Inhibition of MDM2 homodimerization by XIAP IRES stabilizes MDM2, influencing cancer cell survival.
Inhibition of MDM2 homodimerization by XIAP IRES stabilizes MDM2, influencing cancer cell survival.
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DOI：
10.1186/s12943-015-0334-0
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发表时间：
2015-03-26
期刊：
影响因子：
37.3
通讯作者：
Zhou M
中科院分区：
文献类型：
--
作者：
Liu T;Zhang H;Xiong J;Yi S;Gu L;Zhou M
众所周知，MDM2蛋白与其伴侣MDM4形成异源二聚体时是稳定的，但MDM2蛋白在其同型二聚体形式下的稳定性尚不清楚。MDM2蛋白含有一个c端RING结构域，该结构域不仅作为E3连接酶调节p53和MDM2本身的泛素化，而且能够结合几种特定的细胞mrna来调节基因表达。在这项研究中，我们评估了MDM2蛋白的稳定性是否受到特定小RNA （XIAP IRES mRNA）结合的调节。我们通过化学交联和双分子荧光互补（BiFC）实验来检测人MDM2蛋白在其同二聚体形式下的稳定性，以及XIAP IRES对MDM2同二聚体和蛋白稳定性的影响。采用泛素化和脉冲追踪法检测MDM2的自泛素化和蛋白转化率。采用荧光滴定法和ITC检测MDM2 RING蛋白与XIAP IRES的结合。Western blot法检测蛋白表达。采用克隆实验、WST和流式细胞术检测XIAP IRES、siXIAP和IR对癌细胞生长和凋亡的影响。我们发现MDM2的自结合（同二聚化）通过MDM2的c端环结构域发生，并且MDM2蛋白在同二聚化时变得不稳定。MDM2同二聚化导致MDM2自泛素化的RING结构域功能增加。XIAP IRES与RING结构域的结合抑制了MDM2的二聚化和自泛素化，从而稳定了MDM2，增加了XIAP的表达。XIAP和MDM2的上调导致XIAP IRES抑制p53，导致p53正常和p53缺陷癌细胞的细胞生长和存活。我们的研究发现了一种新的IRES RNA，它可以与MDM2蛋白相互作用并调节其稳定性，这表明通过破坏MDM2蛋白-RNA相互作用来靶向MDM2可能是开发新型抗癌疗法的有用策略。
It is known that the MDM2 protein is stabilized when it forms a heterodimer with its partner MDM4, but MDM2 protein stability in its homodimer form is not known. The MDM2 protein contains a C-terminal RING domain that not only functions as an E3 ligase to regulate ubiquitination of p53 and MDM2 itself, but also is characterized to be able to bind several specific cellular mRNAs to regulate gene expression. In this study, we evaluate whether the MDM2 protein stability is regulated by the binding of a specific small RNA (XIAP IRES mRNA). We performed chemical cross-linking and bimolecular fluorescence complementation (BiFC) assay to measure the human MDM2 protein stability in its homodimer form and the effect of XIAP IRES on MDM2 homodimerization and protein stabilization. Ubiquitination and pulse-chase assays were used to detect MDM2 self-ubiquitination and protein turn-over. Fluorescent titration and ITC were used to examine the binding between MDM2 RING protein and XIAP IRES. Western blot assay was used for determining protein expression. Clonogenic assay, WST and flow cytometry were used to test the effects of XIAP IRES, siXIAP and IR on cancer cell growth and apoptosis. We found that self-association (homodimerization) of MDM2 occurs through the C-terminal RING domain of MDM2 and that the MDM2 protein becomes unstable when it is homodimerized. MDM2 homodimerization resulted in an increased function of the RING domain for MDM2 self-ubiquitination. Binding of XIAP IRES to the RING domain inhibited MDM2 homodimerization and self-ubiquitination, which resulted in stabilization of MDM2, as well as increased XIAP expression. Upregulation of XIAP and MDM2 that led to inhibition of p53 by the XIAP IRES resulted in cell growth and survival in both p53-normal and -deficient cancer cells. Our study identified a new IRES RNA that interacts with MDM2 protein and regulates its stabilization, which suggested that targeting of MDM2 through disruption of MDM2 protein-RNA interaction might be a useful strategy for developing novel anti-cancer therapeutics.
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