Inhibitory Effect of mTOR Activator MHY1485 on Autophagy: Suppression of Lysosomal Fusion

Abstract

Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.

Improved autophagic flux is correlated with mTOR activation in the later recovery stage of experimental acute pancreatitis

“…Inhibition of mTOR by rapamycin has been reported to increase the clearance of aggregated mutant proteins through autophagy degradation pathway in many neurodegenerative diseases [43e45]. Accordingly, several recent studies suggest that mTOR inactivation results in the activation of lysosomal function including lysosomal degradation and fusion, whereby autophagic degradation may be enhanced [46,47]. However, increased lysosome activity in HeLa cells under mTOR suppression requires the fusion of autophagosomes with lysosomes [46].…”

Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development

“…Is it possible, for example, to directly target the mTOR system, below the broadly regulatory phosphatidylinositol-3-kinase/protein kinase B stage (Figure 8)? Activation via agents such as the small experimental drugs, MHY 1485 293 or 3BDO 294 might confine the effects to a smaller set of endpoints than use of IGF-1. Will it be possible to selectively target functions below the level of mTOR, for example modulating amino acid transporter expression via changes in microtubule organization?…”

The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress

“…To provide evidence that on C2C12 cells stress granules are cleared through autophagy, we incubated cells either for 3 hours with 12 mmol/L MHY-1485, an mTOR activator that potently inhibits autophagy by suppression of fusion between autophagosomes and lysosomes, 28 or for 24 hours with 20 mmol/L leupeptin, a protease inhibitor that severely impairs lysosomal function. Cells exposed to arsenite and …”