Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo
Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo
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DOI：
10.1016/s0960-9822(06)00374-5
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发表时间：
1997-11-01
期刊：
影响因子：
9.2
通讯作者：
Lane, DP
中科院分区：
文献类型：
--
作者：
Bottger, A;Bottger, V;Lane, DP
背景：p53肿瘤抑制蛋白的转录激活功能是由DNA损伤诱导的，并导致生长停滞和/或凋亡反应。这种反应的一个关键组成部分是p53蛋白浓度的急剧上升，这是由于蛋白质稳定性的增加造成的。最近有研究表明，与Mdm2蛋白的相互作用可能会使p53快速降解。我们设计了一种编码小蛋白的基因，该蛋白与Mdm2蛋白上的p53结合袋紧密结合。我们通过将噬菌体展示优化的mdmp结合肽克隆到硫氧还蛋白的活性位点环中构建了该基因。结果：当引入含有低水平野生型p53的细胞时，该蛋白引起内源性p53蛋白的显著积累，p53应答报告基因的激活，细胞周期阻滞，模拟这些细胞暴露于紫外线或电离辐射后的效果。显微注射Mdm2上p53结合位点的单克隆抗体也达到了类似的效果，从而确定了其特异性。结论：这些结果表明，在正常细胞中，p53反应受Mdm2的组成性调节，仅破坏这种相互作用就足以稳定p53蛋白并激活p53反应。我们的迷你蛋白方法提供了一种强大的新方法来激活p53而不造成DNA损伤。更广泛地说，它建立了一个强大的通用方法来确定细胞中蛋白质-蛋白质相互作用的特定破坏的生物学后果。
Background: The transcriptional activation function of the p53 tumour suppressor protein is induced by DNA damage and results in growth arrest and/or apoptotic responses. A key component of this response is the dramatic rise in p53 protein concentration resulting from an increase in the protein's stability. Very recently, it has been suggested that interaction with the Mdm2 protein may target p53 for rapid degradation. We have designed a gene encoding a small protein that binds tightly to the p53-binding pocket on the Mdm2 protein. We have constructed the gene by cloning a phage display optimised MdmP-binding peptide into the active-site loop of thioredoxin.Results: When introduced into cells containing low levels of wild-type p53, this protein causes a striking accumulation of the endogenous p53 protein, activation of a p53-responsive reporter gene, and cell cycle arrest mimicking the effects seen in these cells after exposure to UV or ionising radiation. Microinjection of a monoclonal antibody to the p53-binding site on Mdm2 achieves a similar effect, establishing its specificity.Conclusions: These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.