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.

项目摘要 Bcl-2家族蛋白是细胞凋亡的关键调节剂，并放松其蛋白质相互作用网络 驱动肿瘤发生和化学抗性。 Bax是一种促凋亡的Bcl-2蛋白，用作红衣主教 死亡路径的execution子手。在体内稳态期间，Bax居住在细胞质中的潜在单体直到 由细胞应力触发以进行重大构象变化，导致其转移到 线粒体和自组装成透明线粒体外膜的寡聚物种。 癌细胞篡夺途径的生存部门，过表达抗凋亡成员，例如Bcl-2和 Mcl-1，可以捕获Bax的激活单体，防止寡聚组件，从而保存 线粒体完整性。在我们理解Bax介导的凋亡中的一个严重缺失的联系 稳态和癌症是寡聚Bax的结构，被称为凋亡研究的“圣杯”。 Walensky实验室最近产生了第一个稳定且均匀的寡聚物种 长度可容纳结构功能分析。在表征了这种Bax低聚物的特征，称为Baxo 小角度X射线散射，负脂质体的负不锈钢显微镜（EM），以及 脂质体，线粒体和细胞中野生型和BAX突变体的比较功能研究，接下来是关键的 步骤是部署Baxo来解决确定的结构并询问其功能接口。我假设这一点 通过对Baxo产生潜水员纳米体，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。 具体而言，我的目的是（1）发展和表征与寡聚Bax结合的纳米体和（2）线束Baxo- 结合纳米体以确定寡聚Bax的结构和对膜至关重要的接口 透化功能。为了实现我的目标，我将追求一个跨学科的工作流，该工作流程结合了一个 酵母展示纳米发现平台，蛋白质工程，模型膜中的生化测定 线粒体，氢 - 居民交换质谱法，X射线晶体学，冷冻显微镜，显微镜， 和癌细胞凋亡的机理分析。通过开发和部署Baxo结合 我的目标 表征Bax介导的细胞凋亡的执行阶段，并发现新颖且潜在的吸毒 癌症热益处的表面。我很高兴能在进行严格的研究生培训计划 达纳 - 法伯癌症研究所和哈佛医学院的洛伦·沃尔伦斯基博士的实验室，看看 在生物化学界面上以独立和创新的身体科学家发展发展， 结构生物学，癌症生物学和临床肿瘤学。