In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGF Serine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties.

Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.

Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining.

Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.

Chemistry and biology of cyclotides: circular plant peptides outside the box.

Cyclotides stand out as the largest family of circular proteins of plant origin hitherto known, with more than 280 sequences isolated at peptide level and many more predicted from gene sequences. Their unusual stability resulting from the signature cyclic cystine knot (CCK) motif has triggered a broad interest in these molecules for potential therapeutic and agricultural applications. Since the time of the first cyclotide discovery, our laboratory in Uppsala has been engaged in cyclotide discovery as well as the development of protocols to isolate and characterize these seamless peptides. We have also developed methods to chemically synthesize cyclotides by Fmoc-SPPS, which are useful in protein grafting applications. In this review, experience in cyclotide research over two decades and the recent literature related to their structures, synthesis, and folding as well the recent proof-of-concept findings on their use as "epitope" stabilizing scaffolds are summarized.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design.

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.

Global cyclotide adventure: a journey dedicated to the discovery of circular peptides from flowering plants.

Circular peptides and proteins of great number and diversity have been discovered in bacteria, plants, and animals. Cyclotides--disulfide-knotted and head-to-tail cyclized plant peptides that exhibit various bioactivities--are by far the largest group of circular proteins. Since their first discovery over three decades ago, there has been a lot of progress in the elucidation of structural characteristics and applications of cyclotides as novel peptide drug grafting frameworks, but there is a lack of information about their native occurrence in various plant families. The "global cyclotide adventure" was initiated as a plant collection and analysis project to advance our understanding of the origin and distribution of cyclotides in flowering plants. Here, I will provide a chronological overview of the preparation of this project, including background information on plant taxonomy and morphology, summarize, and comment on the recent progress about the discovery of cyclotide-producing plants and will give an outlook on the future of cyclotide analysis and further discoveries to be made.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability.

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.