Тионины растений: строение, биологические функции и перспективы использования в биотехнологии

Антимикробные пептиды (АМП) – важнейшие компоненты  защитной системы растений и животных, они представляют собой древний механизм врожденной устойчивости, обеспечивающий «первую линию обороны» против патогенов. Выделяют несколько семейств АМП растений: тионины, дефензины, неспецифические липид-переносящие  белки (ЛПБ), гевеино- и ноттиноподобные пептиды, гарпинины, а также макроциклические пептиды (циклотиды). Обзор посвящен характеристике семейства тионинов. Тионины – характерное только для растений семейство АМП, состоящее из коротких (~5 кДа) цистеинбогатых пептидов (с шестью или восемью остатками цистеина в молекуле), которые обладают антимикробными и токсическими свойствами. На основании сходства амино- кислотных последовательностей и расположения дисульфидных связей выделяют пять структурных классов тионинов. Установлена пространственная структура ряда тионинов. Показано, что амфипатическая молекула тионина имеет форму греческой буквы Г, у которой длинное плечо  образовано двумя антипараллельными α-спиралями, а короткое – двумя параллельными β-тяжами. Выявлены аминокислотные остатки, ответственные за антимикробную активность тионинов. Тионины синтезируются в виде предшественников, состоящих из сигнального пептида, зрелого пептида и С-концевого продомена. Тионины являются защитными пептидами растений против патогенных бактерий и грибов, которые действуют в микромолярных концентрациях непосредственно на мембраны микроорганизмов, хотя детальный механизм действия этих АМП до конца не выяснен. Помимо патогенов растений, тионины подавляют рост ряда патогенных и условно патогенных микроорганизмов человека, таких как Candida spp., Saccharomyces cerevisiae, Fusarium solani, Staphylococcus aureus, Escherichia coli. Тионины токсичны для различного типа клеток, включая линии раковых клеток млекопитающих. Трансгенные растения, в которых экспрессируются гены тионинов, обладают повышенной устойчивостью к патогенам. Широкий спектр антимикробной и токсической активности тионинов открывает возможности их практического использования в сельском хозяйстве и медицине.

антимикробные петиды, тионины, иммунитет растений

1. Asano T., Miwa A., Maeda K., Kimura M., Nishiuchi T. The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum. PLoS Pathog. 2013;9(8):e1003581. DOI 10.1371/ journal. ppat.1003581.

2. Balls A.K., Hale W.S., Harris T.H. A crystalline protein from a lipoprotein of wheat flour. Cereal Chem. 1942;19:279-288.

3. Berrocal-Lobo M., Molina A., Rodriguez-Palenzuela P., Garcia-Olmedo F., Rivas L. Leishmania donovani: thionins, plant antimicrobial peptides with leishmanicidal activity. Exp. Parasitol. 2009;122: 247-249.

4. Bohlmann H. The role of thionins in plant protection. Crit. Rev. Plant Sci. 1994;13:1-16.

5. Bohlmann H., Vignutelli A., Hilpert B., Miersch O., Wasternack C., Apel K. Wounding and chemicals induce expression of the Arabidopsis thaliana gene Thi2.1, encoding a fungal defense thionin, via the octadecanoid pathway. FEBS Lett. 1998;437(3):281-286.

6. Carmona M.J., Molina A., Fernandez J.A., Lopez-Fando J.J., GarciaOlmedo F. Expression of the α-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens. Plant J. 1993; 3(3):457-462.

7. Carrasco I., Vazquez D., Hernandez-Lucas C., Carbonero P., GarciaOlmedo F. Thionins: plant peptides that modify membrane permeability in cultured mammalian cells. Eur. J. Biochem. 1981;116(1): 185-189.

8. Castagnaro A., Maraña C., Carbonero P., García-Olmedo F. Extreme divergence of a novel wheat thionin generated by a mutational burst specifically affecting the mature protein domain of the precursor. J. Mol. Biol. 1992;224(4):1003-1009.

