Protein L-isoaspartyl Methyltransferase Activity Research Articles

Arabidopsis ABSCISIC ACID INSENSITIVE4 targets PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 in seed.

Arabidopsis ABSCISIC ACID INSENSITIVE4 (ABI4) positively regulates the protein repairing enzyme (PRE) PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 (PIMT1) in seed for its implication in seed vigor and longevity. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme (PRE) and is implicated in seed vigor and longevity. PIMT has been shown to be induced by ABA, however, its detailed regulation by ABA signaling components is unknown. Herein, we report that ABSCISIC ACID INSENSITIVE4 (ABI4) directly binds to the PIMT1 promoter and regulates its expression in Arabidopsis seeds. AtPIMT1 promoter analysis demonstrated the presence of putative ABI4 binding sites. Our Y1H analysis revealed that AtABI4 transcription factor binds to the AtPIMT1 promoter. Dual luciferase assay also demonstrated the binding of the AtABI4 transcription factor to the AtPIMT1 promoter. Subsequently, we have generated AtPIMT1 promoter GUS lines and revealedthat ABA induced expression of GUS in Arabidopsis thaliana. Expression analyses exhibited reduced accumulation of PIMT1 protein and transcript with significant reduction in total PIMT activity in abi4-1 mutants as compared to that of the wild type. The AtPIMT1 promoter GUS expression in abi4-1 mutants was also found to be severely affected in both the control and ABA treatment. Hence, through molecular and genetic evidences we show that the AtABI4 plays a central role in regulating the expression of AtPIMT1 to impart seed vigor and longevity to orthodox seeds.

ABI transcription factors and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE module mediate seed desiccation tolerance and longevity in Oryza sativa.

In contrast to desiccation-tolerant orthodox seeds, recalcitrant seeds are desiccation sensitive and are unable to survive for a prolonged time. Here, our analyses of Oryza species with contrasting seed desiccation tolerance reveals that PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT), an enzyme that repairs abnormal isoaspartyl (isoAsp) residues in proteins, acts as a key player that governs seed desiccation tolerance to orthodox seeds but is ineffective in recalcitrant seeds. We observe that, unlike the orthodox seed of Oryza sativa, desiccation intolerance of the recalcitrant seeds of Oryza coarctata are linked to reduced PIMT activity and increased isoAsp accumulation due to the lack of coordinated action of ABA and ABI transcription factors to upregulate PIMT during maturation. We show that suppression of PIMT reduces, and its overexpression increases, seed desiccation tolerance and seed longevity in O. sativa. Our analyses further reveal that the ABI transcription factors undergo isoAsp formation that affect their functional competence; however, PIMT interacts with and repairs isoAsp residues and facilitates their functions. Our results thus illustrate a new insight into the mechanisms of acquisition of seed desiccation tolerance and longevity by ABI transcription factors and the PIMT module.

Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance.

Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity.

We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1' position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

AbstractWe have established a coupled assay system targeting protein l‐isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1′ position of the caspase‐3 recognition sequence. Following PIMT‐catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase‐3 to give fluorescence activation. High‐throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

Accelerated protein damage in brains of PIMT+/− mice; a possible model for the variability of cognitive decline in human aging

Isoaspartate formation is a common type of protein damage normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). Mice with a knockout of the gene (Pcmt1) for this enzyme (KO, −/−) exhibit a pronounced neuropathology with fatal epileptic seizures at 30–60 days. Heterozygous (HZ, +/−) mice have 50% of the PIMT activity found in wild-type (WT, +/+) mice, but appear normal. To see if HZ mice exhibit accelerated aging at the molecular level, we compared brain extracts from HZ and WT mice at 8 months and 2 years with regard to PIMT activity, isoaspartate levels, and activity of an endogenous PIMT substrate, creatine kinase B. PIMT activity declined modestly with age in both genotypes. Isoaspartate was significantly higher in HZ than WT mice at 8 months and more so at 2 years, rising 5× faster in HZ males and 3× faster in females. Creatine kinase activity decreased with age and was always lower in the HZ mice. These findings suggest the individual variation of human PIMT levels may significantly influence the course of age-related central nervous system dysfunction.

