title: Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.
abstract: The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia m.bleackley@latrobe.edu.au m.anderson@latrobe.edu.au.
authors: Mark R
authors: MR
authors: Bleackley
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors: Jennifer A E
authors: JA
authors: Payne
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors: Brigitte M E
authors: BM
authors: Hayes
authors: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors: Thomas
authors: T
authors: Durek
authors: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors: David J
authors: DJ
authors: Craik
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors: Thomas M A
authors: TM
authors: Shafee
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors: Ivan K H
authors: IK
authors: Poon
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors: Mark D
authors: MD
authors: Hulett
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors: Nicole L
authors: NL
authors: van der Weerden
authors: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia m.bleackley@latrobe.edu.au m.anderson@latrobe.edu.au.
authors: Marilyn A
authors: MA
authors: Anderson
mesh_terms: D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D023082:Defensins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005670:Fusarium / Q000187:drug effects; D006801:Humans; D008081:Liposomes; D009355:Neomycin / Q000494:pharmacology; D010539:Permeability; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D000935:Antifungal Agents; D000972:Antineoplastic Agents, Phytogenic; D023082:Defensins; D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D009355:Neomycin