Protein target identifi cation of ginsenosides in skeletal muscle tissues: discovery of natural smallmolecule activators of muscle-type creatine kinase

Feiyan Chen,Kexuan Zhu,Lin Chen,Liufeng Ouyang,Cuihua Chen,Ling Gu,Yucui Jiang,Zhongli Wang,Zixuan Lin,Qiang Zhang,Xiao Shao,Jianguo Dai,Yunan Zhao 고려인삼학회 2020 Journal of Ginseng Research Vol.44 No.3

Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, themechanism 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 potentialtarget 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 thestudy, was selected as a representative to confirm direct binding and its biological importance. Biolayerinterferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPDspecifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by moleculardocking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activityin vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the functionof the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delayingexercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginsengreduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can helpin further developing better CK-MM activators based on the dammarane-type triterpenoid structure.

Discovery and validation of PURA as a transcription target of 20(S)-protopanaxadiol: Implications for the treatment of cognitive dysfunction

Feiyan Chen,Wenjing Zhang,Shuyi Xu,Hantao Zhang,Lin Chen,Cuihua Chen,Zhu Zhu,Yunan Zhao 고려인삼학회 2023 Journal of Ginseng Research Vol.47 No.5

Background: 20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for thecentral nervous system, especially in improving learning and memory. However, its transcriptionaltargets in brain tissue remain unknown. Methods: In this study, we first used mass spectrometry-based drug affinity responsive target stability(DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcriptionfactor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayerinterferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of targetgenes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used toanalyze the potential biological features of these target proteins. Results: The results showed three overlapping transcription factors between the proteomics of DARTSand transcription factor library. BLI analysis further showed that PPD had a higher direct interaction withPURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations inkey amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showedthat PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformaticsanalysis showed that these target proteins were involved in learning and memory function. Conclusion: The above-mentioned findings indicate that PURA is a transcription target of PPD in brain,and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by bindingPURA, which could provide a chemical and biological basis for the study of treating cognitive impairmentby targeting PURA.

Protein target identification of ginsenosides in skeletal muscle tissues: discovery of natural small-molecule activators of muscle-type creatine kinase

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 The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.3

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.

A possible mechanism to the antidepressant-like effects of 20 (S)-protopanaxadiol based on its target protein 14-3-3 ζ

Chen, Lin,Li, Ruimei,Chen, Feiyan,Zhang, Hantao,Zhu, Zhu,Xu, Shuyi,Cheng, Yao,Zhao, Yunan The Korean Society of Ginseng 2022 Journal of Ginseng Research Vol.46 No.5

Background: Ginsenosides and their metabolites have antidepressant-like effects, but the underlying mechanisms remain unclear. We previously identified 14-3-3 ζ as one of the target proteins of 20 (S)-protopanaxadiol (PPD), a fully deglycosylated ginsenoside metabolite. Methods: Corticosterone (CORT) was administered repeatedly to induce the depression model, and PPD was given concurrently. The tail suspension test (TST) and the forced swimming test (FST) were used for behavioral evaluation. All mice were sacrificed. Golgi-cox staining, GSK 3β activity assay, and Western blot analysis were performed. In vitro, the kinetic binding analysis with the Biolayer Interferometry (BLI) was used to determine the molecular interactions. Results: TST and FST both revealed that PPD reversed CORT-induced behavioral deficits. PPD also ameliorated the CORT-induced expression alterations of hippocampal Ser9 phosphorylated glycogen synthase kinase 3β (p-Ser9 GSK 3β), Ser133 phosphorylated cAMP response element-binding protein (p-Ser133 CREB), and brain-derived neurotrophic factor (BDNF). Moreover, PPD attenuated the CORT-induced increase in GSK 3β activity and decrease in dendritic spine density in the hippocampus. In vitro, 14-3-3 ζ protein specifically bound to p-Ser9 GSK 3β polypeptide. PPD promoted the binding and subsequently decreased GSK 3β activity. Conclusion: These findings demonstrated the antidepressant-like effects of PPD on the CORT-induced mouse depression model and indicated a possible target-based mechanism. The combination of PPD with the 14-3-3 ζ protein may promote the binding of 14-3-3 ζ to p-GSK 3β (Ser9) and enhance the inhibition of Ser9 phosphorylation on GSK 3β kinase activity, thereby activating the plasticity-related CREBeBDNF signaling pathway.

