Regulation of Mdmx stability and subcellular localization by ubiquitination

描述(申请人提供)：P53肿瘤抑制因子是调控电路的主要传感器，该电路监测来自不同来源的信号通路，包括DNA损伤、致癌事件、核糖体应激和其他异常细胞过程(Vousedon和Prives，2009；Qian和Chen，2010；熊等，2011；Zhang和Lu，2009)。众所周知，MDM2及其相关蛋白MDMX(也称为MDM4)在特异性抑制癌细胞中P53活性的范围内发挥着重要作用。尽管它有很高的序列同源性 在MDM2和环状结构域的存在下，MDMX不具有固有的P53的E3连接酶活性，而是被证明通过它们的相互作用抑制P53诱导的转录；然而，MDMX基因敲除小鼠即使在MDM2存在的情况下也会死亡，这种致命性也可以通过P53的失活来挽救(Maine等人，2007年)。因此，MDMX在抑制p53功能中的作用与MDM2一样重要，并且需要生理水平的MDMX以非冗余的方式调节体内的p53活性。在保留野生型p53的几种人类肿瘤中发现了MDMX的过度表达(Maine等人，2007；Danovi等人，2004；Li等人，2010；Gilkes和Chen，2007)。值得注意的是，MDMX在大多数人类癌细胞中是一种非常稳定的蛋白质，在正常情况下它主要定位于癌细胞的细胞质中。MDMX在体内被调控的分子机制是一个极其重要的问题，但需要进一步阐明。P53和MDMX都可以被相同的E3连接酶(MDM2)泛素化和降解；然而，有趣的是，虽然P53被MDM2迅速降解，但MDMX在非应激的癌细胞中是一种非常稳定的蛋白质，这提出了一个关键问题，即如何在不影响P53降解的情况下特定地保护MDM2的MDMX降解。在我们的初步研究中，我们已经确定非编码的5S rRNA是人类细胞MDMX相关复合体的主要成分。我们发现5S rRNA在癌细胞中作为MDMX降解的天然抑制剂。5S rRNA的表达抑制MDM2介导的MDMX在人细胞中的降解，但对MDM2对P53的降解无明显影响。这部分工作揭示了非编码5S rRNA在调节MDMX稳定性方面的意外作用。此外，为了了解MDMX亚细胞定位在细胞中的调控机制，我们通过蛋白质纯化鉴定了Pellino 1(Peli1)，它是一种新的MDMX调节因子。我们发现Peli1在体外和体内都与MDMX相互作用。Peli1的表达不影响MDMX的水平，但可诱导MDMX的胞浆定位。因此，我们从MDMX蛋白复合体中鉴定出两个关键的辅因子(非编码的5S rRNA和Peli1)，它们是调节MDMX功能的关键因素。有待检验的中心假设是，MDMX需要多层(稳定性和亚细胞定位)的调控网络，以确保对肿瘤细胞中P53活性的时间和空间控制。它包括以下两个具体目标，我们已经获得了大量的初步数据，以支持每个目标的可行性。在Aim1中，我们将阐明非编码5S rRNA在人癌细胞中对MDMX稳定性的控制。在目标2中，我们将研究新发现的MDMX相关蛋白Peli1在调节MDMX的亚细胞定位以及随后对P53功能的影响中的调节作用。为了进一步研究Peli1-MDMX在体内的相互作用，我们将使用Peli1突变小鼠来剖析Peli1在MDMX介导的发病机制中的作用。 公共卫生相关性：恢复P53活性仍然是寻求更有效的癌症治疗方法的一个重要目标。由于在MDM2或MDMX过度表达的癌症中，p53基因通常保持野生型，所以长期以来人们一直认为，针对这些p53抑制因子可以恢复p53的活性。有趣的是，MDM2和MDMX的过度表达在癌细胞中是相互排斥的，这表明主要抑制子的失调足以灭活导致肿瘤发展的P53。这项研究将阐明控制MDMX在调节P53介导的肿瘤抑制中的稳定性和亚细胞定位的新的分子机制，并为如何在癌症治疗中靶向这一途径提供重要的见解。

DESCRIPTION (provided by applicant): The p53 tumor suppressor acts as the major sensor for a regulatory circuit that monitors signaling pathways from diverse sources, including DNA damage, oncogenic events, ribosomal stress and others abnormal cellular processes (Vousedn and Prives, 2009;Qian and Chen, 2010; Xiong et al., 2011; Zhang and Lu, 2009). It is well accepted that Mdm2 and its related protein Mdmx (also called Mdm4) play a major part in the scope of specifically inhibiting p53 activities in cancer cells. Despite its high sequence homology with Mdm2 and the presence of a RING domain, Mdmx does not have intrinsic E3-ligase activity for p53 but was instead shown to inhibit p53- induced transcription via their interactions; nevertheless, Mdmx knockout mice die even in the presence of Mdm2 and this lethality is also rescued by inactivation of p53 (Maine et al., 2007). Thus, the role of Mdmx in repressing p53 function is as critical as that of Mdm2 and physiological levels of Mdmx are required in a non-redundant manner to regulate p53 activity in vivo. Mdmx over expression is found in several types of human tumors retaining wild type p53 (Maine et al., 2007; Danovi et al., 2004; Li et al., 2010; Gilkes and Chen, 2007). Notably, Mdmx is a very stable protein in most human cancer cells and it is predominantly localized