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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要 Bcl-2家族蛋白是凋亡的关键调节剂，并放松其蛋白质相互作用有助于癌症的发展和化学抗性。细胞死亡的红衣主教执行者Bax 和bak，分别居住在细胞的细胞质和线粒体中，直至被压力激活刺激以自我关联和刺激线粒体外膜，导致凋亡细胞死亡。挫败化学疗法诱导的凋亡和强化细胞永生，癌症篡夺了细胞存活臂 Bcl-2途径通过过表达抗凋亡构件，该抗凋亡构件可以与Bax和Bak结合并防止其转化为有毒线粒体孔。长期无法产生稳定和同质的全长bax和bak的寡聚形式排除了其孔隙结构的确定，将告知线粒体细胞凋亡的执行阶段，并揭示治疗性的新表面 Bax和Bak激活癌症。 Walensky实验室最近报告了一种新的生成策略可容纳一系列结构功能研究的BAX低聚物。与Bax相反，Bak 线粒体的组成性残留物是由独特的结合位点触发的，并表现出差异哺乳动物组织和人类癌症中的表达模式。此外，全长bax的长度很长尤其是重组单体形式的表达，尤其是Bak的产生具有挑战性的。沃尔士斯基实验室开发了一种三突变构建体，允许表达全长表现出生理激活和膜状活性的单体BAK。在初步研究中，我已经从单体BAK和低聚Bax的生产中应用了我们的学习来产生全长首次Bak低聚物。在这里，我建议优化和验证此的稳定性和同质性严格的生化和结构表征的物种（SA1）。通过突变离散功能区域我进一步建议确定激活途径每个步骤的结构决定因素，并评估机械发现及其在BAK依赖性白血病细胞中的功能意义（SA2）。到实现我的目标，我将采用包括蛋白质工程，生化测定在内的多学科方法在模型膜和线粒体中，氢 - 居民交换质谱法，冷冻电子显微镜和细胞凋亡分析。我渴望着手进行严格的培训计划对于我的研究生学习，并期待作为一名独立和创新的医师科学家发展化学生物学，癌症生物学和临床肿瘤学的界面。