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See detailNew models to study the cross-talk between the protein repair L-isoaspartyl methyltransferase and cell signalling
Soliman, Remon UL

Doctoral thesis (2017)

Isomerization of L-aspartyl and L-asparaginyl residues to form L-isoaspartyl residues in proteins is one type of protein damage that can occur under physiological conditions and can potentially lead to ... [more ▼]

Isomerization of L-aspartyl and L-asparaginyl residues to form L-isoaspartyl residues in proteins is one type of protein damage that can occur under physiological conditions and can potentially lead to conformational change, loss of function and enhanced protein degradation. Protein L-isoaspartyl methyltransferase (PCMT or PIMT) is a repair enzyme, which allows the reconversion of L-isoaspartyl residues to L-aspartyl residues in protein. Although the catalytic function of PCMT is known, its physiological roles remain less well understood. Pcmt1 gene knockout in mice leads for example, via molecular mechanisms that remain mostly obscure, to activation of insulin/IGF-1 and MAPK signalling pathways in the brain, and premature death due to massive epileptic seizure events. In this doctoral research project, we have used both mammalian cells and zebrafish models to investigate the impact of PCMT deficiency on insulin/IGF-1, MAPK and calcium signalling as well as how PCMT may be involved in epilepsy. In mammalian cells we used shRNA and CRISPR/Cas9 technology to reduce or completely silence PCMT expression, with the main objective being to mimic, in cell culture, the activation of the IGF-1 and MAPK signalling pathways observed in Pcmt1 knockout mice in the hope to thereby increase the chances to elucidate the underlying molecular mechanisms. In zebrafish we used an antisense morpholino-based strategy to knock down both PCMT homologs and thereby establish a new whole organism model to further study the physiological functions of PCMT, more particularly in the brain. Our results indicate that insulin/IGF-1 signalling is not affected by PCMT knockdown or knockout in mammalian cells whereas a time-dependent MAPK pathway activation could be detected in a Pcmt1 knockout mouse hippocampal cell line. In zebrafish, we showed that the two PCMT homologs Pcmt and Pcmtl (Pcmt/l) possess isoaspartyl methyltransferase activity. In pcmt/l knockdown (or morphant) zebrafish larvae we did not detect abnormal electrical activity in the brain, but we identified movement impairment and strongly perturbed brain calcium fluxes. Abnormal calcium responses were also observed in the Pcmt1 knockout mouse hippocampal cell line. We concluded that the interplay between PCMT and growth signalling pathways is highly dependent on experimental model and may not be amenable to investigation in cell culture. Importantly, our results clearly show that PCMT plays a pivotal role in calcium signalling and suggest that PCMT-dependent repair mechanisms may be important to prevent calcium-related neurological disorders. [less ▲]

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