ABSTRACT
Cyclotides are head-to-tail cyclized peptides with a unique cystine-knot motif. Their structure provides exceptional resistance against enzymatic, chemical, or thermal degradation compared to other peptides. Peptide-based therapeutics promise high specificity, selectivity and lower immunogenicity, making them safer alternatives to small molecules or large biologicals. Cyclotides were researched due to their anti-cancer properties by inducing apoptosis in tumor cells in the past, but the impact of cyclotides on cytotoxic immune cells was poorly studied. Natural Killer (NK) cells are cytotoxic innate lymphoid cells and play an important role in the defense against infected, stressed and transformed cells. NK cells do not need prior sensitization and act in an antigen independent manner, holding promising potential in the field of immunotherapy. To investigate the effect of immunomodulatory cyclotides on NK cells, we evaluated several peptide-enriched plant extracts on NK cell mediated cytotoxicity. We observed that the extract samples derived from Carapichea ipecacuanha (Brot.) L. Andersson augments the killing potential of mouse NK cells against different tumor targets in vitro. Subsequent isolation of cyclotides from C. ipecacuanha extracts led to the identification of a primary candidate that enhances cytotoxicity of both mouse and human NK cells. The augmented killing is facilitated by the increased degranulation capacity of NK cells. In addition, we noted a direct toxic effect of caripe 8 on tumor cells, suggesting a dual therapeutic potential in cancer treatment. This study offers novel insights how natural peptides can influence NK cell cytotoxicity. These pre-clinical findings hold significant promise for advancing current immunotherapeutic approaches.
Subject(s)
Cyclotides , Killer Cells, Natural , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Animals , Cyclotides/pharmacology , Cyclotides/chemistry , Cyclotides/isolation & purification , Mice , Humans , Cell Line, Tumor , Plant Extracts/pharmacology , Mice, Inbred C57BL , Antineoplastic Agents/pharmacologyABSTRACT
Cyclotides are a type of defense peptide most commonly found in the Violaceae family of plants, exhibiting various biological activities. In this study, we focused on the Viola japonica as our research subject and conducted transcriptome sequencing and analysis using high-throughput transcriptomics techniques. During this process, we identified 61 cyclotides, among which 25 were previously documented, while the remaining 36 were designated as vija 1 to vija 36. Mass spectrometry detection showed that 21 putative cyclotides were found in the extract of V. japonica. Through isolation, purification and tandem mass spectrometry, we characterized and investigated the activities of five cyclotides. Our results demonstrated inhibitory effects of these cyclotides on the growth of Acinetobacter baumannii and Bacillus subtilis, with minimum inhibitory concentrations (MICs) of 4.2 µM and 2.1 µM, respectively. Furthermore, time killing kinetic assays revealed that cyclotides at concentration of 4 MICs achieved completely bactericidal effects within 2 h. Additionally, fluorescence staining experiments confirmed that cyclotides disrupt microbial membranes. Moreover, cytotoxicity studies showed that cyclotides possess cytotoxic effects, with IC50 values ranging from 0.1 to 3.5 µM. In summary, the discovery of new cyclotide sequences enhances our understanding of peptide diversity and the exploration of their activity lays the foundation for a deeper investigation into the mechanisms of action of cyclotides.
Subject(s)
Acinetobacter baumannii , Bacillus subtilis , Cyclotides , Microbial Sensitivity Tests , Viola , Cyclotides/pharmacology , Cyclotides/chemistry , Cyclotides/isolation & purification , Viola/chemistry , Acinetobacter baumannii/drug effects , Bacillus subtilis/drug effects , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Infective Agents/pharmacology , Anti-Infective Agents/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistryABSTRACT
Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.
Subject(s)
Cyclotides , Drosophila melanogaster , Insecticides , Plant Proteins , Violaceae , Animals , Humans , Amino Acid Sequence , Cyclotides/chemistry , Cyclotides/isolation & purification , Cyclotides/pharmacology , Drosophila melanogaster/drug effects , Insecticides/chemistry , Insecticides/isolation & purification , Insecticides/pharmacology , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Violaceae/chemistry , Erythrocytes/drug effectsABSTRACT
Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.
Subject(s)
Biological Products , Cyclotides , Plant Proteins , Violaceae , Biological Products/chemistry , Biological Products/isolation & purification , Biological Products/pharmacology , Brazil , Cell Line, Tumor , Cyclotides/chemistry , Cyclotides/isolation & purification , Cyclotides/pharmacology , Humans , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Tandem Mass Spectrometry , Violaceae/chemistryABSTRACT
Cyclotides are plant-derived peptides characterized by an â¼30-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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cyclotides/pharmacology , Drug Discovery , Plants, Medicinal/chemistry , Violaceae/chemistry , Amino Acid Sequence , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cell Line, Tumor , Cyclotides/chemistry , Cyclotides/isolation & purification , Drug Screening Assays, Antitumor , Humans , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Surface Plasmon Resonance , Tandem Mass SpectrometryABSTRACT
Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.
