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The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides., (© 2024. The Author(s).)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Asparaginyl endopeptidases (AEPs) are cysteine proteases that control a myriad of cellular functions in plants, including maturation of seed storage proteins and programmed cell death. Recently, several noteworthy AEPs have been discovered that primarily function as transpeptidases rather than hydrolases, to instead catalyse the formation of new peptide bonds. These AEPs appear to have evolved for the cyclisation of a large class of plant defence peptides called cyclotides. Here we describe recent insights into the structural differences between AEPs that preference peptide ligation over hydrolysis. This knowledge is instrumental for the deployment of AEP ligases as biotechnological tools for in vitro applications such as protein labelling and or cyclization, and for plant molecular farming applications., (Copyright © 2020. Published by Elsevier Inc.)

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis , and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.

This Review explores the class of plant-derived macrocyclic peptides called cyclotides. We include an account of their discovery, characterization, and distribution in the plant kingdom as well as a detailed analysis of their sequences and structures, biosynthesis and chemical synthesis, biological functions, and applications. These macrocyclic peptides are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and cystine knot motif, which render them to be exceptionally stable, with resistance to thermal or enzymatic degradation. Routes to their chemical synthesis have been developed over the past two decades, and this capability has facilitated a wide range of mutagenesis and structure-activity relationship studies. In turn, these studies have both led to an increased understanding of their mechanisms of action as well as facilitated a range of applications in agriculture and medicine, as ecofriendly crop protection agents, and as drug leads or scaffolds for pharmaceutical design. Our overall objective in this Review is to provide readers with a comprehensive overview of cyclotides that we hope will stimulate further work on this fascinating family of peptides.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1 b . These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

Cyclotides are plant defense peptides that have been extensively investigated for pharmaceutical and agricultural applications, but key details of their posttranslational biosynthesis have remained elusive. Asparaginyl endopeptidases are crucial in the final stage of the head-to-tail cyclization reaction, but the enzyme(s) involved in the prerequisite steps of N-terminal proteolytic release were unknown until now. Here we use activity-guided fractionation to identify specific members of papain-like cysteine proteases involved in the N-terminal cleavage of cyclotide precursors. Through both characterization of recombinantly produced enzymes and in planta peptide cyclization assays, we define the molecular basis of the substrate requirements of these enzymes, including the prototypic member, here termed kalatase A. The findings reported here will pave the way for improving the efficiency of plant biofactory approaches for heterologous production of cyclotide analogs of therapeutic or agricultural value., Competing Interests: The authors declare no conflict of interest.

Orbitides are small cyclic peptides with a diverse range of therapeutic bioactivities. They are produced by many plant species, including those of the Jatropha genus. Here, the objective was to provide new structural information on orbitides to complement the growing knowledge base on orbitide sequences and activities by focusing on three Jatropha orbitides: ribifolin (1), pohlianin C (7), and jatrophidin (12). To determine three-dimensional structures, racemic crystallography, an emerging structural technique that enables rapid crystallization of biomolecules by combining equal amounts of the two enantiomers, was used. The high-resolution structure of ribifolin (0.99 Å) was elucidated from its racemate and showed it was identical to the structure crystallized from its l-enantiomer only (1.35 Å). Racemic crystallography was also used to elucidate high-resolution structures of pohlianin C (1.20 Å) and jatrophidin (1.03 Å), for which there was difficulty forming crystals without using racemic mixtures. The structures were used to interpret membrane permeability data in PAMPA and a Caco-2 cell assay, showing they had poor permeability. Overall, the results show racemic crystallography can be used to obtain high-resolution structures of orbitides and is useful when enantiopure samples are difficult to crystallize or solution structures from NMR are of low resolution.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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.

We recently isolated and described the evolutionary origin of a diverse class of small single-disulfide bonded peptides derived from Preproalbumin with SFTI-1 (PawS1) proteins in the seeds of flowering plants (Asteraceae). The founding member of the PawS derived peptide (PDP) family is the potent trypsin inhibitor SFTI-1 (sunflower trypsin inhibitor-1) from Helianthus annuus, the common sunflower. Here we provide additional structures and describe the structural diversity of this new class of small peptides, derived from solution NMR studies, in detail. We show that although most have a similar backbone framework with a single disulfide bond and in many cases a head-to-tail cyclized backbone, they all have their own characteristics in terms of projections of side-chains, flexibility and physiochemical properties, attributed to the variety of their sequences. Small cyclic and constrained peptides are popular as drug scaffolds in the pharmaceutical industry and our data highlight how amino acid side-chains can fine-tune conformations in these promising peptides.

Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets., (© 2016 Wiley Periodicals, Inc.)

Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins., (© 2016 Wiley Periodicals, Inc.)

Cyclotides are cyclic peptides from plants in the Violaceae, Rubiaceae, Fabaceae, Cucurbitaceae, and Solanaceae families. They are sparsely distributed in most of these families, but appear to be ubiquitous in the Violaceae, having been found in every plant so far screened from this family. However, not all geographic regions have been examined and here we report the discovery of cyclotides from a Viola species from South-East Asia. Two novel cyclotides (Visu 1 and Visu 2) and two known cyclotides (kalata S and kalata B1) were identified in V. sumatrana. NMR studies revealed that kalata S and kalata B1 had similar secondary structures. Their biological activities were determined in cytotoxicity assays; both had similar cytotoxic activity and were more toxic to U87 cells compared with other cell lines. Overall, the study strongly supports the ubiquity of cyclotides in the Violaceae and adds to our understanding of their distribution and cytotoxic activity., (© 2016 Wiley Periodicals, Inc.)

Cyclotides are plant-derived cyclic peptides that have a head-to-tail cyclic backbone and three conserved disulphide bonds that form a cyclic cystine knot motif. They occur in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, typically with 10-100 cyclotides in a given plant species, in a wide range of tissues, including flowers, leaves, stems, and roots. Some cyclotides are expressed in large amounts (up to 1g kg(-1) wet plant weight) and their natural function appears to be to protect plants from pests or pathogens. This article provides a brief overview of their discovery, distribution in plants, and applications. In particular, their exceptional stability has led to their use as peptide-based scaffolds in drug design applications. They also have potential as natural 'ecofriendly' insecticides, and as protein engineering frameworks., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases., (© 2015 Authors; published by Portland Press Limited.)

Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used (18)O-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cysteine (Cys)-rich proteins (CRPs) are frequently associated with plant defense and stress resistance. Viola baoshanensis is a cadmium (Cd) hyper-accumulating plant whose CRPs-based defense systems are so far poorly understood. Next generation sequencing (NGS) techniques and a specialist searching tool, CrpExcel, were employed for identifying CRPs in V. baoshanensis. The transcriptome sequences of V. baoshanensis were assembled primarily from 454FLX/Hiseq2000 reads of plant cDNA sequencing libraries. CrpExcel was then used to search the ORFs and 9687 CRPs were identified, and included zinc finger (ZF) proteins, lipid transfer proteins, thaumatins and cyclotide precursors. Real-time PCR results showed that all CRP genes tested are constitutively expressed, but the genes of defensive peptides showed greater up-regulated expression than those of ZF-proteins in Cd- and/or wounding (Wd) treatments of V. baoshanensis seedlings. The NGS-derived sequences of cyclotide precursor genes were verified by RT-PCR and ABI3730 sequencing studies, and 32 novel cyclotides were identified in V. baoshanensis. In general, the metal-binding sites of ZF-containing CRPs also represented the potential vulnerable targets of toxic metals. This study provides broad insights into CRPs-based defense systems and stress-vulnerable targets in V. baoshanensis. It now brings the number of cyclotide sequences in V. baoshanensis to 53 and based on projections from this work, the number of cyclotides in the Violaceae is now conservatively estimated to be >30000., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Plant defensins are small (45-54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined., Results: By transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation., Conclusions: In this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops.

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

Background: Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone., Results: Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity., Conclusion: An analog of MCoTI-II is one of the most potent inhibitors of matriptase., Significance: These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.

Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor cystine knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3-MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cyclotides are a family of plant-derived circular proteins with potential therapeutic applications arising from their remarkable stability, broad sequence diversity, and range of bioactivities. Their membrane-binding activity is believed to be a critical component of their mechanism of action. Using isothermal titration calorimetry, we studied the binding of the prototypical cyclotides kalata B1 and kalata B2 (and various mutants) to dodecylphosphocholine micelles and phosphoethanolamine-containing lipid bilayers. Although binding is predominantly an entropy-driven process, suggesting that hydrophobic forces contribute significantly to cyclotide-lipid complex formation, specific binding to the phosphoethanolamine-lipid headgroup is also required, which is evident from the enthalpic changes in the free energy of binding. In addition, using a combination of dissipative quartz crystal microbalance measurements and neutron reflectometry, we elucidated the process by which cyclotides interact with bilayer membranes. Initially, a small number of cyclotides bind to the membrane surface and then insert first into the outer membrane leaflet followed by penetration through the membrane and pore formation. At higher concentrations of cyclotides, destabilization of membranes occurs. Our results provide significant mechanistic insight into how cyclotides exert their bioactivities.

