网络蛋白质组学在计算机中应用,蛋白质组学分析灵芝酸D的细胞毒性机制和计算机预测其可能的作用网络...

灵芝三萜可以抑制多种肿瘤细胞的生长并被认为是灵芝抗肿瘤作用的基础之一。灵芝酸D(GAD)是灵芝三萜的主要成分之一。用GAD处理48小时可以抑制HeLa人宫颈瘤细胞的生长，作用的IC50-值为17.3 ± 0.3 μM。流式细胞仪分析和DNA断裂分析表明GAD诱导了G2/M期细胞周期阻滞和细胞凋亡。为了寻找GAD作用的蛋白质靶点，用蛋白质双向电泳分析了GAD 处理的细胞和对照细胞的蛋白质表达谱，用质谱鉴定了差异表达的蛋白质点。并用Western blotting 进行了验证。找到了21个差异表达蛋白。用计算机软件INVDOCK分析了其中的可能直接靶点，结果表明GAD可以直接结合6种14-3-3蛋白，annexin A5 和aminopeptidase B。GAD和14-3-3 zeta 的直接结合能力用体外Surface Plasmon Resonance (SPR) Biosensor Analysis 方法进行了验证。此外，功能分析表明这21个蛋白中有14个蛋白在蛋白质相互作用网络中联系紧密。当网络向外扩展后，所有的21个蛋白可以落入一个网络中。这样，我们就建立了与GAD相关的蛋白质靶点的作用网络，并对网络中的每个蛋白在GAD细胞毒性中的可能作用进行了讨论。

该项研究由果德安课题组完成，论文发表Mol Cell Proteomics上。

Proteomic characterization of the cytotoxic mechanism of ganoderic acid D and computer automated estimation of the possible drug-target network.

Qing-Xi Yue, Zhi-Wei Cao, Shu-Hong Guan, Xiao-Hui Liu, Lin-Tao, Wan-Ying Wu, Yi-Xue Li, Peng-Yuan Yang, Xuan Liu*, De-An Guo*

Mol Cell Proteomics (IF: 9.62)

2007 Dec 31; [Epub ahead of print].

Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of Ganoderma lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50-value of 17.3 ± 0.3 μM. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G2/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, 2-DE was performed and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore, these possible GAD-target related proteins were evaluated by an in silico drug target searching program INVDOCK. The INVDOCK analysis results suggested that GAD could bind 6 isoforms of 14-3-3 protein family, annexin A5 and aminopeptidase B. The direct binding affinity of GAD towards 14-3-3 zeta was confirmed in vitro using Surface Plasmon Resonance (SPR) Biosensor Analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly, or associate with each other via only one intermediate protein from previous PPI experimental results. When the network was expanded to a further interaction outward, total 21 proteins can fall into one network. In this way, the possible network associate with GAD-target related proteins was constructed and the possible contribution of these proteins to the cytotoxicity of GAD was discussed in this paper.

(供稿部门：中药现代化中心；供稿人：刘璇)