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.

Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus.

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.

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

Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a repair enzyme that catalyzes the conversion of isomerized aspartic acid (iso-Asp) residues into their normal structure, thereby restoring the configuration and function of proteins. Studies have shown that PCMT1 is overexpressed in several tumors and affects patients' prognosis. However, there are few reports on the role of PCMT1 in prostate cancer (PCa). In the present research, with the assistance of The Cancer Genome Atlas Program (TCGA) database, we found that PCMT1 was overexpressed in PCa tissues. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry staining also showed that PCMT1 expression was significantly increased in PCa tissues and cell lines. In PCa clinical samples, PCMT1 expression was closely related to Gleason score, clinical stage, lymph node metastasis and bone metastasis. The experiments of overexpression and knockdown of PCMT1 in vitro or in vivo showed that PCMT1 can significantly promote the proliferation, migration and invasion of PCa cells, inhibit cell apoptosis, and promote the growth of PCa. We furthermore confirmed that PCMT1 regulated the migration, invasion and apoptosis of PCa cells by modulating the phosphatidylinositol 3-kinase/AKT kinase/glycogen-synthase kinase-3ß (PI3K/AKT/GSK-3ß) signaling pathway. Collectively, PCMT1 plays a cancer-facilitative role in PCa by promoting the proliferation, migration and invasion of PCa cells, and inhibiting apoptosis. Therefore, PCMT1 is considered to represent a novel target for treating PCa.

PCMT1 is a potential target related to tumor progression and immune infiltration in liver cancer.

BACKGROUND: Liver cancer is a prevalent and deadly form of cancer with high incidence and mortality rates. The PCMT1 protein has been linked to cell anti-apoptosis and tumor metastasis, but its significance in liver hepatocellular carcinoma (LIHC) remains largely unexplored. METHODS: We conducted a pan-cancer analysis to examine the expression differences of PCMT1. Kaplan-Meier curves were employed to assess the prognostic impact of PCMT1 on LIHC patients, and we investigated the association between PCMT1 and clinical features, which we validated using a GEO therapeutic dataset. Gene enrichment analysis helped identify signaling pathways associated with PCMT1 expression. Moreover, we evaluated the relationship between PCMT1 and immune cell infiltration, as well as the differences in gene mutations between high-expression and low-expression groups. In vitro and in vivo experiments were performed to assess the effect of PCMT1 on tumor cell lines and mouse tumor models, and potential pathways were explored through gene sequencing. RESULT: PCMT1 is highly expressed in most tumors and exhibits a significant association with prognosis in LIHC patients. Pathway enrichment analysis revealed that PCMT1 is involved in cell cycle regulation, immunity, and other processes. Further immune analysis demonstrated that high expression of PCMT1 could reduce tumor-killing immune cell infiltration. In vitro experiments indicated that PCMT1 knockdown could inhibit cancer cell proliferation and migration while promoting apoptosis. In vivo experiments showed that PCMT1 knockdown significantly reduced tumor growth rate, enhanced CD8+T cell infiltration, and increased caspase-3 expression in the tumor area. Gene sequencing suggested that PCMT1 may function through the PI3K-AKT pathway. CONCLUSION: Our findings suggest that PCMT1 acts as a promoter of liver cancer progression and may serve as a novel prognostic indicator and therapeutic target for patients with LIHC.

The role of PIMT in Alzheimer's disease pathogenesis: A novel hypothesis.

