The overall goal of the proposed research is to understand the function of the enzyme, protein carboxyl methyltransferase (PCMT), in mammalian brain. Recent evidence indicates that PCMT selectively and stoichiometrically modifies only proteins and peptides which contain L-isoaspartate, i.e., aspartate which is coupled through its side-chain beta-carboxyl group, rather than via the typical alpha-carboxyl linkage. The most likely source of isoaspartate in proteins is via spontaneous deamidation of labile asparagine sites. We propose that PCMT modifies deamidation- damaged proteins in order to facilitate their efficient degradation or repair. In testing this hypothesis, our first goal will be to study the metabolic fate of radio-labeled isoaspartate-containing peptides and proteins in brain extracts under conditions which favor or suppress PCMT activity. This should allow us to determine if they are degraded or repaired in a methylation-dependent manner. Our second goal is determine if the previously demonstrated abundance of methylation sites in brain particulate fraction (compared with other tissues) is due to a relative enrichment in brain of isoaspartate-bearing proteins. Our third goal is to compare partial amino acid sequences of the two major isoforms of brain PCMT to determine if they are distinct isozymes. Our final goal is to clone and sequence the cDNA for bovine brain PCMT. This will ultimately allow us to determine if PCMT resembles any proteins of known function and will provide the foundation for future studies designed to alter levels of PCMT activity in vivo. The proposed studies may provide important new insights into the molecular mechanisms of cell damage in the brain and the enzymatic processes evolved to deal with them.
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