Correction: The Interplay between Protein L-Isoaspartyl Methyltransferase Activity and Insulin-Like Signaling to Extend Lifespan in Caenorhabditis elegans

Correction: The Interplay between Protein L-Isoaspartyl Methyltransferase Activity and Insulin-Like Signaling to Extend Lifespan in Caenorhabditis elegans

The Interplay between Protein L-Isoaspartyl Methyltransferase Activity and Insulin-Like Signaling to Extend Lifespan in Caenorhabditis elegans

The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS) pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair.

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE1 (CaPIMT1) from chickpea mitigates oxidative stress-induced growth inhibition of Escherichia coli

PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs deleterious L-isoaspartyl residues synthesized spontaneously in proteins due to aging or stressful environments and is widespread in living organisms including plants. Even though PIMT activity has been detected from various plant sources, detailed studies are limited to a few species. Our present study on a chickpea (Cicer arietinum) PIMT reveals that apart from seed, PIMT activity is present in other organs and noticeably enhanced during stressful conditions. Using degenerate oligonucleotides and RACE strategy, a full length cDNA (CaPIMT1) was cloned and sequenced. The cDNA is 920 bp in length and contains only one open reading frame of 690 bp encoding 229 amino acids. Genomic structure reveals that the CaPIMT1 gene spans about 2,050 bp in length and contains four exons and three introns. By quantitative real-time RT-PCR, we demonstrate that the transcript of CaPIMT1 is distributed across the organs with maximum levels in seed and is also enhanced under various environmental stress conditions. Purified bacterially expressed protein is further characterized for its catalytic properties. The activity is found to be elevated towards high temperature and pH conditions. Escherichia coli expressing CaPIMT1 show greater tolerance to oxidative stress than E. coli without CaPIMT1. Taken together, our results suggest that PIMT from chickpea shows a distinct pattern of expression and may have a specific role in stress adaptation apart from seed.

Protein repair L-isoaspartyl methyltransferase 1 is involved in both seed longevity and germination vigor in Arabidopsis.

The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments.

Formation, Localization, and Repair of l-Isoaspartyl Sites in Histones H2A and H2B in Nucleosomes from Rat Liver and Chicken Erythrocytes

Formation of l-isoaspartyl (isoAsp) peptide bonds is a major source of protein damage in vivo and in vitro. Accumulation of isoAsp in cells is limited by a ubiquitous repair enzyme, protein l-isoaspartyl methyltransferase (PIMT). Reduction of PIMT activity in mouse brain or rat PC12 cells leads to a dramatic and selective accumulation of isoAsp sites in histone H2B. To learn more about the mechanism and specificity of isoAsp formation in histones, we purified mononucleosomes from rat liver and chicken erythrocytes and subjected them to in vitro aging for 0-16 days. In rat nucleosomes, the pattern of isoAsp accumulation duplicated that observed in vivo; only H2B accumulated significant isoAsp that we have now localized to the Asp25-Gly26 bond in the N-terminal tail. In chicken nucleosomes, isoAsp accumulated mainly in histone H2A and, to a lesser extent, in histone H2B. Minor sequence differences are consistent with the species-specific patterns of isoAsp accumulation and suggest that, in chicken, isoAsp occurs at the Asp121-Ser122 bond in the flexible C-terminal tail of H2A and at the Asp26-Lys27 bond in the N-terminal tail of H2B. The aging-induced accumulation of isoAsp in rat and chicken nucleosomes is repaired upon incubation of the damaged nucleosomes with PIMT and AdoMet. Our findings suggest that in vivo generation of isoAsp sites in histones occurs as a self-catalyzed process at the level of the nucleosome and is driven by the same structural features that have been shown to promote isoAsp formation in purified proteins and synthetic peptides.

Accumulation of proteins bearing atypical isoaspartyl residues in livers of alcohol-fed rats is prevented by betaine administration: Effects on protein- l-isoaspartyl methyltransferase activity

