Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

2020; Elsevier BV; Volume: 295; Issue: 32 Linguagem: Inglês

10.1074/jbc.ra120.012627

ISSN

1083-351X

Autores

Qingdan Du, Lai Yue Chan, Edward K. Gilding, Sónia Troeira Henriques, Nicholas D. Condon, Anjaneya S. Ravipati, Quentin Kaas, Yen‐Hua Huang, David J. Craik,

Tópico(s)

Phytoplasmas and Hemiptera pathogens

Resumo

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid–long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide–membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity. Cyclotides are plant-derived peptides characterized by an ∼30-amino acid–long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide–membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity. Cyclotides are a large family of cysteine-rich peptides, ∼30 amino acids in size, derived from plants (1de Veer S.J. Kan M.-W. Craik D.J. Cyclotides: From structure to function.Chem. Rev. 2019; 119 (31829013): 12375-1242110.1021/acs.chemrev.9b00402Crossref PubMed Scopus (88) Google Scholar). They have attracted great attention from both academia and industry since they were first reported in the 1990s. In 2017, Sero-X, which is a cyclotide-based eco-friendly pesticide extracted from Clitoria ternatea (2Oguis G.K. Gilding E.K. Jackson M.A. Craik D.J. Butterfly pea (Clitoria ternatea), a cyclotide-bearing plant with applications in agriculture and medicine.Front. Plant Sci. 2019; 10 (31191573): 64510.3389/fpls.2019.00645Crossref PubMed Scopus (53) Google Scholar), was approved to market in Australia for the protection of cotton and macadamia nut crops (https://innovate-ag.com.au/). In 2019, a single-residue mutated variant of cyclotide kalata B1, [T20K]kalata B1, entered phase I clinical trials for multiple sclerosis (3Gründemann C. Stenberg K.G. Gruber C.W. T20K: An immunomodulatory cyclotide on its way to the clinic.Int. J. Pept. Res. Ther. 2019; 25: 9-1310.1007/s10989-018-9701-1Crossref Scopus (39) Google Scholar). These examples foreshadow the great potential of cyclotides in both agriculture and medicine. Cyclotides have a role in plant defense, acting as insecticides or antipathogenic agents (4Jennings C. West J. Waine C. Craik D. Anderson M. Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis.Proc. Natl. Acad. Sci. U. S. A. 2001; 98 (11535828): 10614-1061910.1073/pnas.191366898Crossref PubMed Scopus (403) Google Scholar, 5Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Isolation, solution structure, and insecticidal activity of kalata B2, a circular protein with a twist: Do Möbius strips exist in nature?.Biochemistry. 2005; 44 (15654741): 851-86010.1021/bi047837hCrossref PubMed Scopus (194) Google Scholar). They also exhibit a diverse range of other biological properties (6Huang Y.-H. Du Q. Craik D.J. Cyclotides: Disulfide-rich peptide toxins in plants.Toxicon. 2019; 172 (31682883): 33-4410.1016/j.toxicon.2019.10.244Crossref PubMed Scopus (26) Google Scholar), including antifouling (7Göransson U. Sjögren M. Svangård E. Claeson P. Bohlin L. Reversible antifouling effect of the cyclotide cycloviolacin O2 against barnacles.J. Nat. Prod. 2004; 67 (15332843): 1287-129010.1021/np0499719Crossref PubMed Scopus (124) Google Scholar), molluscicidal (8Plan M.R.R. Saska I. Cagauan A.G. Craik D.J. Backbone cyclised peptides from plants show molluscicidal activity against the rice pest Pomacea canaliculata (golden apple snail).J. Agric. Food Chem. 2008; 56 (18557620): 5237-524110.1021/jf800302fCrossref PubMed Scopus (141) Google Scholar), uterotonic (9Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide kalata B1.Biochemistry. 