title: Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1.
abstract: Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val10 residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe2 was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe2 plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe6 is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val10, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC)  = 1.6 µM) to Pis-1 (average MIC  = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys10-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity.
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Eunjung
authors.initials: E
authors.lastname: Lee
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Areum
authors.initials: A
authors.lastname: Shin
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Ki-Woong
authors.initials: KW
authors.lastname: Jeong
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Bongwhan
authors.initials: B
authors.lastname: Jin
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Hum Nath
authors.initials: HN
authors.lastname: Jnawali
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Soyoung
authors.initials: S
authors.lastname: Shin
authors.affiliation: Research Center for Proteineous Materials and Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju, Korea.
authors.forename: Song Yub
authors.initials: SY
authors.lastname: Shin
authors.affiliation: Department of Bioscience and Biotechnology, Bio-Molecular Informatics Center, Konkuk University, Seoul, Korea.
authors.forename: Yangmee
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D016207:Cytokines / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology; D029941:Fish Proteins / Q000737:chemistry* / Q000494:pharmacology*; D006461:Hemolysis; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008081:Liposomes / Q000737:chemistry; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008826:Microbial Sensitivity Tests; D041681:NIH 3T3 Cells; D010649:Phenylalanine / Q000737:chemistry; D017433:Protein Structure, Secondary; D014633:Valine / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D016207:Cytokines; D029941:Fish Proteins; D008070:Lipopolysaccharides; D008081:Liposomes; C449802:moronecidin protein, Morone saxatilis; D010649:Phenylalanine; D014633:Valine