Dynamic roles of HAUSP in p53/Mdm2 regulation

描述（由申请人提供）：p53蛋白被称为“基因组的监护人”，因为它在协调细胞对遗传毒性应激的反应中起着至关重要的作用。Mdm 2是一种环指E3泛素连接酶，它诱导p53泛素化，并在p53调控的范围内发挥重要作用。最初，泛素-蛋白酶体途径被认为具有从底物泛素化到被26 S蛋白酶体降解的单向方向。然而，去泛素化酶（DUBs）的发现和出现改变了酶促过程的整体观点，并迅速显示了这一途径令人难以置信的动态。我们早期发现疱疹病毒相关的泛素特异性蛋白酶（HAUSP）通过去泛素化作用与p53相互作用并稳定p53，这是DUB在p53通路中表现出特异性作用的首批迹象之一。令人惊讶的是，简单的线性模型被掩盖，然而，随后的发现，HAUSP去泛素化Mdm 2，是至关重要的控制Mdm 2在体内的稳定性。除了泛素化p53之外，Mdm 2还引起高水平的自身泛素化，这使得Mdm 2本身在细胞中非常容易。我们的研究表明，HAUSP表达可以拯救Mdm 2从自我泛素化。此外，内源性HAUSP的siRNA介导的失活导致Mdm 2的不可管理的自身泛素化和去稳定化，这间接导致p53活化。这些发现得到了Bert Vogelstein实验室中体细胞HAUSP敲除人类细胞（HCT 116-HAUSP-/-）研究的进一步支持，最近我们实验室在小鼠HAUSP（-/-）胚胎中证实了这一点。因此，我们的研究表明HAUSP介导的去泛素化在调节p53通路中的动态作用;然而，它也提出了关于HAUSP在体内的精确功能的更有趣的问题。例如，1）HAUSP在p53活化和肿瘤发生中的生理作用是什么，以及2）HAUSP/Mdm 2和HAUSP/p53相互作用是如何动态调节的？为了了解这一途径的动态性质，我们将首先试图阐明HAUSP/Mdm 2相互作用以及HAUSP/p53相互作用在体内通过翻译后修饰（Aim 1）的调节。为了完全理解HAUSP在p53调控中的生物化学作用，我们计划鉴定HAUSP复合物（Aim 2）的新的相关因子/或调节剂。最后，在目标3中，我们将开发一种条件性HAUSP缺失小鼠，以获得HAUSP在p53/Mdm 2通路中的生理作用。

DESCRIPTION (provided by applicant): The p53 protein is known as a "guardian of the genome" because of its crucial role in coordinating cellular responses to genotoxic stress. It is well accepted that Mdm2, a RING-finger E3 ubiquitin ligase, induces p53 ubiquitination and plays a major part in the scope of p53 regulation. Originally, the ubiquitin-proteasome pathway was thought to have a one way direction from substrate ubiquitination to degradation by the 26S proteasome. However, the discovery and emergence of deubiquitination enzymes (DUBs) changed the global view of the enzymatic process and quickly showed the incredible dynamics of this pathway. Our early finding that the Herpesvirus-Associated Ubiquitin-Specific Protease (HAUSP) interacts and stabilizes p53 by deubiquitination, was one of the first indications thatDUBs exhibited a specific role in the p53 pathway. Surprisingly, the simple linear model was obscured however with the subsequent findings that HAUSP deubiquitinates Mdm2 and is essential for controlling the Mdm2 stability in vivo. In addition to ubiquitinating p53, Mdm2 elicits high levels of self-ubiquitination which makes Mdm2 itself very liable in cells. Our studies demonstrate that HAUSP expression can rescue Mdm2 from self-ubiquitination. Moreover, SiRNA-mediated inactivation of endogenous HAUSP leads to unmanageable self-ubiquitination and destabilization of Mdm2, which indirectly results in p53 activation. These findings were further supported by the study of somatic HAUSP-knock out human cells (HCT116-HAUSP-/-) in Bert Vogelstein's lab and more recently confirmed in mouse HAUSP (-/-) embryos by our lab. Thus, our studies suggest a dynamic role of HAUSP-mediated deubiquitination in regulating the p53 pathway; however, it also raises more interesting questions regarding the precise function of HAUSP in vivo. For example, 1) what is the physiological role of HAUSP in p53 activation and tumorigenesis and 2) how are the HAUSP/Mdm2 and HAUSP/p53 interactions dynamically regulated? To understand the dynamic nature of this pathway, we will first try to elucidate the regulation of the HAUSP/Mdm2 interaction as well as the HAUSP/p53 interaction in vivo by posttranslational modifications (Aim1). To completely understand the biochemical role of HAUSP in p53 regulation, we plan to identify novel associated factor/ or regulators of the HAUSP complexes (Aim2). Finally, in Aim 3, we will develop a conditional HAUSP-null mouse to access the physiological role of HAUSP in the p53/Mdm2 pathway.