Protein-L-isoaspartyl methyltransferase (PIMT) is a methyltransferase that plays a crucial role in the repair of damaged proteins. In this study, we investigated whether ethanol exposure causes an accumulation of modified proteins bearing atypical isoaspartyl residues that may be related to impaired PIMT activity. We further sought to determine whether betaine administration could prevent the accumulation of these types of damaged proteins. Livers of male Wistar rats, fed the Lieber DeCarli control, ethanol or 1% betaine-supplemented diets for 4 weeks, were processed for PIMT-related analyses. We observed a significant increase in the accumulation of modified proteins bearing isoaspartyl residues, i.e. the substrates for PIMT, in homogenate samples and various subcellular fractions of livers from ethanol-fed rats. Betaine supplementation prevented this accumulation of damaged proteins. In contrast, ethanol exposure induced no changes in the PIMT enzyme activity levels as compared to controls. The accumulation of damaged proteins negatively correlated with hepatic S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratios. Ethanol consumption results in the accumulation of modified proteins bearing atypical isoaspartyl residues via impaired in vivo PIMT activity. Betaine administration prevents the ethanol-induced accumulation of isoaspartyl-containing proteins by restoring the PIMT-catalyzed protein repair reaction through normalizing the hepatocellular SAM:SAH ratios.

Protein L-Isoaspartyl Methyltransferase Catalyzes in Vivo Racemization of Aspartate-25 in Mammalian Histone H2B

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (D/L < or = 0.01). Racemization of Asp25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (D/L = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age.

Expression and activity of l-isoaspartyl methyltransferase decrease in stage progression of human astrocytic tumors

Protein l-isoaspartyl methyltransferase (PIMT) functions as a repair enzyme that acts upon damaged proteins bearing abnormal aspartyl residues. We previously reported that PIMT expression and activity are reduced by half in human epileptic hippocampus. Here we investigated PIMT regulation in astrocytic tumors, which are the most common human brain tumors. PIMT expression and enzyme activity were significantly decreased in all grades of human astrocytic tumors. More precisely, PIMT levels were significantly lower by 76% in pilocytic astrocytomas (grade I), 46% in astrocytomas (grade II), 69% in anaplastic astrocytomas (grade III), and a marked 80% in glioblastomas (grade IV) as compared to normal brains. RT-PCR analysis showed that levels of type I PIMT mRNA were up-regulated while those of type II PIMT mRNA were down-regulated in glioblastomas. Furthermore, the reduced PIMT levels correlated closely with a decrease in the number of neuron cells in astrocytic tumors as assessed by measuring the neuron-specific enolase level. Many proteins with abnormal aspartyl residues accumulated in brain tumors and some were specific to individual grades of astrocytic tumors. Similar results were obtained, either by measuring the reduction in PIMT activity and expression or by measuring the formation of abnormal proteins, in an orthotopic rat brain tumor model implanted with invasive CNS-1 glioma cells. The novelty of these findings was to provide the first evidence for a marked reduction of PIMT expression and activity during stage progression of astrocytic tumors in humans.

Down-regulation of protein L-isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin.

Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component beta-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.

Human Erythrocyte Proteinl-Isoaspartyl Methyltransferase: Heritability of Basal Activity and Genetic Polymorphism for Thermal Stability

Proteinl-isoaspartyl methyltransferase (PIMT) is believed to play an important role in the disposition of age-damaged proteins by catalyzing the repair of abnormal isoaspartyl linkages resulting from the spontaneous deamidation of asparaginyl residues or isomerization of aspartyl residues. As a step toward testing the hypothesis that human disease- or age-related pathology might be associated with a deficiency in PIMT, we investigated basal activity and thermal stability of PIMT in erythrocyte lysates from 299 U.S. family members. Thermal stability was measured because it is a sensitive measure of variation in amino acid sequence. Basal activity was normally distributed with a mean ± SD of 558 ± 43 units/ml erythrocytes. Statistical analysis of the data revealed that basal PIMT activity exhibited a high degree of heritability. Enzyme thermal stability showed a skewed bimodal frequency distribution, and segregation analysis of family member pedigrees was consistent with Mendelian inheritance of two major alleles. No DNA was available from the family samples, so we tested two additional population samples for a known Ile/Val polymorphism at codon 119 and for PIMT activity and thermal stability, using blood donated by 25 Norwegians and by 20 Koreans. Single-stranded conformational polymorphism analysis using polymerase chain reaction revealed a 100% correlation between thermal stability grouping and this polymorphism. The high thermal stability samples were all homozygous Ile, the low thermal stability samples were all homozygous Val, and the intermediate thermal stability samples were all heterozygous. Furthermore, this polymorphism was responsible, in part, for the variation observed in basal erythrocyte PIMT activity. These results will help provide a foundation for future studies aimed at correlating levels of PIMT activity, or other properties of this enzyme, with human disease.