Importance of the disulfide bridges in the antibacterial activity of human hepcidin
Importance of the disulfide bridges in the antibacterial activity of human hepcidin
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DOI：
10.1016/j.peptides.2012.06.001
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发表时间：
2012-08-01
期刊：
影响因子：
3
通讯作者：
Garbay, Bertrand
中科院分区：
文献类型：
--
作者：
Hocquellet, Agnes;le Senechal, Caroline;Garbay, Bertrand
铁调素首先被鉴定为存在于人血清和尿液中的抗微生物肽。后来证明，铁调素是长期寻求的调节哺乳动物铁稳态的激素。25个氨基酸的天然肽（Hepc 25）含有维持β-发夹基序的四个二硫键。本研究的目的是评估分子内二硫键是否是Hepc 25抗菌活性所必需的。我们发现，合成的肽对应于人Hepc 25。并且含有四个二硫键，对几种革兰氏阳性和革兰氏阴性细菌菌株具有抗菌活性。相反，其中所有半胱氨酸被丙氨酸替代的合成肽（Hepc 25-Ala）对相同的细菌菌株没有可检测的活性。在进一步的步骤中，研究了Hepc 25对大肠杆菌的作用模式。使用SYTOX绿色吸收来评估细菌膜完整性。用Hepc 25没有观察到膜的透化，表明该肽不会通过破坏细菌的膜来杀死细菌。凝胶阻滞实验表明Hepc 25与DNA的结合效率很高，并且这种结合能力依赖于分子内二硫键的存在。Hepc 25的还原或八个半胱氨酸被丙氨酸残基取代导致肽在体外测定中不再能够结合DNA。总之，这些结果表明，Hepc 25应该采用由分子内二硫键稳定的三维结构，以具有抗菌活性。(C)2012 Elsevier Inc. All rights reserved.
Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a beta-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25. and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity. (C) 2012 Elsevier Inc. All rights reserved.