Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.     

The effect of urea exposure on isoaspartyl content and protein l-isoaspartate methyltransferase activity in Drosophila melanogaster

Urea is a protein unfolding agent that can accumulate to locally high concentrations in tissues of many organisms. We used Drosophila melanogaster to test the hypothesis that urea loading would promote formation of isoaspartate (β-carboxyl-linked aspartate), a common form of protein damage that occurs most readily in unstructured polypeptides and flexible regions of folded proteins. Ten populations of flies were tested; five control populations of urea-sensitive flies and five previously selected urea-tolerant populations. We measured the effects of urea consumption on levels of both isoaspartate and protein l-isoaspartate methyltransferase (PIMT), an enzyme believed to function in the repair or removal of isoaspartyl proteins. For both sets of populations, urea feeding for 6 days increased isoaspartyl levels by approximately 60%, supporting the idea that disruption of protein secondary and tertiary structures can accelerate the formation of isoaspartate in vivo. Urea feeding tended to increase PIMT activity in both control and urea-tolerant populations. There were no significant differences in PIMT activities or isoaspartyl levels between the control and urea-tolerant flies raised on normal or urea food. The latter findings indicate that urea tolerance evolved in the selected populations without any significant change in PIMT expression or activity.     

The effect of urea exposure on isoaspartyl content and protein L-isoaspartate methyltransferase activity in Drosophila melanogaster

Urea is a protein unfolding agent that can accumulate to locally high concentrations in tissues of many organisms. We used Drosophila melanogaster to test the hypothesis that urea loading would promote formation of isoaspartate (beta-carboxyl-linked aspartate), a common form of protein damage that occurs most readily in unstructured polypeptides and flexible regions of folded proteins. Ten populations of flies were tested; five control populations of urea-sensitive flies and five previously selected urea-tolerant populations. We measured the effects of urea consumption on levels of both isoaspartate and protein L-isoaspartate methyltransferase (PIMT), an enzyme believed to function in the repair or removal of isoaspartyl proteins. For both sets of populations, urea feeding for 6 days increased isoaspartyl levels by approximately 60%, supporting the idea that disruption of protein secondary and tertiary structures can accelerate the formation of isoaspartate in vivo. Urea feeding tended to increase PIMT activity in both control and urea-tolerant populations. There were no significant differences in PIMT activities or isoaspartyl levels between the control and urea-tolerant flies raised on normal or urea food. The latter findings indicate that urea tolerance evolved in the selected populations without any significant change in PIMT expression or activity.     

Isoaspartate in ribosomal protein S11 of Escherichia coli

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.     

Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins.

Protein L-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl delta sleep-inducing peptide was methylated under a variety of conditions. We found that 1 microM methyltransferase was required to obtain stoichiometric modification of 2 microM peptide in 40-min reactions at pH 6.2 and 30 degrees C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 microM. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 microM S-adenosylmethionine is the minimum needed for complete modification of 10 microM peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of isoaspartate in peptides and proteins without the use of radioisotopes

A rapid and sensitive HPLC-based method for quantitating isoaspartate levels in peptides and proteins is described. The analyte is incubated for 40 min with S-adenosyl-l-methionine and the commercially available enzyme protein l-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric production of S-adenosyl-l-homocysteine that is separated from the other components of the reaction by reversed-phase HPLC and quantitated online by absorbance at 260 nm. This method can accurately detect 5 pmol or less of isoaspartate and works with tryptic digests as well as intact proteins. Using a commercially available isoaspartyl peptide, the relationship between isoaspartate levels and S-adenosyl-l-homocysteine production was found to be linear and stoichiometric over a range of 5-250 pmol. Compared to methods that measure [(3)H]methanol production after methylation with S-adenosyl-l-[methyl-(3)H]methionine, the HPLC method is safer, faster, less expensive, and equally sensitive.     

Crystal structure of protein isoaspartyl methyltransferase: a catalyst for protein repair

BACKGROUND: Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation. RESULTS: We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 A resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed beta sheet with topology 6 upward arrow7 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow, the central sheet of PIMT is reorganized to 7 upward arrow6 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich beta strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site. CONCLUSIONS: The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.     

