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Cited by 192Autophagy in Disease and TherapyCalcium signaling and nucleotide metabolismPI3K/AKT/mTOR signaling in cancer

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

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Choi, Yeon Ja, Park, Yun Jung, Park, Ji Young, Jeong, Hyoung Oh, Kim, Dae Hyun, Ha, Young Mi, Kim, Ji Min, Song, Yu Min, Heo, Hyoung-Sam, Yu, Byung Pal, Chun, Pusoon, Moon, Hyung Ryong, Chung, Hae Young·2012·PLoS ONE
DOI 10.1371/journal.pone.0043418PMC PMC3425474
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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.

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Figures

Figure 1 Brief scheme of the synthetic process of MHY1485.
Figure 1 Brief scheme of the synthetic process of MHY1485.CC BY · PubMed Central
Figure 2 Increase of the LC3II/LC3I ratio and LC3II-positive vacuoles by MHY1485. Western blot analysis was performed to detect LC3II and LC3I. Ac2F cells were treated with different concentrations of MHY1485 and rapamycin 5 µM as a positive control for 6 h. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated or rapamycin-treated samples with the LC3II/LC3I ratios from untreated samples (A). Control cells treated with same volume of vehicle or MHY1485 (2 µM) for 1, 6 or 12 h were collected. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated samples with LC3II/LC3I ratio from control samples at 1 hour (B). β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, **p<0.01, ***p<0.001; n = 3). Live-cell confocal microscopic images of AdGFP-LC3-transfected Ac2F cells treated with 2 µM MHY1485 for 1, 6 or 12 h are shown. The images show the GFP-LC3-positive vacuoles (upper, green), corresponding phase contrast images (middle) and merged images (bottom). Scale bar, 20 µm.
Figure 2 Increase of the LC3II/LC3I ratio and LC3II-positive vacuoles by MHY1485. Western blot analysis was performed to detect LC3II and LC3I. Ac2F cells were treated with different concentrations of MHY1485 and rapamycin 5 µM as a positive control for 6 h. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated or rapamycin-treated samples with the LC3II/LC3I ratios from untreated samples (A). Control cells treated with same volume of vehicle or MHY1485 (2 µM) for 1, 6 or 12 h were collected. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated samples with LC3II/LC3I ratio from control samples at 1 hour (B). β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, **p<0.01, ***p<0.001; n = 3). Live-cell confocal microscopic images of AdGFP-LC3-transfected Ac2F cells treated with 2 µM MHY1485 for 1, 6 or 12 h are shown. The images show the GFP-LC3-positive vacuoles (upper, green), corresponding phase contrast images (middle) and merged images (bottom). Scale bar, 20 µm.CC BY · PubMed Central
Figure 3 Failure of the increase of autophagic flux. Western blot analysis was performed with samples from cells treated in 2 µM MHY1485 for 6 h. The lysosomotropic agents bafilomycin A1 (bafA1, 10 nM) and chloroquine (100 µM) were applied 1 h before the cell harvest to measure the autophagic flux (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (**p<0.01; n = 3). C and D show the immunoblots of p62 and beclin-1 in samples from the cells treated with MHY1485 or the same amount of vehicle for different times. The blots were quantified by densitometry and were normalized with the control sample at 1 h expressed as mean±SD (*p<0.05; n = 3).
Figure 3 Failure of the increase of autophagic flux. Western blot analysis was performed with samples from cells treated in 2 µM MHY1485 for 6 h. The lysosomotropic agents bafilomycin A1 (bafA1, 10 nM) and chloroquine (100 µM) were applied 1 h before the cell harvest to measure the autophagic flux (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (**p<0.01; n = 3). C and D show the immunoblots of p62 and beclin-1 in samples from the cells treated with MHY1485 or the same amount of vehicle for different times. The blots were quantified by densitometry and were normalized with the control sample at 1 h expressed as mean±SD (*p<0.05; n = 3).CC BY · PubMed Central
Figure 4 Inhibition of starvation-induced autophagic flux by MHY1485. Western blot analysis was performed to measure autophagic flux. Ac2F cells under starvation or co-treatment of 2 µM MHY1485 for 6 h were treated 1 h before the cell harvest with lysosomotropic agents bafilomycin A1 (10 nM) and chloroquine (100 µM) (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, ***p<0.001; n = 3).
Figure 4 Inhibition of starvation-induced autophagic flux by MHY1485. Western blot analysis was performed to measure autophagic flux. Ac2F cells under starvation or co-treatment of 2 µM MHY1485 for 6 h were treated 1 h before the cell harvest with lysosomotropic agents bafilomycin A1 (10 nM) and chloroquine (100 µM) (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, ***p<0.001; n = 3).CC BY · PubMed Central
Figure 5 MHY1485 inhibition of starvation-induced colocalization between autophagosomes and lysosomes. Live-cell confocal microscopic images of AdGFP-LC3 (green) and lysosomes (red) stained by LysoTracker were collected from Ac2F cells under starvation or co-treatment of 2 µM MHY1485 for 6 h. Merged images show the co-localization of LC3-positive autophagosomes and lysosomes (yellow). Scale bar, 20 um. Bars represent the level of overlap index.
Figure 5 MHY1485 inhibition of starvation-induced colocalization between autophagosomes and lysosomes. Live-cell confocal microscopic images of AdGFP-LC3 (green) and lysosomes (red) stained by LysoTracker were collected from Ac2F cells under starvation or co-treatment of 2 µM MHY1485 for 6 h. Merged images show the co-localization of LC3-positive autophagosomes and lysosomes (yellow). Scale bar, 20 um. Bars represent the level of overlap index.CC BY · PubMed Central
Figure 6 Activation of mTOR by MHY1485. Western blot analysis was performed to detect the change of total protein level and levels of phosphorylated forms of mTOR and 4E-BP1 reflecting the activity of mTOR. Ac2F cells were treated with MHY1485 of different concentrations and rapamycin 5 µM as a positive control for 1 h. Bars represent the phospho-mTOR(Ser2448)/mTOR ratio and the phospho-4E-BP1(Thr37/46)/4E-BP1 ratio normalized with the ratio from untreated samples, respectively. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05; n = 3).
Figure 6 Activation of mTOR by MHY1485. Western blot analysis was performed to detect the change of total protein level and levels of phosphorylated forms of mTOR and 4E-BP1 reflecting the activity of mTOR. Ac2F cells were treated with MHY1485 of different concentrations and rapamycin 5 µM as a positive control for 1 h. Bars represent the phospho-mTOR(Ser2448)/mTOR ratio and the phospho-4E-BP1(Thr37/46)/4E-BP1 ratio normalized with the ratio from untreated samples, respectively. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05; n = 3).CC BY · PubMed Central
Figure 7 Three-dimensional model of mTOR and docking simulation between mTOR and PP242 and MHY1485. A three-dimensional model of mTOR (NCBI GI number: 4826730) was built using SWISS-MODEL program (A). Homologue structural template used was 1E8X (18% sequence identity and 6.9E-44 e-value). The Z-score of the predicted model was −4.8. We checked the quality of each residue on the mTOR structure model. The internal region, which bound ATP and target compounds, was more reliable (blue) than the external region (red) of mTOR. The estimated residue error was visualized using a color gradient from blue (more reliable regions) to red (potentially unreliable regions, estimated error above 3.5 Å). B shows the docking simulation between mTOR and PP242 and MHY1485. The magenta compound is PP242, which was used as a control compound. The cyan compound is MHY1485, and the yellow compound is ATP. The binding energies of the compounds were −7.28 kcal/mol (PP242) and −7.55 kcal/mol (MHY1485).
Figure 7 Three-dimensional model of mTOR and docking simulation between mTOR and PP242 and MHY1485. A three-dimensional model of mTOR (NCBI GI number: 4826730) was built using SWISS-MODEL program (A). Homologue structural template used was 1E8X (18% sequence identity and 6.9E-44 e-value). The Z-score of the predicted model was −4.8. We checked the quality of each residue on the mTOR structure model. The internal region, which bound ATP and target compounds, was more reliable (blue) than the external region (red) of mTOR. The estimated residue error was visualized using a color gradient from blue (more reliable regions) to red (potentially unreliable regions, estimated error above 3.5 Å). B shows the docking simulation between mTOR and PP242 and MHY1485. The magenta compound is PP242, which was used as a control compound. The cyan compound is MHY1485, and the yellow compound is ATP. The binding energies of the compounds were −7.28 kcal/mol (PP242) and −7.55 kcal/mol (MHY1485).CC BY · PubMed Central