There are multiple theories of Alzheimer's disease pathogenesis. One major theory is that oxidation of amyloid beta (Aß) promotes plaque deposition that directly contributes to pathology. A competing theory is that hypomethylation of DNA (due to altered one carbon metabolism) results in pathology through altered gene regulation. Herein, we propose a novel hypothesis involving L-isoaspartyl methyltransferase (PIMT) that unifies the Aß and DNA hypomethylation hypotheses into a single model. Importantly, the proposed model allows bidirectional regulation of Aß oxidation and DNA hypomethylation. The proposed hypothesis does not exclude simultaneous contributions by other mechanisms (e.g., neurofibrillary tangles). The new hypothesis is formulated to encompass oxidative stress, fibrillation, DNA hypomethylation, and metabolic perturbations in one carbon metabolism (i.e., methionine and folate cycles). In addition, deductive predictions of the hypothesis are presented both to guide empirical testing of the hypothesis and to provide candidate strategies for therapeutic intervention and/or nutritional modification. HIGHLIGHTS: PIMT repairs L-isoaspartyl groups on amyloid beta and decreases fibrillation. SAM is a common methyl donor for PIMT and DNA methyltransferases. Increased PIMT activity competes with DNA methylation and vice versa. The PIMT hypothesis bridges a gap between plaque and DNA methylation hypotheses.

PIMT is a novel and potent suppressor of endothelial activation.

Proinflammatory agonists provoke the expression of cell surface adhesion molecules on endothelium in order to facilitate leukocyte infiltration into tissues. Rigorous control over this process is important to prevent unwanted inflammation and organ damage. Protein L-isoaspartyl O-methyltransferase (PIMT) converts isoaspartyl residues to conventional methylated forms in cells undergoing stress-induced protein damage. The purpose of this study was to determine the role of PIMT in vascular homeostasis. PIMT is abundantly expressed in mouse lung endothelium and PIMT deficiency in mice exacerbated pulmonary inflammation and vascular leakage to LPS(lipopolysaccharide). Furthermore, we found that PIMT inhibited LPS-induced toll-like receptor signaling through its interaction with TNF receptor-associated factor 6 (TRAF6) and its ability to methylate asparagine residues in the coiled-coil domain. This interaction was found to inhibit TRAF6 oligomerization and autoubiquitination, which prevented NF-κB transactivation and subsequent expression of endothelial adhesion molecules. Separately, PIMT also suppressed ICAM-1 expression by inhibiting its N-glycosylation, causing effects on protein stability that ultimately translated into reduced EC(endothelial cell)-leukocyte interactions. Our study has identified PIMT as a novel and potent suppressor of endothelial activation. Taken together, these findings suggest that therapeutic targeting of PIMT may be effective in limiting organ injury in inflammatory vascular diseases.

Protein l-isoAspartyl Methyltransferase (PIMT) and antioxidants in plants.

All life forms, including plants, accumulate reactive oxygen species (ROS) as a byproduct of metabolism; however, environmental stresses, including abiotic stresses and pathogen attacks, cause enhanced accumulation of ROS in plants. The increased accumulation of ROS often causes oxidative damage to cells. Organisms are able to maintain levels of ROS below permissible limits by several mechanisms, including efficient antioxidant systems. In addition to antioxidant systems, recent studies suggest that protein l-isoaspartyl methyltransferase (PIMT), a highly conserved protein repair enzyme across evolutionary diverse organisms, plays a critical role in maintaining ROS homeostasis by repairing isoaspartyl-mediated damage to antioxidants in plants. Under stress conditions, antioxidant proteins undergo spontaneous isoaspartyl (isoAsp) modification which is often detrimental to protein structure and function. This reduces the catalytic action of antioxidants and disturbs the ROS homeostasis of cells. This chapter focuses on PIMT and its interaction with antioxidants in plants, where PIMT constitutes a secondary level of protection by shielding a primary level of antioxidants from dysfunction and permitting them to guard during unfavorable situations.

[Prognostic Value of PCMT1 Expression in Gastric Cancer and Its Regulatory Effect on Spindle Assembly Checkpoints]. / 胃癌中PCMT1è¡¨è¾¾çš„é¢„åŽä»·å€¼åŠå ¶å¯¹çººé”¤ä½“ç»„è£ æ£€æŸ¥ç‚¹çš„è°ƒæŽ§ä½œç”¨.

