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At least 19 recordsLinked to original sources

PubMed2005

Isolation and characterization of novel cyclotides from Viola hederaceae: solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide.

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 (vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional (1)H NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded beta-sheet, a short 3(10) helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and approximately 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC(50) value of 0.87 microm.

Acquired Immunodeficiency Syndrome↗

PubMed2005

A continent of plant defense peptide diversity: cyclotides in Australian Hybanthus (Violaceae).

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Amino Acid Sequence↗

PubMed2004

Anti-HIV cyclotides.

The cyclotides are a recently discovered, structurally unique family of bioactive plant peptides. Their discovery spawned a series of structural analyses, synthetic efforts, and studies to define the biosynthesis and biological properties of these novel peptide metabolites. Cyclotides have a head-to-tail cyclized amino acid backbone and a conserved cystine knot motif that provides an extremely stable structural framework. They all share a common global fold and are highly resistant to denaturation and to cleavage by proteolytic enzymes. However, these macrocyclic peptides are quite permissive to amino acid substitutions or additions in several peripheral loop regions, since changes in these loops do not alter the core cyclotide structure. These features make cyclotides attractive templates for incorporating desired amino acid sequences and then delivering these peptide sequences in a well defined, highly stable framework. Cyclotides likely function in a defensive role in the source plants since they exhibit a broad spectrum of antimicrobial activity and are detrimental to the growth and survival of herbivorous insects. Cyclotides are gene-encoded polypeptides that are cleaved from larger precursor proteins and then cyclized. This review summarizes research done on a subset of cyclotides that were discovered due to their HIV inhibitory properties. It details the isolation and characterization of these compounds and describes this work in the context of our current state of knowledge of the entire cyclotide family.

Animals↗

PubMed2001

Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis.

Several members of the Rubiaceae and Violaceae families produce a series of cyclotides or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata B1 as well as three other clones for related cyclotides from the African plant Oldenlandia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cyclotides have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Amino Acid Sequence↗

PubMed2006

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins.

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.

Amino Acid Sequence↗

PubMed2004

Variations in cyclotide expression in viola species.

Cyclotides, a family of approximately 50 mini-proteins isolated from various Violaceae and Rubiaceae plants, are characterized by their circular peptide backbone and six conserved cysteine residues arranged in a cystine knot motif. Cyclotides show a wide range of biological activities, making them interesting targets for both pharmaceutical and agrochemical research, but little is known about their natural function and the events that trigger their expression. An investigation of the geographical and seasonal variations of cyclotide profiles has been performed, using the native Australian violet, Viola hederacea, and the Swedish sweet violet, Viola odorata, as model plants. The results showed that in the Australian violet the relative peptide levels of some cyclotides remained almost constant throughout the year, while other cyclotides were present only at certain times of the year. Therefore, it appears that V. hederacea expresses a basic armory of cyclotides as well as special "add-ons" whose levels are influenced by external factors. In the Swedish violet, cyclotide levels were increased up to 14 times during the warmest period of the year. The larger variation in expression levels of the Swedish plants may be a reflection of a greater climatic variation.

Amino Acid Sequence↗

PubMed2004

Novel strategies for isolation and characterization of cyclotides: the discovery of bioactive macrocyclic plant polypeptides in the Violaceae.

This review focuses on the discovery of cyclotides in the Violaceae, their isolation and their anti-cancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solvent-solvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.

Animals↗

PubMed2004

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot.

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

Amino Acid Sequence↗

PubMed2006

Cycloviolacin H4, a hydrophobic cyclotide from Viola hederaceae.

Cycloviolacin H4, a new macrocyclic miniprotein comprising 30 amino acid residues, was isolated from the underground parts of the Australian native violet Viola hederaceae. Its sequence, cyclo-(CAESCVWIPCTVTALLGCSCSNNVCYNGIP), was determined by nanospray tandem mass spectrometry and quantitative amino acid analysis. A knotted disulfide arrangement, which was designated as a cyclic cystine knot motif and characteristic to all known cyclotides, is proposed for stabilizing the molecular structure and folding. The cyclotide is classified in the bracelet subfamily of cyclotides due to the absence of a cis-Pro peptide bond in the circular peptide backbone. A model of its three-dimensional structure was derived based on the template of the homologous cyclotide vhr1 (Trabi et al. Plant Cell 2004, 16, 2204-2216). Cycloviolacin H4 exhibits the most potent hemolytic activity in cyclotides reported so far, and this activity correlates with the size of a surface-exposed hydrophobic patch. This work has thus provided insight into the factors that modulate the cytotoxic properties of cyclotides.

Amino Acid Sequence↗

PubMed2004

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1.

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V. hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.

Amino Acid Sequence↗

PubMed2004

Solution structure of the cyclotide palicourein: implications for the development of a pharmaceutical framework.

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.

Amino Acid Sequence↗

PubMed2006

The cyclotide family of circular miniproteins: nature's combinatorial peptide template.

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The structural features that contribute to their remarkable stability are described in this review.

Amino Acid Sequence↗

PubMed2003

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation.

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Cyclotides↗

PubMed2003

Disulfide mapping of the cyclotide kalata B1. Chemical proof of the cystic cystine knot motif.

The cyclotides are a recently discovered family of plant proteins that have the fascinating structural feature of a continuous cyclic backbone and, putatively, a knotted arrangement of their three conserved disulfide bonds. We here show definite chemical proof of the I-IV, II-V, III-VI knotted disulfide connectivity of the prototypic cyclotide kalata B1. This has been achieved by a new approach for disulfide analysis, involving partial reduction and stepwise alkylation including introduction of charges and enzymatic cleavage sites by aminoethylation of cysteines. The approach overcomes the intrinsic difficulties for disulfide mapping of cyclotides, i.e. the cyclic amide backbone, lack of cleavage sites between cysteines, and a low or clustered content of basic amino acids, and allowed a direct determination of the disulfide bonds in kalata B1 using analysis by mass spectrometry. The established disulfide connectivity is unequivocally shown to be cystine knotted by a topological analysis. This is the first direct chemical determination of disulfides in native cyclotides and unambiguously confirms the unique cyclic cystine knot motif.

Amino Acid Motifs↗

PubMed2004

Discovery, structure and biological activities of the cyclotides.

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.

Animals↗

PubMed2002

Cyclotides: a novel type of cytotoxic agents.

Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin 02, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin 02 was the most potent in all cell lines (IC50 0.1-0.3 microM), followed by varv A (IC50 2.7-6.35 microM) and varv F (IC50 2.6-7.4 microM), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents.

Amino Acid Sequence↗

PubMed2006

The cyclotides and related macrocyclic peptides as scaffolds in drug design.

The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Cyclotides are macrocyclic cystine-knotted mini-proteins that have a broad range of bioactivities and are exceptionally stable, being resistant to chemical, thermal and enzymatic degradation. The general limitations of peptides as drugs can potentially be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides and related peptide scaffolds for drug design is evaluated herein, with reference to increasing knowledge of the structures and sequence diversity of natural cyclotides and the emergence of new approaches in protein engineering.

Amino Acid Sequence↗

PubMed2003

Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation.

The expression of cyclotides-macrocyclic plant peptides-was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis, V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single species containing >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation of cysteines. This overcomes a number of problems intimately associated with the cyclotide core structure-that is, their joined N and C termini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result, charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with tryptic digestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown by the sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B (cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined by nanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of other peptides and proteins displaying similar structural problems for MS analysis.

Amino Acid Sequence↗