Background: Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results: MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion: The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.

Osmotin, a plant defense protein, has functional similarity to adiponectin, an insulin sensitizingsensitising hormone secreted by adipocytes. We speculated that Piper colubrinum Osmotin (PcOSM) could have functional roles in obesity-related cancers, especially breast cancer. Immunofluorescence assays, flow cytometry, cell cycle analysis and a senescence assay were employed to delineate the activity in MDAMB231 breast cancer cell line. PcOSM pre-treated P. nigrum leaves showed significant reduction in disease symptoms correlated with high ROS production. In silico analysis predicted that PcOSM has higher binding efficiency with adiponectin receptor compared to adiponectin. PcOSM was effectively taken up by MDAMB231 cancer cells which resulted in marked increase in intracellular ROS levels leading to senescence and cell cycle arrest in G2/M stage. This study provides evidence on the ROS mediated direct inhibitory activity of the plant derived osmotin protein on the phytopathogen Phytophthora capsici, and the additional functional roles of this plant defense protein on cancer cells through inducing ROS associated senescence. The strong leads produced from this study could be pursued further to obtain more insights into the therapeutic potential of osmotin in human cancers.