The long term objective of Dr. Hudig's research is to determine how serine proteases control lymphocyte-mediated killing. She hypothesizes that the proteases cleave their natural substrates during calcium initiation of granule-mediated killing and that these natural substrates regulate perforin damage.
The specific aims of the project are: 1) To isolate, to characterize biochemically, and to inhibit each serine protease of rat RNK-16 granules, focusing particularly on the chymases required for lysis. Percoll gradients will be used to obtain RNK-16 leukemia cell granules. Isoelectric focusing will remove proteoglycan while cation exchange and heparin, benzamidine and other affinity columns will be used to separate proteins. The Mr, pI, N terminal amino acid sequence, substrate subsite map and inactivation rates with irreversible inhibitors will be determined for each protease. 2) To complete nucleotide sequencing cDNA clones of the serine proteases of rat RNK-16 cells, new clones will be sequenced and additional genes will be isolated in addition to a new, NK associated serine protease already sequenced by the applicant. Derived amino acid sequences will be used for protease identification, prediction of substrate binding sites, and comparison with reported T cell proteases. 3) To block lysis of proteases with cytolytic function with antibodies to the protease S' substrate binding sites. Granule proteases vary uniquely in a 13-15 residue region that binds the part of a protein substrate which becomes the new amino terminus after cleavage. Antibodies to S' peptides should prevent protease access to protein substrates and slow granule lysis if the S' protease participates in killing. 4) To isolate RNK-16 granule proteins that may regulate or control lysis mediated by unfractionated granules. Dr. Hudig will purify a protein that may limit granule-mediated lysis, is inactivated by granules in the presence of Ca++, is protected from inactivation by protease inhibitors and may be a natural substrate. 5) To determine the natural substrates of the RNK-16 proteases. It will be determined if RNK-16 perforin is reduced in size during lysis by unfractionated granule proteins or after incubation with purified RNK-16 proteases. It will also be determined whether lysis-restricting proteins are cleaved during granule-mediated lysis. (animals: rats and rabbits)
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