Nanobodies for Dissecting the Structure and Function of Oligomeric BAX

用于剖析寡聚 BAX 结构和功能的纳米抗体

• 批准号: 10677287
• 负责人: Ellen Yu
• 金额: $ 4.01万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677287
• 关键词: Affinity; Affinity Chromatography; Apoptosis; Apoptosis Regulation Gene; Apoptotic; BAX gene; BCL-2 Protein; BCL1 Oncogene; BCL2 gene; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Cancer Biology; Cell Death; Cell Membrane Permeability; Cells; Cellular Stress; Cessation of life; Chemoresistance; Choline; Clinical Oncology; Complex; Cryoelectron Microscopy; Cytosol; Dana-Farber Cancer Institute; Detergents; Deuterium; Development; Dissection; Electron Microscopy; Epitopes; Escherichia coli; Exposure to; Family; Goals; Homeostasis; Homo; Human; Hydrogen; In Vitro; Incubated; Induction of Apoptosis; Laboratories; Length; Leukemic Cell; Link; Liposomes; MCL1 gene; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Methods; Microscopy; Mitochondria; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Sieve Chromatography; Monitor; Mutagenesis; Negative Staining; Outer Mitochondrial Membrane; Pathway interactions; Permeability; Phase; Physicians; Protein Engineering; Protein Family; Proteins; Protocols documentation; Recombinant Proteins; Relapse; Research; Resolution; Roentgen Rays; Rupture; Scientist; Site-Directed Mutagenesis; Stress; Structure; Surface; Technology; Therapeutic; Training Programs; X-Ray Crystallography; Yeasts; arm; cancer cell; comparative; cytochrome c; innovation; insight; medical schools; member; membrane model; monomer; multidisciplinary; mutant; nanobodies; next generation sequencing; novel; overexpression; preservation; prevent; programs; refractory cancer; response; self assembly; structural biology; structural determinants; tool; tumorigenesis

PROJECT SUMMARY BCL-2 family proteins are critical regulators of apoptosis and deregulation of their protein interaction network drives oncogenesis and chemoresistance. BAX is a pro-apoptotic BCL-2 protein that serves as a cardinal executioner of the death pathway. During homeostasis, BAX resides as a latent monomer in the cytosol until triggered by cellular stress to undergo a major conformational change, leading to its translocation to the mitochondria and self-assembly into oligomeric species that permeabilize the mitochondrial outer membrane. Cancer cells usurp the survival arm of the pathway, overexpressing anti-apoptotic members such as BCL-2 and MCL-1, which can trap activated monomers of BAX, prevent oligomeric assembly, and thereby preserve mitochondrial integrity. A critical missing link in our understanding of BAX-mediated apoptosis during homeostasis and cancer is the structure of oligomeric BAX, referred to as the “holy grail” of apoptosis research. The Walensky laboratory has recently generated the first stable and homogeneous oligomeric species of full- length BAX amenable to structure-function analyses. Having characterized this BAX oligomer, termed BAXo, by small-angle X-ray scattering, negative stain electron microscopy (EM) of BAX-porated liposomes, and comparative functional studies of wild-type and BAX mutants in liposomes, mitochondria, and cells, a critical next step is to deploy BAXo to solve a definitive structure and interrogate its functional interfaces. I hypothesize that by generating diverse nanobodies against BAXo, I will be able to obtain high-resolution structures of this elusive “death channel” and generate fresh insight into the mechanism of BAX-mediated mitochondrial apoptosis. Specifically, I aim to (1) develop and characterize nanobodies that bind to oligomeric BAX and (2) harness BAXo- binding nanobodies to determine the structure of oligomeric BAX and the interfaces critical to membrane- permeabilizing function. To accomplish my goals, I will pursue a multidisciplinary workflow that incorporates a yeast display nanobody discovery platform, protein engineering, biochemical assays in model membranes and mitochondria, hydrogen-deuterium exchange mass spectrometry, X-ray crystallography, cryo- EM microscopy, and mechanistic analyses of apoptosis in cancer cells. Thus, by developing and deploying BAXo-binding nanobodies in comprehensive structure-function studies with built-in alternative approaches, I aim to both characterize the execution-phase of BAX-mediated apoptosis and uncover novel and potentially druggable surfaces for therapeutic benefit in cancer. I am excited to be pursuing a rigorous graduate training program in the laboratory of Dr. Loren Walensky at the Dana-Farber Cancer Institute and Harvard Medical School, and look forward to developing as an independent and innovative physician-scientist at the interface of biochemistry, structural 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网络失控的关键调节因子 促进肿瘤发生和化疗耐药。Bax是一种促凋亡的bcl2蛋白，它是一种 死亡之路的刽子手。在动态平衡期间，Bax以潜伏单体的形式存在于细胞质中，直到 由细胞应激触发，经历重大构象变化，导致其移位到 线粒体和自我组装成低聚物物种，使线粒体外膜通透。 癌细胞侵占了该途径的生存臂，过度表达抗凋亡成员，如bcl2和 MCL-1，它可以捕获激活的bax单体，阻止寡聚组装，从而保存 线粒体的完整性。我们在理解Bax介导的细胞凋亡过程中的一个关键缺失环节 动态平衡与癌症是结构寡聚的Bax，被称为细胞凋亡研究的“圣杯”。 瓦伦斯基实验室最近产生了第一个稳定和均匀的全低聚物种- 长度BAX服从结构-功能分析。在对这种名为BAXo的Bax低聚物进行表征后， Bax脂质体的小角X射线散射、负染色电子显微镜(EM)和 脂质体、线粒体和细胞中野生型和Bax突变体的功能比较研究，关键的下一步 步骤是部署BAXo来解决最终的结构和询问其功能接口。我假设 通过产生不同的针对BAXO的纳米抗体，我将能够获得这种难以捉摸的高分辨率结构 “死亡通道”，并对Bax介导的线粒体凋亡的机制产生新的见解。 具体地说，我的目标是(1)开发和表征与寡聚体Bax结合的纳米体，以及(2)利用BAXo- 结合纳米体以确定寡聚体Bax的结构和对膜至关重要的界面- 透气功能。为了实现我的目标，我将追求一种多学科的工作流程，其中包括 酵母展示纳米体发现平台、蛋白质工程、模型膜中的生化分析和 线粒体、氢-氚交换质谱仪、X射线结晶学、低温电子显微镜、 以及对癌细胞凋亡的机制分析。因此，通过开发和部署BAXo绑定 在全面的结构-功能研究中，通过内置的替代方法，我的目标是既 表征Bax介导的细胞凋亡的执行阶段并发现新的和潜在的可药物 用于癌症治疗的表面。我很高兴能参加一个严格的研究生培训项目 Dana-Farber癌症研究所和哈佛医学院的Loren Walensky博士的实验室，看看 期待着在生物化学领域发展成为一名独立和创新的内科医生-科学家， 结构生物学、癌症生物学和临床肿瘤学。