Endogenous Substrates for Protein Carboxyl Methyltransferase in Cytosolic Fractions of Bovine Brain

Aswad, Dana W.; Deight, Elizabeth A.

doi:10.1111/j.1471-4159.1983.tb00883.x

Cited by 36 publications

(20 citation statements)

References 33 publications

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“…We show here that the production of membrane protein D-Asp,3Me by the highly purified brain methyltransferase I is decreased when the synthetic isopeptide L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala is present in the methylation mixture. Coupled with previous data that demonstrate the broad substrate specificity of the enzyme (1,5,6), these findings suggest that there may be a fair amount of flexibility in the structure surrounding the active site, which allows the enzyme to accommodate a very heterogeneous group of protein substrates.…”

Section: Resultssupporting

confidence: 73%

“…In vitro, these enzymes methylate a large variety of both heterologous and endogenous proteins in a substoichiometric fashion (1,(4)(5)(6). The "nonspecific" methyltransferase is widely distributed in mammalian tissues (1) and has been purified from both mammalian brain (1,(5)(6)(7) and erythrocyte (8,9) tissues. In brain, there are at least two functionally similar isozymes that can be separated by DEAE-cellulose chromatography (5).…”

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confidence: 99%

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Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.

O’Connor¹,

Aswad²,

Clarke³

1984

Proc. Natl. Acad. Sci. U.S.A.

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Two purified isozymes of protein carboxyl methyltransferase from bovine brain catalyze the substoichiometric transfer of methyl groups in vitro from S-adenosyl-L-[methyl-3H]methionine to several erythrocyte membrane proteins, which include bands 2.1, 3, and 4.1, as well as several integral membrane polypeptides. D-Aspartic acid f3-[3H]methyl ester has been isolated from proteolytic digests of these methylated proteins, suggesting that protein D-aspartyl residues can serve as methyl-acceptor sites for the two brain enzymes. This formation of D-aspartic acid 3-[3H]methyl ester is competitively inhibited by the peptide L-Val-L-Tyr-L-Pro-LisoAsp-Gly-L-Ala, which contains an L-aspartyl residue in an unusual /-peptide linkage. Since this peptide is a stoichiometric substrate for the brain methyltransferases, it appears that one enzymatic activity can catalyze methyl ester formation at both D-aspartyl and L-isoaspartyl sites. In these respects, the activity of both brain isozymes closely resembles those previously described for the erythrocyte enzyme. The results are discussed in terms of a model in which derivatized aspartyl residues in proteins, arising by either racemization or isomerization, are recognized by the methyltransferase; the enzyme may function in either the metabolism or correction of the altered structures. The presence of a similar enzyme in both translationally active (brain) and inactive (erythrocyte) tissues suggests that the reactions are of general importance to cellular integrity.Protein carboxyl methyltransferases are widely distributed in both bacteria and eukaryotic tissues (1). In bacteria, very specific carboxyl methyltransferases regulate the bacterial chemotactic response by the methylation of several glutamyl residues in membrane chemoreceptor proteins (2, 3). The eukaryotic enzymes, on the other hand, have very different characteristics. In vitro, these enzymes methylate a large variety of both heterologous and endogenous proteins in a substoichiometric fashion (1, 4-6). The "nonspecific" methyltransferase is widely distributed in mammalian tissues (1) and has been purified from both mammalian brain (1, 5-7) and erythrocyte (8,9) tissues. In brain, there are at least two functionally similar isozymes that can be separated by DEAE-cellulose chromatography (5).The precise function of eukaryotic protein carboxyl methylation reactions has not been determined. It has been proposed that the brain enzyme regulates several cellular processes, including, among others, the storage and release of neuroendocrine substances (10, 11), the modulation of receptor function (12, 13), and the regulation of calmodulin activity (14, 15). However, the data supporting such roles have not been compelling (4,16,17). In particular, the broad substrate specificity and substoichiometric nature of the reactions catalyzed by the enzyme are unusual features for regulatory post-translational modifications. Alternative roles for eukaryotic methylation reactions in the repair or metabolism of damaged proteins ha...

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Section: Resultssupporting

confidence: 73%

mentioning

confidence: 99%

Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.

