Sunday, November 24, 2024
11/24/2024
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.
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