Tear proteins play key roles in combating viral and bacterial infections and in supporting cornea and conjunctiva. A primary function of the acinar cells of the lacrimal gland is the production and release of a variety of proteins including hormones, lactoferrin and lysosomal hydrolases into tear fluid. Many of these proteins are stored in large sub-apical secretory vesicles which release their contents at the apical membrane once appropriate intracellular signaling pathways have been activated. We have found that transduction of primary cultured rabbit lacrimal acini with adenovirus serotype 5 (Ad5) vector decreases the secretagogue-stimulated release of tear proteins in parallel with depletion of rab3D-enriched mature secretory vesicles in the sub-apical cytoplasm. The Ad5 capsid proteins, penton and fiber, mediate virus binding and endocytosis through interactions with `v integrins and coxsackievirus and adenovirus receptor, respectively. Integrin ligation is associated with major changes in intracellular signaling pathways including those which may regulate biosynthetic/secretory membrane traffic. Penton proteins may also participate in the intracellular trafficking of internalized virus by recruitment of host membrane trafficking proteins, diminishing the availability of these proteins for other essential membrane trafficking functions.
Two specific aims are proposed utilizing primary cultured rabbit lacrimal acini as our experimental system: 1) Is Ad5-mediated impairment of lacrimal acinar secretion caused by capsid proteins and 2) Is Ad5-mediated impairment of lacrimal acinar secretion due to sequestration of clathrin and adaptor proteins by the penton dileucine motif? These unique hypotheses will be tested using recombinant assembly-competent wild type and mutant Ad5penton and fiber proteins, and will utilize a variety of techniques including confocal fluorescence and electron microscopy analysis of secretory vesicle content, subcellular membrane fractionation, immunoprecipitation and measurements of stimulated protein secretion. Considerable recent interest has focused on ocular gene therapy using Ad5 or Ad5-derived materials. However, exposure of the lacrimal gland to Ad-derived materials may severely compromise its ability to maintain adequate protein secretory capacity. The studies proposed here will be essential in evaluating the feasibility of ocular gene therapy using Ad-derived materials and in improving the safety of such delivery systems.
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