Subject(s)
Cloning, Molecular/methods , Cyclotides/biosynthesis , Protein Engineering/methods , Recombinant Fusion Proteins/biosynthesis , Chromatography, Affinity/methods , Chromatography, Agarose/methods , Chromatography, High Pressure Liquid , Cyclization , Cyclotides/chemistry , Cyclotides/genetics , Cyclotides/isolation & purification , Cystine/chemistry , Cystine Knot Motifs , Disulfides/chemistry , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Inteins , Plant Proteins/biosynthesis , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Protein Folding , Protein Processing, Post-Translational , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Acyclotides are plant-based, acyclic miniproteins with cystine knot motif formed by three conserved disulfide linkages and lack head to tail ligation. Acyclotides may not necessarily be less stable, even though they lack cyclic backbone, as the conserved cystine knot feature provides the required stability. Violacin A was the first acyclotide, isolated from Viola odorata in 2006. Until now, acyclotides have been reported from five dicot families (Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae, Fabaceae) and one monocot family (Poaceae). In Poaceae, only acyclotides have been found whereas in dicot families both cyclotides and acyclotides have been isolated. In last 15 years, several acyclotides with antimicrobial, cytotoxic and hemolytic bioactivities have been discovered. Thus, although many naturally expressed acyclotides do exhibit bioactivities, the linearization of the cyclic peptides may result in loss of bioactivities. Although, bioactivities of acyclotides are comparable to their cyclic counterparts, the numbers of isolated acyclotides are still few. Further, those discovered, have the scope to be screened for agriculturally important activities (insecticidal, anti-helminthic, molluscicidal) and pharmaceutical properties (anticancer, anti-HIV, immuno-stimulant). The feasibility of application of acyclotides is because of their relatively less complex biological synthesis compared to cyclotides, as the cyclization step is not needed. This attribute facilitates the production of transgenic crops and/or its expression in heterologous organisms, lacking cyclization machinery. Keeping in view the bioactivities and the wide array of emerging potential applications of acyclotides, the present review discusses their distribution in plants, gene and protein structure, biosynthesis, bioactivities and mechanism of action. Further, their potential applications and future perspectives to exploit them in agriculture and pharmaceutical industries have been highlighted.
Subject(s)
Adjuvants, Immunologic/pharmacology , Anti-HIV Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cyclotides/pharmacology , Insecticides/pharmacology , Phytochemicals/pharmacology , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/isolation & purification , Anti-HIV Agents/chemistry , Anti-HIV Agents/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cyclotides/chemistry , Cyclotides/isolation & purification , Humans , Insecticides/chemistry , Insecticides/isolation & purification , Phytochemicals/chemistry , Phytochemicals/isolation & purificationABSTRACT
Peptides with pharmaceutical activities are attractive drug leads, and knowledge of their mode-of-action is essential for translation into the clinic. Comparison of native and enantiomeric peptides has long been used as a powerful approach to discriminate membrane- or receptor-mediated modes-of-action on the basis of the assumption that interactions with cell membranes are independent of peptide chirality. Here, we revisit this paradigm with the cyclotide kalata B1, a drug scaffold with intrinsic membrane-binding activity whose enantiomer is less potent than native peptide. To investigate this chirality dependence, we compared peptide-lipid binding using mirror image model membranes. We synthesized phospholipids with non-natural chirality and demonstrate that native kalata B1 binds with higher affinity to phospholipids with chirality found in eukaryotic membranes. This study shows for the first time that the chiral environment of lipid bilayers can modulate the function of membrane-active peptides and challenges the view that peptide-lipid interactions are achiral.
Subject(s)
Cell Membrane/chemistry , Cyclotides/chemistry , Phospholipids/chemistry , Binding Sites , Cyclotides/chemical synthesis , Cyclotides/isolation & purification , Eukaryotic Cells/cytology , Healthy Volunteers , Humans , Leukocytes, Mononuclear/cytology , Models, MolecularABSTRACT
Cyclotides are cyclic peptides, present in several plant families, that show diverse biological properties. Structurally, cyclotides share a distinctive head-to-tail circular knotted topology of three disulfide bonds. This framework provides cyclotides with extraordinary resistance to thermal and chemical denaturation. There is increasing interest in the therapeutic potential of cyclotides, which combine several promising pharmaceutical properties, including binding affinity, target selectivity, and low toxicity towards healthy mammalian cells. Recently, cyclotides have been reported to be orally bioavailable and have proved to be amenable to modifications. Here, we provide an overview of the structure, properties, and pharmaceutical applications of cyclotides.
