title: AmyI-1-18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system.
abstract: In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4 × 10(-8) M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4 × 10(-6) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system.
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan; Center for Transdisciplinary Research, Niigata University, Niigata 950-2181, Japan. Electronic address: mtanig@eng.niigata-u.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Taniguchi
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Ochiai
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Fukuda
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Sato
authors.affiliation: Graduate School of Technology, Niigata Institute of Technology, Niigata 945-1195, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Saitoh
authors.affiliation: Department of Chemistry, Tokyo Dental College, Tokyo 101-0062, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry* / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology; D002412:Cations / Q000737:chemistry / Q000494:pharmacology; D002474:Cell-Free System; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D019476:Insect Proteins / Q000494:pharmacology; D008156:Luciferases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D017510:Protein Folding / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D000516:alpha-Amylases / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D002412:Cations; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D019476:Insect Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; C000616654:amyl-1-18 octadecapeptide; D049452:Green Fluorescent Proteins; C087710:pyrrhocoricin protein, Pyrrhocoris apterus; D008156:Luciferases; D000516:alpha-Amylases; C063842:dnaK protein, E coli
keywords: Antimicrobial peptide; Cell-free translation system; Inhibition of refolding process; Inhibition of translation process; Intracellular target