A Cell-Based HT Imaging Assay for Palmitoylation(RMI)

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DESCRIPTION (provided by applicant): Many proteins are concentrated on the plasma membrane at specialized subcellular regions like synapses and caveolae by virtue of their lipid modifications. S-palmitoylation, a common form of lipid modification, is unique in that it is reversible and dynamic, suggesting a modulatory role in signal transduction similar to phosphorylation. Addition of this lipid moiety may alter 1 or more properties of the recipient protein such as trafficking to a membrane, redistribution specialized microdomains within a membrane, protein-protein interactions, and enzymatic activity. Very recently a putative signature motif for enzymes capable of catalyzing the addition of paimitate to proteins (palmitoyl acyl transferases or PATs) has been identified. Genes encoding putative PATs have been linked to human diseases including schizophrenia and Huntington's disease but further proof will be required to show that the proteins encoded by these genes are bona fide PATS. Unfortunately, pharmacological tools to study palmitoylation are almost non-existent. Those used most commonly are poorly characterized in terms of their efficacy, toxicity and mechanism of action leaving a conspicuous hole in our ability to characterize and understand fully palmitoylation-related phenomena. The key to discovering and developing such reagents lies in the creation and validation of a robust and reliable high-throughput screening assay that can be used to screen a large compound library. We propose to fill this need by designing and validating a cell-based, fluorescent, high throughput imaging assay using a state-of-the-art high throughput imaging workstation and a novel image analysis algorithm. We will exploit the predominant characteristic of palmitoylation, i. e. its ability to cause a protein to be localized to the cellular plasma membrane. By simultaneously quantifying the subcellular distribution of multiple protein based reporters we will be able to derive important information about the molecular mechanism of action as well as the efficacy of compounds that modulate palmitoylation.