Molecular characterization of the hdm2-p53 interaction
Molecular characterization of the hdm2-p53 interaction
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DOI：
10.1006/jmbi.1997.1078
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发表时间：
1997-06-27
影响因子：
5.6
通讯作者：
Lane, DP
中科院分区：
文献类型：
--
作者：
Bottger, A;Bottger, V;Lane, DP
许多病毒癌基因靶向肿瘤抑制蛋白p53并使其功能失活。这是肿瘤发生的重要一步。细胞癌基因hdm2通过类似的机制起作用。它结合p53的N端，从而干扰p53转录激活基因毒性损伤后负责生长停滞或凋亡的基因的能力。因此，破坏这两种蛋白质的相互作用构成了一个有希望的治疗靶点，用于治疗这种途径活跃的人类癌症子集。在本文中，我们试图描述p53-hdm2相互作用的生物化学特征。我们分析了一系列肽抑制剂的潜力，来源于先前描述的mdm2结合肽展示噬菌体，在ELISA检测中破坏这种相互作用。我们得出结论，p53的F19、W23和L26是p53与hdm2结合的关键接触点。此外，我们还在ELISA检测中显示了单克隆抗体3G5干扰p53与hdm2结合的潜力。因此，我们使用从hdm2的N端和噬菌体展示文库中获得的重叠肽来确定3G5在hdm2上的结合位点。结果表明hdm2上的L66、Y67和E69是3G5的关键结合点。在电泳迁移迁移试验中，我们证明了hdm2-p53复合物的形成，可以在3G5或抑制肽的存在下被破坏。最后，我们描述了NEM和DTT在ELISA检测中对两分子相互作用的影响。我们所有的结果都是根据最近发表的mdm2-p53复合物的晶体结构来讨论的。我们的发现与晶体结构惊人地吻合。(C) 1997学术出版社有限公司
A number of viral oncogenes target the tumour suppressor protein p53 and inactivate its function. This is an important step in tumourogenesis. The cellular oncogene hdm2 acts through a similar mechanism. It binds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or apoptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target for treatment of the subset of human cancers in which this pathway is active.In this paper we attempt to characterize the p53-hdm2 interaction biochemically. We analyse the potential of a series of peptide inhibitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2.Furthermore, ave show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Consequently, we define the binding site of 3G5 on hdm2 using overlapping peptides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding points for 3G5.In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NEM and DTT on the interaction between the two molecules in ELISA assays.All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed. (C) 1997 Academic Press Limited.