Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/6422
Title: Structural and functional studies of the Beclin1-UVRAG complex in autophagy and endosomal trafficking
Authors: He, Yunjiao
Keywords: Autophagic vacuoles.
Proteins -- Physiological transport.
Hong Kong Polytechnic University -- Dissertations
Issue Date: 2013
Publisher: The Hong Kong Polytechnic University
Abstract: Autophagy and endosomal trafficking are two functionally distinct cellular processes with closely intertwined pathways and some shared molecular machineries. Individually and collaboratively, these two systems play crucial roles in many essential functions such as homeostasis, development and immunity. Beclin1 (Bcl-2 binding myosin like protein 1) is a protein that is intimately involved in both processes. Beclin1 forms an endogenous complex with VPS34 (Vacuolar Protein Sorting 34) that is indispensible for the biogenesis of autophagosomes and certain endosomal vesicles. UVRAG (UV radiation Resistance Associated Gene) is a Beclin1-binding protein that can associate with the core Beclin1-VPS34 complex and enhance its activity. Interestingly this UVRAG-containing Beclin1-VPS34 subcomplex co-exists with another autophagy-promoting Beclin1-VPS34 subcomplex that contains Atg14L (Autophagy 14 Like), a protein that competes with UVRAG for binding to Beclin1. The exact role of UVRAG in the VPS34-dependent autophagy and endosomal trafficking is an area of intensive investigation. The aim of this thesis is to conduct biochemical and structural analysis of the Beclin1-UVRAG interaction to help delineate its complex functional profile. We have focused on the coiled coil domains within these two proteins because they are exclusively responsible for the Beclin1-UVRAG interaction. Our biophysical and biochemical characterizations reveal that the Beclin1-UVRAG coiled coil domain complex is remarkably stable with the binding affinity (Kd) of ~0.1 μM and significantly more stable than the Beclin1-Atg14L complex (Kd ~10μM). Formation of the Beclin1-UVRAG complex readily outcompetes that of the Beclin1-Atg14L complex in an in vitro competitive ITC assay.
The structure of the Beclin1-UVRAG complex has been determined by x-ray crystallography. The crystal structure reveals a classic parallel coiled coil assembly with one strand of the Beclin1 coiled coil domain tightly wrapped along the coiled coil domain of UVRAG. The interface of the Beclin1-UVRAG complex contains a series of hydrophobic residues closely packed in pairs, one from Beclin1 and the other from UVRAG, to stabilize the coiled coil structure. The highly hydrophobic nature of the Beclin1-UVRAG interface explains the remarkable stability of this complex as observed in our biochemical studies. Using the Beclin1-UVRAG structure as guidance we generated a series of Beclin1 and UVRAG mutants to perturb the Beclin1-UVRAG interaction and characterized their impact on autophagy and endosomal trafficking using cell-based assays. First of all our data show that the Beclin1-UVRAG interaction is indispensible for the integrity of the endomembrane system because the mis-shaped endosomes and organelles found in Beclin1-/- MEF cell line can be rescued by WT Beclin1 but not Beclin1 mutants that have lost their interaction with UVRAG. Secondly our data suggest that the Beclin1-UVRAG interaction can affect cellular autophagy response because over-expression of UVRAG leads to accelerated degradation of Atg14L, a competitor against UVRAG for Beclin1 binding and a factor essential for autophagy induction. In summary our biochemical and structural studies reveal that the remarkably stable Beclin1-UVRAG interaction ensures in vivo formation of the UVRAG-containing Beclin1-VPS34 complex that is essential for the integrity of the endomembrane system and endosomal trafficking. This stable interaction can also impact autophagy through its competition with Atg14L.
Description: xvi, 139 leaves : ill. (some col.) ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577P ABCT 2013 He
URI: http://hdl.handle.net/10397/6422
Rights: All rights reserved.
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