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Rapid changes in the ATG5-ATG16L1 complex following nutrient deprivation measured using NanoLuc Binary Technology (NanoBIT)

Crowley, Emily; Leung, Euphemia; Reynisson, Johannes; Richardson, Alan


Emily Crowley

Euphemia Leung


Autophagy plays a role in several human diseases, but each of the current methods to measure autophagy have significant drawbacks. ATG5 and ATG16L1 are regulators necessary for autophagy therefore, drugs which inhibit the interaction of these proteins may be therapeutically useful. To evaluate the interaction of ATG5 and ATG16L1 in cells, their cDNAs were fused to the coding sequences of SmBIT and LgBIT, two components of Nanoluc luciferase. This generated a luminescent signal when SmBIT and LgBIT interacted to form a functional luciferase as a result of their co-localization which was brought about by the binding of ATG5 and ATG16L1. The assay measures the interaction in real time and can be used in microplate format to allow for multiple experimental conditions to be assessed. The interaction of ATG5 and ATG16L1 is not significantly altered by inhibition of lysosomal function, or inhibitors of Ulk1, vps34 or mTORC1. Although there was constitutive interaction of ATG5 and ATG16L1 and luminescence was stimulated within 3 minutes, by up to 500%, when the cells are deprived of nutrients. When the nutrients are returned, the complex returns to its basal status equally rapidly. Sphingosine-1-Phosphate and CYM-5541 partially repressed the effects of nutrient starvation. Furthermore, we identified a small molecule inhibitor that interferes with the interaction of ATG5 and ATG16L1 in cells. This assay provides a novel tool for researchers to measure autophagy and can be potentially applied to many cell types.

Journal Article Type Article
Acceptance Date Jan 28, 2020
Online Publication Date Mar 4, 2020
Publication Date 2020-11
Publicly Available Date May 26, 2023
Journal The FEBS Journal
Print ISSN 1742-464X
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 287
Issue 22
Pages 4917-4932
Keywords Autophagy, ATG5, ATG16L1, NanoBIT
Publisher URL