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

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

Abstract

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.

Citation

Crowley, E., Leung, E., Reynisson, J., & Richardson, A. (2020). Rapid changes in the ATG5-ATG16L1 complex following nutrient deprivation measured using NanoLuc Binary Technology (NanoBIT). The FEBS Journal, 287(22), 4917-4932. https://doi.org/10.1111/febs.15275

• 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
• DOI: https://doi.org/10.1111/febs.15275
• Keywords: Autophagy, ATG5, ATG16L1, NanoBIT
• Publisher URL: https://febs.onlinelibrary.wiley.com/doi/abs/10.1111/febs.15275