Journal of Ginseng Research

The Korean Society of Ginseng (고려인삼학회)
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Identification and confirmation of 14-3-3 ζ as a novel target of ginsenosides in brain tissues

  • Chen, Feiyan (Research and Innovation Center, College of Traditional Chinese Medicine Integrated Chinese and Western Medicine College, Nanjing University of Chinese Medicine) ;
  • Chen, Lin (Department of Physiology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Liang, Weifeng (Department of Cell Biology and Medical Genetics, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Zhang, Zhengguang (Department of Cell Biology and Medical Genetics, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Li, Jiao (Department of Cell Biology and Medical Genetics, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Zheng, Wan (Department of Cell Biology and Medical Genetics, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Zhu, Zhu (Department of Pharmacology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Zhu, Jiapeng (Department of Cell Biology and Medical Genetics, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine) ;
  • Zhao, Yunan (Department of Pathology and Pathophysiology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine)
  • Received : 2020.02.29
  • Accepted : 2020.12.23
  • Published : 2021.07.01
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Abstract

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.

Keywords

Acknowledgement

The study was financially supported by National Natural Science Foundation of China (Nos.81703732, 81873025), Natural Science Foundation of Jiangsu Provincial (BK20181423), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine); We thank the staff of Experiment Center for Science and Technology, Nanjing University of Chinese Medicine for assistance.

