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

详细信息    查看全文

• 作者：Masayuki Taniguchi¹ ; ² ; ^{mtanig@eng.niigata-u.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Akihito Ochiai¹ ; Shun Fukuda¹ ; Teppei Sato¹ ; Eiichi Saitoh³ ; Tetsuo Kato⁴ ; Takaaki Tanaka¹
• 关键词：Antimicrobial peptide ; Intracellular target ; Cell-free translation system ; Inhibition of translation process ; Inhibition of refolding process
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：122
• 期：4
• 页码：385-392
• 全文大小：740 K

文摘

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 K_D value of 1.4 × 10⁻⁸ M but not to DNA and that AmyI-1–18 specifically bound to DnaK with a K_D 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.