1995; 34 (7703226): 4147-415810.1021/bi00013a002Crossref PubMed Scopus (371) Google Scholar), anti-HIV (10Wang C.K. Colgrave M.L. Gustafson K.R. Ireland D.C. Goransson U. Craik D.J. Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis.J. Nat. Prod. 2008; 71 (18081258): 47-5210.1021/np070393gCrossref PubMed Scopus (147) Google Scholar), antimicrobial (11Ivanova A. Delcheva I. Tsvetkova I. Kujumgiev A. Kostova I. GC-MS analysis and anti-microbial activity of acidic fractions obtained from Paeonia peregrina and Paeonia tenuifolia roots.Z. Naaturforsch. C. J. Biosci. 2002; 57 (12240987): 624-62810.1515/znc-2002-7-813Crossref PubMed Scopus (15) Google Scholar), cytotoxic anticancer (12Herrmann A. Burman R. Mylne J.S. Karlsson G. Gullbo J. Craik D.J. Clark R.J. Göransson U. The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity.Phytochemistry. 2008; 69 (18191970): 939-95210.1016/j.phytochem.2007.10.023Crossref PubMed Scopus (114) Google Scholar), immunosuppressive (13Thell K. Hellinger R. Schabbauer G. Gruber C.W. Immunosuppressive peptides and their therapeutic applications.Drug Discov. Today. 2014; 19 (24333193): 645-65310.1016/j.drudis.2013.12.002Crossref PubMed Scopus (65) Google Scholar), and protease-inhibitory activities (14Hernandez J.-F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.-P. Heitz A. Trinh Hong T. Pham T.T.C. Le Nguyen D. Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure.Biochemistry. 2000; 39 (10801322): 5722-573010.1021/bi9929756Crossref PubMed Scopus (296) Google Scholar, 15Quimbar P. Malik U. Sommerhoff C.P. Kaas Q. Chan L.Y. Huang Y.-H. Grundhuber M. Dunse K. Craik D.J. Anderson M.A. Daly N.L. High-affinity cyclic peptide matriptase inhibitors.J. Biol. Chem. 2013; 288 (23548907): 13885-1389610.1074/jbc.M113.460030Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Cyclotides are characterized by a head-to-tail cyclic backbone and a cystine knot motif, which renders them ultrastable against degradation by harsh conditions and are also amenable to residue substitutions (16Craik D.J. Daly N.L. Bond T. Waine C. Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif1.J. Mol. Biol. 1999; 294 (10600388): 1327-133610.1006/jmbi.1999.3383Crossref PubMed Scopus (644) Google Scholar, 17Göransson U. Burman R. Gunasekera S. Strömstedt A.A. Rosengren K.J. Circular proteins from plants and fungi.J. Biol. Chem. 2012; 287 (22700984): 27001-2700610.1074/jbc.R111.300129Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Colgrave M.L. Craik D.J. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot.Biochemistry. 2004; 43 (15147180): 5965-597510.1021/bi049711qCrossref PubMed Scopus (453) Google Scholar). These properties make cyclotides ideal scaffolds for grafting bioactive epitopes (19Craik D.J. Du J. Cyclotides as drug design scaffolds.Curr. Opin. Chem. Biol. 2017; 38 (28249194): 8-1610.1016/j.cbpa.2017.01.018Crossref PubMed Scopus (80) Google Scholar), with potential applications in the agricultural and pharmaceutical industries. To date, cyclotides have been identified in five major plant families, namely the Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae, and Fabaceae (20Koehbach J. Attah A.F. Berger A. Hellinger R. Kutchan T.M. Carpenter E.J. Rolf M. Sonibare M.A. Moody J.O. Wong G.K.-S. Dessein S. Greger H. Gruber C.W. Cyclotide discovery in gentianales revisited—identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.Biopolymers. 2013; 100 (23897543): 438-45210.1002/bip.22328Crossref PubMed Scopus (82) Google Scholar, 21Kaas Q. Craik D.J. Analysis and classification of circular proteins in CyBase.Biopolymers. 