PROJECT SUMMARY Chronic obstructive pulmonary disease (COPD) is a growing health concern in the United States with no curative treatments. The development of new therapeutics has been stagnant due to the difficulty of finding new essential biology and protective pathways in the complex tissue of the human lung. Hence, the Robinson lab was interested in using a model organism, the social amoeba Dictyostelium discoideum, as a discovery tool to find new therapeutic targets and pathways that will protect against cigarette smoke (CS), one of the main causes of COPD. With Dictyostelium, a genetic screen was conducted to find these target genes. Overexpression of two genes encoding for adenine nucleotide translocase (ANT) and actin-interacting protein 1 (AIP1) offered the most robust protection in cell growth. Interestingly, we see the same protective effects from these genes in human bronchial epithelial cells exposed to CS. The focus of this proposal will be to mechanistically understand how these proteins negate the effects of CS injury. Beginning with ANT, an ATP/ADP transporter in the inner membrane of the mitochondria, we expected that its overexpression would enhance cellular metabolism. Interestingly, some preliminary data suggested that ANT was protective through different mechanisms. The canonical mitochondria protein was surprisingly found at cilia and modulated ciliary function, which is known to be altered by CS. In ciliated primary human bronchial epithelial cells (NHBEs), ANT2 (one of the paralogs of ANT) enhanced ciliary function by increasing airway hydration and maintaining normal ciliary beat frequency in the presence of CS. Based on this preliminary data and the idea that extracellular ATP is released to increase airway hydration, we hypothesize that ANT is one of the elusive cell surface transporters of extracellular ATP. This idea will be tested in aim 1 of this proposal through immunofluorescence and super-resolution imaging, surface biotinylation assays, and the measurement of extracellular ATP on ANT gain- or loss- of function NHBEs. Since protective phenotypes of ANT were found, a preliminary drug screen will also be conducted to find activators of ANT. In aim 2, we will focus on AIP1. We will similarly find how its overexpression protects against CS. CS was found to affect actin dynamics and cellular mechanics, which caused increased airway barrier permeability. Considering its role as a regulator of actin depolymerization, we expect that AIP1 will negate the effects of CS on actin dynamics, which will tighten cell-cell interactions and fortify airway barrier function. Experiments to study this will include cytoskeletal fractionation to evaluate actin assembly via F/G-actin ratios and confocal imaging to assess whether AIP1 changes the expression and localization of cell junction proteins. Trans-epithelial resistance (TEER) measurements and a FITC-dextran permeability assay will be used to assess epithelial barrier tightness. ...