Structural and functional characterization of the membrane‐permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis

What is it about?

Defensins are small cationic peptides ubiquitously found in the plant and animal kingdoms. These peptides are important host defense peptides and have broad antimicrobial activity against bacteria, fungus and enveloped virus. Some defensins also have the ability to selectively target and kill mammalian tumourogenic cells. As such, defensins are attracting significant interest as novel antibiotics and anticancer molecules. Of major interest is whether the mechanism of defensin action can be better understood and harnessed for an alternative to conventional cancer chemotherapy. In this manuscript, we have identified and characterised a novel plant defensin from Nicotiana occidentalis in regards to its structure, ability to bind phospholipids and kill human tumour cells in vitro and in vivo. Using X-ray crystallography, we have determined a high resolution structure of NoD173 that provides insight into how this defensin dimerizes and forms higher ordered oligomers. We demonstrate that NoD173 specifically binds the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), is able to kill a panel of human tumour cell lines by membrane permeabilization, as well as show that direct injection of the defensin into solid melanoma tumours grown subcutaneously in mice results in inhibition of tumour growth. In addition, we have used this information to engineer by mutagenesis a more potent form of the defensin. These data provide insights into the mechanism of how defensins target membranes to kill tumour cells and provide the first proof-of-concept that defensins are able to inhibit tumour growth in vivo.

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