Plant cyclotides are the largest family of gene-encoded cyclic proteins. They act as host defense molecules to protect plants and are promising candidates as insecticidal and nematocidal agents in agriculture. For this promise to be realized a greater understanding of the post-translational processing of these proteins is needed. Cyclotides are cleaved from precursor proteins with subsequent ligation of the N and C termini to form a continuous peptide backbone. This cyclization step is inefficient in transgenic plants and our work aims to shed light on the specificity requirements at the excision sites for cyclic peptide production. Using the prototypic cyclotide kalata B1 (kB1) expressed from the Oak1 gene, MALDI-TOF mass spectrometry was used to examine the cyclization efficiency when mutants of the Oak1 gene were expressed in transgenic Nicotiana benthamiana. Cleavage at the N terminus of the cyclotide domain occurs rapidly with no strict specificity requirements for amino acids at the cleavage site. In contrast, the C-terminal region of the cyclotide domain in the P2, P1, P1', and P2' positions is highly conserved and only specific amino acids can occupy these positions. The cyclization reaction requires an Asn at position P1 followed by a small amino acid (Ala, Gly, Ser) at the P1' position. The P2' position must be filled by Leu or Ile; in their absence an unusual post-translational modification occurs. Substitution of the P2' Leu with Ala leads to hydroxylation of the neighboring proline. Through mutational analysis this novel proline hydroxylation motif was determined to be Gly-Ala-Pro-Ser.

Cyclotides are a large family of plant peptides that are structurally defined by their cyclic backbone and a trifecta of disulfide bonds, collectively known as the cyclic cystine knot (CCK) motif. Structurally similar cyclotides have been isolated from plants within the Rubiaceae, Violaceae, and Fabaceae families and share the CCK motif with trypsin-inhibitory knottins from a plant in the Cucurbitaceae family. Cyclotides have previously been reported to be encoded by dedicated genes or as a domain within a knottin-encoding PA1-albumin-like gene. Here we report the discovery of cyclotides and related non-cyclic peptides we called "acyclotides" from petunia of the agronomically important Solanaceae plant family. Transcripts for petunia cyclotides and acyclotides encode the shortest known cyclotide precursors. Despite having a different precursor structure, their sequences suggest that petunia cyclotides mature via the same biosynthetic route as other cyclotides. We assessed the spatial distribution of cyclotides within a petunia leaf section by MALDI imaging and observed that the major cyclotide component Phyb A was non-uniformly distributed. Dissected leaf midvein extracts contained significantly higher concentrations of this cyclotide compared with the lamina and outer margins of leaves. This is the third distinct type of cyclotide precursor, and Solanaceae is the fourth phylogenetically disparate plant family to produce these structurally conserved cyclopeptides, suggesting either convergent evolution upon the CCK structure or movement of cyclotide-encoding sequences within the plant kingdom.

Cyclotides are plant mini-proteins whose natural function is thought to be to protect plants from pest or pathogens, particularly insect pests. They are approximately 30 amino acids in size and are characterized by a cyclic peptide backbone and a cystine knot arrangement of three conserved disulfide bonds. This article provides an overview of the reported pesticidal or toxic activities of cyclotides, discusses a possible common mechanism of action involving disruption of biological membranes in pest species, and describes methods that can be used to produce cyclotides for potential applications as novel pesticidal agents.

Cyclotides are disulfide-rich cyclic peptides produced by plants with the presumed natural function of defense agents against insect pests. They are present in a wide range of plant tissues, being ribosomally synthesized via precursor proteins that are posttranslationally processed to produce mature peptides with a characteristic cyclic backbone and cystine knot motif associated with their six conserved cysteine residues. Their processing is not fully understood but involves asparaginyl endoproteinase activity. In addition to interest in their defense roles and their unique topologies, cyclotides have attracted attention as potential templates in peptide-based drug design applications. This chapter provides protocols for the isolation of cyclotides from plants, their detection and sequencing by mass spectrometry, and their structural analysis by NMR, as well as describing methods for the isolation of nucleic acid sequences that encode their precursor proteins. Assays to assess their membrane-binding interactions are also described. These protocols provide a "starter kit" for researchers entering the cyclotide field., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.

Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). A single native cyclotide sequence, kalata B12, has been discovered that has an aspartic acid in this otherwise conserved position. Previous studies have determined that methylation of the glutamate or substitution with alanine abolishes the membrane disrupting activity that is characteristic of the family. To further understand the role of this conserved structural feature, we studied the folding, structure, stability, and activity of the natural aspartic acid variant kalata B12 and compared it to the prototypical cyclotide kalata B1, along with its glutamate to alanine or aspartate mutants. We show that the overall fold of kalata B12 is similar to the structure of other cyclotides, confirming that the cyclotide framework is robust and tolerant to substitution, although the structure appears to be more flexible than other cyclotides. Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.