Objective: The study was conducted to investigate the expression of protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) in gastric cancer and its effect on the prognosis, and to analyze its potential mechanism. Methods: UALCAN, a cancer data analysis platform, was used to conduct online analysis of the expression of PCMT1 in gastric cancer tissues. Through the Database for Annotation, Visualization and Integrated Discovery (DAVID), Gene Ontology (GO) annotation and signaling pathway enrichment by Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the possible functions and signaling pathways. A total of 120 patients who underwent radical gastrectomy for gastric cancer between January 2014 and December 2017 in our hospital were enrolled for the study. Immunohistochemical staining was performed to determine the expression of PCMT1 and Ki67 in gastric cancer tissues. Cox regression, Kaplan-Meier curve, and receiver operating characteristic (ROC) curves were used for prognostic analysis of 5-year survival in gastric cancer patients after surgery. Lentivirus was used to construct PCMT1-interfering or PCMT1-overexpressing vectors, which were then used to transfect human gastric cancer cell lines of MGC-803 and HGC-27 cells. The interfering empty vector (sh-NC) group, the interfering PCMT1 vector (sh-PCMT1) group, the overexpressing empty vector (LV-Vec) group, and the overexpressing PCMT1 vector (LV-PCMT1) group were set up. Western blot was performed to determine the protein expression levels of PCMT1, CyclinB1, and CDC20. CCK-8 assay was performed to measure the proliferation of gastric cancer cells. Flow cytometry was performed to determine the cell cycle. MGC-803 cells were injected in four groups of nude mice to construct a subcutaneous xenograft tumor model, with three nude mice in each group. The body mass of the nude mice was measured. The nude mice were sacrificed after 14 days and the tumor volume was monitored. The expression levels of CyclinB1 and CDC20 proteins in the tumor tissues were determined by Western blot assay. Results: Analysis with UALCAN showed that PCMT1 was highly expressed in gastric cancer tissues. Moreover, elevated expression was found in gastric tumor tissues of different pathological stages and grades and those with lymph node metastasis (P<0.05). GO and KEGG enrichment analyses showed that PCMT1 was mainly involved in the signal regulation of mitosis, spindle assembly checkpoints, and cell cycle. The immunohistochemical results showed that PCMT1 and Ki67 were highly expressed in gastric cancer tissues and that they were positively correlated with each other (P<0.05). Cox multivariate analysis showed that high PCMT1 expression (hazard ratio [HR]=2.921, 95% confidence interval [CI]:1.628-5.239) was one of the independent risk factors affecting the 5-year survival rate of gastric cancer patients after surgery. Kaplan-Meier curve showed that patients with high PCMT1 expression had a lower 5-year survival after surgery (16.7%, HR=4.651, 95% CI: 2.846-7.601) than patients with low PCMT1 expression (70.0%, HR=0.215, 95% CI: 0.132-0.351) did. The ROC curve showed that PCMT1 had an area under the curve (AUC) of 0.764 (95% CI: 0.674-0.854) for predicting 5-year patient survival after surgery. Western blot results showed that lentiviral interference or overexpression of PCMT1 cell lines was successfully constructed. The results of CCK-8 showed that the proliferative ability of MGC-803 and HGC-27 cells was weakened with the downregulation of PCMT1, and the overexpression of PCMT1 promoted cell proliferation (P<0.05). With the interference of PCMT1, the expression of CDC20 protein was decreased, the expression of CyclinB1 protein was increased, and the cell cycle was arrested in the G2/M phase. In contrast, the overexpression of PCMT1 led to the opposite trends (P<0.05). In the sh-PCMT1 group, the tumor volume and mass were decreased and the expression of CDC20 protein was decreased and the expression of CyclinB1 protein was increased in the tumor tissues of the nude mice (P<0.05, compared with those of the sh-NC group. In contrast, the LV-PCMT1 group showed the opposite trends (P<0.05, compared with those of the LV-Vec group). Conclusion: The high expression of PCMT1 in gastric cancer tissues is associated with poor prognosis in patients and may affect tumor cell malignant proliferation via regulating spindle checkpoints in the process of mitosis.

Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide.

Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain-side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the L-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

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.

The Protein L-Isoaspartyl (D-Aspartyl) Methyltransferase Regulates Glial-to-Mesenchymal Transition and Migration Induced by TGF-ß1 in Human U-87 MG Glioma Cells.

The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-ß1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-ß1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-ß1. In contrast, overexpressed wild-type PIMT and TGF-ß1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-ß1, while Snail stimulation by TGF-ß1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-ß1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-ß1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT.

PCMT1 Is a Potential Prognostic Biomarker and Is Correlated with Immune Infiltrates in Breast Cancer.

Background: Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is involved in the occurrence and development of a variety of malignant tumors. However, the prognostic value of PCMT1 in breast cancer remains unclear. Methods: Based on the Cancer Genome Atlas database, we assessed the correlation between the expression of PCMT1 and prognosis, immune invasion, and tumor mutation burden in a variety of cancers. The expression level, mutation, immune correlation, and coexpression of PCMT1 in breast cancer were studied using the following databases: UALCAN database, Human Protein Atlas database, cBioPortal database, TIMER database, and LinkedOmics database. Kaplan-Meier Plotter was used for survival analysis. Receiver operating characteristic (ROC) curves and nomograms were drawn using the R software package. P < 0.05 was considered statistically significant. Results: Pancancer analysis showed that PCMT1 is highly expressed in a variety of cancers and is significantly related to the prognosis of a variety of cancers. PCMT1 is significantly related to the tumor mutation burden of a variety of cancers. PCMT1 is significantly high in breast cancer, and it is significantly related to the abundance of immune infiltration. Survival analysis revealed that high PCMT1 expression is significantly associated with shorter overall survival (OS), relapse-free survival (RFS), and postprogression survival (PPS) in breast cancer patients. ROC curves and nomograms verify the effectiveness of PCMT1 as a prognostic biomarker for breast cancer. Conclusions: PCMT1 can be used as a potential prognostic biomarker of breast cancer, and it is significantly related to the abundance of breast cancer immune infiltration.

PIMT/TGS1: An evolving metabolic molecular switch with conserved methyl transferase activity.

Transcriptional coactivators play a crucial role in regulating gene expression. PRIP interacting protein with methyl transferase domain (PIMT)/trimethyl guanosine synthase 1 (TGS1) is a co-activator interacting protein with an RNA methyl transferase domain. PIMT serves as a bridge between HAT and non-HAT coactivators and differentially modulates gene expression. Disruption of PIMT is embryonic lethal. PIMT regulates hepatic gluconeogenesis and TNF-α-induced insulin resistance in the skeletal muscle. As a methyl transferase, PIMT controls post-transcriptional regulation of HIV-1 and is essential for human telomerase RNA biogenesis. This review comprehensively describes the dual role of PIMT, which promises to be a putative target in metabolic disorders.

Differential expression of microRNAs associated with neurodegenerative diseases and diabetic nephropathy in protein l-isoaspartyl methyltransferase-deficient mice.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from the spontaneous decomposition of normal l-aspartyl and l-asparaginyl residues during aging, has been revealed to be involved in neurodegenerative diseases (NDDs) and diabetes. However, the molecular mechanisms for a putative association of PIMT dysfunction with these diseases have not been clarified. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the brain and kidneys of PIMT-deficient mice and uncover the epigenetic mechanism of PIMT-involved NDDs and diabetic nephropathy (DN). Differentially expressed miRNAs by sequencing underwent target prediction and enrichment analysis in the brain and kidney of PIMT knockout (KO) mice and age-matched wild-type (WT) littermates. Sequence analysis revealed 40 differentially expressed miRNAs in the PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In the PIMT KO mouse kidney, there were 80 differentially expressed miRNAs including 40 upregulated miRNAs and 40 downregulated miRNAs. Enrichment analysis and a systematic literature review of differentially expressed miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expressed miRNAs between the brain and kidney were quantitatively assessed in the brain, kidney, and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating the pathological hallmarks and identify the potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN.

