Ras, Cycling and Inhibition.

描述（由申请人提供）：gtp结合蛋白是调节关键细胞功能（如生长和分化）的信号通路中的关键开关。Ras作为gtp结合的原型，在包括胰腺、膀胱癌、结肠癌和肺癌在内的许多癌症中被组成性激活。这项研究的长期目标是通过了解其循环来干扰人类疾病中Ras的致癌形式。由于致癌Ras依赖于其内在的水解和交换GTP的能力，因此本研究计划的主要目标是通过揭示GTP水解和鸟嘌呤核苷酸交换反应的中间体结构来更好地了解Ras在活性和非活性状态之间的循环。筛选稳定非信号构象的致癌Ras的药物也将进行。这一建议是基于这样的发现：改变铰链区域的柔韧性可以稳定通常是短暂的Ras结构。利用这种方法，GTP水解路径上的两个中间体结构以及一个开放的非信号构象被稳定下来，这也是天然蛋白所采用的。本提案的第一个目的是测试Ras的开放构象模拟核苷酸交换中间体的结构和未被发现的天然构象的结构-e的假设。通过改变另一个铰链区域的柔韧性，可以产生Ras循环的额外中间体结构。生成的突变体将使用结构、分子动力学、生化和体内技术的组合进行研究。第二个目的是验证Rho-家族成员和三聚体g蛋白不遵循GTP水解和核苷酸交换的Ras路径的假设，尽管序列同源性很强。第三个目标是使用生物物理、结构和细胞方法来研究通过筛选NCI数据库识别的小分子如何与开放的非信号构象相互作用，并抑制胰腺癌模型系统中的致癌Ras。拟议研究的结果将提高我们对关键信号蛋白调控的理解，以及我们干预它们在人类疾病中的作用的能力。

DESCRIPTION (provided by applicant): GTP-binding proteins are key switches in signaling pathways that regulate critical cellular functions such as growth and differentiation. Ras, which serves as a prototype for GTP-binding is constitutively activated in a large number of cancers including those of the pancreas, bladder, colon, and lung. The long-term goal of this research is to interfere with the oncogenic forms of Ras in human disease by understanding its cycling. Because oncogenic Ras relies on its intrinsic ability to hydrolyze and exchange GTP, the primary goal of this research proposal is to better our understanding of Ras cycling between the active and inactive states by revealing structures of intermediates of the reactions of GTP hydrolysis and guanine nucleotide exchange. Screening of drugs that will stabilize a non-signaling conformation of oncogenic Ras will be also performed. This proposal is based on findings that altering the flexibility of a hinge region stabilizes structures of Ras that are normally transient. Using this approach, two structures of intermediates along the path for GTP hydrolysis were stabilized as well as an open non-signaling conformation, which is also adopted by the native protein. The first aim of this proposal tests the hypothesis that the open conformation of Ras mimics the structure of an intermediate for nucleotide exchange and the structu-e of an unappreciated native conformation. Additional structures of intermediates for Ras cycling will be generated by altering the flexibility of another hinge region. The generated mutants will be studied using a combination of structural, molecular dynamics, biochemical, and in vivo techniques. The second aim tests the hypothesis that Rho- family members and trimeric G-proteins do not follow the Ras path of GTP hydrolysis and nucleotide exchange despite strong sequence homology. The third aim uses biophysical, structural, and cellular approaches to study how small molecules identified by screening the NCI database interact with the open non-signaling conformation and inhibit oncogenic Ras in a pancreatic cancer model system. The outcome of the proposed research should improve our understanding of the regulation of key signaling proteins and our ability to interfere with their action in human diseases.