Cyclotides are plant proteins whose defining structural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea (Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternatea and discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in planta cyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternatea suggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.

The cyclotides are a family of plant-derived proteins that occur in plants from the Violaceae (violet), Rubiaceae (coffee) and Cucurbitaceae (cucurbit) families and have a diverse range of biological activities, including uterotonic, anti-HIV, antimicrobial, and insecticidal activities; the latter suggests their natural function lies in plant defense. Individual plants express suites of 10-100 cyclotides. Cyclotides comprise ∼30 amino acids, contain a head-to-tail cyclised backbone, and incorporate three disulfide bonds arranged in a cystine knot topology. The combination of a knotted and strongly braced structure with a circular backbone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. The cyclotides are the largest of several groups of naturally occurring circular proteins that have been discovered in bacteria, plants and animals over recent years. This article describes the discovery of the cyclotides in plants, their structural characterisation, evolutionary relationships and their applications in drug design., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Many plants of the Violaceae plant family have been used in traditional remedies, and these plants often contain cyclotides, a particular type of plant cyclopeptide that is distinguished by a cyclic cystine knot motif. In general, bioactive plant cyclopeptides are interesting candidates for drug development. In the current study, a suite of 14 cyclotides, which includes seven novel cyclotides [vitri B, C, D, E, F, varv Hm, and He], together with seven known cyclotides [varv A, D, E, F, H, vitri A, and cycloviolacin O2], was isolated from Viola tricolor, a common flower. A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, U251, MDA-MB-231, A549, DU145, and BEL-7402. Three cyclotides, vitri A, vitri F, and cycloviolacin O2, were the most cytotoxic. The cytotoxic activity of the cyclotides did not correlate well with their hemolytic activity, indicating that different interactions, most likely with membranes, are involved for cytotoxic and hemolytic activities. Homology modeling of the structures was used in deriving structure-activity relationships., (Copyright 2010 Elsevier Inc. All rights reserved.)

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K(i) of <5 nM, C4A/C41A-T1 has a weaker K(i) of approximately 350 nM, and the potency of the C8A/C37A variant is further decreased to a K(i) of approximately 1.8 microM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Native chemical ligation methodology developed in the laboratory of Stephen Kent is a versatile approach to the linkage of peptide fragments using a native peptide bond. It is readily adaptable to the task of joining the N- and C-termini of peptides to produce cyclic molecules and we have used it for the cyclization of a range of disulfide-rich peptides. Specifically, it has been valuable for the synthesis of cyclotides, naturally occurring peptides characterized by a head-to-tail cyclized backbone and a knotted arrangement of three conserved disulfide bonds. Cyclotides have a diverse range of biological activities, including anti-HIV, antimicrobial, and insecticidal activities. They are ultrastable owing to their cyclic cystine knot motif, and native chemical ligation methodology has been invaluable in the synthesis of a range of native and modified cyclotides to explore their structure-activity relationships and applications in drug design. Similar studies have also been applied to a smaller cyclic peptide produced in sunflower seeds, sunflower trypsin inhibitor-1, which also shows promise as a template in drug design applications. We have also found native chemical ligation to be a valuable methodology for the cyclization of conotoxins, small disulfide-rich peptides from the venoms of marine cone snails. Conotoxins target a range of ions channels and receptors and are exciting leads in drug design applications. The synthetic cyclization of conotoxins with peptide linkers stabilizes them and improves their biopharmaceutical properties. In summary, this article illustrates the use of native chemical ligation technology in the cyclization of cyclotides, sunflower trypsin inhibitor-1, and conotoxins in our laboratory., (Copyright (c) 2010 Wiley Periodicals, Inc.)

Cyclotides are a large family of plant-derived proteins typified by their head-to-tail cyclic backbone and knotted arrangement of three disulfide bonds. Although they display a diverse range of biological activities, their native function is thought to be plant defense. Here we characterized the expression, three-dimensional structure, and hemolytic activity of the cyclotide kalata B5 from the African plant Oldenlandia affinis. Kalata B5 shows an interesting seasonal variation in its expression and can only be isolated during certain times of the year, when the plant is flowering. It displays a typical tightly folded cyclic Scystine knot structure. A range of pH and temperature titrations reveal that a conserved glutamic acid in loop 1 Sof the structure forms a key hydrogen bond network, similar to that reported previously for other cyclotides. However, specific line broadening in the NMR spectra of kalata B5 suggests that the hydrogen bonding network in this peptide is less rigid than in other cyclotides. Notably, the pK9a) of Glu6 of 4.5 is higher than the values for other cyclotides studied so far, which range from 3.0 to 4.0, providing a further indication of a weaker hydrogen bond network. Kalata B5 has only moderate hemolytic activity compared with other highly expressed cyclotides, and this reduced activity probably reflects its more flexible structure. As is the case with other cyclotides, kalata B5 has an exposed hydrophobic region on its surface, supporting suggestions that this hydrophobic patch is a key feature for membrane binding and biological activity of cyclotides.