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  1. [21]Tanida I, Waguri S (2010) Measurement of autophagy in cells and tissues. Methods in molecular biology 648: 193– 214. 20700714

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Metadata

Authors
Choi, Y. J., Park, Y. J., Park, J. Y., Jeong, H. O., Kim, D. H., Ha, Y. M., Kim, J. M., Song, Y. M., Heo, H.-S., Yu, B. P., Chun, P., Moon, H. R., & Chung, H. Y.
Affiliations
  • Pusan National University (KR)
  • Korea Research Institute of Bioscience and Biotechnology (KR)
  • The University of Texas at San Antonio Health Science Center (US)
  • Inje University (KR)
Corresponding author
  • Moon, Hyung Ryong
  • Chung, Hae Young
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-08-22
Volume
7
Issue
8
Pages
e43418
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0043418
PMC
PMC3425474
OpenAlex
W1985820389
Cited by
192
References
47
Citations by year
2026: 18 · 2025: 13 · 2024: 28 · 2023: 16 · 2022: 14 · 2021: 16 · 2020: 17 · 2019: 10 · 2018: 17 · 2017: 15 · 2016: 10 · 2015: 9 · 2014: 6 · 2013: 3
FWCI
4.29
Type
journal article
Language
en
Update notices
correction (2013-01-29)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • KRIBB Repository (submittedVersion)
Funders
  • National Research Foundation
  • National Research Foundation of Korea (20090083538, No. 2009-0083538)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
Keywords
MHY1485, autophagy inhibition, mTOR activation, lysosome fusion, autophagosome-lysosome fusion, starvation-induced autophagy, autophagic flux, hepatocytes, p62, Beclin-1, mTOR inhibitors
MeSH terms
Animals, Autophagy, Liver, Lysosomes, Microtubule-Associated Proteins, Morpholines, Protein Conformation, Triazines, Vacuoles, Rats, TOR Serine-Threonine Kinases, Molecular Docking Simulation

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