9. Chan Y.L., Prasad V., Sanjaya, Chen K.H., Liu P.C., Chan M.T., Cheng C.P. Transgenic tomato plants expressing an Arabidopsis thionin (Thi2.1) driven by fruit-inactive promoter battle against phytopathogenic attack. Planta. 2005;221(3):386-393. DOI 10.1007/s00425-004-1459-3.

10. Coulon A., Berkane E., Sautereau A.M., Urech K., Rouge P., Lopez A. Modes of membrane interaction of a natural cysteine-rich peptide: viscotoxin A3. Biochim. Biophys. Acta. 2002;1559(2):145-159. DOI 10.1016/S0005-2736(01)00446-1.

11. Coulson E.J., Harris T.H., Axelrod B. Effect on small laboratory animals of the injection of the crystalline hydrochloride of a sulfur protein from wheat flour. Cereal Chem. 1942;19:301-307.

12. de Souza Cândido E., e Silva Cardoso M.H., Sousa D.A., Viana J.C., de Oliveira-Júnior N.G., Miranda V., Franco O.L. The use of versatile plant antimicrobial peptides in agribusiness and human health. Peptides. 2014;55:65-78. DOI 10.1016/j.peptides.2014.02.003.

13. Diaz I., Carmona M.J., Garcia-Olmedo F. Effects of thionins on betaglucuronidase in vitro and in plant protoplasts. FEBS Lett. 1992; 296(3):279-282. DOI 10.1016/0014-5793(92)80304-Y.

14. Egorov T.A., Odintsova T.I. Defense peptides of plant immune system. Russ. J. Bioorg. Khim. 2012;38(1):1-9. DOI 10.1134/ S1068162012010062.

15. Epple P., Apel K., Bohlmann H. Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. Plant Cell. 1997;9(4):509-520. DOI 10.1105/tpc.9.4.509.

16. Escudero-Martinez C.M., Morris J.A., Hedley P.E., Bos J.I.B. Barley transcriptome analyses upon interaction with different aphid species identify thionins contributing to resistance. Plant Cell Environ. 2017;40(11):2628-2643. DOI 10.1111/pce.12979.

17. Fernandez de Caleya R., Gonzalez-Pascual B., Garcia-Olmedo F., Carbonero P. Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro. Appl. Microbiol. 1972;23(5):998-1000.

18. Guzmán-Rodríguez J.J., Ochoa-Zarzosa A., López-Gómez R., LópezMeza J.E. Plant antimicrobial peptides as potential anticancer agents. Biomed. Res. Int. 2015;2015:735087. DOI 10.1155/2015/735087.

19. Huang W., Vernon L.P., Bell J.D. Enhancement of adenylate cyclase activity in S49 lymphoma cell membranes by the toxin thionin from Pyrularia pubera. Toxicon. 1994;32(7):789-797.

20. Hughes P., Dennis E., Whitecross M., Llewellyn D., Gage P. The cytotoxic plant protein, β-purothionin, forms ion channels in lipid membranes. J. Biol. Chem. 2000;275(2):823-827. DOI 10.1074/jbc. 275.2.823.

21. Iwai T., Kaku H., Honkura R., Nakamura S., Ochiai H., Sasaki T., Ohashi Y. Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants overproducing an oat cell-wall-bound thionin. Mol. Plant Microbe Interact. 2002;15(6):515-521. DOI 10.1094/ MPMI.2002.15.6.515.

22. Ji H., Gheysen G., Ullah C., Verbeek R., Shang C., De Vleesschauwer D., Höfte M., Kyndt T. The role of thionins in rice defence against root pathogens. Mol. Plant Pathol. 2015;16(8):870-881. DOI 10.1111/mpp.12246.

23. Johansson S., Gullbo J., Lindholm P., Ek B., Thunberg E., Samuelsson G., Larsson R., Bohlin L., Claeson P. Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells. Cell. Mol. Life Sci. 2003; 60(1):165-175. DOI 10.1007/s000180300011.

