Using Chemical Biology to Study the Small GTPase Ra(RMI)

描述(由申请人提供)：该项目的目标是开发将应用于高通量小分子筛查的分析方法，旨在针对小GTP酶RAN及其相关蛋白进行设计。Ran调节间期核质运输，最近研究表明，Ran还调节有丝分裂中的关键事件，如纺锤体组装、核膜组装和核孔复合体组装。在间期和有丝分裂中，RanGTP在染色体附近富含。在通过核孔导入的最后阶段，RanGTP与货物受体Importin-beta结合，导致货物分子释放到核质中。RanGTP很可能在有丝分裂过程中以类似的方式发挥作用，导致Importin-beta释放在染色质附近具有重要有丝分裂活动的货物。越来越多的证据支持这一假设，这些证据来自于使用非洲爪哇卵提取液进行的实验，然而，关于这一过程如何在活细胞中发挥作用的细节却知之甚少。此外，RAN很可能在有丝分裂中调节尚未确定的事件。我们的目标是使用化学生物学来识别靶向RAN途径的小分子。我们提出了以下具体目标：目的1)优化筛选程序，以确定破坏RAN和运输受体Importin-beta之间相互作用的小分子。目的2)建立一种用于高通量筛选的方法来鉴定抑制RanGAP的化合物，RanGAP是一种调节RAN核苷酸状态的蛋白质。目的3)将非洲爪哇卵提取系统改造成384孔的形式，用于高通量筛选，以识别RAN途径中的新靶点。我们的每一种分析方法都利用了荧光共振能量转移(FRET)。在涉及这些检测的高通量筛选中，我们将根据抑制剂影响FRET信号的能力来选择它们。我们的分析既依赖于纯蛋白质溶液，也依赖于复杂的提取物，每一种都有可能发现不同类型的抑制剂。在这些筛选中发现的抑制物将成为剖析活细胞有丝分裂过程中RAN途径作用的宝贵工具。使用高通量方法识别的小分子可以被开发用于治疗人类疾病。由于RAN途径被认为在有丝分裂中起着重要的调节作用，因此破坏这一途径的抑制剂将有可能被开发为治疗癌症的药物。

DESCRIPTION (provided by applicant): The goal of this project is to develop assays that will be applied to high throughput small molecule screens designed to target the small GTPase Ran and its associated proteins. Ran regulates nucleocytoplasmic transport during interphase and recently it has been demonstrated that Ran also regulates key event in mitosis such as spindle assembly, nuclear envelope assembly and nuclear pore complex assembly. In interphase and in mitosis RanGTP is enriched near the chromosomes. In the final stage of import through a nuclear pore, RanGTP binds the cargo receptor importin-beta, causing the release of cargo molecules into the nucleoplasm. It is likely that RanGTP functions in a similar manner during mitosis by causing importin-beta to release cargoes with important mitotic activities in the vicinity of chromatin. There is a growing body of evidence to support this hypothesis from experiments performed using extracts from the eggs of Xenopus laevis, however the details of how this process works in living cells are poorly understood. In addition, it is likely that Ran regulates events in mitosis that have yet to be identified. Our goal is to use chemical biology to identify small molecules that target the Ran pathway. We are proposing the following specific aims: Aim 1) To optimize a screening procedure to identify small molecules that disrupt the interaction between Ran and the transport receptor importin-beta. Aim 2) To develop an assay for use in a high throughput screen to identify compounds that inhibit RanGAP, a protein that regulates the nucleotide state of Ran. Aim 3) To adapt the Xenopus laevis egg extract system to a 384-well format for use in a high throughput screen to identify novel targets in the Ran pathway. Each of our assays takes advantage of fluorescence resonance energy transfer (FRET). In high throughput screens involving these assays we will select inhibitors based on their ability to affect the FRET signal. Our assays rely on both pure protein solutions and complex extracts, each of which has the potential to uncover different types of inhibitors. Inhibitors identified in these screens would serve as invaluable tools for dissecting the role of the Ran pathway during mitosis in live cells. Small molecules identified using high throughput approaches can be developed for the treatment of human disease. Because the Ran pathway is thought to plav an important regulatory role in mitosis, inhibitors that disrupt this pathway would have the potential to be developed as therapeutics for the treatment of cancer.