title: 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.
authors.affiliation: Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan, Korea.
authors.forename: Yeon Ja
authors.initials: YJ
authors.lastname: Choi
authors.forename: Yun Jung
authors.initials: YJ
authors.lastname: Park
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Park
authors.forename: Hyoung Oh
authors.initials: HO
authors.lastname: Jeong
authors.forename: Dae Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Young Mi
authors.initials: YM
authors.lastname: Ha
authors.forename: Ji Min
authors.initials: JM
authors.lastname: Kim
authors.forename: Yu Min
authors.initials: YM
authors.lastname: Song
authors.forename: Hyoung-Sam
authors.initials: HS
authors.lastname: Heo
authors.forename: Byung Pal
authors.initials: BP
authors.lastname: Yu
authors.forename: Pusoon
authors.initials: P
authors.lastname: Chun
authors.forename: Hyung Ryong
authors.initials: HR
authors.lastname: Moon
authors.forename: Hae Young
authors.initials: HY
authors.lastname: Chung
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D008099:Liver / Q000166:cytology; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D062105:Molecular Docking Simulation; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D058570:TOR Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D014227:Triazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
chemical_list: D008869:Microtubule-Associated Proteins; D009025:Morpholines; D058570:TOR Serine-Threonine Kinases; D014227:Triazines; C577756:4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine; C526109:Anxa3 protein, rat