24. Kong J.L., Du X.B., Fan C.X., Xu J.F., Zheng X.J. Determination of primary structure of a novel peptide from mistletoe and its antitumor activity. Acta Pharmaceutica Sinica. 2004;39(10):813-817.

25. Kramer K.J., Klassen L.W., Jones B.L., Speirs R.D., Kammer A.E. Toxi city of purothionin and its homologues to the tobacco hornworm, Manduca sexta (L.) (Lepidoptera: Sphingidae). Toxicol. Appl. Pharmacol. 1979;48:179-183.

26. Krens F.A., Schaart J.G., Groenwold R., Walraven A.E.J., Hesselink T., Thissen J.T.N.M. Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines. Transgenic Res. 2011;20:1113-1123. DOI 10.1007/s11248-011-9484-z. Li S.-S., Gullbo J., Lindholm P., Larsson R., Thunberg E., Samuelsson G., Bohlin L., Claeson P. Ligatoxin B, a new cytotoxic protein with a novel helix-turn-helix DNA-binding domain from the mistletoe Phoradendron liga. Biochem. J. 2002;366(2):405-413. DOI 10.1042/bj20020221. Loeza-Ángeles H., Sagrero-Cisneros E., Lara-Zárate L., VillagómezGómez E., López-Meza J.E., Ochoa-Zarzosa A. Thionin Thi2.1 from Arabidopsis thaliana expressed in endothelial cells shows antibacterial, antifungal and cytotoxic activity. Biotechnol. Lett. 2008; 30(10):1713-1719. DOI 10.1007/s10529-008-9756-8. Molina A., Goy P.A., Fraile A., Sanchez-Monge R., Garcia-Olmedo F. Inhibition of bacterial and fungal plant pathogens by thionins of types I and II. Plant Sci. 1993;92:169-177. Muramoto N., Tanaka T., Shimamura T., Mitsukawa N., Hori E., Koda K., Otani M., Hirai M., Nakamura K., Imaeda T. Transgenic sweet potato expressing thionin from barley gives resistance to black rot disease caused by Ceratocystis fimbriata in leaves and storage roots. Plant Cell Rep. 2012;31(6):987-997. DOI 10.1007/s00299011-1217-5. Oard S.V. Deciphering a mechanism of membrane permeabilization by α-hordothionin peptide. Biochim. Biophys. Acta. 2011;1808(6): 1737-1745. DOI 10.1016/j.bbamem.2011.02.003. Oard S.V., Enright F.M. Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi. Plant Cell Rep. 2006;25(6):561-572. DOI 10.1007/s00299-005-0102-5. Oard S., Karki B., Enright F. Is there a difference in metal ion-based inhibition between members of thionin family: molecular dynamics simulation study. Biophys. Chem. 2007;130(1-2):65-75. DOI 10.1016/j.bpc.2007.07.005. Oard S., Rush M.C., Oard J.H. Characterization of antimicrobial peptides against a US strain of the rice pathogen Rhizoctonia solani. J. Appl. Microbiol. 2004;97(1):169-180. DOI 10.1111/j.1365-2672. 2004.02291.x. Ochoa-Zarzosa A., Loeza-Angeles H., Sagrero-Cisneros E., Villagómez-Gómez E., Lara-Zárate L., López-Meza J.E. Antibacterial activity of thionin Thi2.1 from Arabidopsis thaliana expressed by bovine endothelial cells against Staphylococcus aureus isolates from bovine mastitis. Vet. Microbiol. 2008a;127(3-4):425-430. DOI 10.1016/j.vetmic.2007.08.031. Ochoa-Zarzosa A., Loeza-Lara P.D., Torres-Rodríguez F., Loeza-Angeles H., Mascot-Chiquito N., Sánchez-Baca S., López-Meza J.E. Antimicrobial susceptibility and invasive ability of Staphylococcus aureus isolates from mastitis from dairy backyard systems. Antonie Van Leeuwenhoek. 2008b;94(2):199-206. DOI 10.1007/s10482008-9230-6. Orru S., Scaloni A., Giannattasio M., Urech K., Pucci P., Schaller G. Amino acid sequence, S-S bridge arrangement and distribution in plant tissues of thionins from Viscum album. Biol. Chem. 1997; 378(9):989-996. Osório e Castro V.R., Vernon L.P. Stimulation of prothrombinase activity by the nonapeptide Thr-Trp-Ala-Arg-Asn-Ser-Tyr-Asn-Val, a segment of a plant thionin. Peptides. 2003;24(4):515-521. DOI 10.1016/S0196-9781(03)00115-3. Plattner S., Gruber C., Stadlmann J., Widmann S., Gruber C.W., Altmann F., Bohlmann H. Isolation and characterization of a thionin proprotein-processing enzyme from barley. J. Biol. Chem. 2015; 290(29):18056-18067. DOI 10.1074/jbc.M115.647859. Rao U., Teeter M.M. Improvement of turn structure prediction by molecular dynamics: a case study of alpha 1-purothionin. Protein Eng. 1993;6(8):837-847. Rayapuram C., Wu J., Haas C., Baldwin I.T. PR­13/Thionin but not PR­1 mediates bacterial resistance in Nicotiana attenuata in nature, and neither influences herbivore resistance. Mol. Plant Microbe Interact. 2008;21(7):988-1000. DOI 10.1094/MPMI-21-7-0988.

