The trabecular meshwork (TM) at the chamber angle of the eye is the major site for regulation of the aqueous humor outflow. Cells in this tissue are essential for the maintenance of normal aqueous outflow. Aberrations of cell integrity may be a key step toward obstruction of the outflow, intraocular pressure (IOP) elevation, and glaucomatous conditions. Glaucoma, a major cause of blindness in the United States, is a heterogeneous group of diseases generally characterized by IOP elevation, and neural and visual loss. Recent genetic analyses have linked TIGR (Trabecular Meshwork Inducible Glucocorticoid Response) or myocilin gene to both juvenile and adult-onset open angle glaucomas. Multiple mutations were identified. These advances triggered intense interest on myocilin/TIGR. To date, however, the exact properties and functions of this protein remain elusive. It is also unclear why pathology develops with certain mutations in the gene. Preliminary studies from our laboratory revealed the possible association of myocilin/TIGR with mitochondria and cytoskeletal structures. Yeast two-hybrid screening further suggested that myosin light chain-2 may be an interacting partner of myocilin/TIGR. We propose, in this application, to systematically investigate the nature and function of myocilin/TIGR and to evaluate the impact of mutations. We will 1) study the glycosylation and phosphorylation patterns of myocilin/TIGR and produce full length, mutated, and truncated myocilin/TIGR by recombinant DNA techniques for exploration of their binding with extracellular matrix elements; 2) examine the association of myocilin/TIGR with cytoskeletal proteins and mitochondria and its interaction with myosin light chain. Cell biology/molecular biology methods including immunogold labeling, binding and motility assays and microinjections will be used; 3) assess the influence on cell activities such as adhesion, migration, and phagocytosis through over- and under-expression of wild type and mutated myocilin/TIGR in human TM cells; and 4) investigate the modulation of myocilin/TIGR expression in human TM cells after phagocytic challenges, oxidative stress and after cytokine treatments to provide insights into gene regulation. The overall objectives are to provide a better understanding of the functions and protein-protein interactions of myocilin/TIGR and to define the role of this novel molecule in the cellular and biochemical mechanisms in TM cells. The ultimate goal is to reveal the pathogenic processes of glaucomas.
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