Role of Otubain 1 in the p53 Pathway

描述（由申请人提供）：p53肿瘤抑制因子在维持基因组完整性和预防肿瘤发生中起关键作用。MDM 2是一种环指泛素E3连接酶，在调节p53水平中起主要作用。MDM 2泛素化p53并靶向其进行蛋白酶体介导的降解。MDMX是一种MDM 2同系物，在控制p53中也起着不可或缺的作用。MDMX协助MDM 2抑制p53活性和水平，而MDM 2介导MDMX泛素化和降解以响应DNA损伤。由于MDM 2以及可能的MDMX可以由p53转录诱导，因此它们形成p53-MDM 2-MDMX反馈环。这个环维持这些蛋白质在细胞中的正常稳态。最近的研究还表明，p53-MDM 2-MDMX环是通过来自遍在蛋白特异性蛋白酶（USP）家族的几种去遍在蛋白酶（DUB）的反作用而通过去遍在蛋白化来调节的。USP 7去泛素化并稳定所有三个参与者，而USP 10仅去泛素化p53，USP 2a去泛素化MDM 2和MDMX，为p53途径提供另一层精确和动态调节。然而，该环是否由USP家族以外的DUB调节尚不清楚。我们最近发现，OTU结构域蛋白酶（OTU）家族成员Otubain 1（Otub 1）是p53-MDM 2-MDMX环的一种新型调节因子。我们发现过表达Otub 1逆转MDM 2介导的p53泛素化，稳定细胞中的p53，并显著诱导p53依赖性凋亡和细胞生长抑制。过表达Otub 1的显性负突变体或通过siRNA消除内源性Otub 1显著减弱了DNA损伤引起的p53激活。这些结果表明Otub 1在调节p53信号传导中起着至关重要的作用。为了进一步了解调控，我们将研究Otub 1在调节Aim 1中p53通路中的分子和生化机制。由于Otub 1还抑制MDMX泛素化并稳定细胞中的MDMX，有趣的是，Otub 1稳定的MDMX是高度磷酸化的，我们将进一步表征MDMX的Otub 1调节机制及其在Aim 2中的意义。最后，由于Otub 1在细胞培养系统中显著诱导依赖于p53的细胞生长抑制，我们将研究是否 在Aim 3中，使用小鼠异种移植和敲除模型，Otub 1抑制体内肿瘤生长。我们将研究Otub 1表达是否失调和/或Otub 1基因在人类癌症中突变。我们还将研究Otub 1调节p53信号对DNA损伤的反应的机制。这些目标的完成不仅将进一步了解p53功能如何通过动态泛素化和去泛素化进行适当调节，以及这种动力学的失调如何有助于肿瘤发生，而且还有助于寻找新的癌症治疗方法来重新激活野生型p53癌症中的p53。

DESCRIPTION (provided by applicant): The p53 tumor suppressor plays a key role in maintaining genomic integrity and preventing tumorigenesis. MDM2, a RING-finger ubiquitin E3 ligase, plays a major role in the regulation of p53 levels. MDM2 ubiquitinates p53 and targets it for proteasome-mediated degradation. MDMX, an MDM2 homolog, also plays an indispensible role in controlling p53. MDMX assists MDM2 in suppressing p53 activity and levels, while MDM2 mediates MDMX ubiquitination and degradation in response to DNA damage. As MDM2, and possibly MDMX as well, can be transcription ally induced by p53, they form a p53-MDM2-MDMX feedback loop. This loop ensues a normal homeostasis of these proteins in cells. Recent studies have also shown that the p53-MDM2-MDMX loop is regulated via deubiquitination by counteraction of several deubiquitinating enzymes (DUBs) from the ubiquitin specific protease (USP) family. USP7 deubiquitinates and stabilizes all the three players, whereas USP10 deubiquitinates p53 only and USP2a deubiquitinates MDM2 and MDMX, providing another layer of precise and dynamic regulation of the p53 pathway. However, whether this loop is regulated by DUBs other than USP family is not known. We recently discovered that an OTU-domain containing protease (OTU) family member, Otubain 1 (Otub1), as a novel regulator of the p53-MDM2-MDMX loop. We show that over expression of Otub1 reverses MDM2-mediated p53 ubiquitination, stabilize p53 in cells, and drastically induce p53- dependent apoptosis and cell growth inhibition. Over expression of a dominant-negative mutant of Otub1 or ablation of endogenous Otub1 by siRNA significantly attenuated p53 activation in response to DNA damage. These results suggest a crucial role for Otub1 in the regulation of p53 signaling. To further gain insight into the regulation, we will investigate the molecular and biochemical mechanisms underlying the role of Otub1 in regulating the p53 pathway in Aim 1. As Otub1 also suppresses MDMX ubiquitination and stabilizes MDMX in cells and, intriguingly, the stabilized MDMX by Otub1 is highly phosphorylated, we will further characterize the mechanism underlying Otub1 regulation of MDMX as well as its significance in Aim 2. Finally, as Otub1 drastically induces p53-dependent cell growth inhibition in cell culture system, we will examine if Otub1 suppresses tumor growth in vivo using mouse xenograft and knockout models in Aim 3. We will examine whether Otub1 expression is deregulated and/or the Otub1 gene is mutated in human cancers. We will also examine the mechanism underlying the Otub1 regulation of p53 signaling in response to DNA damage. Completion of these aims would not only further our understanding of how p53 function is properly regulated through dynamic ubiquitination and deubiquitination and how deregulation of this dynamics contributes to tumorigenesis, but also aid the search for novel cancer therapeutics to re-activate p53 in wild-type p53 containing cancers.