During the aging process """"""""altered"""""""" proteins acumulate in the cell. This is believed to be due to a decreased rate of degradation of these """"""""altered"""""""" proteins. To understand these changes, which are occuring in aging cells, it is necessary to understand the mechanisms by which native proteins in the cell become """"""""altered"""""""" and then degraded by intracellular proteases. At least 10 different mechanisms have been proposed for how native proteins become """"""""altered"""""""". However, in only one or two cases have these mechanisms of alteration of the protein been shown to be the signal for degradation by the intracellular proteases. To fully understand the events occuring in the aging process it is necessary to elucidate the mechanisms of protein turnoverin the cell. Our long term goal is to determine if the nonenzymatic deamidation of asparagine and glutamine residues in proteins is one of the signals which converts a native protein into an """"""""altered"""""""" protein and makes the protein susceptible to degradation by intracellular proteases. Previous work by other investigators suggets that this is an important mechanism. Our approach will be to extend previous studies on the mechanism of noneznymatic deamidation of asparagine and glutamine residues in model peptides with respect to nearest neighbor effects, general acid and base catalysis, and effect of ionic strength. A new assay based on C- 13 NMR of asparagine residues will be developed to accurately measure the deamidation reaction. The goal of these experiments is to generate a set of predictive rules to help in identifying sequences near Asn and Gln residues which deamidate rapidly. Using the extensive sequence and structure data bases, all existing protein sequences will be surveyed for Asn and Gln primary and secondary structure environment, and avialable structures analyzed by computer tertiary graphics to provide a structural basis for these rules. We will also study the role of deamidation on the structure and function of the enzymes serine hydroxymethyltransferase and alkaline phosphatase. Using the technique of cellular microinjection the effect of deamidation of these proteins on their intracellular turnover will be determined.