2010; 94 (20564021): 584-59110.1002/bip.21424Crossref PubMed Scopus (58) Google Scholar, 22Craik D.J. Malik U. Cyclotide biosynthesis.Curr. Opin. Chem. Biol. 2013; 17 (23809361): 546-55410.1016/j.cbpa.2013.05.033Crossref PubMed Scopus (60) Google Scholar). They are categorized into three subfamilies (bracelet, Möbius, and trypsin inhibitor), with archetypical cyclotides (23Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. Twists, knots, and rings in proteins. Structural definition of the cyclotide framework.J. Biol. Chem. 2003; 278 (12482868): 8606-861610.1074/jbc.M211147200Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 24Wang C.K. Colgrave M.L. Ireland D.C. Kaas Q. Craik D.J. Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes.Biophys. J. 2009; 97 (19720036): 1471-148110.1016/j.bpj.2009.06.032Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25Heitz A. Hernandez J.-F. Gagnon J. Hong T.T. Pham T.T.C. Nguyen T.M. Le-Nguyen D. Chiche L. Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins.Biochemistry. 2001; 40 (11434766): 7973-798310.1021/bi0106639Crossref PubMed Scopus (150) Google Scholar) shown in Fig. 1. A distinctive structural feature of the Möbius subfamily is a 180° twist in the main chain structure, which originates from a Pro in loop 5 adopting a cis conformation (16Craik D.J. Daly N.L. Bond T. Waine C. Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif1.J. Mol. Biol. 1999; 294 (10600388): 1327-133610.1006/jmbi.1999.3383Crossref PubMed Scopus (644) Google Scholar). The majority of cyclotides discovered to date are from the bracelet and Möbius subfamilies; trypsin inhibitor cyclotides have been found only in the Momordica genus from the Cucurbitaceae family (14Hernandez J.-F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.-P. Heitz A. Trinh Hong T. Pham T.T.C. Le Nguyen D. Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure.Biochemistry. 2000; 39 (10801322): 5722-573010.1021/bi9929756Crossref PubMed Scopus (296) Google Scholar, 26Chiche L. Heitz A. Gelly J.-C. Gracy J. Chau P.T. Ha P.T. Hernandez J.-F. Le-Nguyen D. Squash inhibitors: From structural motifs to macrocyclic knottins.Curr. Protein Pept. Sci. 2004; 5 (15551519): 341-34910.2174/1389203043379477Crossref PubMed Scopus (149) Google Scholar). In contrast to other plant families, every species in the Violaceae family seems to produce cyclotides (27Bobey A. Pinto M. Cilli E. Lopes N. da Silva Bolzani V. A cyclotide isolated from Noisettia orchidiflora (Violaceae).Planta Med. 2018; 84 (29843182): 947-95210.1055/a-0632-2204Crossref PubMed Scopus (4) Google Scholar, 28Gruber C.W. Elliott A.G. Ireland D.C. Delprete P.G. Dessein S. Göransson U. Trabi M. Wang C.K. Kinghorn A.B. Robbrecht E. Craik D.J. Distribution and evolution of circular miniproteins in flowering plants.Plant Cell. 2008; 20 (18827180): 2471-248310.1105/tpc.108.062331Crossref PubMed Scopus (213) Google Scholar). Of the 433 cyclotides described in CyBase (RRID:SCR_012925), 259 occur in violaceous plants (29Wang C.K. Kaas Q. Chiche L. Craik D.J. CyBase: A database of cyclic protein sequences and structures, with applications in protein discovery and engineering.Nucleic Acids Res. 2008; 36 (17986451): D206-D21010.1093/nar/gkm953Crossref PubMed Scopus (220) Google Scholar, 30Mulvenna J.P. Wang C. Craik D.J. Cybase: A database of cyclic protein sequence and structure.Nucleic Acids Res. 2006; 34 (16381843): D192-D19410.1093/nar/gkj005Crossref PubMed Scopus (131) Google Scholar). The violaceous herb Hybanthus enneaspermus Linn F. Muell (H. enneaspermus) is an Indian traditional medicinal herb with a wide range of medicinal applications, including anticonvulsant (31Hemalatha S. Wahi A. Singh P. Chansouria J. Anticonvulsant and free radical scavenging activity of Hybanthus enneaspermus: A preliminary screening.Indian J. Trad. Knowledge. 