Molecular mechanism by which Epstein-Barr Virus-encoded BHRF1 blocks BECN1-mediated autophagy

Epstein-Barr病毒编码的BHRF1阻断BECN1介导的自噬的分子机制

• 批准号: 10439285
• 负责人: Sangita Sinha
• 金额: $ 43.5万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10439285
• 关键词: Adult; Allosteric Site; Apoptotic; Artificial Intelligence; Autophagocytosis; BCL-2 Protein; BCL2 gene; BH3 Domain; Binding; Biochemical; Biological Assay; Biophysics; Calorimetry; Cardiovascular Diseases; Cardiovascular system; Cell Death; Cell physiology; Cells; Cellular Assay; Communicable Diseases; Complex; Coronavirus; Coupled; Crystallization; Data; Defect; Development; Disease; Down-Regulation; Embryo; Epithelial; Goals; HIV; Herpesviridae; Homeostasis; Homologous Gene; Human; Human Herpesvirus 4; Human Herpesvirus 8; Infectious Mononucleosis; Knock-out; Lytic Phase; Malignant Neoplasms; Malignant lymphoid neoplasm; Mediating; Membrane; Methods; Molecular; Molecular Conformation; Molecular and Cellular Biology; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Pathway interactions; Pharmaceutical Preparations; Play; Population; Proteins; Regulation; Research; Role; Signal Transduction; Site; Structure; Surface; Testing; Thermodynamics; Tissues; Titrations; Viral Proteins; Virus; Yeasts; base; experimental study; flexibility; gammaherpesvirus; human disease; insight; latent infection; macromolecule; new therapeutic target; simulation; therapeutic development; therapeutic target; virus envelope

PROJECT ABSTRACT Autophagy is a conserved eukaryotic, cellular catabolic pathway responsible for the sequestration and degradation of intracellular macromolecules and assemblies that are unnecessary, defective or harmful to cells. Beclin 1 / BECN1, a highly conserved, key autophagy protein serves as an interaction hub to integrate numerous signals that regulate autophagy. Defects or deficiencies in BECN1-mediated autophagy play a role in a wide range of diseases such as cancer, cardiovascular diseases, neurodegenerative diseases, embryonic defects, as well as several infectious diseases, especially those caused by viruses. BECN1 is a complex, multi-domain, conformationally-flexible protein and the mechanisms by which its diverse interactions regulate autophagy is poorly understood. The long-term goal of the proposed research is to understand how the BECN interactome regulates autophagy and cellular homeostasis. Epstein-Barr virus (EBV), an obligate intracellular ɣ-herpesvirus (ɣHV), infects more than 95% of the global adult population. EBV establishes latent and lytic infections, causing infectious mononucleosis and is associated with many diverse malignancies of lymphoid and epithelial tissues. EBV initially blocks autophagy, and later, after lytic cycle induction, up-regulates autophagy incorporating autophagic membranes into the final EBV envelope, but the mechanism(s) by which EBV modulates autophagy is unknown. Like other ɣHVs, EBV encodes an anti-apoptotic BCL2 homolog, BHRF1. BCL2s encoded by other ɣHVs bind to a non-conserved BH3 domain (BH3D) within BECN1 to block autophagy, thereby helping ɣHVs evade autophagic degradation. Our hypothesis is that BHRF1 binds to and impacts the function of BECN1 domains beyond the BH3D essential for up-regulating autophagy, and this interaction causes substantial conformational changes in both BECN1 and BHRF1. The objective of this proposal is to obtain a structural and mechanistic understanding by which BHRF1 binds to and regulates human BECN1, while simultaneously elucidating binding-induced structural changes in BHRF1. This objective will be accomplished by three specific aims: (1) To demonstrate that BHRF1 blocks BECN1-mediated autophagy, and identify the minimal BECN1 region required to bind BHRF1. (2) To elucidate the mechanism by which BHRF1-binding blocks BECN1-mediated autophagy. (3) To understand BECN1-binding associated structural changes in BHRF1. A wide range of computational, structural, biophysical, biochemical, cellular and molecular biology methods will be used to accomplish these aims. This research will provide the first biochemical and structural information of the mechanism by which EBV BHRF1 blocks autophagy; the role of BECN1 domains beyond the BH3D in binding BCL2s; and conformational changes and allosteric sites in BHRF1. This detailed information will lay the groundwork to elucidating similarities and differences in the binding of BECN1 to BHRF1 and human BCL2s to potentially identify “drug-able” sites on BHRF1 to enable development of therapeutics that selectively target EBV BHRF1.

项目摘要 自噬是一种保守的真核，细胞分解代谢途径，负责隔离和 不必要，有缺陷或有害的细胞内大分子和组件的降解 细胞。 Beclin 1 / becN1，一种高度保守的密钥自噬蛋白，用作集成的相互作用中心 许多调节自噬的信号。 BECN1介导的自噬的缺陷或缺陷起作用 在多种疾病中，例如癌症，心血管疾病，神经退行性疾病，胚胎 缺陷以及几种传染病，尤其是病毒引起的疾病。 BECN1是一个复杂的 多域，构象的蛋白质及其潜水员相互作用的机制 自噬知之甚少。拟议研究的长期目标是了解BECN 相互作用组调节自噬和细胞稳态。 Epstein-Barr病毒（EBV），专性细胞内 ɣ疱疹病毒（ɣHV），感染超过95％的全球成年人口。 EBV建立潜在和裂解 感染，引起感染性单核细胞增多症，并与许多淋巴的潜水恶性肿瘤有关 和上皮组织。 EBV最初阻止自噬，然后在裂解周期感应后，上调 将自噬机制编码为最终的EBV包膜的自噬，但是 EBV调节自噬是未知的。像其他ɣHV一样，EBV编码抗凋亡的Bcl2同源物， BHRF1。由其他ɣHV编码的Bcl2s与BECN1中的非保存BH3域（BH3D）结合到阻止 自噬，从而帮助ɣHV逃避自噬降解。我们的假设是BHRF1与 影响BECN1域的功能以外的BH3D对于上调自噬所必需的，这 相互作用会导致BECN1和BHRF1的实质构象变化。这个目的 建议是获得BHRF1与结合并调节的结构和机械理解 人类BECN1同时阐明了结合诱导的BHRF1的结构变化。这个目标 将通过三个特定目标来完成：（1）证明BHRF1阻止BECN1介导 自噬，并确定结合BHRF1所需的最小BECN1区域。 （2）通过 哪个BHRF1结合可以阻止BECN1介导的自噬。 （3）了解与BECN1结合相关的 BHRF1的结构变化。广泛的计算，结构，生物物理，生化，细胞和 分子生物学方法将用于实现这些目标。这项研究将提供第一个 EBV BHRF1阻止自噬的机制的生化和结构信息；的作用 在结合Bcl2s中，BH3D以外的BECN1域；以及构象变化和变构位点 BHRF1。这些详细信息将为阐明相似性和差异的基础 BECN1与BHRF1和人BCl2的结合，以识别BHRF1上的“可吸毒”位点以启用 理论的发展有选择地针对EBV BHRF1。