Novel Functions for Ras family GTPases

项目摘要： Ras蛋白在生物学的许多方面发挥着核心作用。这些单体GTPases在一个 一个是GDP绑定状态，另一个是GDP绑定状态。Ras的突变激活发生在 大约30%的具有某些癌症（如胰腺癌）的人类肿瘤在其基因组中具有Ras突变。 >90%的肿瘤。这些与癌症相关的突变将Ras捕获在GTP结合状态，导致慢性炎症。 Ras调节的信号通路的激活和致癌转化。因此，RasGTP已被 被认为是该分子唯一具有生物学意义的形式。然而，我们发现了一个新的角色， 无核苷酸Ras（nfRas）在细胞信号传导的负调控中的作用。这种形式的Ras，形成为 从RasGDP到RasGTP的过渡中间体，负调节磷脂酰肌醇3-激酶， 2 β型（PI 3 KC 2 β）。这些发现对于理解Ras介导的 信号和转换。我们提出nfRas结合靶点，例如，PI 3 KC 2 β，导致相互 抑制Ras和相关靶标。我们的模型预测Ras的致癌激活导致 这些靶点的抑制丧失，导致它们在不结合RasGTP的情况下活化。因此，我们的研究结果表明， 对于Ras驱动的肿瘤发生重要但不结合激活的Ras的一类新分子。更好地 为了了解nfRas在细胞信号传导中的作用，我们已经产生了高亲和力，高特异性的单抗体， 选择性结合不同状态的Ras的试剂。使用这些试剂，我们证明nfRas 代表总细胞Ras的显著池。此外，我们的Ras单体之一，它结合所有 Ras的三种形式（GTP、GDP和无核苷酸）作为致癌Ras的高效抑制剂发挥作用， 介导的信号传导。使用这些试剂，我们将表征nfRas在体内的调节，并确定nfRas在体内的表达。 nfRas在调节特定靶点中的作用。最后，我们将利用这些新的单体试剂来干扰 利用一种新的化学-遗传学方法干扰胰腺癌发展的体内Ras功能 和进步。鉴于Ras突变在人类癌症中的普遍性，了解Ras突变在癌症中的作用至关重要。 Ras参与肿瘤发生的机制。我们的工作代表了一个全新的概念 在Ras生物学中，它定义了一类参与Ras介导的信号传导的新靶点， 转型这些研究将为Ras介导的肿瘤发生机制提供新的见解 因此具有很高的翻译意义。这项工作将有利于退伍军人以及 一般人群，两者都患有癌症。然而，某些癌症的发病率，如肺癌， 胰腺癌Ras突变发生率高（<90%），据报道， 在退伍军人群体中高达5- 7倍，使这些研究与退伍军人特别相关。此外，本发明还 我们建议的目标与最近建立的国家癌症登月计划是一致的， 加快研究工作，以改善癌症治疗。最后，考虑到Ras家族成员在中国的普遍存在， 真核生物及其在生物学许多方面的作用，这些结果将具有广泛的科学意义， 对许多生物医学研究领域的重要性。

Project Summary: Ras proteins play a central role in many aspects of biology. These monomeric GTPases cycle between an inactivate GDP-bound state and an active GTP-bound state. Mutational activation of Ras occurs in approximately 30% of human tumors with some cancers such as pancreatic cancer having Ras mutations in >90% of tumors. These cancer associated mutations trap Ras in the GTP-bound state, leading to chronic activation of Ras-regulated signaling pathways and oncogenic transformation. Thus, RasGTP has been considered the only biologically important form of the molecule. However, we have discovered a novel role for nucleotide-free Ras (nfRas) in the negative regulation of cell signaling. This form of Ras, formed as an intermediate in the transition from RasGDP to RasGTP, negatively regulates phosphatidylinositol 3-kinase, class 2beta (PI3KC2β). These finding have profound implications for the understanding of Ras-mediated signaling and transformation. We propose that nfRas binds targets, e.g., PI3KC2β, leading to the mutual inhibition of both Ras and the associated target. Our model predicts that oncogenic activation of Ras leads to loss of repression of these targets resulting in their activation without binding RasGTP. Thus, our findings point to a new class of molecules important for Ras-driven tumorigenesis yet do not bind activated Ras. To better understand the role of nfRas in cell signaling, we have generated high affinity, high specificity monobody reagents that selectively bind different states of Ras. Using these reagents we demonstrate that nfRas represents a significant pool of total cellular Ras. In addition, one of our Ras monobodies which binds to all three forms of Ras (GTP, GDP, and nucleotide free) acts as a highly potent inhibitor of oncogenic Ras- mediated signaling. Using these reagents we will characterize the regulation of nfRas in vivo and determine the role of nfRas in regulating specific targets. Finally, we will utilize these novel monobody reagents to perturb Ras function in vivo using a novel chemical-genetic approach to interfere with pancreatic cancer development and progression. Given the prevalence of Ras mutations in human cancers, it is critical to understand the mechanisms through which Ras contributes to tumorigenesis. Our work represents an entirely new concept in Ras biology that defines a new class of targets that participate in Ras-mediated signaling and transformation. These studies will provide new insights into the mechanisms of Ras-mediated tumorigenesis and are therefore of high translational significance. This work will be beneficial to Veterans as well as the general population, both of which suffer from cancer. However, the incidence for certain cancers, such as lung and pancreatic cancer the latter of which has a high incidence of Ras mutations (<90%), has been reported to be up to 5-7x higher in Veteran populations making these studies particularly relevant to Veterans. Additionally, the goals of our proposal are consistent with the recently established National Cancer Moonshots Program to accelerate research efforts to improve cancer treatment. Finally, given the ubiquity of Ras family members in eukaryotes and their roles in many aspects of biology these results will be of broad scientific interest and importance to many areas of biomedical research.