The immune system uses granule-mediated apoptosis to eliminate foreign cells. The lethal hit involves delivery of granule associated serine proteases such as granzyme B (GrB) to the target cell cytosol by perforin, a pore forming protein. Perforin is postulated to deliver granzymes by acting as a conduit. Experimental support for this model, however, remains elusive. They have accumulated data supporting the concept that perforin acts intracellularly to deliver the granzymes. In this model, endocytosis of both granzyme and perforin by the target cell are necessary for susceptibility to cytotoxic cell-mediated apoptosis. Subsequently, GrB is released to the cytosol by PFN where the granzyme specifically activates executioner procaspases-3 and -7 through a novel two-step mechanism. Furthermore, related work indicates a target cell is exposed to multiple granzyme molecules complexed to its cognate granule-associated proteoglycan, serglycin. The binding, internalization, intracellular trafficking and proteolytic specificity of GrB-serglycin may differ greatly from the results obtained with the free, cationic granzyme. Conceptually they suggest that granule mediated apoptosis mimics strategies adopted by viruses to enter nucleated cells where PFN delivers a macromolecular signaling complex-an array of granzymes toxically linked to serglycin. The overall goal of our application is to learn whether PFN undergoes endocytosis to deliver GrB and to determine whether GrB-serglycin complexes display the same caspase-activating potential as cytotoxic cells using newly described procaspase-3 deficient MCF-7 cells and a stable transfectant expressing this member.
The Specific Aims are: 1. To show a target cell requires normal endocytic function for susceptibility to PFN/GrB- as well as CTL-mediated apoptosis. 2. To characterize GRB-SG complexes isolated from granules of YT cells. 3. To determine whether isolated PFN or the form secreted by a cytotoxic cell induces apoptosis in a target cell containing only vesicle-associated GrB/SG. 4. To study the vesicular trafficking of GRB-SG complexes. 5. To learn whether delivery of GrB/SG mimics the ordered caspase activation induced by a cytotoxic cell secreting only GrB. The information will provide novel insights to the phenomenon of intracellular protein delivery as well as the biologic function of multimeric enzyme complexes in intracellular proteolysis. Notably, the system described here will clarify for the first time how PFN delivers granule proteins and GrB activates the caspase cascade in whole cells to initiate death program.
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