References

  1. Smith I, Williamson EM, Putnam S, Farrimond J, Whalley BJ. Effects and mechanisms of ginseng and ginsenosides on cognition. Nutr Rev 2014;72(5): 319-33. https://doi.org/10.1111/nure.12099
  2. Shi ZY, Zeng JZ, Wong AST. Chemical structures and pharmacological profiles of ginseng saponins. Molecules 2019;24(13):2443. https://doi.org/10.3390/molecules24132443
  3. Chen FY, Zhu KX, Chen L, Ouyang LF, Chen CH, Gu L, Jiang YC, Wang ZL, Lin ZX, Zhang Q, et al. Protein target identification of ginsenosides in skeletal muscle tissues: discovery of natural small-molecule activators of muscle-type creatine kinase. J Ginseng Res 2020;44:461-74. https://doi.org/10.1016/j.jgr.2019.02.005
  4. Li XX, Zhang CC, Xiong YK. Advances in pharmacokinetics of ginsenosides. Chin Pharm J 2012;47(14):1101-4.
  5. Yang XW. Pharmacokinetic studies of chemical constituents of ginseng. Mod Chin Med 2016;18(1):16-35.
  6. Qi LW, Wang CZ, Du GJ, Zhang ZY, Calway T, Yuan CS. Metabolism of ginseng and its interactions with drugs. Curr Drug Metab 2011;12(9):818-22. https://doi.org/10.2174/138920011797470128
  7. Peng D, Wang H, Qu C, Xie L, Wicks SM, Xie J. Ginsenoside Re: its chemistry, metabolism and pharmacokinetics. Chin Med 2012;7:2. https://doi.org/10.1186/1749-8546-7-2
  8. Liu MY, Wang HT, Zhao SH, Shi XW, Zhang YF, Xu HH, Wang YF, Li XJ, Zhang LT. Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze-dried powder. Biomed Chromatogr 2011;25(11):1260-72. https://doi.org/10.1002/bmc.1600
  9. Chen C, Wang LS, Cao FR, Miao XQ, Chen TK, Chang Q, Zheng Y. Formulation of 20(S)-protopanaxadiol nanocrystals to improve oral bioavailability and brain delivery. Int J Pharm 2016;497:239-47. https://doi.org/10.1016/j.ijpharm.2015.12.014
  10. Jin S, Jeon JH, Lee S, Kang WY, Seong SJ, Yoon YR, Choi MK, Song IS. Detection of 13 ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1, compound K, 20(S)-Protopanaxadiol, and 20(S)-Protopanaxatriol) in human plasma and application of the analytical method to human pharmacokinetic studies following two week-repeated administration of red ginseng extract. Molecules 2019;24(14):2618. https://doi.org/10.3390/molecules24142618
  11. Rokot NT, Kairupan TS, Cheng KC, Runtuwene J, Kapantow NH, Amitani M, Morinaga A, Amitani H, Asakawa A, Inui A. A role of ginseng and its constituents in the treatment of central nervous system disorders. Evid Based Complement Alternat Med 2016;2016:2614742.
  12. Zheng MM, Xin YZ, Li YJ, Xu FX, Xi XZ, Guo H, Cui XW, Cao H, Zhag X, Han CC. Ginsenosides: a potential neuroprotective agent. Biomed Res Int 2018;2018: 8174345. https://doi.org/10.1155/2018/8174345
  13. Wang PP, Wei YJ, Fan Y, Liu QF, Wei W, Yang CS, Zhang L, Zhao GP, Yue JM, Yan X, et al. Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metab Eng 2015;29:97-105. https://doi.org/10.1016/j.ymben.2015.03.003
  14. Zheng SW, Xiao SY, Wang J, Hou W, Wang YP. Inhibitory effects of ginsenoside ro on the growth of B16F10 melanoma via its metabolites. Molecules 2019;24(16):2985. https://doi.org/10.3390/molecules24162985
  15. Zhang HL, Li Z, Zhou ZL, Yang HY, Zhong ZY, Lou CX. Antidepressant-like effects of ginsenosides: a comparison of ginsenoside Rb3 and its four deglycosylated derivatives, Rg3, Rh2, compound K, and 20(S)-protopanaxadiol in mice models of despair. Pharmacol Biochem Behav 2016;140:17-26. https://doi.org/10.1016/j.pbb.2015.10.018
  16. Chen W, Guo YJ, Yang WJ, Zheng P, Zeng JS, Tong WS. Protective effect of ginsenoside Rb1 on integrity of blood-brain barrier following cerebral ischemia. Exp Brain Res 2015;233(10):2823-31. https://doi.org/10.1007/s00221-015-4352-3
  17. Zhu G, Wang Y, Li J, Wang J. Chronic treatment with ginsenoside Rg1 promotes memory and hippocampal long-term potentiation in middle-aged mice. Neuroscience 2015;292:81-9. https://doi.org/10.1016/j.neuroscience.2015.02.031
  18. Hou JG, Xue JJ, Lee M, Liu L, Zhang DL, Sun MQ, Zheng YN, Sung CK. Ginsenoside Rh2 improves learning and memory in mice. J Med Food 2013;16(8): 772-6. https://doi.org/10.1089/jmf.2012.2564
  19. Lomenick B, Olsen RW, Huang J. Identification of direct protein targets of small molecules. ACS Chem Biol 2011;6(1):34-46. https://doi.org/10.1021/cb100294v
  20. Zhao YN, Wang ZL, Dai JG, Chen L, Huang YF. Preparation and quality assessment of high-purity ginseng total saponins by ion exchange resin combined with macroporous adsorption resin separation. Chin J Nat Med 2014;12(5):382-92. https://doi.org/10.1016/S1875-5364(14)60048-0
  21. Ouyang LF, Wang ZL, Dai JG, Chen L, Zhao YN. Determination of total ginsenosides in ginseng extracts using charged aerosol detection with post-column compensation of the gradient. Chin J Nat Med 2014;12(11):857-68. https://doi.org/10.1016/S1875-5364(14)60129-1
  22. Joo EJ, Ha YW, Shin H, Son SH, Kim YS. Generation and characterization of monoclonal antibody to ginsenoside rg3. Biol Pharm Bull 2009;32(4):548-52. https://doi.org/10.1248/bpb.32.548
  23. Wang YS, Zhang B, Zhu J, Yang CL, Guo Y, Liu CL, Liu F, Huang HQ, Zhao SW, Liang Y, et al. Molecular basis for the final oxidative rearrangement steps in chartreusin biosynthesis. J Am Chem Soc 2018;140(34):10909-14. https://doi.org/10.1021/jacs.8b06623