in the cytoplasm of cancer cells under normal conditions. The molecular mechanism by which Mdmx is regulated in vivo is an extremely important issue but needs further elucidation. Both p53 and Mdmx can be ubiquitinated and degraded by the same E3 ligase (Mdm2); interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a very stable protein in unstressed cancer cells, raising a critical issue regarding how Mdmx degradation by Mdm2 is specifically protected without affecting the degradation of p53. In our preliminary studies, we have identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation in cancer cells. 5S rRNA expression inhibits Mdm2- mediated degradation of Mdmx but has no obvious effects on p53 degradation by Mdm2 in human cells. This part of the work reveals an unexpected role of noncoding 5S rRNA in modulating the stability of Mdmx. Moreover, to understand the mechanism by which Mdmx sub cellular localization is regulated in cells, we have indentified Pellino 1 (Peli1), as a novel Mdmx regulator through protein purification. We found Peli1 interacts with Mdmx both in vitro and in vivo. Peli1 expression does not affect the Mdmx levels but induces cytoplasmic localization of Mdmx. Thus, we have indentified two critical cofactors (the noncoding 5S rRNA and Peli1) from the Mdmx protein complex that are critically involved in regulating Mdmx function. The central hypothesis to be tested is that Mdmx requires multiple layers (the stability and sub cellular localization) of regulatory networks to ensure temporal and spatial controls of p53 activity in tumor cells. It includes the following two specific aims and we have obtained substantial preliminary data to support the feasibility of each aim. In Aim1, we will elucidate the stability control of Mdmx by th noncoding 5S rRNA in human cancer cells. In Aim 2, we will investigate the regulatory role of Peli1, a newly identified Mdmx-associated protein, in modulating the sub cellular localization of Mdmx as well as subsequent effects on p53 functions. To further investigate the role of the Peli1-Mdmx interplays in vivo, we will use Peli1 mutant mice to dissect the role of Peli1 in Mdmx-mediated pathogenesis. PUBLIC HEALTH RELEVANCE: Restoration of p53 activity remains an important goal in the quest for more effective cancer therapeutics. Because the p53 gene often remains wild-type in Mdm2- or Mdmx- over expressing cancers, it has long been conceived that targeting these p53 repressors could restore p53 activity. Interestingly, over expression of MDM2 and MDMX are mutually exclusive in cancer cells, suggesting that dysregulation of a major repressor is sufficient to inactivate p53 leading to tumor development. This study will elucidate novel molecular mechanisms for controlling the stability and sub cellular localization of Mdmx in regulating p53-mediated tumor suppression and yield crucial insights regarding how to target this pathway in cancer therapy.