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， 细胞内大分子和组装体的降解，这些大分子和组装体是不必要的、有缺陷的或有害的， 细胞Beclin 1 /BECN 1是一种高度保守的关键自噬蛋白，作为整合的相互作用中心 许多调节自噬的信号BECN 1介导的自噬的缺陷或不足发挥作用 在广泛的疾病如癌症、心血管疾病、神经退行性疾病、胚胎性疾病、 缺陷，以及一些传染病，特别是那些由病毒引起的。BECN 1是一种复合物， 多结构域，构象灵活的蛋白质及其多种相互作用调节的机制 对自噬的了解很少。拟议研究的长期目标是了解BECN如何 相互作用体调节自噬和细胞内稳态。EB病毒（EBV），一种专性细胞内 疱疹病毒（HSV）感染全球超过95%的成年人群。EBV建立潜伏性和溶解性 感染，引起传染性单核细胞增多症，并与许多不同的淋巴系统恶性肿瘤有关。 和上皮组织。EBV最初阻断自噬，随后，在裂解周期诱导后， 自噬将自噬膜结合到最终的EBV包膜中，但自噬的机制 EBV调节自噬是未知的。与其他HBV一样，EBV编码抗凋亡BCL 2同源物， BHRF 1.由其他HBV编码的BCL 2与BECN 1内的非保守BH 3结构域（BH 3D）结合， 自噬，从而帮助抗HIV病毒逃避自噬降解。我们的假设是，BHRF 1结合， 影响BECN 1结构域的功能，而BH 3D是上调自噬所必需的， 相互作用引起BECN 1和BHRF 1两者的实质性构象变化。的目的 我们的建议是获得BHRF 1结合和调节的结构和机制的理解， 人BECN 1，同时阐明BHRF 1中结合诱导的结构变化。这一目标 将通过三个具体目标来实现：（1）证明BHRF 1阻断BECN 1介导的 自噬，并确定最小的BECN 1区域所需的结合BHRF 1。(2)为了阐明其机制， BHRF 1结合阻断BECN 1介导的自噬。(3)了解BECN 1结合相关 BHRF 1的结构变化。广泛的计算，结构，生物物理，生物化学，细胞和 将利用分子生物学方法来实现这些目标。这项研究将首次提供 EBV BHRF 1阻断自噬机制的生化和结构信息; BECN 1结构域在结合BCL 2中超出BH 3D;以及BECN 1结构域中的构象变化和变构位点。 BHRF 1.这些详细的信息将为阐明 BECN 1与BHRF 1和人BCL 2的结合，以潜在地鉴定BHRF 1上的“可药物化”位点， 开发选择性靶向EBV BHRF 1的治疗剂。

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.