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IDENTIFICATION AND CHARACTERIZATION OF HUMAN AIRWAY EPITHELIAL CELL PROTEINS PHOSPHORYLATED IN RESPONSE TO PARTICULATE MATTER (PM) EXPOSURE.
Impact/Purpose:
Specifically, this work will support efforts to (1) Identify mechanisms of toxicity for particulate matter (PM) constituents/sources and (2) Define the molecular basis of transition metal-induced effects in human lung cells.
Description:
Multiple studies conducted by NHEERL scientists in recent years have shown that acute exposure to metals found associated with combustion-derived particulate matter (PM) alters phosphoprotein metabolism in human airway epithelial cells causing intracellular signaling. This disregulation of tyrosine, serine and threonine phosphorylation is believed to be the result of phosphatase inhibition and, possibly, kinase activation. Mechanistic details of the interaction between metal ions with kinases and phosphatases are currently the subject of intense investigation by researchers on both sides of the HSD-CEMLB Co-op. A major challenge faced by researchers in the field of intracellular signaling is the identification of proteins that serve as substrates for phosphorylation by kinases. This is due in part to the sheer number of potential protein targets available for phosphorylation that are known, as well as a larger number that is believed to be undiscovered. Direct methods for the screening and identification of proteins that undergo post- translational modification in response to cellular activation have benefitted significantly from recent technological advances in the field of mass spectrometry, Edman sequencing and the advent of phospho-state-specific antibodies. New approaches now permit the use of Aprotein chip@ technology to screen for changes in protein modification with greatly improved efficiency. Spectroscopic techniques that until recently were the exclusive domain of chemistry departments are now available in a friendlier form to the non-specialist. In addition, an increasing number or biotech companies offer an array of kits, antibodies and pre-packaged reagents that greatly facilitate protein research. This project is aimed at identifying and characterizing human airway epithelial cell proteins that are phosphorylated in response to exposure to PM components such as metallic ions. The approach will be to expose transformed cells (BEAS) or primary cultures of cells (grown on plastic or air-liquid interface) to relevant PM stimuli, extract cellular proteins, purify/enrich proteins with the characteristic of interest (e.g., tyrosine phosphorylation) and subject them to analysis. The analytical methods to be used include 1- and 2-dimensional protein electrophoresis, affinity chromatography, HPLC, chemical and enzymatic protein and peptide digestion, SELDI mass spectrometry, time-of-flight mass spectrometry, tandem quadruple mass spectrometry and Edman sequencing. We expect that this work will result in the identification of novel proteins that are phosphorylated upon exposure of the cell to PM, and thereby produce information that complements current pathway-specific projects on mechanisms of PM toxicity in the human lung.