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis., (2009 Elsevier Ltd. All rights reserved.)

Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v., 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v., 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC5o values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2-3 days; lower doses were without complications.

Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4 expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon.

In recent years, the discovery of a large family of macrocyclic peptides, the cyclotides, has revealed Natures ingenuity in molecular drug design. The incorporation of a cyclic peptide backbone and a knotted arrangement of disulfide bridges into their structures confers extraordinary chemical, thermal, and enzymatic stability on these biologically active peptides. However, these structural attributes present challenges in the identification of cyclotides. Until now, the sequencing of cyclotides has been slow and inefficient owing to inherent difficulties in the separation of these hydrophobic peptides from plants, the multiple chemical and enzymatic derivatization steps required to make them amenable to mass spectrometric sequencing, and the lack of software tools to efficiently deal with these circular permutants. The current bottleneck slowing the speed of cyclotide sequencing is the requirement for multiple HPLC purification steps before analysis. Here, we have applied proteomic strategies to fast-track the discovery of known, modified and novel sequences. Using four fractions from a previously well-characterized cyclotide-containing plant species, Viola odorata, 11 new sequences, as well as a plethora of known and modified cyclotides, were uncovered. In addition, the methodology was validated through analysis of crude leaf extracts ofOldenlandia affinis and Arabidopsis thaliana. The unambiguous identification of a suite of cyclotides in the Oldenlandia affinis extract provided the ultimate proof-of-concept for this application. Major advances in methodology include the use of optimized LC-MS/MS conditions and design of a custom-built cyclotide database, in which mature cyclotide sequences are excised, replicated and appended, marking a new "era" for cyclotide sequencing.

Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 x 10(3) M(-1) and 6.0 x 10(3) M(-1), respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a beta-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides.

Cyclotides are ultra-stable plant proteins that have a circular peptide backbone crosslinked by a cystine knot of disulfide bonds. They are produced in large quantities by plants of the Violaceae and Rubiaceae families and have a role in plant defence against insect predation. As I discuss here, recent studies have begun to reveal how their unique circular topology evolved. Cyclization is achieved by hijacking existing plant proteolytic enzymes and operating them in 'reverse' to form a peptide bond between the N- and C-termini of a linear precursor. Such studies suggest that circular proteins are more common in the plant kingdom than was previously thought, and their exceptional stability has led to their application as protein-engineering templates in drug design.

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids ( approximately 125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

Progress in understanding the biosynthetic pathway of the cyclotides has been hampered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r(2)>0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.

The cyclotides are a novel family of backbone-cyclized cystine-knot containing peptides from plants that have been shown to possess insecticidal activity against Helicoverpa larvae, an important pest of corn and cotton. In the current study, we investigated the in vitro effects of the cyclotides on the viability of egg, larval, and adult life stages of two species of economically important gastrointestinal nematode parasites of livestock, Hemonchus contortus and Trichostrongylus colubriformis. The cyclotides showed significant activity in inhibiting development of nematode larvae and motility of adult worms. Activities were comparable to some currently used anthelmintic compounds in these in vitro assay systems. A series of alanine mutants of the prototypic cyclotide kalata B1 were assayed against larvae to determine regions of the peptide responsible for activity. It was observed that anthelmintic activity was dramatically reduced as a consequence of the mutation of a large number of residues that are found clustered on one surface. Activities toward larvae were equivalent in the naturally occurring L-isomer of kalata B1 and a synthetic all-D-isomer, indicating that there is no chiral requirement for anthelmintic activity. The clustering of important residues and the lack of chiral selectivity further support the proposed mode of action of the cyclotides, which involves a membrane-based interaction rather than an interaction at a specific receptor. The cyclotide-induced leakage of a fluorescent dye from vesicles used as a model membrane mimetic further confirms the membrane lytic ability of cyclotides. The relative potency of kalata B1 and kalata B2 in causing membrane leakage is consistent with the order of their anthelmintic activity. These results demonstrate that the cyclotides show potential for use in the control of gastrointestinal nematode parasites.