Centro de Análises Proteômicas e Bioquímicas-Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Distrito Federal, Brazil.
PLoS One. 2012;7(10):e47047. doi: 10.1371/journal.pone.0047047. Epub 2012 Oct 8.
Recently, defense peptides that are able to act against several targets have been characterized. The present work focuses on structural and functional evaluation of the peptide analogue Pa-MAP, previously isolated as an antifreeze peptide from Pleuronectes americanus. Pa-MAP showed activities against different targets such as tumoral cells in culture (CACO-2, MCF-7 and HCT-116), bacteria (Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923), viruses (HSV-1 and HSV-2) and fungi (Candida parapsilosis ATCC 22019, Trichophyton mentagrophytes (28d&E) and T. rubrum (327)). This peptide did not show toxicity against mammalian cells such as erythrocytes, Vero and RAW 264.7 cells. Molecular mechanism of action was related to hydrophobic residues, since only the terminal amino group is charged at pH 7 as confirmed by potentiometric titration. In order to shed some light on its structure-function relations, in vitro and in silico assays were carried out using circular dichroism and molecular dynamics. Furthermore, Pa-MAP showed partial unfolding of the peptide changes in a wide pH (3 to 11) and temperature (25 to 95°C) ranges, although it might not reach complete unfolding at 95°C, suggesting a high conformational stability. This peptide also showed a conformational transition with a partial α-helical fold in water and a full α-helical core in SDS and TFE environments. These results were corroborated by spectral data measured at 222 nm and by 50 ns dynamic simulation. In conclusion, data reported here show that Pa-MAP is a potential candidate for drug design against pathogenic microorganisms due to its structural stability and wide activity against a range of targets.
最近，已经鉴定出能够针对多个靶点发挥作用的防御肽。本工作重点研究了先前从美洲比目鱼 Pleuronectes americanus 中分离出的抗冻肽 Pa-MAP 的结构和功能。Pa-MAP 对不同的靶标具有活性，如培养中的肿瘤细胞（CACO-2、MCF-7 和 HCT-116）、细菌（大肠杆菌 ATCC 8739 和金黄色葡萄球菌 ATCC 25923）、病毒（HSV-1 和 HSV-2）和真菌（近平滑念珠菌 ATCC 22019、毛癣菌属（28d&E）和红色毛癣菌（327））。该肽对哺乳动物细胞如红细胞、Vero 和 RAW 264.7 细胞没有毒性。作用机制与疏水性残基有关，因为只有在 pH 7 时，末端氨基才带电荷，这一点通过电位滴定得到了证实。为了阐明其结构-功能关系，进行了体外和计算研究，使用了圆二色性和分子动力学。此外，Pa-MAP 显示出在宽 pH（3 至 11）和温度（25 至 95°C）范围内肽变化的部分展开，尽管在 95°C 时它可能不会完全展开，表明其具有较高的构象稳定性。该肽在水相中有部分α-螺旋折叠构象，在 SDS 和 TFE 环境中有完整的α-螺旋核心构象，表现出构象转变。这些结果得到了在 222nm 处测量的光谱数据和 50ns 动态模拟的证实。总之，这里报告的数据表明，Pa-MAP 是一种有前途的药物设计候选物，可用于针对致病微生物，因为它具有结构稳定性和对多种靶标的广泛活性。
