Structure-Function Analysis of a Novel Inhibitory Mechanism for Pro-Apoptotic BAK

促凋亡 BAK 的新型抑制机制的结构-功能分析

• 批准号: 9333068
• 负责人: Zachary Joel Hauseman
• 金额: $ 4.4万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-02 至 2020-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333068
• 关键词: Apoptosis; Apoptotic; BAX gene; BCL1 Oncogene; BCL2 gene; BH4 Domain; Binding; Biochemical; Biology; C-terminal; Cell Death; Cellular Stress; Cessation of life; Chemicals; Complex; Dana-Farber Cancer Institute; Disease; Ensure; Environment; Equilibrium; Face; Family; Goals; Homeostasis; Human; Laboratories; Length; Libraries; Life; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Mitochondria; Modeling; Molecular; Molecular Conformation; Monitor; Oncogenic; Outer Mitochondrial Membrane; Pathologic; Pathway interactions; Photoaffinity Labels; Preparation; Protein Family; Protein Overexpression; Proteins; Regulation; Research Personnel; Research Proposals; Role; Site; Structure; Surface; Time; Training Programs; X-Ray Crystallography; alpha helix; analog; anticancer research; cancer cell; career; clinical efficacy; drug development; inhibitor/antagonist; insight; interdisciplinary approach; medical schools; member; monomer; novel; novel therapeutic intervention; prevent; research and development; response; tetrahydrobiopterin; tumorigenesis

Project Summary Mitochondrial apoptosis is a form of programmed cell death regulated by BCL-2 family proteins. BAX and BAK are the executioner proteins of the pathway and, in response to cellular stress, transform from inactive monomers to toxic oligomers that porate the mitochondrial outer membrane, leading to cell death. Anti-apoptotic members of the BCL-2 family bind and block BAX and BAK to prevent unwanted cell death, and the delicate balance between BAX/BAK activation and inhibition is required for homeostasis. Cancer cells usurp the anti-apoptotic suppression pathway to ensure their immortality. The classic mechanism of suppression involves sequestration of the BH3 “killer domain” of pro-apoptotic members in a surface groove located at the C-terminal face of anti- apoptotic members. The BCL-2 inhibitor ABT-199, which is showing clinical efficacy in BCL-2 dependent cancers, blocks the anti-apoptotic groove and thereby restores apoptosis. This year, the Walensky lab discovered a distinct mechanism for apoptotic suppression by direct interaction between the BH4 domain of anti- apoptotic BCL-2 and a novel inhibitory interaction site on BAX. Whether or not BAK, the mitochondrial resident analog of BAX, is also subject to BH4 regulation is unknown. Using newly generated stabilized alpha-helices of BCL-2 domains (SAHBs) modeled after the BH4 domains of anti-apoptotic BCL-2 family proteins, I recently detected specific BH4/BAK binding interactions. Thus, I hypothesize that negative regulation by the BH4 domain regions of anti-apoptotic proteins may be a general phenomenon and represents a previously unappreciated mechanism for pathologic suppression of BAK-mediated apoptosis. To elucidate this novel structure-function mechanism, I will apply multidisciplinary approaches in pursuit of the following aims: (1) Generate a library of stabilized alpha-helices of BCL-2 domains (SAHBs) modeled after BH4 motifs to characterize their functional interactions with full-length BAK, and (2) Apply diverse structural methods to both determine the effect of BH4 engagement on BAK conformational activation and define the BH4/BAK binding interface. Thus, the goal of my research proposal is to apply a unique constellation of chemical, biochemical, and structural approaches to characterize a novel mechanism for suppression of pro-apoptotic BAK activation by BH4 domain interaction. Given the oncogenic role of anti-apoptotic blockade of BAK activation, my results could inform a new strategy to restore BAK-mediated apoptosis in human cancer. I look forward to pursuing a comprehensive interdisciplinary training program for my graduate studies at Harvard Medical School and the Dana-Farber Cancer Institute, in preparation for a scientific career as an independent investigator who can operate at the interface of chemical biology, cancer research, and drug development.

项目摘要 线粒体凋亡是一种受bc1-2家族蛋白调控的程序性细胞死亡。BAX和BAK 是该途径的执行者蛋白，在对细胞压力的反应中，从不活跃的单体转化为 到破坏线粒体外膜，导致细胞死亡的有毒寡聚体。抗凋亡成员 BCL-2家族的成员结合和阻断Bax和BAK，以防止不必要的细胞死亡，并保持微妙的平衡 BAX/BAK的激活和抑制之间是动态平衡所必需的。癌细胞篡夺抗细胞凋亡作用 抑制途径，以确保他们的不朽。经典的抑制机制包括封存 在位于抗凋亡分子C-端面的表面凹槽中，具有促凋亡成员的BH3“杀手结构域” 细胞凋亡者。Bcl2抑制剂ABT-199对bcl2依赖的临床疗效 癌症，阻断抗细胞凋亡的沟槽，从而恢复细胞凋亡。今年，瓦伦斯基实验室 发现了一种独特的机制，通过直接相互作用的BH4结构域之间的抗 凋亡的bcl2和bax上一个新的抑制相互作用部位。不管BAK是不是线粒体居民 BAX类似物，是否也受BH4调控未知。使用新产生的稳定的α-螺旋 BCL-2结构域(SAHBs)是根据抗凋亡的BCL-2家族蛋白的BH4结构域模拟的，I最近 检测到特定的BH4/BAK结合作用。因此，我假设BH4结构域的负调控 抗凋亡蛋白的区域可能是一种普遍现象，代表着以前未被认识到的 BAK介导的细胞凋亡的病理抑制机制。为了阐明这种新的结构-功能 为了实现以下目标，我将采用多学科方法：(1)建立一个图书馆 BCL-2结构域的稳定α-螺旋(SAHBs)模拟BH4基序以表征其功能 与全长BAK的相互作用，以及(2)应用不同的结构方法来确定BH4的效果 参与BAK构象激活并定义BH4/BAK结合界面。因此，我的目标是 研究建议是应用一种独特的化学、生化和结构方法来 描述一种通过BH4结构域相互作用抑制促凋亡BAK激活的新机制。 考虑到BAK激活的抗凋亡阻断的致癌作用，我的结果可能为新的策略提供参考 恢复BAK介导的人类肿瘤细胞凋亡。我期待着追求一种全面的跨学科 我在哈佛医学院和达纳-法伯癌症研究所攻读研究生的培训计划 作为一名能够在化学界面上操作的独立研究员，为科学生涯做准备 生物学、癌症研究和药物开发。