Single Cell Analysis of EGFR Activity in Human Airway Cells Exposed to Pollutants

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DESCRIPTION (provided by applicant): Cardiovascular disease is the leading cause of death in the US for both men and women, causing 34% of deaths in 2006. Studies have shown that the small particles released into the air by processes such as diesel fuel combustion can deposit in the airways and lead to increases in blood pressure, changes in heart rhythm, and significantly increased risk of heart attack and stroke. While the mechanism underlying these health effects is not fully understood, airway inflammation seems to play an important role. The epidermal growth factor receptor (EGFR) is a cell surface protein that helps regulate cell growth and response to external stimuli, including the inflammatory response. While it is thought that EGFR plays an important role in lung inflammation due to pollutant inhalation, direct measurements of EGFR activity in live human airway samples are not currently feasible. This research will develop a new technology capable of measuring EGFR activity that will permit analysis of such samples, and will provide important information about the link between air pollution, lung inflammation, and heart disease. The technology developed in this proposal involves putting a small, fluorescent protein fragment, or peptide, into living cells, allowing it to be modified by EGFR by the addition of a phosphate group (phosphorylation), and then measuring the rate of this process using capillary electrophoresis, which allows the separation and measurement of molecules based on differences in size and charge. The aim of this project is to compare EGFR activity in healthy cells and those exposed to diesel exhaust particles, first using cells grown in the lab, then in biopsy specimens removed directly from human subjects, which was not previously feasible due to the small number of living cells that can be obtained. The technique proposed is capable of analyzing single cells, making it ideally suited to such an application. To effectively measure EGFR activity in cells, the peptide will need to be efficiently phosphorylated by EGFR but not related enzymes. Also, it must be able to resist degradation by enzymes within the cell known as proteases, which break down proteins for recycling and to eliminate foreign bodies. To assure these requirements are met, the structure of the peptide will be modified using an established library-based method. This project will solidify the link between air pollution and lung inflammation leading to heart disease, and provide strong evidence to support environmental regulations that are in the best interest of human health. Additionally, once developed, this technique will also be readily adapted to other areas of research, including cancer and infectious disease.