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

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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.