x
To verify the simulation results, we performed an in vitro kinase assay with various concentrations of ATP. PP242, a kinase inhibitor of mTOR, was used as a positive control for mTOR inhibition. We found that resveratrol-induced mTOR inhibition was restored by the addition of ATP. To clarify whether resveratrol inhibits mTOR through direct interaction, we generated a resveratrol-resistant mTOR mutant. Based on the resolved mTOR-ATP structure and our computational simulation. Asp 2195 was deemed to be a resveratrol-binding residue that does not affect the mTOR-ATP interaction.
Thereafter, we generated an mTOR mutant in which Asp 2195 was substituted with alanine (hereafter, referred to as D2195A) and performed an in vitro kinase assay. As expected, resveratrol inhibited wild-type (WT) mTOR but not D2195A mTOR. The basal activity level of the D2195A mutant was lower than that of WT mTOR, possibly due to the fact that the mutated residue resides in the kinase domain of the protein. We observed that PP242 also inhibited DA mTOR, albeit the inhibitory potency was less than WT mTOR. Although Asp 2195 was suggested as a residue for the interaction between mTOR and PP242, other residues also participate the interaction between them and not all residues are shared between PP242 and resveratrol for mTOR binding.
We speculate that other residues but not D2105 may be essential for the PP242 binding to mTOR and that is why D2195A mTOR was not completely resistant to PP242. To examine whether the resveratrol resistant mTOR blocks the resveratrol-induced autophagy, we measured the autophagy levels upon resveratrol treatment in the WT or D2195A mTOR along with Raptor transfected cells. We found that resveratrol induced autophagy in the WT mTOR transfected cells, but not in the D2109A mTOR transfected cells. Taken together, the results from the experiments using resveratrol resistant mTOR suggest that resveratrol induces autophagy by directly inhibits mTOR through ATP competition.
Article Produced By
Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 689-798, Republic of Korea
Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-Dong, Kwangjin-Ku, Seoul 143-747, Republic of Korea
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763238/
(All these Articles can be read with this URL:
https://markethive.com/group/ama/blog/the-role-of-resveratrol-in-achieving-optimal-health-p5 )
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