Abstract
Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish PCMT homologous genes pcmt and pcmtl. Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the pcmt and pcmtl genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for Pcmt1. This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time in vivo, that PCMT plays a pivotal role in the regulation of calcium fluxes.
Highlights
Protein L-isoaspartyl methyltransferase (PCMT) is an enzyme that recognizes isoaspartyl residues in proteins, methylates them and initiates their reconversion to normal aspartyl residues (Johnson et al, 1987b)
For the in vitro activity assay, we used the nonapeptide KASAisoDLAKY, a high affinity substrate for PCMT1 used before to characterize the activity of PCMT homologs from many different species (Ichikawa and Clarke, 1998)
All amino acid residues of the three AdoMet domains and the two isoaspartyl methyltransferase domains are strictly conserved between Pcmt and Pcmtl except for Ile180 in the AdoMet-III domain, which is replaced by a Val in Pcmtl
Summary
Protein L-isoaspartyl methyltransferase (PCMT) is an enzyme that recognizes isoaspartyl residues in proteins, methylates them and initiates their reconversion to normal aspartyl residues (Johnson et al, 1987b). Isoaspartyl residues are formed through a nucleophilic attack of the nitrogen atom in the C-flanking peptide bond on the side chain carbonyl carbon atom of an PCMT and Calcium Signaling in Zebrafish aspartyl or asparaginyl residue. This leads to the formation of an unstable succinimide intermediate, which hydrolyses to a mixture of aspartyl and isoaspartyl residues, with the latter being the major product (70%) (Geiger and Clarke, 1987). A deeper understanding of the implications of PCMTmediated protein repair may suggest new routes for disease modulation, especially in these neurological disorders
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