PROTEIN CARBOXYL METHYLATION IN BRAIN

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DESCRIPTION (Adapted from applicant's abstract): The overall goal of this project is to elucidate the biological role of protein L-isoaspartyl methyltransferase (PIMT), a protein methylating enzyme that is enriched in brain and catalyzes selective methylation of atypical L-isoaspartyl residues. These unusual structures are characterized by an abnormal isopeptide bond in which an aspartyl residue is linked to its carboxyl-flanking neighbor via its side-chain beta-carboxyl group, rather than through the normal alpha-linkage. Isoaspartyl sites are believed to arise from spontaneous alterations of proteins during aging and/or from errors in protein synthesis. In vitro, PIMT has been demonstrated to catalyze isoaspartate repair by converting beta-linkage to normal alpha linkages. The aims of this continuation application are focused on two areas: characterization of several proteins which appear to be important targets of PIMT action in cells, and an attempt to demonstrate directly that PIMT functions as a repair enzyme in vivo. This will be accomplished by addressing 4 specific aims. The first will focus on histone H2B, a nuclear protein recently shown to undergo a selective and dramatic increase in isoaspartate content when PIMT activity is inhibited in rat PC12 cells. The second will involve the purification and characterization of a recently discovered high molecular weight methyl acceptor protein (HMAP) that is found only in brain and appears to have an unusually high content of isoaspartyl sites, as well as a high affinity for PIMT. The third specific aim is to determine if synapsin-1, a protein involved in regulating the availability of synaptic vesicles, forms isoaspartate in vivo. The final aim is to characterize an unusual isoaspartate-rich protein in E. coli that serves as an excellent substrate for rat brain PIMT. Together these investigations should provide important new insights into the significance of protein damage and repair systems in normal cell aging and in human disease.