Cell Death Regulation by Pro-Apoptotic BOK During Hematopoiesis

描述（由申请方提供）：程序性细胞死亡是组织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，例如在血细胞减少症（例如血小板减少症）或病理性细胞存活中，如在血液学癌症（例如滤泡性淋巴瘤）中所见。细胞的生与死由BCL-2蛋白的一个广泛的家族调节，该家族包括一个错综复杂的监护人和刽子手蛋白的网络。多结构域促凋亡成员BAX和巴克充当细胞死亡的最终守门人，并且一旦被激活，就不可逆地诱导细胞死亡。尽管BAX和巴克在细胞凋亡中起着重要作用，但双敲除小鼠发育正常的胸部和腹部器官，具有适度过量的造血细胞积累。这些数据表明，额外的促凋亡多结构域蛋白质可能subserve在这些组织中的BAX和巴克的功能。在线粒体凋亡的执行中起核心作用的候选者中的主要是BOK，其是BAX和巴克的广泛表达的密切同源物。我提出了一个多学科的方法，采用合成化学技术，生物化学和细胞实验，和小鼠建模，以调查的生理作用，BOK在维持组织稳态，重点是造血。我的目标是：（1）设计、合成和纯化重组BOK蛋白和钉合肽调节剂以研究BH 3触发的BOK活化;（2）定义配体诱导的BOK寡聚化的功能活性;和（3）确定BOK介导的细胞凋亡在造血系统的发育和稳态中的生理作用。保留天然BH 3死亡配体的特异性并直接激活BOK的BCL-2结构域的稳定化α-螺旋（SAHB）将用作独特的分子探针和原型治疗剂，以在体外和体内研究和调节BCL-2家族相互作用。因此，所提出的BOK活性的机制解剖将推进我们的目标，即开发新的药理学方法来调节BOK，以在病理性细胞存活或过早细胞死亡的血液疾病中获得治疗益处。该研究计划将在一个全面的职业发展计划，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咨询委员会，和一个有成就的多学科医生科学家导师的背景下执行。这些组成部分将推动该项目的成功完成，并推进研究者的独立职业生涯，作为一个专门从事造血细胞凋亡生物学及其临床应用的血液病理学家。 公共卫生相关性：在决定生命和死亡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，它独特地准备触发细胞死亡。解开BOK活性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过度细胞存活的条件下重新激活细胞死亡。我建议生成基于肽的化合物和新型小鼠模型，以探讨蛋白质相互作用如何触发BOK在血细胞发育过程中调节细胞死亡的关键问题，并最终将这些见解应用于生成BOK介导的细胞死亡的药理学调节剂以获得治疗益处。

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.