  24. Lountos GT, Zhao XZ, Kiselev E, Tropea JE, Needle D, Pommier Y, Burke TR, Waugh DS. Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening. Nucleic Acids Res 2019;47(19):10134-50. https://doi.org/10.1093/nar/gkz515
  25. Zhang B, Wang KB, Wang W, Wang X, Liu F, Zhu J, Shi J, Li LY, Han H, Xu K, et al. Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products. Nature 2019;568(7750):122-6. https://doi.org/10.1038/s41586-019-1021-x
  26. Curran EC, Wang H, Hinds TR, Zheng N, Wang EH. Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy. Sci Rep 2018;8(1): 4630. https://doi.org/10.1038/s41598-018-22879-5
  27. Wang YS, Lin Y, Li H, Li Y, Song Z, Jin YH. The identification of molecular target of (20S) ginsenoside Rh2 for its anti-cancer activity. Sci Rep 2017;7(1):12408. https://doi.org/10.1038/s41598-017-12572-4
  28. Lu Y, Ding S, Zhou R, Wu J. Structure of the complex of phosphorylated liver kinase B1 and 14-3-3zeta. Acta Crystallogr F Struct Biol Commun 2017;73: 196-201. https://doi.org/10.1107/S2053230X17003521
  29. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 2011;67:271-81. https://doi.org/10.1107/S0907444910048675
  30. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr 2007;40:658-74. https://doi.org/10.1107/S0021889807021206
  31. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010;66:213-21. https://doi.org/10.1107/S0907444909052925
  32. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 2010;66:486-501. https://doi.org/10.1107/S0907444910007493
  33. Moustakim M, Riedel K, Schuller M, Gehring AP, Monteiro OP, Martin SP, Fedorov O, Heer J, Dixon DJ, Elkins JM, et al. Discovery of a novel allosteric inhibitor scaffold for polyadenosine-diphosphate-ribose polymerase 14 (PARP14) macrodomain 2. Bioorg Med Chem 2018;26(11):2965-72. https://doi.org/10.1016/j.bmc.2018.03.020
  34. Furukawa A, Kakita K, Yamada T, Ishizuka M, Sakamoto J, Hatori N, Maeda N, Ohsaka F, Saitoh T, Nomura T, et al. Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRalpha immune cell receptor. J Biol Chem 2017;292(51):21128-36. https://doi.org/10.1074/jbc.M117.799239
  35. Park KD, Kim D, Reamtong O, Eyers C, Gaskell SJ, Liu R, Kohn H. Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 zeta. J Am Chem Soc 2011;133(29):11320-30. https://doi.org/10.1021/ja2034156
  36. Hage DS, Matsuda R. Affinity chromatography: a historical perspective. Methods Mol Biol 2015;1286:1-19. https://doi.org/10.1007/978-1-4939-2447-9_1
  37. Futamura Y, Muroi M, Osada H. Target identification of small molecules based on chemical biology approaches. Mol Biosyst 2013;9(5):897-914. https://doi.org/10.1039/c2mb25468a
  38. Huber KV, Olek KM, Muller AC, Tan CS, Bennett KL, Colinge J, Superti-Furga G. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Nat Methods 2015;12(11):1055-7. https://doi.org/10.1038/nmeth.3590
  39. Pai MY, Lomenick B, Hwang H, Schiestl R, McBride W, Loo JA, Huang J. Drug affinity responsive target stability (DARTS) for small-molecule target identification. Methods Mol Biol 2015;1263:287-98. https://doi.org/10.1007/978-1-4939-2269-7_22
  40. Chang J, Kim Y, Kwon HJ. Advances in identification and validation of protein targets of natural products without chemical modification. Nat Prod Rep 2016;33(5):719-30. https://doi.org/10.1039/c5np00107b
  41. Zhou XY, Hu DX, Chen RQ, Chen XQ, Dong WL, Yi CL. 14-3-3 isoforms differentially regulate NFkappaB signaling in the brain after ischemia-reperfusion. Neurochem Res 2017;42(8):2354-62. https://doi.org/10.1007/s11064-017-2255-3
  42. Dai JG, Murakami K. Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain. J Neurochem 2003;84(1): 23-34. https://doi.org/10.1046/j.1471-4159.2003.01254.x
  43. Toyo-oka K, Wachi T, Hunt RF, Baraban SC, Taya S, Ramshaw H, Kaibuchi K, Schwarz QP, Lopez AF, Wynshaw-Boris A. 14-3-3-psilon and zeta regulate neurogenesis and differentiation of neuronal progenitor cells in the developing brain. J Neurosci 2014;34(36):12168-81. https://doi.org/10.1523/JNEUROSCI.2513-13.2014
  44. Deng Y, Jiang B, Rankin CL, Toyo-Oka K, Richter ML, Maupin-Furlow JA, Moskovitz J. Methionine sulfoxide reductase A (MsrA) mediates the ubiquitination of 14-3-3 protein isotypes in brain. Free Radic Biol Med 2018;129: 600-7. https://doi.org/10.1016/j.freeradbiomed.2018.08.002
  45. Mackie S, Aitken A. Novel brain 14-3-3 interacting proteins involved in neurodegenerative disease. Febs J 2005;272(16):4202-10. https://doi.org/10.1111/j.1742-4658.2005.04832.x
  46. Li T, Paudel HK. 14-3-3zeta facilitates GSK3beta-catalyzed tau phosphorylation in HEK-293 cells by a mechanism that requires phosphorylation of GSK3beta on Ser9. Neurosci Lett 2007;414(3):203-8. https://doi.org/10.1016/j.neulet.2006.11.073
  47. Cromm PM, Wallraven K, Glas A, Bier D, Furstner A, Ottmann C, Grossmann TN. Constraining an irregular peptide secondary structure through ring-closing alkyne metathesis. Chembiochem 2016;17(20):1915-9. https://doi.org/10.1002/cbic.201600362