title: Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.
abstract: Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 μM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília, DF, Brazil; S-inova Biotech, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS, Brazil.
authors.forename: Ludovico
authors.initials: L
authors.lastname: Migliolo
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Mário R
authors.initials: MR
authors.lastname: Felício
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília, DF, Brazil; S-inova Biotech, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS, Brazil; Programa de Pós-Graduação em Patologia Molecular, Faculdade de Medicina, Universidade de Brasília, Brasília, DF, Brazil.
authors.forename: Marlon H
authors.initials: MH
authors.lastname: Cardoso
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília, DF, Brazil.
authors.forename: Osmar N
authors.initials: ON
authors.lastname: Silva
authors.affiliation: Departamento de Biologia Celular, Laboratório de Biofísica, UnB, Brasília, DF, Brazil.
authors.forename: Mary-Ann E
authors.initials: MA
authors.lastname: Xavier
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília, DF, Brazil; Research Laboratory of Electronics, Massachusetts Institute of Technology - MIT, Cambridge, MA, United States.
authors.forename: Diego O
authors.initials: DO
authors.lastname: Nolasco
authors.affiliation: Instituto de Salud Global, CRESIB - Hospital Clinic, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Adeliana Silva
authors.initials: AS
authors.lastname: de Oliveira
authors.affiliation: Instituto de Salud Global, CRESIB - Hospital Clinic, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Ignasi
authors.initials: I
authors.lastname: Roca-Subira
authors.affiliation: Instituto de Salud Global, CRESIB - Hospital Clinic, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Vila Estape
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília, DF, Brazil.
authors.forename: Leandro D
authors.initials: LD
authors.lastname: Teixeira
authors.affiliation: Departamento de Biologia Celular, Laboratório de Biofísica, UnB, Brasília, DF, Brazil.
authors.forename: Sonia M
authors.initials: SM
authors.lastname: Freitas
authors.affiliation: Laboratorio de Immunoanalitica y péptidos antimicrobianos, Faculdad de Biología, Centro de estudios de Proteínas, Universidad de La Habana, Cuba.
authors.forename: Anselmo J
authors.initials: AJ
authors.lastname: Otero-Gonzalez
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Sónia
authors.initials: S
authors.lastname: Gonçalves
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Nuno C
authors.initials: NC
authors.lastname: Santos
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, UCB, Brasília, DF, Brazil; S-inova Biotech, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS, Brazil; Programa de Pós-Graduação em Patologia Molecular, Faculdade de Medicina, Universidade de Brasília, Brasília, DF, Brazil. Electronic address: ocfranco@gmail.com.
authors.forename: Octavio L
authors.initials: OL
authors.lastname: Franco
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000302:isolation & purification; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D004927:Escherichia coli Infections / Q000188:drug therapy* / Q000382:microbiology / Q000401:mortality; D005432:Flounder / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D057786:Peptidomimetics / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016019:Survival Analysis; D053835:Unilamellar Liposomes / Q000737:chemistry; D014709:Vero Cells
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D008070:Lipopolysaccharides; D057786:Peptidomimetics; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D053835:Unilamellar Liposomes; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D002784:Cholesterol; D000409:Alanine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: Molecular dynamics; Pa-MAP2; Pleuronectes americanus; Secondary structure; Synthetic palindromic peptide