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

项目摘要 线粒体凋亡是一种由BCL-2家族蛋白调控的程序性细胞死亡。BAX和巴克 是该途径的执行蛋白，并且响应于细胞应激，从无活性单体转化 有毒的寡聚体，穿孔线粒体外膜，导致细胞死亡。抗细胞凋亡成员 BCL-2家族中的一个结合并阻断BAX和巴克，以防止不必要的细胞死亡， BAX/巴克激活和抑制之间的平衡是体内平衡所必需的。癌细胞篡夺了抗凋亡基因 以确保他们的不朽。经典的抑制机制包括隔离 促凋亡成员的BH 3“杀伤结构域”位于抗凋亡蛋白C-末端表面的表面沟中， 凋亡成员。BCL-2抑制剂ABT-199，其在BCL-2依赖性 癌症，阻断抗凋亡沟，从而恢复凋亡。今年，瓦伦斯基实验室 发现了一个独特的机制，通过直接相互作用的BH 4结构域之间的抗- 凋亡BCL-2和BAX上的一个新的抑制性相互作用位点。无论巴克，线粒体的居民 BAX的类似物，也受到BH 4的调节是未知的。使用新产生的稳定的α-螺旋， BCL-2结构域（SAHB）模仿抗凋亡BCL-2家族蛋白的BH 4结构域，我最近 检测到特定的BH 4/巴克结合相互作用。因此，我假设BH 4结构域的负调控作用 抗凋亡蛋白的区域可能是一种普遍现象， 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 我将采用多学科方法，追求以下目标：（1）建立一个图书馆， BCL-2结构域的稳定化α-螺旋（SAHB）在BH 4基序之后建模以表征其功能 与全长巴克的相互作用，以及（2）应用不同的结构方法来确定BH 4 BH 4/巴克结合界面的定义。因此，我的目标是 一项研究计划是应用一种独特的化学、生物化学和结构方法， 表征通过BH 4结构域相互作用抑制促凋亡巴克活化的新机制。 考虑到巴克激活的抗凋亡阻断的致癌作用，我的研究结果可以提供一种新的策略， 在人类癌症中恢复BAK介导的细胞凋亡。我期待着追求一个全面的跨学科 我在哈佛医学院和丹娜-法伯癌症研究所的研究生学习培训计划， 作为一个独立的调查员谁可以在化学界面操作的科学生涯的准备 生物学、癌症研究和药物开发。

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.