Complex interactions of the protein L-isoaspartyl methyltransferase and calmodulin revealed with the yeast two-hybrid system.
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 Km values of 3.6 and 8.6 microM 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.
O'Connor
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MB
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02167-3811, USA.
O'Connor
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CM
D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004789:Enzyme Activation; D006801:Humans; D011485:Protein Binding; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D002147:Calmodulin; D018076:DNA, Complementary; D011994:Recombinant Proteins; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase