It is apparent that MARCKS protein is a major signaling molecule in a variety of cellular functions. In the ung, MARCKS serves as a crossbridge between stimulation at the cell surface and subsequent secretion of mucin from epithelial secretory cells. MARCKS also can serve as a mediator of leukocyte degranulation as well as leukocyte migration, suggesting it also has an important role in inflammation. One of the key findings of the previous funding period is that it appears to be the N-terminus of MARCKS that is involved in these functions, as a peptide identical to the MARCKS N-terminus has potent effects on these parameters when cells are treated with it. The studies in the next funding period will now investigate and further nail down the exact molecular mechanism(s) by which MARCKS interacts with other cellular proteins in each of these cell types in order to carry out its function. We will use some new imaging techniques as well as develop new methods to assay for secretion and epithelial function. We plan to use proteomic approaches to identify novel proteins associated with mucin granule membranes and investigate their interactions with MARCKS during the secretory process. We also plan to expand our collaborations with investigators at Duke Medical enter to move into translational studies utilizing airway epithelial cells derived from patients with asthma, COPD and CF and the role(s) of MARCKS in secretion by these individuals. In addition, we anticipate moving to several in vivo models of mucus hypersecretion, airway inflammation and cell migration to show that these mechanisms are operative in vivo and thus could represent some important new therapeutic targets. Relatedly, the aims of the next period are: I. Determine the specific protein-protein interactions that influence MARCKS protein function in mucin secretion in vitro and in vivo. II. Determine the role of MARCKS in neutrophil migration and inflammation. . Initiate translational studies utilizing human cells from donors with asthma and COPD, with and without viral infections.
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