Defining the in vivo role of Huwe1 in p53 regulation

摘要 肿瘤抑制蛋白p53在约50%的人类肿瘤中是基因失活的。在肿瘤细胞中， 然而，p53型p53活性通常以各种其它方式被抑制。在这方面，泛素E3连接酶 Mdm 2及其同系物MdmX对于抑制p53的活性至关重要。Mdm 2和MdmX形式 异二聚体催化p53蛋白降解并阻断其转录活性。因此，委员会认为， Mdm 2和MdmX的消除对于p53的完全激活是必需的。尽管有许多关于 调节p53活性，介导Mdm 2-MdmX异二聚体降解的分子机制 尤其是在体内仍然知之甚少。我们最近获得的结果强烈表明， HECT结构域泛素E3连接酶Huwe 1可能是长期寻找的负责DNA损伤的E3连接酶。 诱导Mdm 2和MdmX降解。重要的是，Huwe 1最初被发现是一种E3连接酶， 泛素化p53用于降解。然而，我们的初步数据表明：（a）Huwe 1与两者相互作用， 不依赖于p53的Mdm 2和MdmX蛋白;（B）DNA损伤诱导的Mdm 2和MdmX的降解 在Huwe 1 siRNA介导的敲低后显著抑制;和（c）Huwe 1敲低使细胞 通过稳定Mdm 2和MdmX，对DNA损伤诱导的p53活化具有高度抗性。我们假设 在非应激条件下抑制稳态p53水平的同时，Huwe 1通过促进 Mdm 2和MdmX响应于遗传毒性应激的降解，这是p53活化的关键事件。测试 在体内，我们建立了条件性Huwe 1基因敲除小鼠模型。使用小鼠模型，我们 将确定Huwe 1的急性缺失如何影响DNA损伤前后的p53激活（目的1）， 确定DNA损伤诱导的胸腺淋巴瘤发生中Huwe 1缺失的影响（目的2）。成功 完成拟议的体内研究将为Huwe 1作为一种抗肿瘤药物的作用提供概念验证证据。 p53在DNA损伤中的关键激活剂。这些结果将改变人们对这一有争议的 E3连接酶，也将为修改Mdm 2/MdmX稳定性的调节和促进Mdm 2/MdmX的表达提供理论基础。 进一步探讨Huwe 1的调控机制。

Abstract The tumor suppressor protein p53 is genetically inactivated in ~50% of human tumors. In tumor cells with wild type p53, however, p53 activity is often suppressed in various other ways. In this regard, the ubiquitin E3 ligase Mdm2 and its homolog, MdmX, are critical for the suppression of the activity of p53. Mdm2 and MdmX form heterodimers which catalyze p53 protein degradation and block its transcriptional activity. Consequently, elimination of both Mdm2 and MdmX is essential for full p53 activation. Despite numerous studies on the regulation of p53 activity, the molecular mechanism that mediates the Mdm2-MdmX heterodimer degradation remains poorly understood particularly in vivo. We have recently obtained results strongly indicating that the HECT-domain ubiquitin E3 ligase Huwe1 may be the long-sought E3 ligase responsible for DNA damage- induced degradation of both Mdm2 and MdmX. Importantly, Huwe1 was initially discovered as an E3 ligase that ubiquitinates p53 for degradation. Nevertheless, our preliminary data show that: (a) Huwe1 interacts with both Mdm2 and MdmX proteins independently of p53; (b) DNA damage-induced degradation of Mdm2 and MdmX was markedly inhibited upon Huwe1 siRNA-mediated knockdown; and (c) Huwe1 knockdown renders cells highly resistant to DNA damage-induced p53 activation by stabilizing both Mdm2 and MdmX. We hypothesize that while suppressing steady-state p53 levels under non-stressed conditions, Huwe1 activates p53 by promoting the degradation of Mdm2 and MdmX in response to genotoxic stress, an event critical for p53 activation. To test this hypothesis in vivo, we created conditional Huwe1 knockout mouse models. Using the mouse models, we will determine how acute deletion of Huwe1 impacts p53 activation before and after DNA damage (Aim 1) and identify the impact of Huwe1 loss in DNA damage-induced thymic lymphomagenesis (Aim 2). The successful completion of the proposed in vivo studies will provide the proof-of-concept evidence for the role of Huwe1 as a crucial activator of p53 upon DNA damage. These outcomes will change the prevailing view of this controversial E3 ligase and will also provide a rationale for revising the regulation of Mdm2/MdmX stability and impetus for investigating the mechanism of Huwe1 regulation in the future.