Lillian Sando

Engineering Stabilized Vascular Endothelial Growth Factor-A Antagonists : Synthesis, Structural Characterization, and Bioactivity of Grafted Analogues of Cyclotides

Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclized backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Taking advantage of this stable framework we developed novel VEGF-A antagonists by grafting a peptide epitope involved in VEGF-A antagonism onto the stable cyclotide framework. Antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. A grafted analogue showed biological activity in an in vitro VEGF-A antagonism assay at low micromolar concentration and the in vitro stability of the target epitope was markedly increased using this approach. In general, the stabilization of bioactive peptide epitopes is a significant problem in medicinal chemistry and in the current study we have provided insight into one approach to stabilize these peptides in a biological environment.

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.

Alanine Scanning Mutagenesis of the Prototypic Cyclotide Reveals a Cluster of Residues Essential for Bioactivity

The cyclotides are stable plant-derived mini-proteins with a topologically circular peptide backbone and a knotted arrangement of three disulfide bonds that form a cyclic cystine knot structural framework. They display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. To determine the influence of individual residues on structure and activity in the prototypic cyclotide kalata B1, all 23 non-cysteine residues were successively replaced with alanine. The structure was generally tolerant of modification, indicating that the framework is a viable candidate for the stabilization of bioactive peptide epitopes. Remarkably, insecticidal and hemolytic activities were both dependent on a common, well defined cluster of hydrophilic residues on one face of the cyclotide. Interestingly, this cluster is separate from the membrane binding face of the cyclotides. Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities.

A Synthetic mirror image of kalata B1 reveals that cyclotide activity is independent of a protein receptor.

Featuring a circular, knotted structure and diverse bioactivities, cyclotides are a fascinating family of peptides that have inspired applications in drug design. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anti‐cancer, anti‐HIV, and hemolytic activities. In all of these activities, cell membranes appear to play an important role. However, the question of whether the activity of cyclotides depends on the recognition of chiral receptors or is primarily modulated by the lipid‐bilayer environment has remained unknown. To determine the importance of lipid membranes on the activity of the prototypic cyclotide, kalata B1, we synthesized its all‐D enantiomer and assessed its bioactivities. After the all‐D enantiomer had been confirmed by 1H NMR to be the structural mirror image of the native kalata B1, it was tested for anti‐HIV activity, cytotoxicity, and hemolytic properties. The all‐D peptide is active in these assays, albeit with less efficiency; this reveals that kalata B1 does not require chiral recognition to be active. The lower activity than the native peptide correlates with a lower affinity for phospholipid bilayers in model membranes. These results exclude a chiral receptor mechanism and support the idea that interaction with phospholipid membranes plays a role in the activity of kalata B1. In addition, studies with mixtures of L and D enantiomers of kalata B1 suggested that biological activity depends on peptide oligomerization at the membrane surface, which determines affinity for membranes by modulating the association–dissociation equilibrium.

Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides

Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.

Structural and biochemical characteristics of the cyclotide kalata B5 from Oldenlandia affinis

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.

STRUCTURE AND FUNCTION OF PLANT TOXINS (WITH EMPHASIS ON CYSTINE KNOT TOXINS)

Plant toxins are substances produced and secreted by plants to defend themselves against predators. In a broad sense, this includes all substances that have a toxic effect on targeted organisms, whether they are microbes, other plants, insects, or higher animals. Plant toxins have a diverse range of structures, from small organic molecules through to proteins. This review gives an overview of the various classes of plant toxins but focuses on an interesting class of protein-based plant toxins containing a cystine knot motif. This structural motif confers exceptional stability on proteins containing it and is associated with a wide range of biological activities. The biological activities and structural stability offer many potential applications in the pharmaceutical and agricultural fields. One particularly exciting prospect is in the use of protein-based plant toxins as molecular scaffolds for displaying pharmaceutically important bioactivities. Future applications of plant toxins are likely to involve genetic engineering techniques and “molecular pharming” approaches.

Structure-Function Studies of the Plant Cyclotides: The Role of a Circular Protein Backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide‐circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

Threaded Rings and Complex Topologies in Antimicrobial Peptides: Nature’s Engineering Templates

Introduction Over recent years several families of naturally occurring antimicrobial peptides that contain a head-to-tail macrocyclic backbone have been discovered in bacteria, plants and animals [1]. Many of these peptides contain a unique additional topological constraint in the form of a covalent bond or peptide chain that threads through an embedded ring in the structure. The macrocyclic backbone and threaded structures make these peptides particularly stable and resistant to proteolysis, leading to suggestions of their use as stable peptide templates for drug design applications [2]. The largest family of such molecules is the cyclotides [3-4], currently estimated to comprise hundreds of members, each containing approximately 30-amino acids surrounding a cystine knot motif. They are present in plants as host defense molecules but have a range of potentially useful pharmacological activities, including anti-HIV activity [5]. Microcin J25 is another topologically complex antimicrobial molecule that has comparable stability to the cyclotides but lacks the disulfide bonding network [6]. Microcin J25 has attracted interest as a potential lead molecule for the design of novel antibacterial peptides because it has a unique mode of action that involves targeting bacterial RNA polymerase, and has an unusual threaded structure [7-9]. Figure 1 shows the generic structure of the prototypic cyclotide kalata B1 together with a schematic representation of the cyclic cystine knot (CCK) framework that defines the cyclotide family. Understanding Biology Using Peptides Sylvie E. Blondelle (Editor) American Peptide Society, 2005