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

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 降解细胞内不必要的、有缺陷的或有害的大分子和组件 细胞。Beclin 1/BECN1是一种高度保守的关键自噬蛋白，是整合 大量调节自噬的信号。BECN1介导的自噬中的缺陷或缺陷在其中起作用 在一系列疾病中，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，特别是由病毒引起的疾病。BECN1是一个复合体， 多结构域、构象柔性蛋白质及其不同相互作用的调控机制 人们对自噬知之甚少。拟议研究的长期目标是了解BECN是如何 相互作用组能调节自噬和细胞内稳态。EB病毒(EBV)--一种专性细胞内病毒 ɣ疱疹病毒(ɣHV)感染了全球95%以上的成年人。EBV建立潜伏和裂解 感染，导致传染性单核细胞增多症，与许多不同的淋巴系统恶性肿瘤有关 和上皮组织。EBV最初阻止自噬，后来，在溶解周期诱导后，上调 自噬将自噬膜结合到最终的EB病毒包膜中，但其机制(S) EB病毒对自噬的调节尚不清楚。与其他ɣHV一样，EBV编码抗凋亡的bcl2同源物， BHRF1.由其他ɣHV编码的BCL2结合到BECN1中的非保守BH3域(BH3D)以阻止 自噬，从而帮助ɣHV逃避自噬降解。我们的假设是BHRF1与AND结合 影响BECN1结构域的功能，而不是BH3D上调自噬所必需的，这 相互作用导致BECN1和BHRF1的实质性构象变化。这样做的目的是 建议是获得BHRF1约束和调节的结构性和机械性理解 人BECN1，同时阐明结合诱导BHRF1的结构变化。这一目标 将通过三个具体目标来实现：(1)证明BHRF1阻断BECN1介导的 自噬，并确定与BHRF1结合所需的最小BECN1区域。(2)通过以下方式阐明其机制 BHRF1结合阻断BECN1介导的自噬。(3)了解BECN1结合相关 BHRF1基因的结构变化。广泛的计算、结构、生物物理、生化、细胞和 分子生物学方法将被用来实现这些目标。这项研究将提供第一个 EBV BHRF1阻断自噬机制的生化和结构信息； BECN1结构域在BH3D之外结合BCL2；以及构象变化和变构位置 BHRF1.这些详细的信息将为阐明 BECN1与BHRF1和人BCL2的结合，以潜在地识别BHRF1上的“可药物”部位，从而使 选择性靶向EBV BHRF1的治疗药物的发展。

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.