Dynamic roles of HAUSP in p53/Mdm2 regulation

描述(申请人提供)：P53蛋白被称为“基因组的守护者”，因为它在协调细胞对遗传毒性压力的反应中起着至关重要的作用。众所周知，MDM2是一种环指E3泛素连接酶，可诱导P53泛素化，在P53调控范围内发挥重要作用。最初，泛素-蛋白酶体途径被认为是从底物泛素化到26S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对酶过程的全球看法，并迅速展示了这一途径令人难以置信的动力学。我们早期发现，疱疹病毒相关泛素特异蛋白(Hausp)通过去泛素化作用相互作用并稳定P53，这是DUB在P53途径中发挥特殊作用的最早迹象之一。然而，令人惊讶的是，简单的线性模型被随后的发现所掩盖，即Hausp去泛素化MDM2，并且对于控制体内MDM2的稳定性是必不可少的。除了泛素化P53外，MDM2还引起高度的自我泛素化，这使得MDM2本身在细胞中非常容易发生。我们的研究表明，Hausp的表达可以将MDM2从自我泛素化中拯救出来。此外，siRNA介导的内源性Hausp失活导致无法控制的自身泛素化和MDM2的不稳定，从而间接导致p53激活。Bert Vogelstein的实验室对体细胞Hausp基因敲除的人类细胞(HCT116-Hausp-/-)的研究进一步支持了这些发现，最近我们实验室在小鼠Hausp(-/-)胚胎中证实了这些发现。因此，我们的研究表明，Hausp介导的去泛素化在调节P53途径中起着动态作用；然而，它也提出了关于Hausp在体内的确切功能的更有趣的问题。例如，1)Hausp在P53激活和肿瘤发生中的生理作用是什么；2)Hausp/MDM2和Hausp/P53的相互作用是如何动态调节的？为了了解这一途径的动力学性质，我们首先试图通过翻译后修饰(Aim1)来阐明Hausp/MDM2相互作用以及体内Hausp/p53相互作用的调节。为了完全了解Hausp在p53调控中的生化作用，我们计划鉴定Hausp复合体的新的相关因子/或调节因子(AIM2)。最后，在目标3中，我们将发展一个条件性Hausp缺失的小鼠，以了解Hausp在p53/MDM2途径中的生理作用。

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.