A novel autophagy inhibitor, bTBT, disturbs autophagosome formation

TL;DR

This study identifies a new autophagy inhibitor, bTBT, which disrupts a late stage of autophagosome formation in mammalian cells.

Contribution

The paper introduces bTBT as a novel autophagy inhibitor targeting a unique late step in autophagosome formation.

Findings

bTBT suppresses LC3 flux and accumulates early ATG proteins in punctate structures.

bTBT prevents lysosomal marker LAMP1 from co-localizing with LC3, indicating late-stage inhibition.

bTBT causes prolonged accumulation of Stx17 and WIPI2 in large autophagic structures.

Abstract

Macroautophagy (hereafter, autophagy) is a form of intracellular degradation in which autophagosome formation is systematically coordinated by multiple processes involving numerous autophagy-related gene (ATG) proteins. Autophagy-modulating compounds are valuable for understanding the molecular mechanism of autophagy and its clinical application. Although several autophagy inhibitors have been identified, their inhibitory steps during autophagosome formation by the inhibitors are limited. Herein, we identified a novel autophagy inhibitor, bis-tributyltin (bTBT), which inhibits a unique step in autophagosome formation. In mammalian cells, bTBT treatment suppresses LC3 flux and accumulates most of ATG proteins, including LC3 and early ATG proteins (ULK1, ATG16L1, and WIPI2), in punctate structures. On the other hand, LAMP1, a lysosomal marker, did not co-localize with accumulated LC3 after bTBT treatment, indicating bTBT inhibits a late step of autophagosome formation. Stx17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that mediates autophagosome–lysosome fusion, is usually recruited to LC3-positive structures after the dissociation of early ATG proteins. However, bTBT accumulates Stx17 and WIPI2 positive large autophagic structures and maintains the autophagic structures for much longer. In conclusion, we identified a novel type of autophagy inhibitor, bTBT, which disturbs autophagosome formation.

ATG, autophagy-related gene; bTBT, bis-tributyltin; LC3, microtubule-associated protein 1A/1B-light chain 3; MEFs, mouse embryonic fibroblasts; PI3P, phosphatidylinositol 3-phosphate; Stx17, syntaxin 17; ULK1, Unc-51-like autophagy activating kinase1; VPS34, vacuolar sorting protein 34

Figures