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

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.

PCMT1 regulates the migration, invasion, and apoptosis of prostate cancer through modulating the PI3K/AKT/GSK-3β pathway

“…PCMT1 protein contains an RNA binding domain, a methyltransferase domain, and a S-adenosylme- thionine (SAM) binding domain [ 14 ]. PCMT1 catalyzes the methyl groups of SAM to the side chain carboxyl groups of L-isoaspartic acid and D-aspartic acid residues, so that the protein can restore normal conformation and function [ 15 ]. PCMT1 also participates in biological processes including synaptic transmission, cell matrix interaction and apoptosis through the deamidation of asparagine [ 16 , 17 ].…”

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins

“…22 Studies of PIMT in mice and zebrafish confirm the essential nature of this enzyme, as mice lacking PIMT died of epileptic seizures within 4−8 weeks of birth, 23,24 and PIMT knockdown in zebrafish larvae resulted in decreased survivability and disruption of brain calcium homeostasis. 25 PIMTmediated repair of L-isoAsp begins with methylation of the side chain by reaction with S-adenosyl methionine (SAM). The methyl ester intermediate converts readily to a five-membered succinimide ring, which then undergoes hydrolysis and reopens to form one of the four possible aspartic acid isomers as illustrated in Scheme 1.…”

“…PIMT is unusual in its ability to recognize l -isoAsp, but it is widespread throughout many different organisms including Gram-negative bacteria, plants, fungi, invertebrates, and vertebrates and is thought to be essential to most life forms . Studies of PIMT in mice and zebrafish confirm the essential nature of this enzyme, as mice lacking PIMT died of epileptic seizures within 4–8 weeks of birth, , and PIMT knockdown in zebrafish larvae resulted in decreased survivability and disruption of brain calcium homeostasis . PIMT-mediated repair of l -isoAsp begins with methylation of the side chain by reaction with S -adenosyl methionine (SAM).…”