Identification and confirmation of 14-3-3 ζ as a novel target of ginsenosides in brain tissues

Feiyan Chen,Lin Chen,Weifeng Liang,Zhengguang Zhang,Jiao Li,Wan Zheng,Zhu Zhu,Jiapeng Zhu,Yunan Zhao 고려인삼학회 2021 Journal of Ginseng Research Vol.45 No.4

Background: Ginseng can help regulate brain excitability, promote learning and memory, and resist cerebral ischemia in the central nervous system. Ginsenosides are the major effective compounds of Ginseng, but their protein targets in the brain have not been determined. Methods: We screened proteins that interact with the main components of ginseng (ginsenosides) by affinity chromatography and identified the 14-3-3 ζ protein as a potential target of ginsenosides in brain tissues. Results: Biolayer interferometry (BLI) analysis showed that 20(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, exhibited the highest direct interaction to the 14-3-3 ζ protein. Subsequently, BLI kinetics analysis and isothermal titration calorimetry (ITC) assay showed that PPD specifically bound to the 14-3-3 ζ protein. The cocrystal structure of the 14-3-3 ζ protein-PPD complex showed that the main interactions occurred between the residues R56, R127, and Y128 of the 14-3-3 ζ protein and a portion of PPD. Moreover, mutating any of the above residues resulted in a significant decrease of affinity between PPD and the 14-3-3 ζ protein. Conclusion: Our results indicate the 14-3-3 ζ protein is the target of PPD, a ginsenoside metabolite. Crystallographic and mutagenesis studies suggest a direct interaction between PPD and the 14-3-3 ζ protein. This finding can help in the development of small-molecular compounds that bind to the 14-3-3 ζ protein on the basis of the structure of dammarane-type triterpenoid.

Target engagement of ginsenosides in mild cognitive impairment using mass spectrometry-based drug affinity responsive target stability

Zhu, Zhu,Li, Ruimei,Qin, Wei,Zhang, Hantao,Cheng, Yao,Chen, Feiyan,Chen, Cuihua,Chen, Lin,Zhao, Yunan The Korean Society of Ginseng 2022 Journal of Ginseng Research Vol.46 No.6

Background: Mild cognitive impairment (MCI) is a transitional condition between normality and dementia. Ginseng is known to have effects on attenuating cognitive deficits in neurogenerative diseases. Ginsenosides are the main bioactive component of ginseng, and their protein targets have not been fully understood. Furthermore, no thorough analysis is reported in ginsenoside-related protein targets in MCI. Methods: The candidate protein targets of ginsenosides in brain tissues were identified by drug affinity responsive target stability (DARTS) coupled with label-free liquid chromatography-mass spectrometry (LC-MS) analysis. Network pharmacology approach was used to collect the therapeutic targets for MCI. Based on the above-mentioned overlapping targets, we built up a proteineprotein interaction (PPI) network in STRING database and conducted gene ontology (GO) enrichment analysis. Finally, we assessed the effects of ginseng total saponins (GTS) and different ginsenosides on mitochondrial function by measuring the activity of the mitochondrial respiratory chain complex and performing molecular docking. Results: We screened 2526 MCI-related protein targets by databases and 349 ginsenoside-related protein targets by DARTS. On the basis of these 81 overlapping genes, enrichment analysis showed the mitochondria played an important role in GTS-mediated MCI pharmacological process. Mitochondrial function analysis showed GTS, protopanaxatriol (PPT), and Rd increased the activities of complex I in a dose-dependent manner. Molecular docking also predicted the docking pockets between PPT or Rd and mitochondrial respiratory chain complex I. Conclusion: This study indicated that ginsenosides might alleviate MCI by targeting respiratory chain complex I and regulating mitochondrial function, supporting ginseng's therapeutic application in cognitive deficits.

