Edited by F. Anne StephensonProtein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30 -60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80 -100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.Protein L-isoaspartyl methyltransferase (PIMT/PCMT1; product of the pcmt1 gene) 2 functions to repair abnormal isoaspartyl (isoAsp) sites in proteins that occur via isomerization of the peptide bond linking asparagine or aspartate residues to their carboxyl-flanking neighbor (1-3). isoAsp formation, like oxidation, is an extremely common type of spontaneous protein damage that occurs under mild conditions in vivo and in vitro and is known to alter protein function and antigenicity. PIMT utilizes the cofactor S-adenosyl-L-methionine (AdoMet) to transfer a methyl group onto the ␣-carboxyl group of isoAsp sites leading to a succinimidyl intermediate that subsequently hydrolyzes to L-Asp and L-isoAsp (Fig. 1). Continuing cycles of PIMT action efficiently repair L-isoAsp sites in vitro (4 -9), and reduction of PIMT activity in cultured cells or KO mice dramatically increases the level of isoAsp-containing proteins (10 -13). PIMT is widely distributed in mammalian tissues but is particularly rich in the CNS (14 -17). A critical need for PIMT in brain is evident from the overt neurological phenotype of PIMT-deficient mice; increased brain size, abnormal neuroanatomical and electrophysiological properties of hippocampal cells along with reduced cognitive function (18), atypical open field behavior (19), and fatal epileptic seizures beginning at 4 weeks of age (11,12). A proteomic study utilizing the PIMT-KO mouse revealed over 30 neuronal targets for PIMT, including synapsins I and II, CRMP2 (collapsin response mediator protein 2), dynamin 1, ␣-and -tubulin, and creatine kinase B, among others (20). Based on the phenotype of the PIMT-KO mouse, a major reduction of PIMT repair activity in humans would likely contribute to neurodevelopmental disorders, including epilepsy, in the young. Moreover, current evidence suggests that even moderate decrements in PIMT activity accelerate age-related changes in cognition and even lifespan. Brain extracts of PIMT ϩ/Ϫ mice have 50 -55% of the PIM...