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防止肿瘤发生方面起着关键作用。MDM2是一种环指泛素E3连接酶，在P53水平的调节中起着重要作用。MDM2泛素化P53，并针对它进行蛋白酶体介导的降解。MDMX是一种MDM2同源基因，在控制p53方面也起着不可或缺的作用。MDMX协助MDM2抑制P53的活性和水平，而MDM2介导MDMX的泛素化和DNA损伤的降解。由于MDM2，以及可能的MDMX，都可以由p53转录诱导，它们形成了一个p53-MDM2-MDMX反馈环。这个环路保证了这些蛋白质在细胞中的正常内稳态。最近的研究也表明，P53-MDM2-MDMX环是通过泛素特异蛋白酶(USP)家族的几个去泛素化酶(DUB)的去泛素化来调节的。USP7去泛素化和稳定所有这三个角色，而USP10去泛素化只作用于P53，USP2a去泛素化MDM2和MDMX，为P53通路提供另一层精确和动态的调节。然而，该环是否受USP家族以外的DUB调控尚不清楚。我们最近发现了一个含有OTU结构域的蛋白(OTU)家族成员Otubain 1(Otub1)，它是P53-MDM2-MDMX环的一个新的调节因子。我们发现Otub1的过表达逆转了MDM2介导的P53泛素化，稳定了细胞中的P53，并显著诱导了依赖于P53的细胞凋亡和细胞生长抑制。Otub1显性负性突变体的过度表达或内源性Otub1的siRNA消融显著减弱了对DNA损伤的P53激活。这些结果表明Otub1在P53信号的调节中起着至关重要的作用。为了进一步深入了解这一调控，我们将研究Otub1在Aim 1中调控p53途径的分子和生化机制。由于Otub1还抑制细胞中MDMX的泛素化并稳定MDMX，有趣的是，Otub1稳定的MDMX高度磷酸化，我们将进一步表征Otub1调控MDMX的机制及其在Aim 2中的意义。最后，由于Otub1在细胞培养系统中强烈诱导依赖于P53的细胞生长抑制，我们将研究 在AIM 3中，Otub1通过小鼠异种移植和基因敲除模型抑制体内肿瘤生长。我们将检测Otub1在人类癌症中的表达是否被解除调控和/或Otub1基因发生突变。我们还将研究Otub1调控P53信号以应对DNA损伤的机制。这些目标的完成不仅有助于我们进一步了解如何通过动态泛素化和去泛素化来适当地调节p53的功能，以及这种动态调控的解除如何促进肿瘤的发生，而且有助于寻找新的癌症治疗方法来重新激活含有野生型p53的癌症中的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.