Dissecting the Execution Phase of BAK-Mediated Apoptosis in Cancer

项目摘要 BCL-2家族蛋白是细胞凋亡和蛋白质相互作用失调的关键调节因子 有助于癌症的发展和耐药性。细胞死亡的主要执行者，BAX 和巴克分别作为单体存在于细胞的胞质溶胶和线粒体中，直到被应激激活 刺激自缔合并穿孔线粒体外膜，导致凋亡性细胞死亡。挫败 化疗诱导的细胞凋亡和加强细胞永生，癌症篡夺了细胞存活的手臂， BCL-2通路的抑制剂，其通过过表达抗凋亡成员，其可以结合BAX和巴克并阻止它们的凋亡。 转化为有毒的线粒体孔。长期以来无法产生稳定和均匀的 全长BAX和巴克的寡聚体形式已经排除了它们的穿孔结构的确定， 将告知线粒体凋亡的执行阶段，并揭示用于治疗的新表面 在癌症中BAX和巴克的激活。Walensky实验室最近报道了一种新的策略， 一种BAX寡聚体，可用于一系列结构-功能研究。与BAX相比，巴克 在线粒体上的组成型残基，由一个独特的结合位点触发，并表现出不同的 在哺乳动物组织和人类癌症中的表达模式。此外，尽管全长BAX具有长的 巴克的生产特别适合于以重组单体形式表达， 挑战性Walensky实验室开发了一种三重突变构建体，允许表达全长 单体巴克表现出生理活化和膜穿孔活性。在初步研究中，我 我们已经应用我们从单体巴克和寡聚体BAX的生产中获得的知识来生产全长的 巴克低聚物首次。在这里，我建议优化和验证的稳定性和均匀性，这 物种进行严格的生化和结构表征（SA 1）。通过突变离散的功能区域 的巴克，我进一步提出，以确定结构的决定因素，每一步的活化途径， 评估BAK依赖性白血病细胞（SA 2）的机制发现及其功能意义。到 为了实现我的目标，我将应用多学科的方法，包括蛋白质工程，生化分析， 在模型膜和线粒体中，氢-氘交换质谱、低温电子 显微镜和细胞凋亡分析。我很想参加这个严格的训练计划 我的研究生学习，并期待着发展成为一个独立和创新的物理学家，科学家在 化学生物学、癌症生物学和临床肿瘤学的接口。

PROJECT SUMMARY BCL-2 family proteins are critical regulators of apoptosis and deregulation of their protein interactions contributes to the development and chemoresistance of cancer. The cardinal executioners of cell death, BAX and BAK, respectively reside in the cytosol and mitochondria of the cell as monomers until activated by stress stimuli to self-associate and porate the mitochondrial outer membrane, leading to apoptotic cell death. To thwart chemotherapy-induced apoptosis and enforce cellular immortality, cancer usurps the cell survival arm of the BCL-2 pathway by overexpressing anti-apoptotic members, which can bind to BAX and BAK and prevent their transformation into toxic mitochondrial pores. The longstanding inability to generate stable and homogeneous oligomeric forms of full-length BAX and BAK has precluded the determination of their porating structures, which would inform both the execution phase of mitochondrial apoptosis and reveal novel surfaces for therapeutic activation of BAX and BAK in cancer. The Walensky laboratory recently reported a novel strategy for generating a BAX oligomer that was amenable to a battery of structure-function studies. In contrast to BAX, BAK constitutively residues at the mitochondria, is triggered by a distinct binding site, and exhibits differential expression patterns in mammalian tissues and in human cancers. Further, whereas full-length BAX has long been amenable to expression in recombinant monomeric form, production of BAK has been especially challenging. The Walensky lab developed a triple-mutant construct that allowed for the expression of full-length monomeric BAK that exhibited physiologic activation and membrane-porating activity. In preliminary studies, I have applied our learnings from the production of monomeric BAK and oligomeric BAX to produce a full-length BAK oligomer for the first time. Here, I propose to optimize and validate the stability and homogeneity of this species for rigorous biochemical and structural characterization (SA1). By mutating discrete functional regions of BAK, I further propose to identify the structural determinants of each step of the activation pathway, and evaluate the mechanistic findings and their functional implications in BAK-dependent leukemia cells (SA2). To achieve my goals, I will apply multidisciplinary approaches that include protein engineering, biochemical assays in model membranes and mitochondria, hydrogen-deuterium exchange mass spectrometry, cryo-electron microscopy, and cellular apoptosis analyses. I am eager to embark on the rigorous training program proposed for my graduate studies and look forward to developing as an independent and innovative physician-scientist at the interface of chemical biology, cancer biology, and clinical oncology.