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[³H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated ³H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of ³H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.     

Synapsin I is a major endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4-10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 +/- 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme.     

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.     

Inhibition of GTPgammaS-dependent L-isoaspartyl protein methylation by tyrosine kinase inhibitors in kidney

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP. GTPgammaS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by L-isoaspartyl-containing peptides. GTPgammaS was the most potent nucleotide tested, whereas GDPbetaS and ATPgammaS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTPgammaS was reached at a physiological pH in the presence of 10 mM MgCl2. Other divalent cations, such as Cu2+, Zn2+ and Co2+ (100 microM), totally inhibited GTPgammaS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPgammaS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 microM, but increasing the vanadate concentration to more than 40 microM resulted in a dose-dependent inhibition of the GTPgammaS effect. The tyrosine kinase inhibitors genistein (IC50 = 4 microM) and tyrphostin (IC50 = 1 microM) abolished GTPgammaS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPgammaS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPgammaS-stimulated PIMT activity in the kidney cytosol.     

Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage

Isoaspartate-containing versions of sea urchin sperm-activating peptide, delta sleep-inducing peptide, and lactate dehydrogenase (231-242) were cleaved at internal sites by carboxypeptidase Y. Cleavage occurred between the isoaspartate and the preceding amino acid, and it was accompanied by sequential digestion of amino acids from the two resulting carboxyl termini. Because the isoaspartyl bonds were not cleaved, isoaspartyl dipeptides were among the final products. The rate of release of isoaspartyl dipeptides was different for the three peptides, a 24-h digestion yielding 0.32 mol of isoaspartylglycine/mol of isoaspartyl sperm-activating peptide, 0.50 mol of isoaspartylalanine/mol of isoaspartyl delta sleep-inducing peptide, and 1.15 mol of isoaspartylserine/mol of isoaspartyl lactate dehydrogenase (231-242). The different rates could be explained by the slow cleavage of amino acids preceded by glycine. Isoaspartyl dipeptides were not detected in digests of the corresponding aspartate- or asparagine-containing forms of the peptides. Release of isoaspartyl dipeptides by carboxypeptidase Y was used to demonstrate the presence of isoaspartylglycine sequences in deamidated adrenocorticotropin (0.54 mol/mol), in a mixture of trypic fragments of base-treated calmodulin (0.20 mol/mol), and in a mixture of tryptic fragments of base-treated triosephosphate isomerase (0.08 mol/mol). These results confirm earlier work suggesting that isoaspartylglycine formation is prevalent in proteins exposed to alkaline conditions. They also provide a methodology that should prove useful in the characterization of natural substrates for protein L-isoaspartyl methyltransferase.     

Protein L-isoaspartyl methyltransferase catalyzes in vivo racemization of Aspartate-25 in mammalian histone H2B

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (D/L < or = 0.01). Racemization of Asp25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (D/L = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age.     

Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome

Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.     

Structural integrity of histone H2B in vivo requires the activity of protein L-isoaspartate O-methyltransferase, a putative protein repair enzyme

Protein L-isoaspartate O-methyltransferase (PIMT) is postulated to repair beta-aspartyl linkages (isoaspartyl (isoAsp)) that accumulate at certain Asp-Xaa and Asn-Xaa sites in association with protein aging and deamidation. To identify major targets of PIMT action we cultured rat PC12 cells with adenosine dialdehyde (AdOx), a methyltransferase inhibitor that promotes accumulation of isoAsp in vivo. Subcellular fractionation of AdOx-treated cells revealed marked accumulation of isoAsp in a 14-kDa nuclear protein. Gel electrophoresis and chromatography of nuclei (3)H-methylated in vitro by PIMT revealed this protein to be histone H2B. The isoAsp content of H2B in AdOx-treated cells was approximately 18 times that in control cells, although no isoAsp was seen in other core histones, regardless of treatment. To confirm the relevance and specificity of this effect, we measured isoAsp levels in histones from brains of PIMT knockout mice. IsoAsp was found at near stoichiometric levels in H2B extracted from knockout brains and was at least 80 times greater than that in H2B from normal mice. Little or no isoAsp was detected in H2A, H3, or H4 from mice of either genotype. Accumulation of isoAsp in histone H2B may disrupt normal gene regulation and contribute to the reduced life span that characterizes PIMT knockouts. In addition to disrupting protein function, isoAsp has been shown to trigger immunity against self-proteins. The propensity of H2B to generate isoAsp in vivo may help explain why this histone in particular is found as a major antigen in autoimmune diseases such as lupus erythematosus.     

