Identification and structural characterization of a novel cyclotide with activity against an insect pest of sugar cane

Cyclotides are a family of plant-derived cyclic peptides comprising six conserved cysteine residues connected by three intermolecular disulfide bonds that form a knotted structure known as a cyclic cystine knot (CCK). This structural motif is responsible for the pronounced stability of cyclotides against chemical, thermal, or proteolytic degradation and has sparked growing interest in this family of peptides. Here, we isolated and characterized a novel cyclotide from Palicourea rigida (Rubiaceae), which was named parigidin-br1. The sequence indicated that this peptide is a member of the bracelet subfamily of cyclotides. Parigidin-br1 showed potent insecticidal activity against neonate larvae of Lepidoptera (Diatraea saccharalis), causing 60% mortality at a concentration of 1 mu M but had no detectable antibacterial effects. A decrease in the in vitro viability of the insect cell line from Spodoptera frugiperda (SF-9) was observed in the presence of parigidin-br1, consistent with in vivo insecticidal activity. Transmission electron microscopy and fluorescence microscopy of SF-9 cells after incubation with parigidin-br1 or parigidin-br1-fluorescein isothiocyanate, respectively, revealed extensive cell lysis and swelling of cells, consistent with an insecticidal mechanism involving membrane disruption. This hypothesis was supported by in silico analyses, which suggested that parigidin-br1 is able to complex with cell lipids. Overall, the results suggest promise for the development of parigidin-br1 as a novel biopesticide

A novel alpha-amylase inhibitor from amaranth (Amaranthus hypocondriacus) seeds.

The major alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypocondriacus, a variety of the Mexican crop plant amaranth, is a 32-residue-long polypeptide with three disulfide bridges. Purified AAI strongly inhibits the alpha-amylase activity of insect larvae (Tribolium castaneum and Prostephanus truncatus) and does not inhibit proteases and mammalian alpha-amylases. AAI was sequenced with the automated Edman method, and the disulfide bridges were localized using enzymatic and chemical fragmentation methods combined with N-terminal sequencing. AAI is the shortest alpha-amylase inhibitor described so far which has no known close homologs in the sequence data bases. Its residue conservation patterns and disulfide connectivity are related to the squash family of proteinase inhibitors, to the cellulose binding domain of cellobiohydrolase, and to omega-conotoxin, i.e. a group of small proteins termed "knottins" by Nguyen, D. L., Heitz, A., Chiche, L., Castro, B., Boigegrain, R., Favel, A., and Coletti-Previero, M. ((1990) (Biochimie 72, 431-435) The three-dimensional model of AAI was built according to the common structural features of this group of proteins using side-chain replacement and molecular dynamics refinement techniques