The overall goal of this project is to understand the role of protein carboxyl methylation in neuronal function. There are two current hypotheses of methylation function in eucaryotes: (1) that it mediates some aspect of stimulus-response coupling via the reversible modification of protein function, and (2) that it facilitates the repair or degradation of defective proteins which contain abnormal forms of aspartic acid. In testing the regulation hypothesis, we propose to measure the ability of certain neurotransmitters and depolarizing agents to stimulate transient protein methylation (as indicated by the evolution of radiolabeled methanol), to search in purified subfractions of brain for specific protein which serve as preferential substrates in an in vitro transient methylation assay, and, to determine if the in vitro transient methylation reaction is regulated by the second messengers cAMP, cGMP, Ca++ or phosphatidyl inositol. Our strategy in these experiments is derived from recent indications that carboxyl methylation may serve as an initial activation step in a more complex protein modification reaction than hitherto assumed. An alternative role for carboxyl methylation in the repair or degradatiom of damaged proteins is suggested by the recently discovered selectivity of the methyltransferase enzyme for proteins containing abnormal forms of aspartate, particularly L-isoaspartyl and D-aspartyl residues. We propose to determine if the methyl accepting substrates we have located in synaptic membranes and myelin are enriched in these atypical forms of aspartate, and, to determine if the levels of these abnormal proteins and/or the methyltransferase enzyme changes significantly with age in the human or in association with Alzheimer's disease. Establishing a firm role for carboxyl methylation should provide important new insight on fundamental mechanisms of cellular regulation and/or aging processes in the mammalian nervous system.
