A failure to repair self-proteins leads to T cell hyperproliferation and autoantibody production

It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT(-/-) CD4(+) T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4(+) T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT(-/-) bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.     

Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50–300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.     

Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover

At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure.     

Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.     

The influence of protein structure on the products emerging from succinimide hydrolysis

Proteins are vulnerable to spontaneous, covalent modifications that may result in alterations to structure and function. Asparagines are particularly labile, able to undergo deamidation through the formation of a succinimide intermediate to produce either aspartate or isoaspartate residues. Although aspartates cannot undergo deamidation they can form a succinimide and result in the same products. Isoaspartyls are the principal product of succinimide hydrolysis, accounting for 65-85% of the emerging residues. The variability in the ratio of products emerging from succinimide hydrolysis suggests the ability of protein structure to influence succinimide outcome. In the H15D histidine-containing protein (HPr), phosphorylation of the active site aspartate catalyzes the formation of a cyclic intermediate. Resolution of this species is exclusively to aspartate residues, suggestive of either a succinimide with restrained hydrolysis, or an isoimide, from which aspartyl residues are the only possible product. Deletion of the C-terminal residue of this protein does not influence the ability for phosphorylation or ring formation, but it does allow for isoaspartyl formation, verifying a succinimide as the cyclic intermediate in H15D HPr. Isoaspartyl formation in H15D Delta85 is rationalized to occur as a consequence of elimination of steric restrictions imposed by the C terminus on the main-chain carbonyl of the succinimide, the required point of nucleophilic attack of a water molecule for isoaspartyl formation. This is the first reported demonstration of the influence of protein structure on the products emerging from succinimide hydrolysis.     

Metabolism of a synthetic L-isoaspartyl-containing hexapeptide in erythrocyte extracts. Enzymatic methyl esterification is followed by nonenzymatic succinimide formation

The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.     

Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.     

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.     

Determination of Β-isomerized aspartic acid as the corresponding alcohol

The age-related formation of succinimides in proteins, through spontaneous deamidation of asparagine, and through cyclization of aspartic acid, is thought to be followed by the hydrolysis of the succinimide ring, yielding a mixture of normal aspartic acid sites and-isomerized aspartic acid sites (isoaspartic acid). The chemical reduction of an isoaspartyl site to the corresponding amino acid alcohol, isohomoserine, has now been investigated as a general approach to measuring the accumulation of isomerized residues in aging proteins. The methods employed were based on conditions previously found to be successful in reducing protein aspartic acid to homoserine. Borane was employed as the reducing agent, and was found to produce the expected amino acid alcohols in reactions with model peptides. In addition, amino acid analysis revealed a complex pattern of unknown products of these reduction reactions, some of which were also evident when a much stronger reducing agent, lithium aluminum hydride, was used. The correlation of some of these side-products with the isomerization of the peptide suggests, unexpectedly, that the reactivity of reducing agents toward aspartyl residues and perhaps other sites in the peptide may be influenced by steric factors related to aspartyl isomerization. The borane reduction method was also applied to proteins. No detectable isohomoserine was formed either in ovalbumin, a model aged protein, or in human lens proteins of advanced age, with conditions that fully reduced normal aspartyl residues to homoserine. These tests thus indicate that the percentage of aspartic acid in the isomerized form in these proteins is below the limit of detectability (below 5%). These results complement previous experimental results that have indicated a low bulk isoaspartyl content in most natural proteins.     

Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins.

The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp(93) to Leu(94) occurs through the beta-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiological conditions. These post-translationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.     

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.     

Chemical deglycosylation can induce methylation, succinimide formation, and isomerization

Interpretation of deglycosylation studies relies heavily on the absence of modifications to the polypeptide chain. We have found that by using a common chemical deglycosylation technique, one can effect at least three changes in a peptide's structure: methylation, isomerization, and ring formation. It was determined that the conditions of chemical deglycosylation introduce a + 14 Da shift in the masses of our model peptides, RKDVY, RKEVY, and horseradish peroxidase. This shift is localized to acidic functional groups and is interpreted as methylation of the free carboxylates in our models. An additional shift in mass of – 18 Da is found in the model peptide RKDVY consistent with the loss of water associated with succinimide ring formation in this peptide. Chemical treatment induced isomerization of aspartyl residues to isoaspartyl residues in another model peptide, tetragastrin. These results indicate that one should use caution when interpreting the results of chemical deglycosylation experiments.     

Induction of beta-sheet structure in amyloidogenic peptides by neutralization of aspartate: a model for amyloid nucleation.

Amyloid fibril formation is widely accepted as a critical step in all types of amyloidosis. Amyloid fibrils derived from different amyloidogenic proteins share structural elements including beta-sheet secondary structure and similar tertiary structure. While some amyloidogenic proteins are rich in beta-sheet in their soluble form, others, like Alzheimer beta-amyloid peptide (Abeta) or serum amyloid A, must undergo significant structural transition to acquire a high beta-sheet content. We postulate that Abeta and other amyloidogenic proteins undergo a transition to beta-sheet as a result of aging-related chemical modifications of aspartyl residues to the form of succinimide or isoaspartyl methyl ester. We hypothesize that spontaneous cyclization of aspartate residues in amyloidogenic proteins can serve as a nucleation event in amyloidogenesis. To test this hypothesis, we synthesized a series of designed peptides having the sequence VTVKVXAVKVTV, where X represents aspartic acid or its derivatives. Studies using circular dichroism showed that neutralization of the aspartate residue through the formation of a methyl ester or an amide, or replacement of aspartate with glutamate led to an increased beta-sheet content at neutral and basic pH. A higher content of beta-sheet structure correlated with increased propensity for fibril formation and decreased solubility at neutral pH.     

Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.     

Methylation of microinjected isoaspartyl peptides in Xenopus oocytes. Competition with protein carboxyl methylation reactions

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.     

Application of chemical selective cleavage methods to analyze post-translational modification in proteins

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.     

Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

We have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Tyr-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater than the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by [3H]diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.     

Synthesis, aggregation, and neurotoxicity of the Alzheimer's Abeta1-42 amyloid peptide and its isoaspartyl isomers

Amyloid Abeta1-42 peptide (Abeta1-42) and its isomers with an isoaspartyl residue at position 7 or 23 [Abeta1-42(isoAsp7) and Abeta1-42(isoAsp23)] were synthesized in high purity by the Fmoc-solid phase technique, followed by HPLC on a silica-based reversed-phase column under the basic conditions. Importantly, Abeta1-42(isoAsp23) aggregated more strongly than native Abeta1-42 and showed significant neurotoxicity, while the aggregation ability and neurotoxicity of Abeta1-42(isoAsp7) was weak. This suggests that the isomerization of the aspartyl residues plays an important role in fibril formation in Alzheimer's disease.     

Considerations in the identification of endogenous substrates for protein L-isoaspartyl methyltransferase: the case of synuclein

Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl peptide bonds in age-damaged proteins. It has been reported that synuclein, a protein implicated in neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to neurodegenerative diseases, we attempted to determine the stoichiometry of isoaspartate accumulation in synuclein in vivo and in vitro. Brain proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl proteins, and by immunoblotting to confirm the coincident presence of synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl proteins, but no signal in the position of synuclein. This finding was corroborated by immunoprecipitation of synuclein followed by on-blot (3)H-methylation. To assess the propensity of synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-synucleins were aged for two weeks by incubation at pH 7.5 and 37°C. The stoichiometries of isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of isoaspartate per mol of protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl protein methyl ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of proteins to form isoaspartyl sites and the instability of the (3)H-methyl esters used to tag them. The results presented here indicate that synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in proteins.