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KMID : 0352720200440030461
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Journal of Ginseng Research
2020 Volume.44 No. 3 p.461 ~ p.474
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Protein target identification of ginsenosides in skeletal muscle tissues: discovery of natural small-molecule activators of muscle-type creatine kinase
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Chen Feiyan
Zhu Kexuan
Chen Lin
Ouyang Liufeng
Chen Cuihua
Gu Ling
Jiang Yucui
Wang Zhongli
Lin Zixuan
Zhang Qiang
Shao Xiao
Dai Jianguo
Zhao Yunan
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Abstract
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Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown.
Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues.
Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice.
Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.
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KEYWORD
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Affinity chromatography, Creatine kinase, Fatigue, Ginseng, 20(S)-protopanaxadiol
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