Poorebrahim Mansour, Sadeghi Solmaz, Ghorbani Raziyeh, Asghari Matin, Abazari Mohammad Foad, Kalhor Hourieh, Rahimi Hamzeh
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Molecular Medicine Department, Biotechnology Research Center., Pasteur Institute of Iran, Iran.
J Theor Biol. 2017 Apr 7;418:111-121. doi: 10.1016/j.jtbi.2017.01.009. Epub 2017 Jan 16.
Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, has been implicated in some biological processes such as cell proliferation, development and differentiation. High mitogenic activity of this protein has made it very suitable for repairing radiation-and chemotherapy-induced damages. Palifermin, which has been developed from human KGF, is clinically applied to reduce the incidence and duration of cancer therapeutic agents. However, the activity of Palifermin is limited during treatment due to its poor stability. In this study, we have improved the stability and activity of recombinant human KGF (Palifermin) using a computational mutagenesis approach. According to the KGF multiple sequence alignment among different species as well as literature-based information, we have generated several mutations using PyMOL program and evaluated their effects on the stability and activity of KGF in silico. In order to preserve the KGF activity, we did not change the predicted functional residues. Prior to mutagenesis, the 3D structure of rhKGF was predicted by Modeller v9.15 program and quantitative evaluation of predicted models were carried out using VADAR and PROSESS servers. The stability and activity of rhKGF mutants were analyzed using GROMACS molecular dynamics (MD) simulations and docking tools, respectively. The results showed that N159S (N105S in rhKGF sequence) and I172V (I118V in rhKGF) substitutions caused an increased stability and affinity of the rhKGF to Fibroblast growth factor receptor 2 (FGFR2). We will evaluate the effects of favorable mutations on the rhKGF stability and activity in vitro.
角质形成细胞生长因子（KGF）是成纤维细胞生长因子（FGF）家族的成员之一，参与了细胞增殖、发育和分化等一些生物学过程。这种蛋白质的高促有丝分裂活性使其非常适合修复辐射和化疗引起的损伤。从人KGF开发而来的帕利夫明在临床上用于降低癌症治疗药物的发生率和持续时间。然而，由于其稳定性差，帕利夫明在治疗过程中的活性受到限制。在本研究中，我们使用计算诱变方法提高了重组人KGF（帕利夫明）的稳定性和活性。根据不同物种间的KGF多序列比对以及基于文献的信息，我们使用PyMOL程序产生了几个突变，并在计算机上评估了它们对KGF稳定性和活性的影响。为了保留KGF活性，我们没有改变预测的功能残基。在诱变之前，使用Modeller v9.15程序预测了rhKGF的三维结构，并使用VADAR和PROSESS服务器对预测模型进行了定量评估。分别使用GROMACS分子动力学（MD）模拟和对接工具分析了rhKGF突变体的稳定性和活性。结果表明，N159S（rhKGF序列中的N105S）和I172V（rhKGF中的I118V）取代导致rhKGF对成纤维细胞生长因子受体2（FGFR2）的稳定性和亲和力增加。我们将在体外评估有利突变对rhKGF稳定性和活性的影响。
