Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an important role in the immunological surveillance against proliferation of tumor cells, virus-infected cells, and other foreign agents. Work performed in this lab has led to the identification/characterization of several families of mediators, one of which is perforin, a major cytolytic pore-forming protein localized in the granules of killer lymphocytes. Upon contact with target cells, lymphocytes exocytose perforin which, in the presence of calcium, assemble into transmembrane lesions with internal diameters averaging 16 nm. Perforin has now been purified to homogeneity and its primary and genomic structures have been dissected by cDNA cloning. The genes for both mouse and human perforin have now been mapped. Perforin was described earlier by this lab as structurally and functionally homologous to the terminal components of the complement cascade, a finding which was confirmed by cDNA cloning. Several biochemical/biophysical features of perforin have already been elucidated. In this renewal grant proposal the following specific aims will be attained. (1) Functional mapping of regulatory elements for mouse and human perforin genes through deletional and mutational analyses of reporter constructs in lymphocyte cell lines. (2) Protein interactions with these regulatory sequences will be studied by DNAse I hypersensitivity assays, footprinting, and gel retardation analyses of IL-2 and PMA-induced cellular extracts. (3) The domain structure of perforin will be studied by defining the pore-forming, self-aggregation, and membrane-spanning and -associated domains using peptides and recombinant perforin as substrates. (4) The role of perforin and granule serine esterases in immunopathologies in vivo will be assessed by a combination of immunohistochemistry and in situ hybridization. (5) Protectin, a surface molecule of lymphocytes that protects these cells from self-mediated lysis, will be purified by affinity chromatography using perforin as the affinity substrate and will be functionally reconstituted and cloned. (6) The technique of homologous recombination in embryonic stem cells will be used to generate perforin negative mice which will provide powerful tools in assessing the physiological role/relevance of perforin.
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