Limited Accumulation of Damaged Proteins inl-Isoaspartyl (d-Aspartyl)O-Methyltransferase-deficient Mice

Abstract

L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1)can initiate the conversion of damaged aspartyl and asparaginyl residues to normal L-aspartyl residues. Mice lacking this enzyme (Pcmt1؊/؊ mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation ("transgenic Pcmt1؊/؊ mice") had brain PCMT1 activity levels that were 6.5-13% those of wild-type mice but had little or no activity in other tissues. The transgenic Pcmt1؊/؊ mice lived, on average, 5-fold longer than nontransgenic Pcmt1؊/؊ mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concentration of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1؊/؊ mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine from Pcmt1؊/؊ mice contained increased amounts of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.The spontaneous chemical modification of proteins by reaction with oxygen, water, sugars, and other abundant metabolites is unavoidable. The accumulation of such nonenzymatically altered proteins is associated with normal aging as well as atherosclerosis, Alzheimer's disease, and diabetes (1-3). Organisms have several strategies for dealing with damaged proteins, including intracellular proteolysis mediated by proteasome and lysosome action (4 -6). Some types of covalent damage, however, are simple enough to recognize and repair directly (7). Enzymes such as prolyl cis-trans isomerase (8), methionine sulfoxide reductase (9), and disulfide isomerase (8) can restore activity to proteins that have been chemically altered.We are interested in a common type of spontaneous protein damage in which L-aspartyl and L-asparaginyl residues undergo an intramolecular reaction that converts them to L-succinimidyl residues (10, 11). Nonenzymatic hydrolysis of the succinimide ring readily occurs at either carbonyl to generate both normal aspartyl residues and isoaspartyl residues, in which the peptide backbone proceeds through the ␤-carbonyl rather than the ␣-carbonyl moiety (12). The succinimide also racemizes more rapidly than do the open chain forms, and hydrolysis of the D-succinimide produces D-aspartyl and D-isoaspartyl residues (12). Local protein structure causes some L-aspartyl and L-asparaginyl residues to be especially prone to succinimide formation, and the presence of damaged aspartyl residues at these sites can significantly alter the structure, function...

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…Methyltransferase-deficient mice suffer fatal seizures at an early age (19 -21). Interestingly, the effect of methyltransferase loss on the accumulation of damaged substrates in these knockout organisms is quite variable, from small effects seen in worms (22) and bacteria (23) to relatively large effects in mice (21).…”

“…The functional significance of these differences is not clear, although mathematical simulations of the repair reaction clearly show that repair efficiency is directly related to the affinity of the enzyme for the methyl-acceptor (25). It is possible that the complexity of human protein interactions necessitates a more efficient repair to minimize the presence of proteins containing abnormal residues (21,22).…”

“…Methyltransferase-deficient mice suffer fatal seizures at an early age (19 -21). Interestingly, the effect of methyltransferase loss on the accumulation of damaged substrates in these knockout organisms is quite variable, from small effects seen in worms (22) and bacteria (23) to relatively large effects in mice (21).Although all of the L-isoaspartyl methyltransferases characterized so far recognize L-isoaspartyl residues in synthetic peptide substrates, their relative affinity for these methyl-acceptors is also quite variable. For example, the human enzyme recognizes substrates with 40 -1000 times the affinity of the E. coli, nematode, and plant enzymes (24).…”

“…The functional significance of these differences is not clear, although mathematical simulations of the repair reaction clearly show that repair efficiency is directly related to the affinity of the enzyme for the methyl-acceptor (25). It is possible that the complexity of human protein interactions necessitates a more efficient repair to minimize the presence of proteins containing abnormal residues (21,22).In mammalian cells, this methyltransferase can also catalyze the AdoMet-dependent methylation of substrates containing D-aspartyl residues arising from spontaneous protein racemization reactions (15,26,27). The K m for corresponding synthetic peptides containing D-aspartyl residues in place of L-isoaspartyl residues can be 700 -10,000-fold higher, however.…”

“…Additionally, no methylation of D-aspartyl peptides has been detected for the E. coli (28), worm (13), and higher plant (24) enzymes. In mammalian cells, it is possible that the methylation of D-aspartyl residues can lead to eventual L-aspartyl formation in a repair reaction similar to that observed for L-isoaspartyl residues, although considerable amounts of D-isoaspartyl residues would also be expected to accumulate (27).We have been interested in comparing the properties of protein repair methyltransferases from a variety of organisms …”