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In vitro studies of autophagic membrane expansion and cargo recruitment mediated by human lipidated ATG8 proteins

Zhang, Wenxin; (2023) In vitro studies of autophagic membrane expansion and cargo recruitment mediated by human lipidated ATG8 proteins. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Autophagy is a highly conserved bulk degradation pathway, in which cells generate a unique double-membraned vesicle, called “autophagosome”, to engulf cytoplasmic materials for lysosomal degradation. It targets long-lived proteins or damaged organelles under both basal or stressed conditions to maintain cellular homeostasis and cell survival. The core autophagy machinery has been well characterised and is regulated hierarchically by autophagy-related (ATG) proteins. ATG8 is a unique ubiquitin-like protein covalently conjugated on autophagosomes via lipidation reaction. The lipidated ATG8 is a key player along the whole autophagic process, from autophagosome biogenesis, membrane expansion, cargo recruitment, until autophagosome-lysosome fusion. To better understand how lipidated ATG8 mediates autophagosome membrane expansion and cargo recruitment, I employed various in vitro reconstitution approaches using artificial model membranes to investigate human lipidated ATG8 proteins (LC3s/GABARAPs). First, using a real-time lipidation assay, I found that the N-termini of LC3B/GABARAP are highly dynamic and associate with membrane after lipidation. The atomistic MD simulation and FRET assays indicate that N-termini of LC3B/GABARAP associate in cis with the membrane where ATG8 gets lipidated. This cis-membrane association is important to maintain membrane expansion and regulate autophagosome size in cells, consequently, resulting in an efficient degradation of cytosolic cargo p62. Afterwards, I investigated the function of lipidated ATG8 during cargo recruitment. The preliminary results show that p62 is prone to interact with membrane-bound ATG8. This suggests that the lipidated ATG8 may have a higher binding affinity to cytosolic substrates, for example p62, compared to cytosolic ATG8. More experiments are required to better understand the interaction between lipidated ATG8 and p62. Finally, I performed in vitro ATG8 lipidation reactions using the cell membranes derived from autophagic deficient cells, which could be the potential membrane models for the future studies. In this study, I proposed a model integrating the functions of ATG8 lipidation in membrane expansion.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: In vitro studies of autophagic membrane expansion and cargo recruitment mediated by human lipidated ATG8 proteins
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10163621
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