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Title: Investigation of the role of WIPI2 in autophagosome formation : functional characterisation of the WIPI2-Atg16L1 interaction
Author: Dooley, H. C.
ISNI:       0000 0004 5358 2411
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Macroautophagy, here called autophagy, is a catabolic process that is required for cell homeostasis and cell survival under nutrient starvation, as well as development and immunity in higher eukaryotes. Characterised by the formation of double membrane-bound vesicles, termed autophagosomes, macroautophagy results in degradation of intracellular proteins in the lysosome. Autophagy initiation leads to the formation of a double membrane phagophore, which expands and sequesters cytoplasm components before autophagosome closure and fusion with endosomes and lysosomes. Autophagosome formation requires the sequential and concerted action of a number of core autophagy proteins. First identified in yeast, characterisation of mammalian autophagy proteins has shed light on the molecular mechanisms of autophagy initiation and phagophore expansion. However, a number of key questions remain unanswered, including what the functions of the core autophagy proteins mAtg9 and WIPI2 are. mAtg9 is the only transmembrane protein required for autophagy while WIPI2 is a PtdIns(3)P-binding protein. To address the function of these proteins, I used and explored immunoprecipitation - mass spectrometry based approaches to identify and characterise novel interactors. I showed that mAtg9 binds transferrin receptor and this interaction supports a model whereby mAtg9 traffics from a tubular-vesicular mAtg9 compartment, with recycling endosome-like characteristics, to support autophagosome formation. β-propeller proteins such as WIPI2 frequently act in mediating protein-protein interactions and so WIPI2 may recruit autophagy proteins to the PtdIns(3)P-positive site of autophagosome formation. I characterised the Atg16L1-WIPI2b interaction and investigated possible regulation of WIPI2 function by phosphorylation. I used immunoprecipitation to map the interacting regions of WIPI2b and Atg16L1 and to demonstrate that these proteins interact directly. I produced binding mutants of each protein and subsequently characterised the function of Atg16L1-WIPI2b binding, showing that the interaction is required for starvation- and pathogen-induced autophagy. Preliminary data was also obtained on the phosphorylation of the C-terminus of WIPI2 as a possible mechanism of WIPI2 function regulation. I propose a model in which WIPI2b is a PtdIns(3)P effector protein required for recruitment of the Atg12–5-16L1 complex to the site of autophagosome formation and LC3 lipidation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available