METHYLATION OF ATYPICAL PROTEIN ASPARTYL RESIDUES

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The mammalian protein isoaspartyl methyltransferase (PIMT) specifically modifies abnormal protein aspartyl residues that have arisen spontaneously in aging proteins. The experiments of this project test the hypothesis that mammalian PIMT plays a role in repairing or degrading damaged cellular proteins that this function is particularly important for terminally differentiated cells and for cells approaching senescence. In situ hybridization will be used to identify cellular patterns of PIMT gene expression in brain and testis, tissues with large numbers of postmitotic cells and high levels of PIMT activity. The experiments will identify stages of sperm differentiation characterized by increased PIMT expression. The studies with brain tissue will identify groups of neurons with elevated PIMT levels. Similar studies will be done with brain tissue from aging animals to test whether changes in PIMT expression accompany the loss of neuronal function during aging. PIMT levels will be experimentally manipulated in cultured cell models to determine the consequences of PIMT depletion and overexpression on cellular physiology. Permanent cell lines that either overexpress or are deficient in PIMT will be constructed by transfection with high level expression plasmids containing the PIMT gene sequence in either sense or antisense orientations. Experiments will measure the effects of PIMT dosage on cellular protein metabolism, ability to survive nutritional stress, tendency to enter senescence and differentiation to a neuronal phenotype. A biochemical model will be used to identify roles for the PIMT in protein repair or degradation. Radiolabeled calmodulins will be converted to isomerized forms in an artificial aging protocol. These substrates will be injected into Xenopus oocytes and the stability of the protein will be measured in the absence and presence of methylation inhibitors to determine if carboxyl methylation decreases or increases protein stability. Peptide mapping will be used to monitor any repair reactions. By allowing dysfunctional proteins to accumulate in cells, PIMT malfunction could contribute to infertility and neurodegenerative disorders associated with aging. For example, L-isoaspartyl residues have been identified at several locations in beta-amyloid peptides from Alzheimer-diseased brains, raising the possibility that deficiencies in methylation contribute to the aberrant processing reactions in that disease. The elucidation of the methylation-mediated pathway and the identification of effectors that increase its efficiency could have potential value in treating age-related disorders which involve defects in protein metabolism.