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

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dc.contributor.author Crowley, Emily en
dc.contributor.author Leung, Yee Fun en
dc.contributor.author Reynisson, Jóhannes en
dc.contributor.author Richardson, Alan en
dc.date.accessioned 2020-06-15T03:54:26Z en
dc.date.issued 2020-03-04 en
dc.identifier.issn 1742-464X en
dc.identifier.uri http://hdl.handle.net/2292/51556 en
dc.description.abstract Autophagy plays a role in several human diseases, but each of the current methods to measure autophagy has significant drawbacks. ATG5 and ATG16L1 are regulators necessary for autophagy; therefore, drugs that 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 colocalization that 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. However, there was a constitutive interaction of ATG5 and ATG16L1 and luminescence was stimulated within 3 min, by up to 500%, when the cells were 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. DATABASE: Replicate data are available in Figshare database https://doi.org/10.6084/m9.figshare.11798946. en
dc.format.medium Print-Electronic en
dc.language eng en
dc.relation.ispartofseries The FEBS journal en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Rapid changes in the ATG5-ATG16L1 complex following nutrient deprivation measured using NanoLuc Binary Technology (NanoBIT). en
dc.type Journal Article en
dc.identifier.doi 10.1111/febs.15275 en
dc.rights.holder Copyright: The author en
pubs.publication-status Published en
dc.rights.accessrights http://purl.org/eprint/accessRights/RestrictedAccess en
pubs.subtype Journal Article en
pubs.elements-id 797199 en
pubs.org-id Medical and Health Sciences en
pubs.org-id Medical Sciences en
pubs.org-id Auckland Cancer Research en
dc.identifier.eissn 1742-4658 en
pubs.record-created-at-source-date 2020-03-05 en
pubs.dimensions-id 32129924 en

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