2003; 2: 383-388Google Scholar), free radical scavenging (31Hemalatha S. Wahi A. Singh P. Chansouria J. Anticonvulsant and free radical scavenging activity of Hybanthus enneaspermus: A preliminary screening.Indian J. Trad. Knowledge. 2003; 2: 383-388Google Scholar), nephroprotective (32Setty M. Narayanaswamy V. Sreenivasan K. Shirwaikar A. Free radical scavenging and nephroprotective activity of Hybanthus enneaspermus (L) F. Muell.Pharmacologyonline. 2007; 2: 158-171Google Scholar), antiarthritic (33Tripathy S. Sahoo S. Pradhan D. Sahoo S. Satapathy D. Evaluation of anti arthritic potential of Hybanthus enneaspermus.Afr. J. Pharm. Pharmacol. 2009; 3: 611-614Google Scholar), larvicidal (34Suman T.Y. Radhika Rajasree S.R. Jayaseelan C. Mary R.R. Gayathri S. Aranganathan L. Remya R.R. GC-MS analysis of bioactive components and biosynthesis of silver nanoparticles using Hybanthus enneaspermus at room temperature evaluation of their stability and its larvicidal activity.Environ. Sci. Pollut. Res. Int. 2016; 23 (26438369): 2705-271410.1007/s11356-015-5468-5Crossref PubMed Scopus (23) Google Scholar), anti-anemic (35Sampath B. Phytochemical Screening and Evaluation of Anti-Anaemic Activity of Entire Plant of Hybanthus Enneaspermus (Linn.) F. Muell. Madras Medical College, Chennai2016Google Scholar), antidiabetic (36Patel D.K. Kumar R. Prasad S.K. Sairam K. Hemalatha S. Antidiabetic and in vitro antioxidant potential of Hybanthus enneaspermus (Linn) F. Muell in streptozotocin–induced diabetic rats.Asian Pac. J. Trop. Biomed. 2011; 1 (23569783): 316-32210.1016/S2221-1691(11)60051-8Crossref PubMed Scopus (129) Google Scholar), hepatoprotective (37Vuda M. D'Souza R. Upadhya S. Kumar V. Rao N. Kumar V. Boillat C. Mungli P. Hepatoprotective and antioxidant activity of aqueous extract of Hybanthus enneaspermus against CCl4-induced liver injury in rats.Exp. Toxicol. Pathol. 2012; 64 (21478003): 855-85910.1016/j.etp.2011.03.006Crossref PubMed Scopus (73) Google Scholar, 38Bhanu H.S. Meena B. Pallavi C. Suppression of paracetamol toxicity by antitoxidant principles of Hybanthus enneaspermus (L.) F. Muell. in mice blood and liver.Int. J. Pharm. Pharm. Sci. 2011; 3: 90-94Google Scholar), and aphrodisiac activities (39Narayanswamy V. Setty M.M. Malini S. Shirwaikar A. Preliminary aphrodisiac activity of Hybanthus enneaspermus in rats.Pharmacologyonline. 2007; 1: 152-161Google Scholar). A previous study suggested the existence of cyclotides in this plant based on mRNA analysis, but only two partial sequences were reported (40Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Göransson U. Craik D.J. A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae).Plant Cell. 2005; 17 (16199617): 3176-318910.1105/tpc.105.034678Crossref PubMed Scopus (142) Google Scholar). Therefore, we aimed to further explore the cyclotide content of H. enneaspermus and to evaluate their bioactivity, specifically, their cytotoxic activity against mammalian cells. The cytotoxic activity of cyclotides has attracted interest for their potential anticancer applications (41Henriques S.T. Craik D.J. Cyclotides as templates in drug design.Drug Discov. Today. 2010; 15 (19878736): 57-6410.1016/j.drudis.2009.10.007Crossref PubMed Scopus (118) Google Scholar, 42Gerlach S.L. Rathinakumar R. Chakravarty G. Göransson U. Wimley W.C. Darwin S.P. Mondal D. Anticancer and chemosensitizing abilities of cycloviolacin O2 from Viola odorata and psyle cyclotides from Psychotria leptothyrsa.Biopolymers. 