Comparison of the Structure and Biological Activities of Wild-type and Mutant Liver-targeting Peptide Modified Recombinant Human Endostatin (rES-CSP) in Human Hepatocellular Carcinoma HepG2 Cells
Comparison of the Structure and Biological Activities of Wild-type and Mutant Liver-targeting Peptide Modified Recombinant Human Endostatin (rES-CSP) in Human Hepatocellular Carcinoma HepG2 Cells
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DOI：
10.2174/0929866522666150302125218
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发表时间：
2015-01-01
影响因子：
1.6
通讯作者：
Zhu, Jiayong
中科院分区：
文献类型：
--
作者：
Bao, Dongmei;Jin, Xiaobao;Zhu, Jiayong
肝靶向肽CSP I-plus修饰的rEndostatin（rES-CSP）在113位发生了天冬氨酸（Aspartic）突变为天冬酰胺（Asparagine），导致蛋白质二级结构改变，生物活性降低。为了阐明融合蛋白rES-CSP的结构与功能关系，采用基于模板的建模方法构建了野生型和突变型D113 NrES-CSP的三维结构模型。为了评估单突变对rES-CSP稳定性的影响，使用分子动力学模拟来揭示结构和动力学特征。采用体外细胞增殖、迁移、细胞周期和细胞凋亡等生物活性检测方法，对重组蛋白的生物活性进行了分析。结果表明，突变型rES-CSP的稳定性降低，功能丧失，野生型rES-CSP能与正常肝细胞Chang's和肝癌细胞HepG 2结合，但明显高于非靶向rEndostatin。rES-CSP能抑制肝癌细胞的增殖，并呈剂量依赖性，使肝癌细胞G1期比例增加，S期比例减少，促进肝癌细胞凋亡。这些结果进一步补充了融合蛋白rES-CSP的作用机制，为制备肝靶向药物治疗肝病提供了一种可行和方便的方法。
A missense mutation of Aspartic to Asparagine acid in 113 position of liver-targeting peptide CSP I-plus modified rEndostatin (rES-CSP) happened unexpectedly results in the changes of protein secondary structure and a reduced bioactivity. With the aim to clarify the structure-function relationships featuring the fuse protein rES-CSP, the three-dimensional structural models of wild-type and mutant D113NrES-CSP were constructed by template-based modeling approach. To evaluate the effect of the single mutation on rES-CSP stability, the molecular dynamic simulation was used to reveal the structural and dynamic characteristics. Analysis on the bioactivity were conducted using a number of validated in vitro assays including proliferation, migration, cell cycle and apoptosis in HepG2 cells. Results showed that the mutant rES-CSP reduce the stability and loss of function, and the wild-type rES-CSP could both bind to the normal liver cells Chang's and the hepatoma cells HepG2 but significantly higher than non-targeted rEndostatin. rES-CSP could inhibit the proliferation of hepatoma cells in a dose-dependent manner, and increase the proportion of G1 phase, reduce the proportion of S phase, promote the apoptosis on hepatoma cells. These results make a further complement of the mechanisms by which the fuse protein rES-CSP would provide a feasible and convenient approach to produce liver-targeting drugs for treatment of the liver disease.