Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1.

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.

Recombinant production of the cyclotide kalata B1 by conditional split inteins.

This study describes the design, production, and characterization of a novel conditional intein system for the recombinant production of cyclic peptides. The system is based on two key features: (1) a promiscuous extein recognition site allowing cyclization of virtually any peptide, and (2) a secondary split site within the intein itself enabling triggered splicing at will. Two intein precursors were recombinantly expressed, purified, and then self-assembled in vitro to cyclize the model peptide kalata B1 (kB1). Cyclized kB1 was successfully purified, refolded and characterized by mass spectrometry and NMR, demonstrating correct disulfide bond formation and identical structure to synthetic kB1. Importantly, the intein-derived kB1 retained full biological activity as evidenced by insect cell toxicity assays. This work establishes a versatile and efficient approach for intein-mediated protein cyclization with potential applications in bioengineering and peptide discovery.

Cyclotides: The next generation in biopesticide development for eco-friendly agriculture.

Chemical pesticides remain the predominant method for pest management in numerous countries. Given the current landscape of agriculture, the development of biopesticides has become increasingly crucial. The strategy empowers farmers to efficiently manage pests and diseases, while prioritizing minimal adverse effects on the environment and human health, hence fostering sustainable management. In recent years, there has been a growing interest and optimism surrounding the utilization of peptide biopesticides for crop protection. These sustainable and environmentally friendly substances have been recognized as viable alternatives to synthetic pesticides due to their outstanding environmental compatibility and efficacy. Numerous studies have been conducted to synthesize and identify peptides that exhibit activity against significant plant pathogens. One of the peptide classes is cyclotides, which are cyclic cysteine-rich peptides renowned for their wide range of sequences and functions. In this review, we conducted a comprehensive analysis of cyclotides, focusing on their structural attributes, developmental history, significant biological functions in crop protection, techniques for identification and investigation, and the application of biotechnology to enhance cyclotide synthesis. The objective is to emphasize the considerable potential of cyclotides as the next generation of plant protection agents on the global scale.

Mutagenesis of bracelet cyclotide hyen D reveals functionally and structurally critical residues for membrane binding and cytotoxicity.

Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.

Topoly: Python package to analyze topology of polymers.

The increasing role of topology in (bio)physical properties of matter creates a need for an efficient method of detecting the topology of a (bio)polymer. However, the existing tools allow one to classify only the simplest knots and cannot be used in automated sample analysis. To answer this need, we created the Topoly Python package. This package enables the distinguishing of knots, slipknots, links and spatial graphs through the calculation of different topological polynomial invariants. It also enables one to create the minimal spanning surface on a given loop, e.g. to detect a lasso motif or to generate random closed polymers. It is capable of reading various file formats, including PDB. The extensive documentation along with test cases and the simplicity of the Python programming language make it a very simple to use yet powerful tool, suitable even for inexperienced users. Topoly can be obtained from https://topoly.cent.uw.edu.pl.

Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.

Linking molecular evolution to molecular grafting.

Molecular grafting is a strategy for the engineering of molecular scaffolds into new functional agents, such as next-generation therapeutics. Despite its wide use, studies so far have focused almost exclusively on demonstrating its utility rather than understanding the factors that lead to either poor or successful grafting outcomes. Here, we examine protein evolution and identify parallels between the natural process of protein functional diversification and the artificial process of molecular grafting. We discuss features of natural proteins that are correlated to innovability-the capacity to acquire new functions-and describe their implications to molecular grafting scaffolds. Disulfide-rich peptides are used as exemplars because they are particularly promising scaffolds onto which new functions can be grafted. This article provides a perspective on why some scaffolds are more suitable for grafting than others, identifying opportunities on how molecular grafting might be improved.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.

Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization.

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.

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus.

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.