Dissecting the Execution Phase of BAK-Mediated Apoptosis in Cancer

剖析 BAK 介导的癌症细胞凋亡的执行阶段

• 批准号: 10311905
• 负责人: Catherine Elizabeth Newman
• 金额: $ 3.8万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311905
• 关键词: Apoptosis; Apoptosis Regulation Gene; Apoptotic; BAX gene; BCL2 gene; BH3 Domain; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biology; Biophysics; C-terminal; Cancer Biology; Cell Death; Cell Survival; Cells; Cellular biology; Cessation of life; Chemicals; Chemoresistance; Clinical Oncology; Cryoelectron Microscopy; Cytosol; Detergents; Deuterium; Development; Electron Microscopy; Environment; Equilibrium; Exhibits; Family member; Goals; Homeostasis; Homo; Homologous Gene; Human; Hydrogen; Intercept; Laboratories; Learning; Length; Leukemic Cell; Life; Liposomes; Liver Mitochondria; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Mitochondria; Molecular Conformation; Molecular Sieve Chromatography; Mus; Mutagenesis; Mutate; Organism; Outer Mitochondrial Membrane; Pathologic; Pathway interactions; Pattern; Pharmacology; Phase; Physicians; Physiological; Production; Property; Protein Engineering; Protein Family; Proteins; Proteomics; Reagent; Recombinants; Reporting; Resolution; Role; Scientist; Series; Stimulus; Stress; Structure; Surface; Technology; Therapeutic; Time; Tissues; Training; Training Programs; Variant; X-Ray Crystallography; apoptosis deregulation; arm; cancer cell; chemotherapy; combat; comparative; differential expression; innovation; insight; interdisciplinary approach; member; membrane model; mitochondrial membrane; monomer; multidisciplinary; mutant; next generation; novel; novel strategies; overexpression; prevent; pro-apoptotic protein; programs; reconstitution; structural biology; therapeutic target

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.

项目总结 BCL-2家族蛋白是细胞凋亡及其蛋白相互作用解除调控的关键调节因子 有助于癌症的发展和化疗耐药。细胞死亡的主要刽子手，巴克斯 和BAK分别以单体形式存在于细胞的胞浆和线粒体中，直到被胁迫激活 刺激使线粒体外膜自结合和开孔，导致细胞凋亡。阻挠 化疗诱导细胞凋亡并强制细胞永生，癌症夺走了细胞生存的手臂 BCL-2途径通过高表达抗凋亡分子，与Bax和BAK结合，阻止其 转化为有毒的线粒体毛孔。长期以来，无法产生稳定和同质的 全长BAX和BAK的寡聚体形式排除了对其穿孔结构的确定，这 将告知线粒体凋亡的执行阶段，并揭示治疗的新表面 BAX和BAK在癌症中的激活沃伦斯基实验室最近报告了一种新的发电策略 一种适用于一系列结构-功能研究的Bax齐聚物。与BAX相比，BAK 线粒体上的构成残基，由不同的结合部位触发，表现出不同的差异 在哺乳动物组织和人类癌症中的表达模式。此外，全长BAX有很长的 由于BAK易于以重组单体形式表达，因此BAK的生产尤其受到重视 很有挑战性。瓦伦斯基实验室开发了一种三重突变结构，允许表达全长 具有生理活性和膜穿孔活性的单体BAK。在初步研究中，我 已经应用我们从生产单体BAK和寡聚BAX中学到的知识来生产全长 BAK齐聚物首次出现。在这里，我建议优化和验证这个函数的稳定性和同质性 用于严格的生化和结构特征的物种(SA1)。通过突变离散的功能区 对于BAK，我进一步建议确定激活途径每一步的结构决定因素，以及 评估BAK依赖白血病细胞(SA2)的机制发现及其功能意义。至 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-重离子交换质谱仪，冷冻电子 显微镜和细胞凋亡分析。我渴望参加拟议中的严格培训计划。 为我的研究生学习，并期待着发展成为一名独立和创新的内科科学家在 化学生物学、癌症生物学和临床肿瘤学的接口。