T and natural killer (NK) lymphocyte-mediated killing involves release of granules containing lethal proteins. One granule protein, perforin, assembles into pores that lead to cell death. The granules also contain serine- dependent proteases termed granzymes. The PI's laboratory studies the granzymes that function in pore formation, Chymase 1 and Tryptase 3. These granzymes must have at least one natural substrate involved in lysis. Perforin is not a likely substrate. Hudig et al. have discovered another granule protein called perforin enhancing protein (PEPr) that enables fixed amounts of perforin to lyse more cells. PEPr is nonlytic by itself and is effective only when granzymes are present. Its bioactivity makes it an enticing candidate to be a substrate that links granzymes to perforin lysis. Their hypothesis is that PEPr requires proteolytic processing to enhance pore formation and that processing is mediated by Chymase 1 and by Tryptase 3. PEPr may be the rat equivalent of granulysin, a lipid-binding protein of human T cell granules that is naturally processed at chymase and tryptase sites. The PI's laboratory has purified native components of lysis: granzymes, perforin and PEPr. The principal investigator has obtained recombinant perforin and has expressed active recombinant granzymes.
The specific aims are:
Aim 1, to biochemically characterize PEPr and to evaluate granzyme activation.
Aim 2, to purify trypase 3 and determine its role in perforin lysis;
Aim 3, to match chymase 1 with its cDNA clone and to obtain the cDNAs for trypase 3 and PEPr-ases, in order to express these granzymes;
Aim 4, to express recombinant granzymes as active enzymes to use as components of lysis;
Aim 5, to isolate and characterize natural substrates of chymase 1 and trypase 3, if appropriate. The goal is to reconstitute the perforin system of lysis using native components from rat PNK-16 cell granules and then replace each component with its recombinant protein. This research provides a scientific foundation for development of new immunosuppressive drugs.
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