PROTEIN L-ISOASPARTYL METHYLTRANSFERASE protects enolase dysfunction by repairing isoaspartyl-induced damage and is positively implicated in agronomically important seed traits.

The protein-repairing enzyme (PRE) PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) influences seed vigor by repairing isoaspartyl-mediated protein damage in seeds. However, PIMTs function in other seed traits, and the mechanisms by which PIMT affects such seed traits are still poorly understood. Herein, through molecular, biochemical, and genetic studies using overexpression and RNAi lines in Oryza sativa and Arabidopsis thaliana, we demonstrate that PIMT not only affects seed vigor but also affects seed size and weight by modulating enolase (ENO) activity. We have identified ENO2, a glycolytic enzyme, as a PIMT interacting protein through Y2H cDNA library screening, and this interaction was further validated by BiFC and co-immunoprecipitation assay. We show that mutation or suppression of ENO2 expression results in reduced seed vigor, seed size, and weight. We also proved that ENO2 undergoes isoAsp modification that affects its activity in both in vivo and in vitro conditions. Further, using MS/MS analyses, amino acid residues that undergo isoAsp modification in ENO2 were identified. We also demonstrate that PIMT repairs such isoAsp modification in ENO2 protein, protecting its vital cellular functions during seed maturation and storage, and plays a vital role in regulating seed size, weight, and seed vigor. Taken together, our study identified ENO2 as a novel substrate of PIMT, and both ENO2 and PIMT in turn implicate in agronomically important seed traits.

The protein carboxymethyltransferase-dependent aspartate salvage pathway plays a crucial role in the intricate metabolic network of Escherichia coli.

Protein carboxymethyltransferase (Pcm) is a highly evolutionarily conserved enzyme that initiates the conversion of abnormal isoaspartate to aspartate residues. While it is commonly believed that Pcm facilitates the repair of damaged proteins, a number of observations suggest that it may have another role in cell functioning. We investigated whether Pcm provides a means for Escherichia coli to recycle aspartate, which is essential for protein synthesis and other cellular processes. We showed that Pcm is required for the energy production, the maintenance of cellular redox potential and of S-adenosylmethionine synthesis, which are critical for the proper functioning of many metabolic pathways. Pcm contributes to the full growth capacity both under aerobic and anaerobic conditions. Last, we showed that Pcm enhances the robustness of bacteria when exposed to sublethal antibiotic treatments and improves their fitness in the mammalian urinary tract. We propose that Pcm plays a crucial role in E. coli metabolism by ensuring a steady supply of aspartate.

Protective role of madecassoside from Centella asiatica against protein L-isoaspartyl methyltransferase deficiency-induced neurodegeneration.

Protein L-isoaspartyl methyltransferase (PIMT/PCMT1) could repair l-isoaspartate (L-isoAsp) residues formed by deamidation of asparaginyl (Asn) residues or isomerization of aspartyl (Asp) residues in peptides and proteins during aging. Aside from abnormal accumulation of L-isoAsp, PIMT knockout (KO) mice mirrors some neuropathological hallmarks such as anxiety-like behaviors, impaired spatial memory and aberrant synaptic plasticity in the hippocampus of neurodegenerative diseases (NDs), including Alzheimer's disease (AD) and related dementias, and Parkinson's disease (PD). While some reports indicate the neuroprotective effect of madecassoside (MA) as a triterpenoid saponin component of Centella asiatica, its role against NDs-related anxiety and cognitive impairment remains unclear. Therefore, we investigated the effect of MA against anxiety-related behaviors in PIMT deficiency-induced mouse model of NDs. Results obtained from the elevated plus maze (EPM) test revealed that MA treatment alleviated anxiety-like behaviors in PIMT knockout mice. Furthermore, Real-time PCR, electroencephalogram (EEG) recordings, transmission electron microscopy analysis and ELISA were carried out to evaluate the expression of clock genes, sleep and synaptic function, respectively. The PIMT knockout mice were characterized by abnormal clock patterns, sleep disturbance and synaptic dysfunction, which could be improved by MA administration. Collectively, these findings suggest that MA exhibits neuroprotective effects associated with improved circadian rhythms sleep-wake cycle and synaptic plasticity in PIMT deficient mice, which could be translated to ameliorate anxiety-related symptoms and cognitive impairments in NDs.

Evaluation of supplemented protein-L-isoaspartate-O-methyltransferase (PIMT) gene of Carica papaya and Ricinus communis in stress survival of Escherichia coli BL21(DE3) cells.

In growing plant population, effect of stress is a perturb issue affecting its physiological, biochemical, yield loss and developmental growth. Protein-L-isoaspartate-O-methyltransferase (PIMT) is a broadly distributed protein repair enzyme which actuate under stressful environment or aging. Stress can mediate damage converting protein bound aspartate (Asp) residues to isoaspartate (iso-Asp). This spontaneous and deleterious conversion occurs at an elevated state of stress and aging. Iso-Asp formation is associated with protein inactivation and compromised cellular survival. PIMT can convert iso-Asp back to Asp, thus repairing and contributing to cellular survival. The present work describes the isolation, cloning, sequencing and expression of PIMT genes of Carica papaya (Cp pimt) and Ricinus communis (Rc pimt) Using gene specific primers, both the pimts were amplified from their respective cDNAs and subsequently cloned in prokaryotic expression vector pProEXHTa. BL21(DE3) strain of E. coli cells were used as expression host. The expression kinetics of both the PIMTs were studied with various concentrations of IPTG and at different time points. Finally, the PIMT supplemented BL21(DE3) cells were evaluated against different stresses in comparison to their counterparts with the empty vector control.