体内的相互作用。因此，提出的BOK活性的机制解剖将促进我们开发新的药理学方法来调节BOK，以治疗病理性细胞存活或细胞过早死亡的血液疾病。该研究计划将在一个全面的职业发展计划的背景下执行，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和一位有成就的多学科医师科学家导师。这些组成部分将推动该项目成功完成，并推进研究者作为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。

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.