Patel, Bhavini (2020) Molecular functions of ArfGAP3 during autophagy and ageing in Drosophila. PhD thesis, University of Warwick.

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Abstract

Loss of proteostasis is one of the key hallmarks of ageing. Autophagy is one of the two key mechanisms that regulates protein homeostasis by intracellularly degrading cellular components. Functional dysfunctions in autophagy is a mechanistic feature in neurodegeneration. Macroautophagy is closely related to the endosomal trafficking as both have converging steps and common participating molecules. Atg8/LC3, is a key autophagy protein embedded within the autophagosomal membrane. In order to find Atg8a-interacting proteins in Drosophila, a yeast-two hybrid screening was performed, in which ArfGAP3, an endosomal trafficking associated protein, was found to be a novel Atg8a interactor. ArfGAP3 belongs to the ArfGAP sub-family of multi domain proteins with a primary function to hydrolyse GTP-bound Arf proteins. Co-localisation experiments in Drosophila fat body in this study further supports this interaction as strong co-localisation of ArfGAP3 and Atg8a to autophagosomes was observed when cells were subjected to starvation to initiate autophagy. Biochemical analysis also confirmed that endogenous ArfGAP3 is selectively degraded by autophagy as accumulation of endogenous ArfGAP3 was observed in Atg8a mutant flies compared to wild-type. Herein, we show that knockdown of ArfGAP3 seems to disrupt the autophagic flux. We also show that ArfGAP3 co-localises to Rab5 and Rab7, and knockdown disrupts endo-lysosome fusion. Additionally, we show low levels of ArfGAP3 disrupts functional lysosomal degradation and lipid droplet maturation. Moreover, knockdown of ArfGAP3 resulted in an accumulation of Ref(2)P in aged flies. Our study for the first time provides convincing evidence of the interaction between ArfGAP3 and Atg8a suggesting ArfGAP3 potentially plays a key role in the relationship between endosomal trafficking and selective autophagy. Furthermore, our study also suggests possible implications of ArfGPA3 in age-related diseases.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history
Library of Congress Subject Headings (LCSH):	Cellular control mechanisms, Proteins, Drosophila -- Physiological aspects, Phagosomes, Endosomes, Cells -- Aging
Official Date:	October 2020
Dates:	Date Event October 2020 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Nezis, Ioannis P.
Sponsors:	Midlands Integrative Biosciences Training Partnership
Format of File:	pdf
Extent:	xvi, 241 leaves : colour illustrations
Language:	eng

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