2010; 94 (20564026): 617-62510.1002/bip.21435Crossref PubMed Scopus (91) Google Scholar). Two Möbius cyclotides (varv A and varv F), and a bracelet cyclotide (cycloviolacin O2, cyO2), have been identified as having strong cytotoxic activity against 10 different human tumor cell lines (43Lindholm P. Göransson U. Johansson S. Claeson P. Gullbo J. Larsson R. Bohlin L. Backlund A. Cyclotides: A novel type of cytotoxic agents.Mol. Cancer Ther. 2002; 1 (12477048): 365-369Crossref PubMed Scopus (41) Google Scholar). Other cyclotides with antitumor effects include vitri A (44Svangård E. Göransson U. Hocaoglu Z. Gullbo J. Larsson R. Claeson P. Bohlin L. Cytotoxic cyclotides from Viola tricolor.J. Nat. Prod. 2004; 67 (14987049): 144-14710.1021/np030101lCrossref PubMed Scopus (163) Google Scholar), vibi (E, G, and H) (12Herrmann A. Burman R. Mylne J.S. Karlsson G. Gullbo J. Craik D.J. Clark R.J. Göransson U. The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity.Phytochemistry. 2008; 69 (18191970): 939-95210.1016/j.phytochem.2007.10.023Crossref PubMed Scopus (114) Google Scholar), psyle (A, C, and E) (42Gerlach S.L. Rathinakumar R. Chakravarty G. Göransson U. Wimley W.C. Darwin S.P. Mondal D. Anticancer and chemosensitizing abilities of cycloviolacin O2 from Viola odorata and psyle cyclotides from Psychotria leptothyrsa.Biopolymers. 2010; 94 (20564026): 617-62510.1002/bip.21435Crossref PubMed Scopus (91) Google Scholar), viphi (A, D–G) (45He W. Chan L.Y. Zeng G. Daly N.L. Craik D.J. Tan N. Isolation and characterization of cytotoxic cyclotides from Viola philippica.Peptides. 2011; 32 (21723349): 1719-172310.1016/j.peptides.2011.06.016Crossref PubMed Scopus (55) Google Scholar), mram 8, viba (15 and 17), varv (A and E) (44Svangård E. Göransson U. Hocaoglu Z. Gullbo J. Larsson R. Claeson P. Bohlin L. Cytotoxic cyclotides from Viola tricolor.J. Nat. Prod. 2004; 67 (14987049): 144-14710.1021/np030101lCrossref PubMed Scopus (163) Google Scholar, 45He W. Chan L.Y. Zeng G. Daly N.L. Craik D.J. Tan N. Isolation and characterization of cytotoxic cyclotides from Viola philippica.Peptides. 2011; 32 (21723349): 1719-172310.1016/j.peptides.2011.06.016Crossref PubMed Scopus (55) Google Scholar), vitri F (46Tang J. Wang C.K. Pan X. Yan H. Zeng G. Xu W. He W. Daly N.L. Craik D.J. Tan N. Isolation and characterization of cytotoxic cyclotides from Viola tricolor.Peptides. 2010; 31 (20580652): 1434-144010.1016/j.peptides.2010.05.004Crossref PubMed Scopus (58) Google Scholar), mela (1de Veer S.J. Kan M.-W. Craik D.J. Cyclotides: From structure to function.Chem. Rev. 2019; 119 (31829013): 12375-1242110.1021/acs.chemrev.9b00402Crossref PubMed Scopus (88) Google Scholar, 2Oguis G.K. Gilding E.K. Jackson M.A. Craik D.J. Butterfly pea (Clitoria ternatea), a cyclotide-bearing plant with applications in agriculture and medicine.Front. Plant Sci. 2019; 10 (31191573): 64510.3389/fpls.2019.00645Crossref PubMed Scopus (53) Google Scholar, 3Gründemann C. Stenberg K.G. Gruber C.W. T20K: An immunomodulatory cyclotide on its way to the clinic.Int. J. Pept. Res. Ther. 2019; 25: 9-1310.1007/s10989-018-9701-1Crossref Scopus (39) Google Scholar, 4Jennings C. West J. Waine C. Craik D. Anderson M. Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis.Proc. Natl. Acad. Sci. U. S. A. 2001; 98 (11535828): 10614-1061910.1073/pnas.191366898Crossref PubMed Scopus (403) Google Scholar, 5Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Isolation, solution structure, and insecticidal activity of kalata B2, a circular protein with a twist: Do Möbius strips exist in nature?.Biochemistry. 2005; 44 (15654741): 851-86010.1021/bi047837hCrossref PubMed Scopus (194) Google Scholar, 6Huang Y.-H. Du Q. Craik D.J. Cyclotides: Disulfide-rich peptide toxins in plants.Toxicon. 