O’Connor¹,

Aswad²,

Clarke³

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…We first reported the accumulation of isoaspartyl synapsin I in PIMTdeficient mice in 2001 (45 H-methylated synapsins after SDS-PAGE, they may have been foiled by the instability of isoaspartyl methyl esters, which generally do not survive SDS-PAGE at the high pH and temperatures encountered in the commonly used Tris/glycine buffer systems (47,48). In our study, we characterized the 3 H-methylated synapsins after separation at 4°C in the BisTris-based NuPAGE Novex gel system (Invitrogen), which utilizes a substantially lower pH compared with the Tris/glycine-based systems.…”

Section: Discussionmentioning

confidence: 99%

Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

Reissner

Paranandi

Luc

et al. 2006

Journal of Biological Chemistry

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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. Fig. 1, dashed arrows) constitutes a major pathway for spontaneous protein damage at physiological pH and temperature (1-3). The internal ␣-carboxyl characteristic of isoaspartyl sites is selectively methylated by a widely distributed protein L-isoaspartyl O-methyltransferase (PIMT) 2 (4 -6). Considerable evidence suggests that PIMT functions to repair this damage in vivo by the mechanism illustrated in Fig. 1 (solid arrows). PIMT selectively methylates the atypical ␣-carboxyl of the isoAsp site. This is followed immediately by a spontaneous demethylation to form a metastable succinimide. Spontaneous hydrolysis of the succinimide over a time course of 2-4 h generates a normal L-aspartyl site with an efficiency of 15-30%. Purified PIMT catalyzes conversion of isoaspartyl sites to normal Asp-Xaa linkages in synthetic peptides (7-9) and has been shown to restore function to isoaspartate-containing forms of proteins such as calmodulin (10) and the Escherichia coli phosphocarrier protein HPr (11). Pharmacological inhibition of PIMT activity in rat PC12 cells leads to reversible accumulation of isoaspartyl sites in a variety of proteins, including tubulin (12) and histone H2B (13). Formation of isoaspartyl sites (Studies with PIMT-deficient mice further support a repair function for PIMT (14,15). Whereas proteins in extracts from wild-type or heterozygous PIMT mice contain only trace levels of isoaspartate, proteins in extracts from knock-out mice show greatly increased isoaspartate levels in extracts from all tissues examined, w...

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“…The more acidic isozyme of PCMT was purified from calf brain as described by Aswad and Deight (34) and used in all experiments. Assay mixtures (25 l) contained final concentrations of 0.44 mM ovalbumin, 22 nM PCMT, 2.…”

Section: Methodsmentioning

confidence: 99%

Complex Interactions of the Protein l-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System

O'Connor

O’Connor

1998

Journal of Biological Chemistry

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The widely distributed protein-L-isoaspartyl, D-aspartyl carboxylmethyltransferase (EC 2.1.1.77) is hypothesized to play a role in the repair or metabolism of deamidated and isomerized proteins that are spontaneously generated during the aging of proteins in cells. The yeast two-hybrid system was used to identify proteins that potentially interact with the methyltransferase in a cellular processing pathway. Two cDNAs, both encoding calmodulin, were isolated from a human fetal brain cDNA library using the human methyltransferase as the bait. Enzymatic assays with purified components revealed a complex set of interactions between the methyltransferase and calmodulin. Calmodulin weakly stimulated protein carboxylmethyltransferase activity in vitro at concentrations of the two proteins reflecting their representation in mammalian brain. Calmodulin stimulation of methyltransferase was observed in both the presence and absence of calcium, although the effect was greater in the presence of calcium. Native calmodulin was not a substrate for the carboxylmethyltransferase, but deamidated variants of calmodulin act as substrates for the methyltransferase, with calculated K m values of 3.6 and 8.6 M for calcium-liganded and unliganded calmodulin, respectively. Both the effector and substrate interactions of calmodulin with the protein isoaspartyl methyltransferase likely contributed to the positive results obtained with the two-hybrid system. Protein-L-isoaspartyl, D-aspartyl carboxyl methyltransferase (PCMT)1 (E.C. 2.1.1.77), a well conserved enzyme detected in a broad spectrum of organisms from Escherichia coli to humans (1-6), specifically modifies L-isoaspartyl and D-aspartyl residues that arise in proteins due to spontaneous deamidation and racemization reactions involving asparaginyl and aspartyl residues (7,8). Because the formation of L-isoaspartate can adversely affect the stability and biological potency of many proteins (9 -13), it has been proposed that methylation of the damaged protein initiates either the repair or degradation of the substrate. Experimental support for a structural repair function for PCMT has been obtained in purified systems using isoaspartyl peptides as substrates for the PCMT (14 -16). The esterified isoaspartyl peptides formed by PCMT hydrolyze into both aspartyl peptides and isoaspartyl peptides, the latter of which can be remethylated by the PCMT. The nearly quantitative conversion of isoaspartyl to aspartyl forms of the peptides has been interpreted as evidence for a repair function, although the process lacks the efficiency characteristic of enzymatic processes, and no isoaspartyl residues are converted into asparaginyl residues, the likely origin of most isoaspartyl sites in cellular proteins (2).Because of the structural complexity of protein substrates for the PCMT, very little information is available regarding the fate of isoaspartyl residues in protein substrates. Deamidation of some proteins, including calmodulin (11) and the bacterial HPr phosphocarrier protein (17), generate...

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Endogenous Substrates for Protein Carboxyl Methyltransferase in Cytosolic Fractions of Bovine Brain

Cited by 36 publications

References 33 publications

Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.

Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.

Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

Complex Interactions of the Protein l-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System

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