27. Richard J.A., Kelly I., Marion D., Pezolet M., Auger M. Interaction between β-purothionin and dimyristoylphosphatidylglycerol: a 31P- NMR and infrared spectroscopic study. Biophys. J. 2002;83: 2074-2083. DOI 10.1016/S0006-3495(02)73968-4.

28. Romero A., Alamillo J.M., Garcia-Olmedo F. Processing of thionin precursors in barley leaves by a vacuolar proteinase. Eur. J. Biochem. 1997;243(1-2):202-208. DOI 10.1111/j.14321033.1997.0202a.x.

29. Sánchez-Monge R., Delibes A., Hernandéz-Lucas C., Carbonero P., García-Olmedo F. Homoeologous chromosomal location of the genes encoding thionins in wheat and rye. Theor. Appl. Genet. 1979; 54(2):61-63. DOI 10.1007/BF00265470.

30. Sarethy I.P. Plant peptides: bioactivity, opportunities and challenges. Protein Pept. Lett. 2017;24(2):102-108. DOI 10.2174/0929866523 666161220113632.

31. Schrader-Fischer G., Apel K. cDNA-derived identification of novel thio nin precursors in Viscum album that contain highly divergent thio nin domains but conserved signal and acidic polypeptide domains. Plant Mol. Biol. 1993;23(6):1233-1242.

32. Silverstein K.A., Moskal W.A., Jr., Wu H.C., Underwood B.A., Graham M.A., Town C.D., VandenBosch K.A. Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J. 2007;51(2):262-280. DOI 10.1111/j.1365313X.2007.03136.x.

33. Slavokhotova A.A., Shelenkov A.A., Odintsova T.I. Prediction of Leymus arenarius (L.) antimicrobial peptides based on de novo transcriptome assembly. Plant. Mol. Biol. 2015;89(3):203-214. DOI 10.1007/s11103-015-0346-6.

34. Stec B. Plant thionins – the structural perspective. Cell. Mol. Life Sci. 2006;63(12):1370-1385. DOI 10.1007/s00018-005-5574-5.

35. Stec B., Markman O., Rao U., Heffron G., Henderson S., Vernon L.P., Brumfeld V., Teeter M.M. Proposal for molecular mechanism of thio nins deduced from physico-chemical studies of plant toxins. J. Pept. Res. 2004;64(6):210-224. DOI 10.1111/j.1399-3011.2004.00187.x.

36. Stotz H.U., Waller F., Wang K. Innate immunity in plants: The role of antimicrobial peptides. Antimicrobial Peptides and Innate Immunity. Eds. S. Hiemstra, S.A.J. Zaat. Springer, 2013;29-51.

37. Stuart L.S., Harris T.H. Bactericidal and fungicidal properties of a crystalline protein from unbleached wheat flour. Cereal Chem. 1942;19: 288-300.

38. Tabiasco J., Pont F., Fournie J.J., Vercellone A. Mistletoe viscotoxins increase natural killer cell-mediated cytotoxicity. Eur. J. Biochem. 2002;269(10):2591-2600. DOI 10.1046/j.1432-1033.2002.02932.x.

39. Tam J.P., Wang S., Wong K.H., Tan W.L. Antimicrobial peptides from plants. Pharmaceuticals (Basel). 2015;8(4):711-757. DOI 10.3390/ ph8040711.

40. Taveira G.B., Carvalho A.O., Rodrigues R., Trindade F.G., Da Cunha M., Gomes V.M. Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species. BMC Microbiol. 2016;16:12. DOI 10.1186/s12866-016-0626-6.

41. Taveira G.B., Mathias L.S., da Motta O.V., Machado O.L., Rodrigues R., Carvalho A.O., Teixeira-Ferreira A., Perales J., Vasconcelos I.M., Gomes V.M. Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts. Biopolymers. 2014;102(1):30-39. DOI 10.1002/bip.22351.

42. Taveira G.B., Mello É.O., Carvalho A.O., Regente M., Pinedo M., de La Canal L., Rodrigues R., Gomes V.M. Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum an nuum fruits and combinatorial treatment with fluconazole against Fusarium solani. Biopolymers. 2017;108(3). DOI 10.1002/bip.23008.

43. Vernon L.P., Bell J.D. Membrane structure, toxins and phospholipase A2 activity. Pharmacol. Ther. 1992;54(3):269-295. DOI 10.1016/01637258(92)90003-I.

44. Vernon L.P., Evett G.E., Zeikus R.D., Gray W.R. A toxic thionin from Pyrularia pubera: purification, properties, and amino acid sequence. Arch. Biochem. Biophys. 1985;238(1):18-29. DOI 10.1016/00039861(85)90136-5.

45. Vila-Perelló M., Andreu D. Characterization and structural role of disulfide bonds in a highly knotted thionin from Pyrularia pubera. Biopolymers. 2005;80(5):697-707. DOI 10.1002/bip.20270.

46. Vila-Perelló M., Sánchez-Vallet A., García-Olmedo F., Molina A., Andreu D. Synthetic and structural studies on Pyrularia pubera thionin: a single-residue mutation enhances activity against Gram-negative bacteria. FEBS Lett. 2003;536(1-3):215-219. DOI 10.1016/S00145793(03)00053-X.

47. Vila-Perelló M., Sánchez-Vallet A., García-Olmedo F., Molina A., Andreu D. Structural dissection of a highly knotted peptide reveals minimal motif with antimicrobial activity. J. Biol. Chem. 2005;280(2): 1661-1668. DOI 10.1074/jbc.M410577200.

48. Vila-Perelló M., Tognon S., Sánchez-Vallet A., García-Olmedo F., Molina A., Andreu D. A minimalist design approach to antimicrobial agents based on a thionin template. J. Med. Chem. 2006;49(2):448451. DOI 10.1021/jm050882i.

49. Wada K., Ozaki Y., Matsubara H., Yoshizumi H. Studies on purothionin by chemical modifications. J. Biochem. 1982;91(1):257-263.

50. Woynarowski J.M., Konopa J. Interaction between DNA and viscotoxins. Cytotoxic basic polypeptides from Viscum album L. Hoppe Seylers Z. Physiol. Chem. 1980;361:1535-1545.