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

Abstract

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...

Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

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Maize PIMT2 repairs damaged 3‐METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor

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l-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain

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