2019; 172 (31682883): 33-4410.1016/j.toxicon.2019.10.244Crossref PubMed Scopus (26) Google Scholar, 7Göransson U. Sjögren M. Svangård E. Claeson P. Bohlin L. Reversible antifouling effect of the cyclotide cycloviolacin O2 against barnacles.J. Nat. Prod. 2004; 67 (15332843): 1287-129010.1021/np0499719Crossref PubMed Scopus (124) Google Scholar), mech (2 and 3) (47Ravipati A.S. Henriques S.T. Poth A.G. Kaas Q. Wang C.K. Colgrave M.L. Craik D.J. Lysine-rich cyclotides: A new subclass of circular knotted proteins from Violaceae.ACS Chem. Biol. 2015; 10 (26322745): 2491-250010.1021/acschembio.5b00454Crossref PubMed Scopus (31) Google Scholar), vila (A and B) (48Tang J. Wang C.K. Pan X. Yan H. Zeng G. Xu W. He W. Daly N.L. Craik D.J. Tan N. Isolation and characterization of bioactive cyclotides from Viola labridorica.Helv. Chim. Acta. 2010; 93: 2287-229510.1002/hlca.201000115Crossref Scopus (20) Google Scholar), Poca (A and B), cyO4 (49Pinto M.E.F. Najas J.Z.G. Magalhães L.G. Bobey A.F. Mendonça J.N. Lopes N.P. Leme F.M. Teixeira S.P. Trovó M. Andricopulo A.D. Koehbach J. Gruber C.W. Cilli E.M. Bolzani V.S. Inhibition of breast cancer cell migration by cyclotides isolated from Pombalia calceolaria.J. Nat. Prod. 2018; 81 (29757646): 1203-120810.1021/acs.jnatprod.7b00969Crossref PubMed Scopus (27) Google Scholar), and kalata B1 and B2 (50Henriques S.T. Huang Y.-H. Chaousis S. Wang C.K. Craik D.J. Anticancer and toxic properties of cyclotides are dependent on phosphatidylethanolamine phospholipid targeting.ChemBioChem. 2014; 15 (25099014): 1956-196510.1002/cbic.201402144Crossref PubMed Scopus (56) Google Scholar). Previous studies indicate cyclotides exert their cytotoxic activity by disrupting the lipid bilayer of cell membranes (51Henriques S.T. Craik D.J. Cyclotide structure and function: The role of membrane binding and permeation.Biochemistry. 2017; 56 (28085267): 669-68210.1021/acs.biochem.6b01212Crossref PubMed Scopus (38) Google Scholar, 52Svangård E. Burman R. Gunasekera S. Lövborg H. Gullbo J. Göransson U. Mechanism of action of cytotoxic cyclotides: Cycloviolacin O2 disrupts lipid membranes.J. Nat. Prod. 2007; 70 (17378610): 643-64710.1021/np070007vCrossref PubMed Scopus (112) Google Scholar). In particular, cyclotides interact with cells by specifically binding to phospholipids containing phosphatidylethanolamine (PE) headgroups via interaction with a conserved patch of amino acid residues, called a bioactive patch, and insert into the lipid bilayer via a hydrophobic patch on their surface (50Henriques S.T. Huang Y.-H. Chaousis S. Wang C.K. Craik D.J. Anticancer and toxic properties of cyclotides are dependent on phosphatidylethanolamine phospholipid targeting.ChemBioChem. 2014; 15 (25099014): 1956-196510.1002/cbic.201402144Crossref PubMed Scopus (56) Google Scholar, 53Ireland D.C. Wang C.K.L. Wilson J.A. Gustafson K.R. Craik D.J. Cyclotides as natural anti-HIV agents.Biopolymers. 2008; 90 (18008336): 51-6010.1002/bip.20886Crossref PubMed Scopus (122) Google Scholar, 54Henriques S.T. Huang Y.-H. Rosengren K.J. Franquelim H.G. Carvalho F.A. Johnson A. Sonza S. Tachedjian G. Castanho M.A.R.B. Daly N.L. Craik D.J. Decoding the membrane activity of the cyclotide kalata b1: The importance of phosphatidylethanolamine phospholipids and lipid organization on hemolytic and anti-HIV activities.J. Biol. Chem. 2011; 286 (21576247): 24231-2424110.1074/jbc.M111.253393Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 55Huang Y.-H. Colgrave M.L. Clark R.J. Kotze A.C. Craik D.J. Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity.J. Biol. Chem. 2010; 285 (20103593): 10797-1080510.1074/jbc.M109.089854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In this study, we sequenced 12 cyclotides in plant material from H. enneaspermus for structural and activity characterization, of which 11 were novel. Five of the novel cyclotides demonstrated potent cytotoxicity, which correlated with their affinity for lipid membrane bilayers. This work demonstrates that the medicinal plant H. enneaspermus is a rich source of cyclotides with cytotoxic activity. A total of 12 cyclotides were isolated from the leaves, stems, and seeds of H. enneaspermus. Each cyclotide showed a mass increment of 348 Da after reduction and S-alkylation with iodoacetamide, suggesting the existence of three disulfide bonds, and had a mass increment of 18 Da after digestion with endoproteinase Glu-C, indicative of a cyclic backbone. They were fully sequenced using tandem MS (Table 1). Eleven of these cyclotides have novel sequences and were named hyen (Hybanthus enneaspermus) according to a previously reported nomenclature system (40Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Göransson U. Craik D.J. A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae).Plant Cell. 2005; 17 (16199617): 3176-318910.1105/tpc.105.034678Crossref PubMed Scopus (142) Google Scholar, 56Broussalis A.M. Göransson U. Coussio J.D. Ferraro G. Martino V. Claeson P. First cyclotide from Hybanthus (Violaceae).Phytochemistry. 2001; 58 (11524112): 47-5110.1016/S0031-9422(01)00173-XCrossref PubMed Scopus (84) Google Scholar) and designated as hyen C to hyen M in order of their appearance on RP-HPLC (Fig. 2). One of these peptides has an identical sequence to the previously characterized cyclotide cyO2. Hyen A and hyen B are two partial mRNA transcripts reported earlier from the same plant (40Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Göransson U. Craik D.J. A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae).Plant Cell. 2005; 17 (16199617): 3176-318910.1105/tpc.105.034678Crossref PubMed Scopus (142) Google Scholar); however, because of their low expression levels, only the sequence of hyen B could be partially identified in this study. Hyen C was the only Möbius cyclotide isolated from H. enneaspermus. To illustrate the sequencing of hyen peptides, hyen D, the most abundant cyclotide in this plant, is shown as a representative example (Fig. 3).Table 1Cyclotides isolated and sequenced from H. enneaspermusCyclotideExperimental mass (monoisotopic)(Da)FromSequenceUniprotKB accession numbersaThe protein sequence data reported here was submitted to in the UniProt Knowledgebase under these accession numbers.Overall chargehyen AbTwo partial sequences reported in the literature as Hyen A and Hyen B.n.d.RNA(40Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Göransson U. Craik D.J. A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae).Plant Cell. 2005; 17 (16199617): 3176-318910.1105/tpc.105.034678Crossref PubMed Scopus (142) Google Scholar)XXXXCGESCVYIP-CTVTALLGCSCKDKV-CYKNn.d.hyen BcTwo partial sequences reported in the literature as Hyen A and Hyen B.n.d.RNA(40Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Göransson U. Craik D.J. A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae).Plant Cell. 2005; 17 (16199617): 3176-318910.1105/tpc.105.034678Crossref PubMed Scopus (142) Google Scholar)XXXXCGETCKVTKRCSGQG- - -CSCLKGRSCYDn.d.hyen C3316.4st, ldst: stem; l: leaf.GTHPCQETCVTSTRCSTQG- - -CHCNWPI-CFKNeIsobaric residues determined by transcriptome.C0HLN7+1hyen D3154.5st, lGF-PCGESCVYIP-CFTAAIG-CSCKSKV-CYKNfIsobaric residues determined by NMR spectroscopy analysis.C0HLN8+2hyen E3232.2stGV-PCGESCVYIP-CFTGIIN-CSCRDKV-CYNNfIsobaric residues determined by NMR spectroscopy analysis.C0HLN90hyen F3143.2sgs: seed.GL-PCGESCVYIP-CISTVLG-CSCSNKV-CYRNeIsobaric residues determined by transcriptome.C0HLP0+1hyen G3144.2st, lGL-PCGESCVYIP-CISTVLG-CSCSNKV-CYRDhIsobaric residues deduced by sequence homology.C0

Referência(s)