Regulation of Ras by Monoubiquitination

描述(由申请人提供)：RAS基因构成了人类癌症中最常见的癌基因突变类别(33%)。它们的蛋白质对应物起到GDP-GTP调节开关的作用，调节控制细胞生长、分化和凋亡的信号网络。突变激活的RAS蛋白不易被GTPase激活蛋白失活，并且持续与GTP结合，导致慢性下游效应器的激活和信号转导。尽管经过了三十年的密集努力，但还没有一种有效的直接针对突变的RAS蛋白的抑制剂成功应用于临床。因此，过去和目前正在进行的许多努力都采取了间接的方法，但都失败了或缺乏抗肿瘤疗效。在这里，我们研究了一种新的RAS激活机制，涉及泛素化的翻译后修饰。最近的发现表明，通过单核苷酸修饰RAS的Covalen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，单素化对固有RAS功能的直接影响是什么？其次，先前的研究表明，当K-RAS在NIH3T3小鼠成纤维细胞中异位表达时，阻止单泛素化会削弱激活的K-RAS促进肿瘤移植瘤生长的能力。在证实对突变K-RAS和正常K-RAS功能上瘾的人结直肠肿瘤细胞中，这种对内源性突变和正常K-RAS的修饰在细胞增殖中有多重要？我们将通过对单素化的RAS进行生化和结构研究，以及对泛素化缺陷的K-RAS进行生物学研究来解决这些问题，以严格评估其作用 在野生型和突变型K-RAS的生物学活性、效应信号和调控方面。

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.