对Ras蛋白亚型特异性信号传递分子机制的研究

RAS, cycling between active and inactive states by binding to GTP or GDP, is a key switch molecule in the cellular signaling pathway. Some overactive Ras mutants keep signaling pathway on and drive tumorigenesis. Human has four Ras isoforms (Hras, Nras, Kras4a, and Kras4b), which are co-expressed in most cells, with highly overlapping upstream and downstream proteins. Moreover, they are very much similar in structure. These could well offer functional redundancy among Ras isoforms but can be hardly used to explain their specificity in signaling. Recent studies have found that the cancers related to Ras mutations have a high correlation with Ras isoforms, which strongly suggests the specificity of Ras isoforms in signaling. Therefore, exploring the differences in structure and function among Ras isoforms has become a crucial task. In cells, the function of signaling protein is affected by the micro-environment, so it is essential to study those proteins with their intact structures under near-physiological conditions. In this project, we will combine a variety of biochemical methods to prepare samples and use NMR techniques to analyze the conformational dynamics of Ras isoforms on the membrane, and detect how Ras isoforms interact with their effectors. Ultimately, we will explore the mechanism of Ras protein isoform-specific signaling at the atomic level, and contribute to the theoretical foundation for the research of Ras-related cancer.

Ras蛋白通过与GDP或GTP结合来转换活化状态，是细胞信号通路中的关键开关分子。一些持续处于激活状态的Ras突变体会造成信号通路持续开启从而导致癌症发生。人体中有四种Ras蛋白亚型，它们在细胞中共表达；上下游互作蛋白重叠；分子结构也高度相似。这些因素为Ras亚型间功能的可替代性提供了条件，却给解释亚型特异性信号传递带来了问题。近年来的研究不断发现特定癌症类型的发生往往与Ras蛋白亚型的突变有特异性关联，这强烈暗示了Ras各亚型在信号传递上所具有的特异性。因此探究Ras亚型间结构与功能的差异成为一个重要的研究方向。在细胞中，蛋白的信号传递受到其环境的影响，因此在更接近生理条件下研究拥有完整结构的蛋白尤为重要。本项目将结合多种生化手段制备样品并运用核磁技术分析Ras在膜上的构象动力学以及与效应因子的相互作用，从原子水平探索其特异性信号传递的机制，为Ras相关癌症的研究奠定重要的理论基础。

Ras蛋白是癌症研究中的热点蛋白，其在信号传递过程中的非正常活跃度导致多种癌症的发生。由于Ras蛋白不易被小分子结合的结构特征，到目前为止都没有筛选出足够多的药物候选分子。在我们前期的研究中，我们发现用液态核磁动力学手段观测Ras蛋白在微秒制度上的动态特征可以找到隐藏的小分子结合口袋，这为通过计算方法筛选Ras蛋白的药物起到了巨大作用。但是由于Ras蛋白亚型间HVR区域的差异导致细胞膜可能对Ras蛋白亚型的结构动力学产生影响，因此补充Ras蛋白亚型之间的动力学差异以及获取生物膜对蛋白质结构的影响对理解Ras信号传递的机制以及完善Ras药物设计模型都有重要意义。本研究的结果部分得证明了膜系统对Ras亚型的结构影响具有显著差异，同时也从Raf 及 PI3K两个下游因子与Ras蛋白的结合强度及Ras蛋白亚型的GTP水解速度的数据中找到了Ras蛋白亚型特异性信号传递机制的线索。