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.
Highlights
Autophagy is a cellular process responsible for the degradation of cytoplasmic components via the lysosomal pathway [1], [2]
Autophagy is a self-degradation process via the lysosomal pathway that is required for the normal turnover of cellular components and the starvation response [1], [2]
The present study verified that the synthesized compound, MHY1485, inhibited the fusion between autophagosomes and lysosomes leading to accumulation of LC3II protein and enlarged autophagosomes in rat hepatocytes and induced the activity of mammalian target of rapamycin (mTOR)
Summary
Autophagy is a cellular process responsible for the degradation of cytoplasmic components via the lysosomal pathway [1], [2]. As an essential cellular housekeeping system, autophagy occurs at low baseline levels in all cells and contributes to maintaining cellular homeostasis [3], [4]. Autophagy is upregulated beyond basal levels when cells need to utilize intracellular nutrients under certain conditions such as starvation, hypoxia, and growth factor withdrawal [5], [6]. Cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm. Autophagosomes fuse with lysosomes to form autolysosomes, and the sequestered contents undergo degradation by lysosomal hydrolases [7]. During the formation of the autophagosome cytosolic microtubule-associated protein 1 light chain 3-I (LC3I) is cleaved and conjugated with phosphatidylethanolamine (PE), leading to formation of LC3II, an autophagic vacuole-associated form [8]
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