Regulation of Ras by Monoubiquitination

描述（由申请人提供）：RAS基因是人类癌症中最常见的致癌基因突变类型（33%）。它们的对应蛋白作为GDP-GTP调节开关，调节控制细胞生长、分化和凋亡的信号网络。突变激活的Ras蛋白难以被GTP酶激活蛋白失活，并且持续与GTP结合，导致慢性下游效应物激活和信号传导。尽管三十年的密集努力，没有有效的抑制剂直接针对突变的Ras蛋白已成功地达到临床应用。因此，过去和目前正在进行的许多努力都采用间接方法，这些方法失败或缺乏抗肿瘤功效。在此，我们研究了一种涉及翻译后泛素化修饰的Ras激活新机制。最近的研究发现，通过单泛素化修饰Ras的共价蛋白可能是其驱动人类肿瘤发生的关键，这为开发直接Ras抑制剂提供了一个可能的方向。要严格评估这种可能性，需要解决两个关键问题。首先，单泛素化对内在Ras功能的直接影响是什么？其次，先前的研究表明，当NIH3T3小鼠成纤维细胞异位表达活化的K-Ras时，阻止单泛素化会损害其促进肿瘤异种移植物生长的能力。这种对内源性突变K-Ras和正常K-Ras在细胞增殖中具有依赖性的人类结直肠肿瘤细胞的修饰有多重要？我们将解决这些问题，通过对单泛素化Ras进行生化和结构研究，以及对泛素化缺陷K-Ras进行生物学研究，以严格评估其作用

DESCRIPTION (provided by applicant): RAS genes comprise the most commonly mutated class of oncogenes in human cancer (33%). Their protein counterparts function as GDP-GTP regulated switches to modulate signaling networks that control cell growth, differentiation and apoptosis. Mutationally activated Ras proteins are refractory to inactivation by GTPase activating proteins, and are persistently GTP- bound, leading to chronic downstream effectors activation and signaling. Despite three decades of intensive effort, no effective inhibitors that directly target mutant Ras proteins have successfully reached clinical application. Therefore, much of the past and current ongoing efforts have taken indirect approaches, which have met with failure or lack of anti-tumor efficacy. Herein, we investigate a novel mechanism of Ras activation involving posttranslational modification by ubiquitination. Recent findings that covalen modification of Ras by monoubiquitination may be key for its ability to drive human oncogenesis, suggest one possible direction for developing direct Ras inhibitors. To rigorously assess this possibility, two key issues need to be addressed. First, what are the direct consequences of monoubiquitination on intrinsic Ras function? Second, the previous study showed that preventing monoubiquitination impaired the ability of activated K-Ras to promote tumor xenograft growth when ectopically- expressed in NIH3T3 mouse fibroblasts. How important is this modification for endogenous mutant and normal K-Ras in human colorectal tumor cells with validated addiction to mutant K- Ras and normal K-Ras function in cell proliferation? We will address these issues, by performing biochemical and structural studies on monoubiquitinated Ras as well as biological studies with ubiquitination-deficient K-Ras to rigorously assess its role in wild type and mutant K-Ras biological activity, effectors signaling and regulation.