Crystal structure of human L-isoaspartyl-O-methyl-transferase with S-adenosyl homocysteine at 1.6-A resolution and modeling of an isoaspartyl-containing peptide at the active site

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.     

Repair of Isoaspartate Formation Modulates the Interaction of Deamidated 4E-BP2 with mTORC1 in Brain

In eukaryotes, a rate-limiting step of translation initiation is recognition of the mRNA 5′ m⁷GpppN cap structure by the eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex consisting of the cap-binding protein, eIF4E, along with eIF4G, and eIF4A. The eIF4E-binding proteins (4E-BPs) repress translation by disrupting eIF4F formation, thereby preventing ribosome recruitment to the mRNA. Of the three 4E-BPs, 4E-BP2 is the predominant paralog expressed in the mammalian brain and plays an important role in synaptic plasticity and learning and memory. 4E-BP2 undergoes asparagine deamidation, solely in the brain, during early postnatal development. Deamidation spontaneously converts asparagines into a mixture of aspartates or isoaspartates, the latter of which may be destabilizing to proteins. The enzyme protein l-isoaspartyl methyltransferase (PIMT) prevents isoaspartate accumulation by catalyzing the conversion of isoaspartates to aspartates. PIMT exhibits high activity in the brain, relative to other tissues. We report here that 4E-BP2 is a substrate for PIMT. In vitro deamidated 4E-BP2 accrues isoapartyl residues and is methylated by recombinant PIMT. Using an antibody that recognizes 4E-BP2, which harbors isoaspartates at the deamidation sites, Asn⁹⁹ and Asn¹⁰², we demonstrate that 4E-BP2 in PIMT−/− brain lysates contains isoaspartate residues. Further, we show that 4E-BP2 containing isoaspartates lacks the augmented association with raptor that is a feature of deamidated 4E-BP2.     

Overexpression of L-isoaspartate O-methyltransferase in Escherichia coli increases heat shock survival by a mechanism independent of methyltransferase activity

Over time and under stressing conditions proteins are susceptible to a variety of spontaneous covalent modifications. One of the more commonly occurring types of protein damage is deamidation; the conversion of asparagines into aspartyls and isoaspartyls. The physiological significance of isoaspartyl formation is emphasized by the presence of the conserved enzyme L-isoaspartyl O-methyltransferase (PIMT), whose physiological function appears to be in preventing the accumulation of deamidated proteins. Seemingly consistent with a repair function, overexpression of PIMT in Drosophila melanogaster extends lifespan under conditions expected to contribute to protein damage. Based on structural information and sequence homology we have created mutants of residues proposed to be involved in co-factor binding in Escherichia coli PIMT. Both mutants retain S-adenosyl L-methionine binding capabilities but demonstrate dramatically reduced kinetic capabilities, perhaps suggestive of catalytic roles beyond co-factor binding. As anticipated, overexpression of the wild type enzyme in E. coli results in bacteria with increased tolerance to thermal stress. Surprisingly, even greater levels of heat tolerance were observed with overexpression of the inactive PIMT mutants. The increased survival capabilities observed with overexpression of PIMT in E. coli, and possibly in Drosophila, are not due to increased isoaspartyl repair capabilities but rather a temperature-independent induction of the heat shock system as a result of overexpression of a misfolding-prone protein. An alternate hypothesis as to the physiological substrate and function of L-isoaspartyl methyltransferase is proposed.     

Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-(3)H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(+/+) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.