Cytotoxic cell granule-mediated apoptosis is a unique form of cell signaling that entails the intracellular delivery of granule-associated serine proteases (granzymes) by the pore forming protein perforin. The identification of genetic defects in the granule secretion pathway have clearly established that granule- mediated apoptosis is crucial for immune homeostasis. Acquired disruption of cytotoxic cell effector function by viral proteins are likely to trigger lymphoproliferation and autoreactivity as well as diminish host defense responsiveness. A virus-infected or tumor cell may also resist granule mediated apoptosis by inhibiting virtually any aspect of this pathway. Delineating the multiple steps of this poorly understood phenomenon has become increasingly important to comprehend the immunopathogenesis of a number of disease states. To achieve an understanding of granzyme delivery by perforin requires molecular characterization of the most proximal steps - the events that surround the binding, internalization and early trafficking of these granule components. Granzyme B remains bound to the granule-associated proteoglycan, serglycin, after secretion by a cytotoxic cell. Unexpectedly, the granzyme undergoes electrostatic exchange from serglycin to target cell sulfated glycosaminoglycans which then facilitate binding to undefined receptor(s) for optimal internalization. In addition, the granzyme appears to manifest membranolytic activity if a sufficient endosomal concentration is achieved. Entitled the conjoined Perforin-Granzyme delivery hypothesis - both perforin and granzymes are predicted to undergo electrostatic exchange to the cell surface from serglycin and together contribute to endosomolytic delivery process. Extending previous efforts to the glycobiology of granzyme B-cell surface interactions and emphasizing imaging techniques, particularly new methods that directly visualize the interaction of the granzyme and perforin with target cells, the Specific Aims are: 1. Study the transfer of granzyme B from serglycin to the target cell surface 2. Ascertain role of perforin in granzyme delivery - a molecular analysis 3. Assess plasma membrane and endosomal integrity of the target cell during cytotoxic cell-mediated apoptosis

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Cellular and Molecular Immunology - B (CMI)
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Leitner, Wolfgang W
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Northshore University Healthsystem
United States
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Metkar, S S; Marchioretto, M; Antonini, V et al. (2015) Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes. Cell Death Differ 22:74-85
Froelich, Christopher; Metkar, Sunil (2011) Intracellular trafficking of perforin: to thwart a killer. Immunity 34:823-5
Martin, Praxedis; Pardo, Julián; Schill, Natalie et al. (2010) Granzyme B-induced and caspase 3-dependent cleavage of gelsolin by mouse cytotoxic T cells modifies cytoskeleton dynamics. J Biol Chem 285:18918-27
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Froelich, C J; Metkar, S S; Raja, S M (2004) Granzyme B-mediated apoptosis--the elephant and the blind men? Cell Death Differ 11:369-71
Mackewicz, Carl E; Wang, Baikun; Metkar, Sunil et al. (2003) Lack of the CD8+ cell anti-HIV factor in CD8+ cell granules. Blood 102:180-3
Metkar, Sunil S; Wang, Baikun; Ebbs, Michelle L et al. (2003) Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis. J Cell Biol 160:875-85
Odeberg, Jenny; Browne, Helena; Metkar, Sunil et al. (2003) The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins. J Virol 77:4539-45
Vermijlen, David; Luo, Dianzhong; Froelich, Christopher J et al. (2002) Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway. J Leukoc Biol 72:668-76
Metkar, Sunil S; Wang, Baikun; Aguilar-Santelises, Miguel et al. (2002) Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 16:417-28

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