Subject(s)
Cyclotides , Drug Discovery/methods , Amino Acid Sequence , Computer Simulation , Cyclotides/chemical synthesis , Cyclotides/isolation & purification , Cyclotides/pharmacology , Databases, Factual , Humans , Plants/chemistryABSTRACT
The emergence of rapidly evolving multidrug-resistant pathogens and a deficit of new compounds entering the clinical pipeline necessitate the exploration of alternative sources of antimicrobial therapeutics. Cyclotides revealed in Viola spp. are a class of highly stable, cyclic, and disulfide-bound peptides with diverse intrinsic bioactivities. Herein we have identified a novel complement of 42 putative cyclotide masses in the plant species Viola inconspicua. Cyclotide-containing fractions of a V. inconspicua peptide library revealed potent bioactivities against the Gram-negative bacteria Escherichia coli ATCC 25922 and the highly virulent and multidrug-resistant Klebsiella pneumoniae VK148. As such, six previously uncharacterized cyclotides, cycloviolacins I1-6 (cyI1-cyI6), were prioritized for molecular characterization. Cyclotides cyI3-cyI6 contain a novel "TLNGNPGA" motif in the highly variable loop six region, expanding the already substantial sequence diversity of this peptide class. Library fractions comprised of cyclotides cyI3-cyI6 exhibited MIC values of 18 and 35 µM against E. coli and K. pneumoniae, respectively, whereas isolated cyI3 killed â¼50% of E. coli at 60 µM and isolated cyI4 demonstrated no killing at concentrations >60 µM against both pathogens. This work expands the repertoire of bioactive cyclotides found in Viola spp. and highlights the potential of these antibacterial cyclic peptides.
Subject(s)
Anti-Bacterial Agents/pharmacology , Cyclotides/pharmacology , Viola/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Cyclotides/chemistry , Cyclotides/isolation & purification , Escherichia coli/drug effects , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Molecular StructureABSTRACT
Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized VIa Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature's complexity.
Subject(s)
Antifungal Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cyclotides/pharmacology , Fusarium/drug effects , Viola/chemistry , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclotides/chemistry , Cyclotides/isolation & purification , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Fibroblasts/drug effects , Humans , Molecular Structure , Peptide Library , Structure-Activity Relationship , Tandem Mass SpectrometryABSTRACT
Biologically active cyclotides have been found on some flowering plants species and are involved in the role of the plant protection. As part of studies focusing on peptides from Brazilian plant species, we are reporting the detection by LC-MS of several cyclotides from leaves and stems of Noisettia orchidiflora (Violaceae). From stems it was possible to isolate and characterize a cyclotide named Nor A. Its primary structure (amino acid sequence) was established by MALDI-TOF-MS, based on the y- and b-type ion series, after reduction and alkylation reactions, as well as enzymatic digestion using the enzymes endoproteinase glutamic acid (endoGlu-C), trypsin, and chymotrypsin. Furthermore, the amino acid analysis was also described.
Subject(s)
Cyclotides/isolation & purification , Violaceae/chemistry , Amino Acid Sequence , Chromatography, Liquid , Cyclotides/chemistry , Plant Leaves/chemistry , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Stems/chemistry , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Modification of metal surfaces with antimicrobial peptides is a promising approach to reduce bacterial adhesion. Here, cyclic peptides or cycloids, possessing remarkable stability and antimicrobial activities, were extracted and purified from Viola philippica Cav., and identified using mass spectrometry. Cyclotides were subsequently utilized to modify stainless steel surfaces via polydopamine-mediated coupling. The resulting cyclotide-modified surfaces were characterized by Fourier transform infrared (FTIR) spectroscopy and contact angle analysis. The antibacterial capacity of these cyclotides against Staphylococcus aureus was assessed by Alamar blue assay. The antibiofilm capacity of the modified surfaces was assessed by crystal violet assay, and scanning electron microscopy (SEM). A composite of Kalata b1, Varv A, Viba 15 and Viba 17 (P1); Varv E (P2); and Viphi G (P3) were isolated and identified. FTIR analysis of the modified surfaces demonstrated that cyclotides bound to the surfaces and induced reduction of contact angles. Antimicrobial effects showed an order P3 > P1 and P2, with P3-treated surfaces demonstrating the strongest antibiofilm capacity. SEM confirmed reduced biofilm formation for P3-treated surfaces. This study provides novel evidence for cyclotides as a new class for development of antibacterial and antibiofilm agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Cyclotides/pharmacology , Metals/chemistry , Plant Extracts/pharmacology , Viola/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Cyclotides/chemistry , Cyclotides/isolation & purification , Indoles/chemistry , Microscopy, Electron, Scanning , Plant Extracts/chemistry , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Polymers/chemistry , Staphylococcus aureus/drug effects , Staphylococcus aureus/physiologyABSTRACT
Cyclotides are cyclic cystine knotted macrocyclic plant peptides that have several promising applications. This study was undertaken to detect and identify known and new cyclotides in Viola odorata, a commercially important medicinal plant, from three geographical locations in India. The number of cyclotides in the plant varied with the tissue (leaves, petioles, flowers, runners, and roots) and with geographical locations in India. Using liquid chromatography coupled to Fourier transform mass spectrometry (FTMS), 166 cyclotide-like masses were observed to display cyclotide-diagnostic mass shifts following reduction, alkylation, and digestion, and 71 of these were positively identified based on automated spectrum matching. Of the remaining 95 putative cyclotides observed, de novo peptide sequencing of three new cyclotides, namely, vodo I1 (1), vodo I2 (2), and vodo I3 (3), was carried out with tandem mass spectrometry.
