Agrotis, Alexander George; (2018) The regulation of human autophagy by ATG4 proteases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Autophagy is an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane organelles called autophagosomes. Lipidation of ubiquitin- like LC3/GABARAP proteins on the autophagosome membrane is essential for autophagy. The cysteine protease ATG4 executes C-terminal processing (priming) of newly-synthesised LC3/GABARAP to enable subsequent lipidation. ATG4 is also proposed to be important for deconjugating/delipidating LC3/GABARAP from autophagosome membranes, although the ex- act purpose of this is unclear in mammals. Four ATG4 isoforms (ATG4A-D) exist in mammals, however the functional redundancy of these proteins in cells is poorly understood. The aim of this thesis is to characterise the redundancy of human ATG4 proteins and investigate their deconjugation roles in cells. In Chapter 3, I show that human HAP1 and HeLa cells lacking ATG4B exhibit a severe but incomplete defect in LC3/GABARAP processing and autophagy. By further genetic depletion of ATG4 isoforms I uncover that ATG4A, ATG4C and ATGD all contribute to residual priming activity, which is sufficient to enable lipidation of GABARAPL1 on autophagosomes. In Chapter 4, I reveal that delipidation of LC3/GABARAP by ATG4 isoforms is not essential for autophagic degradation of the cargo receptor p62/SQSTM1, arguing that delip- idation by ATG4 has limited importance in mammalian autophagy compared to LC3/GABARAP priming. Finally, I report the discovery of a novel deconjugation function of ATG4 isoforms in the removal of LC3/GABARAP conjugates from endogenous proteins such as ATG3 and ATG7. Thus, I provide the first evidence that LC3/GABARAP can act as a protein modifier and that ATG4 can reverse such modifications, opening up a new avenue of future research to under- stand the functional relevance of this phenomenon.

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