Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice

Abstract

L-Asparaginyl and L-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing L-isoaspartyl and D-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal L-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein L-isoaspartate(D-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and succumbed to fatal seizures at an average of 42 days after birth. These results suggest that the ability of mice to repair L-isoaspartyl-and D-aspartylcontaining proteins is essential for normal growth and for normal central nervous system function.The success of an aging organism depends on its ability to maintain the integrity of its unstable macromolecular machinery over time (1, 2). We have been particularly interested in the consequences of protein damage on aging. Proteins are subject to a variety of spontaneous degradation processes, including oxidation, glycation, deamidation, isomerization, and racemization (1, 3-9). These nonenzymatic modifications can produce functionally damaged species that reflect the action of aging at the molecular level (5, 10).L-Asparagine and L-aspartate are among the most unstable residues in proteins, being particularly susceptible to linked deamidation, isomerization, and racemization reactions (6, 11-13). The initial event in these nonenzymatic reactions at neutral pH is generally the nucleophilic attack of the side-chain carbonyl carbon by the peptide-bond nitrogen to yield an unstable five-membered L-succinimidyl ring (Fig. 1). The spontaneous hydrolysis of the succinimide produces either an L-isoaspartyl residue (in which the peptide backbone is redirected through the ␤-carboxyl group) or a normal L-aspartyl residue. In addition, the ␣-carbon of the succinimidyl residue is racemization-prone, and D-aspartyl and D-isoaspartyl residues can also form, although in lower yields (6,14). Under physiological conditions, succinimide-linked deamidation of asparagine can occur with half-times as short a...

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

“…These mice exhibited slow growth and a fatal seizure disorder leading to death after an average of 42 days of age (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998). In addition, PIMT-/-mice showed aberrant synaptic transmission in the CA3 region of the hippocampus, associated with an abnormal distribution of synaptic vesicles in the mossy fiber terminals (Ikegaya et al 2001).…”

“…It has been previously reported that PIMT activity is important for the maintenance of the central nervous system since PIMT-deficient mice died from progressive epileptic seizures and showed aberrant synaptic transmission in the hippocampus (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998;Ikegaya et al 2001). This suggests that changes in PIMT activity in the human brain during epilepsy, particularly in the hippocampus which is the major seat of temporal lobe epilepsy, may be involved in this disease.…”

“…These mice showed an accumulation of damaged aspartyl residues in all tissues, particularly in the brain (Kim et al 1997). Such knockout mice (PIMT-/-) also showed significantly slower growth as well as premature death, caused by fatal generalized seizures characteristic of fatal progressive epilepsy (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998). Also, these PIMT-/-mice show aberrant neurotransmission in the synapses of mossy fibers in the CA3 region of the hippocampus, associated with an atypical distribution of synaptic vesicles, abnormal microtubule organization in the dendrites of pyramidal neurons and vacuolar degeneration (Yamamoto et al 1998;Ikegaya et al 2001).…”

“…These mice showed an accumulation of damaged aspartyl residues in all tissues, particularly in the brain (Kim et al 1997). Such knockout mice (PIMT-/-) also showed significantly slower growth as well as premature death, caused by fatal generalized seizures characteristic of fatal progressive epilepsy (Kim et al 1997(Kim et al , 1999Yamamoto et al 1998).…”

Human Erythrocyte Proteinl-Isoaspartyl Methyltransferase: Heritability of Basal Activity and Genetic Polymorphism for Thermal Stability

“…We show here that PIMT levels in erythrocytes of healthy individuals fell within a narrow the level of isoaspartate-bearing proteins increases dramatically in rat PC12 cells grown in the presence range, and the level of activity appeared to be governed substantially by inheritance. Thermal stability data of a methyltransferase inhibitor (17) and in mice in which the gene encoding PIMT has been selectively from a population sample of 299 donors suggested the presence of two major alleles which were subsequently disrupted (18). The PIMT-deficient mice grow more slowly than their normal counterparts and undergo shown to be associated with the presence of an Ile/Val polymorphism recently described by Tsai and Clarke fatal seizures between 26 and 60 days after birth.…”