Regultion of p53 Deubiquitination

描述（由申请人提供）：p53是一种重要的肿瘤抑制因子，在超过50%的人类癌症中发生突变。p53的一个主要功能是激活其靶基因p21和PUMA的表达，以应对细胞应激，从而调节细胞周期进程和凋亡。P53的活性主要受翻译后修饰的调控，如泛素化、sumo化、磷酸化、乙酰化和甲基化。其中，mdm2诱导的p53泛素化诱导p53核输出和降解，是调控p53活性的主要细胞机制。然而，逆转p53泛素化的过程，即去泛素化，尚不清楚。我们已经发现泛素特异性蛋白酶USP10在体外和细胞中去泛素化p53。USP10对于稳定非应激细胞和DNA损伤细胞中的p53都是必需的。USP10主要定位于非应激细胞的细胞质中。在DNA损伤的反应中，USP10被稳定并易位到细胞核中，而USP10的atm依赖性磷酸化对于USP10的易位和稳定至关重要。此外，我们发现USP10在野生型p53细胞中抑制肿瘤细胞生长，而在突变型p53细胞中促进肿瘤细胞生长。最后，我们发现USP10在高比例的肾细胞癌病例中表达缺失。基于这些初步结果，我们推测USP10是p53的重要调控因子，USP10通过p53抑制肿瘤发生。因此，进一步研究USP10-p53通路在细胞和模型系统中的调控作用具有重要意义。我们提出以下具体目标：研究Ras和G3BP1对USP10-p53通路的调控作用。G3BP1 （Ras- gtpase激活蛋白sh3结构域结合蛋白）是Ras的下游效应物，先前已在体外证明可抑制USP10。我们假设Ras-G3BP1通路通过USP10调控p53的稳定性，并将对此假设进行验证。2. 研究DNA损伤反应通路对USP10的调控作用。USP10主要定位于非应激细胞的细胞质中。然而，在DNA损伤后，USP10易位到细胞核并在DNA损伤后变得稳定，这有助于p53的激活。我们已经证明，USP10的atm依赖性磷酸化是USP10稳定和易位所必需的，然而这些事件的机制尚不完全清楚。我们发现USP10也以磷酸化依赖的方式被聚合，这表明USP10的聚合可能对USP10的稳定和易位很重要。为此，我们将研究USP10 sumoylation的功能意义和调控。3. 探讨USP10在肿瘤发生中的作用。由于USP10正调控p53，我们假设USP10作为肿瘤抑制因子发挥作用。与这一假设相一致，USP10能够以p53依赖的方式抑制癌细胞的生长，并且在高比例的肾细胞癌样本中USP10的表达下调。我们将使用USP10基因敲除小鼠作为模型系统来研究USP10在体内肿瘤发生中的作用。总之，这些研究将揭示p53调控和肿瘤抑制的新机制。它们也将为未来的分子干预癌症治疗奠定基础。

DESCRIPTION (provided by applicant): p53 is an important tumor suppressor that is mutated in more than 50% of human cancers. A major function of p53 is to activate the expression of its target genes, such as p21 and PUMA in response to cellular stress, thereby regulating cell cycle progression and apoptosis. p53 activity is mainly regulated by posttranslational modifications, such as ubiquitination, sumoylation, phosphorylation, acetylation, and methylation. Among them, Mdm2-induced ubiquitination of p53 induces p53 nuclear export and degradation, which is a major cellular mechanism that regulates p53 activity. However, the process of reversing ubiquitination of p53, i.e. deubiquitination, is not well understood. We have found that the ubiquitin-specific protease USP10 deubiquitinates p53 in vitro and in cells. USP10 is required for the stabilization p53 in both unstressed cells and cells subjected to DNA damage. USP10 predominately localizes in the cytoplasm in unstressed cells. In response to DNA damage, USP10 is stabilized and translocates into the nucleus following DNA damage, and ATM-dependent phosphorylated of USP10 is important for USP10 translocation and stabilization. Furthermore, we found that USP10 suppresses tumor cell growth in cells with wild-type p53, while promoting tumor cell growth in cells with mutant p53. Finally, we found loss of USP10 expression in high percentage of renal cell carcinoma cases. Based on these preliminary results, we hypothesize that USP10 is an important regulator of p53, and USP10 suppresses tumorigenesis through p53. Therefore, it is important to further investigate the regulation of the USP10-p53 pathway in cells and in model systems. We propose the following Specific Aims: 1. Study the regulation of USP10-p53 pathway by Ras and G3BP1. G3BP1 (Ras- GTPase-activating protein SH3-domain-binding protein), a downstream effector of Ras, has previously been shown to inhibit USP10 in vitro. We hypothesize that the Ras-G3BP1 pathway regulates p53 stability through USP10, and will test this hypothesis in this aim. 2. Study the regulation of USP10 by the DNA damage response pathway. USP10 predominately localizes in the cytoplasm of unstressed cells. However, following DNA damage, USP10 translocates to the nucleus and becomes stabilized following DNA damage, which contributes to p53 activation. We have shown that ATM-dependent phosphorylation of USP10 is required for USP10 stabilization and translocation, however the mechanisms underlying these events are not entirely clear. We found that USP10 is also sumoylated in a phosphorylation-dependent manner, suggesting that USP10 sumoylation might be important for USP10 stabilization and translocation. We will study the functional significance and regulation of USP10 sumoylation in this aim. 3. Examine the role of USP10 in tumorigenesis. Since USP10 positively regulates p53, we hypothesize that USP10 functions as a tumor suppressor. Consistent with this hypothesis, USP10 can suppress cancer cell growth in p53-dependent manner and USP10 expression is downregulated in high percentage of renal cell carcinoma samples. We will examine the role of USP10 in tumorigenesis in vivo using USP10 knockout mice as a model system. In summary, these studies will reveal novel mechanisms of p53 regulation and tumor suppression. They will also lay the base for future molecular intervention for cancer therapy. PUBLIC HEALTH RELEVANCE: p53 is a critical tumor suppressor that is mutated in more than 50% of human cancers. Ubiquitination of p53 is a major regulatory mechanism of p53, however, the deubiquitination of p53 is unclear. We will investigate the regulation of p53 by a novel p53 deubiquitinase USP10, which will provide important insights into the molecular mechanism of p53 regulation and tumor suppression.