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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 BCL-2 家族蛋白是细胞凋亡和蛋白相互作用网络失调的关键调节因子 BAX 是一种促凋亡 BCL-2 蛋白，是一种重要蛋白。在体内平衡期间，BAX 作为细胞质中的潜在单体存在。由细胞应激触发，发生重大构象变化，导致其易位至线粒体和自组装成寡聚体，使线粒体外膜通透。癌细胞侵占了该通路的生存臂，过度表达抗凋亡成员，例如 BCL-2 和 MCL-1，可以捕获 BAX 的活化单体，防止寡聚体组装，从而保留线粒体完整性是我们理解 BAX 介导的细胞凋亡的关键缺失环节。体内平衡与癌症的关系是寡聚BAX的结构，被称为细胞凋亡研究的“圣杯”。 Walensky 实验室最近生成了第一个稳定且均质的全寡聚物种。长度 BAX 适合结构功能分析，通过以下方式表征了这种 BAX 低聚物，称为 BAXo。小角 X 射线散射、BAX 穿孔脂质体的负染色电子显微镜 (EM)，以及野生型和 BAX 突变体在脂质体、线粒体和细胞中的比较功能研究，是下一步的关键步骤是部署 BAXo 来解决确定的结构并询问其功能接口。通过针对 BAXo 生成不同的纳米抗体，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 介导的线粒体凋亡机制产生新的见解。具体来说，我的目标是 (1) 开发并表征与寡聚 BAX 结合的纳米抗体，以及 (2) 利用 BAXo- 结合纳米抗体以确定寡聚 BAX 的结构和对膜至关重要的界面为了实现我的目标，我将追求包含以下内容的多学科工作流程：酵母展示纳米抗体发现平台、蛋白质工程、模型膜中的生化测定和线粒体、氢氘交换质谱、X 射线晶体学、冷冻电镜显微镜、因此，通过开发和部署 BAXo 结合来进行癌细胞凋亡的机制分析。纳米抗体在综合结构功能研究中具有内置的替代方法，我的目标是表征 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化的我很高兴能够从事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 Loren Walensky 博士的实验室，看看致力于在生物化学领域发展成为一名独立、创新的医师科学家，结构生物学、癌症生物学和临床肿瘤学。