A fluorescent probe for colorimetric detection of bisulfite and application in sugar and red wine

Haitao Chen,Xiaoming Wu,Jialin Wang,Hao Wang,Feiyan Tao,Shaoxiang Yang,Hongyu Tian,Yongguo Liu,Baoguo Sun 한국식품과학회 2019 Food Science and Biotechnology Vol.28 No.4

A new fluorescent probe made from (E)-2-(benzo[d]thiazol-2-yl)-3-(6-hydroxynaphthalen-2-yl) acrylonitrile(Probe 1) was synthesized for the determination ofbisulfite concentrations in real food samples (red wine andsugar). Adding bisulfite to a Probe 1 solution caused amarked decrease in fluorescence intensity and a visualcolor change from yellow to light yellow. This distinctcolor response indicates that Probe 1 could be used as avisual sensor for bisulfite. Probe 1 can detect bisulfitequantitatively in the range 0–400 lM with a detection limitof 0.10 lM. This makes Probe 1 a convenient signalinginstrument for determining bisulfite levels in sugar and redwine samples.

Iterative Parameter Identification for Time-delay Nonlinear Rational Models via L1-Regularized Least Squares

Qianyan Shen,Jing Chen,Feiyan Sun 제어·로봇·시스템학회 2022 International Journal of Control, Automation, and Vol.20 No.2

This paper develops an unbiased iterative parameter identification algorithm for time-delay nonlinear rational systems. In order to reduce redundant parameters, the time delay is estimated by the iterative estimator based on the L1 regularization technique and the cross-validation strategy first, then a rational system is decomposed into two subsystems by the hierarchical principle. The unbiased parameter estimates are obtained by the least squares iterative technique. The simulation example shows the effectiveness of the proposed algorithm.

Effect of Bacterial Wilt on Fungal Community Composition in Rhizosphere Soil of Tobaccos in Tropical Yunnan

Yuanxian Zheng,Jiming Wang,Wenlong Zhao,Xianjie Cai,Yinlian Xu,Xiaolong Chen,Min Yang,Feiyan Huang,Lei Yu,Yuansheng He 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.3

Bacterial wilt, which is a major soil-borne disease with widespread occurrence, poses a severe danger in the field of tobacco production. However, there is very limited knowledge on bacterial wilt-induced microecological changes in the tobacco root system and on the interaction between Ralstonia solanacearum and fungal communities in the rhizosphere soil. Thus, in this study, changes in fungal communities in the rhizosphere soil of tobaccos with bacterial wilt were studied by 18S rRNA gene sequencing. The community composition of fungi in bacterial wilt-infected soil and healthy soil in two tobacco areas (Gengma and Boshang, Lincang City, Yunnan Province, China) was studied through the paired comparison method in July 2019. The results showed that there were significant differences in fungal community composition between the rhizosphere soil of diseased plants and healthy plants. The changes in the composition and diversity of fungal communities in the rhizosphere soil of tobaccos are vital characteristics of tobaccos with bacterial wilt, and the imbalance in the rhizosphere microecosystem of tobacco plants may further aggravate the disease.

Different Response Mechanisms of Rhizosphere Microbial Communities in Two Species of Amorphophallus to Pectobacterium carotovorum subsp. carotovorum Infection

Min Yang,Ying Qi,Jiani Liu,Penghua Gao,Feiyan Huang,Lei Yu,Hairu Chen 한국식물병리학회 2023 Plant Pathology Journal Vol.39 No.2

Soft rot is a widespread, catastrophic disease caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) that severely damages the production of Amorphophallus spp. This study evaluated the rhizosphere bacterial and fungal communities in Pcc-infected and uninfected plants of two species of Amorphophallus, A. muelleri and A. konjac. Principal component analysis showed that the samples formed different clusters according to the Pcc infection status, indicating that Pcc infection can cause a large number of changes in the bacterial and fungal communities in the Amorphophallus spp. rhizosphere soil. However, the response mechanisms of A. muelleri and A. konjac are different. There was little difference in the overall microbial species composition among the four treatments, but the relative abundances of core microbiome members were significantly different. The relative abundances of Actinobacteria, Chloroflexi, Acidobacteria, Firmicutes, Bacillus, and Lysobacter were lower in infected A. konjac plants than in healthy plants; in contrast, those of infected A. muelleri plants were higher than those in healthy plants. For fungi, the relative abundances of Ascomycota and Fusarium in the rhizosphere of infected A. konjac plants were significantly higher than those of healthy plants, but those of infected A. muelleri plants were lower than those of healthy plants. The relative abundance of beneficial Penicillium fungi was lower in infected A. konjac plants than in healthy plants, and that of infected A. muelleri plants was higher than that of healthy plants. These findings can provide theoretical references for further functional research and utilization of Amorphophallus spp. rhizosphere microbial communities in the future.