Subject(s)
Cyclotides/isolation & purification , Plants, Medicinal/chemistry , Viola/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Cyclotides/chemistry , India , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Plant Leaves/chemistryABSTRACT
Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.
Subject(s)
Cyclotides/isolation & purification , Cystine/isolation & purification , Fabaceae/chemistry , Plant Proteins/isolation & purification , Rubiaceae/chemistry , Solanaceae/chemistry , Violaceae/chemistry , Amino Acid Sequence , Cyclotides/chemistry , Cystine/chemistry , Molecular Structure , Plant Proteins/chemistryABSTRACT
Cyclotides are fascinating microproteins (≈30 residues long) present in several families of plants that share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology. Naturally occurring cyclotides have shown to posses various pharmacologically relevant activities and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes. In this chapter we describe how to express a native folded cyclotide using intein-mediated protein trans-splicing in live Escherichia coli cells.
Subject(s)
Cyclotides , Inteins , Recombinant Fusion Proteins , Cyclotides/biosynthesis , Cyclotides/chemistry , Cyclotides/genetics , Cyclotides/isolation & purification , Escherichia coli/genetics , Escherichia coli/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Möbius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.
Subject(s)
Antibodies/chemistry , Chromatography, Affinity/methods , Cyclotides/chemistry , Cyclotides/isolation & purification , Amino Acid Sequence , Animals , Antibodies/immunology , Cyclotides/immunology , Cystine Knot Motifs/immunology , RabbitsABSTRACT
Plants from the genus Psychotria include species bearing cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with cyclotides in Psychotria leiocarpa and P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpa and P. brachyceras were monitored following herbivore- and pathogen-associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant cyclotide found in the leaves of P. leiocarpa, and also found in P. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity and resulted in increased rates of larval survival in treatments containing indole alkaloids. This suggests that plant alkaloids ingested by larvae might contribute to herbivore oxidative stress detoxification, corroborating, in a heterologous system with artificial oxidative stress stimulation, the antioxidant efficiency of Psychotria alkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpa as a cyclotide-bearing species, and the absence of these peptides in P. umbellata.
Subject(s)
Cyclotides/isolation & purification , Indole Alkaloids/isolation & purification , Insecticides/isolation & purification , Insecticides/pharmacology , Psychotria/chemistry , Amino Acid Sequence , Animals , Antioxidants/chemistry , Brazil , Cyclotides/analysis , Cyclotides/chemistry , Cyclotides/pharmacology , Herbivory , Indole Alkaloids/analysis , Indole Alkaloids/chemistry , Indole Alkaloids/pharmacology , Insecticides/chemistry , Larva/drug effects , Lepidoptera/drug effects , Molecular Sequence Data , Molecular Structure , Oxidative Stress , Plant Leaves/chemistryABSTRACT
Cyclotides are an interesting family of circular plant peptides. Their unique three-dimensional structure, comprising a head-to-tail circular backbone chain and three disulfide bonds, confers them stability against thermal, chemical, and enzymatic degradation. Their unique stability under extreme conditions creates an idea about the possibility of using harsh extraction methods such as microwave-assisted extraction (MAE) without affecting their structures. MAE has been introduced as a potent extraction method for extraction of natural compounds, but it is seldom used for peptide and protein extraction. In this work, microwave irradiation was applied to the extraction of cyclotides. The procedure was performed in various steps using a microwave instrument under different conditions. High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) results show stability of cyclotide structures on microwave radiation. The influential parameters, including time, temperature, and the ratio of solvents that are affecting the MAE potency, were optimized. Optimal conditions were obtained at 20 min of irradiation time, 1200 W of system power in 60 °C, and methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with maceration extraction shows that there are similarities between cyclotide sequences and extraction yields.
Subject(s)
Cyclotides/analysis , Microwaves , Plant Extracts/chemistry , Viola/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Cyclotides/isolation & purification , Molecular Sequence Data , Plant Extracts/isolation & purification , Sequence Alignment , Solid Phase Extraction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150â¯000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.