The enzyme L-isoaspartyl (D-aspartyl) methyltransferase promotes migration and invasion in human U-87 MG and U-251 MG glioblastoma cell lines.

The protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT) recognizes abnormal L-isoaspartyl and D-aspartyl residues in proteins. Among examined tissues, PIMT shows the highest level in the brain. The U-87 MG cell line is a commonly used cellular model to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT amount increased when U-87 MG cells were detached from the extracellular matrix. Recently, we also showed that PIMT possessed pro-angiogenic properties. Together, these PIMT features led us to postulate that PIMT could play a critical role in glioblastoma growth. Here, we investigate PIMT role in U-87 MG cell viability, adhesion, migration, invasion, and colony formation and in the reorganization of the actin and tubulin cytoskeleton. PIMT inhibition by siRNA significantly reduced in vitro cell migration and invasion in various assays, including wound-healing assay, Boyden chambers coated with gelatin and Matrigel invasion assay. Conversely, in stably transfected U-87 MG cells overexpressing wild-type PIMT, cell migration, invasive capacity and colony formation significantly increased. However, in stably transfected cells with the gene encoding for mutated PIMT(D83V), despite of its overexpression, migration and invasion remained similar to those observed in control cells. In all these conditions, cell viability was unaffected. Importantly, overexpressed wild-type PIMT and mutated PIMT(D83V) have opposite effects on the organization of microtubules and actin cytoskeleton and thus on morphology of U-87 cells. These data highlighted the importance of PIMT level and its catalytic activity in migration and invasion of U-87 glioma cells and its possible contribution in cancer invasion during glioma growth.

Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) in plants: regulations and functions.

Proteins are essential molecules that carry out key functions in a cell. However, as a result of aging or stressful environments, the protein undergoes a range of spontaneous covalent modifications, including the formation of abnormal l-isoaspartyl residues from aspartyl or asparaginyl residues, which can disrupt the protein's inherent structure and function. PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT: EC 2.1.1.77), an evolutionarily conserved ancient protein repairing enzyme (PRE), converts such abnormal l-isoaspartyl residues to normal l-aspartyl residues and re-establishes the protein's native structure and function. Although originally discovered in animals as a PRE, PIMT emerged as a key PRE in plants, particularly in seeds, in which PIMT plays a predominant role in preserving seed vigor and viability for prolonged periods of time. Interestingly, higher plants encode a second PIMT (PIMT2) protein which possesses a unique N-terminal extension, and exhibits several distinct features and far more complexity than non-plant PIMTs. Recent studies indicate that the role of PIMT is not restricted to preserving seed vigor and longevity but is also implicated in enhancing the growth and survivability of plants under stressful environments. Furthermore, expression studies indicate the tantalizing possibility that PIMT is involved in various physiological processes apart from its role in seed vigor, longevity and plant's survivability under abiotic stress. This review article particularly describes new insights and emerging interest in all facets of this enzyme in plants along with a concise comparative overview on isoAsp formation, and the role and regulation of PIMTs across evolutionary diverse species. Additionally, recent methods and their challenges in identifying isoaspartyl containing proteins (PIMT substrates) are highlighted.

Effect of amino acids present at the carboxyl end of succinimidyl residue on the rate constants for succinimidyl hydrolysis in small peptides.

Structural alterations of aspartyl and asparaginyl residues in various proteins can lead to their malfunction, which may result in severe health disorders. The formation and hydrolysis of succinimidyl intermediates are crucial in specific protein modifications. Nonetheless, only few studies investigating the hydrolysis of succinimidyl intermediates have been published. In this study, we established a method to prepare peptides bearing succinimidyl residues using recombinant protein l-isoaspartyl methyltransferase and ultrafiltration units. Using succinimidyl peptides, we examined the effect of amino acid residues on succinimidyl hydrolysis at the carboxyl end of succinimidyl residues and determined the rate constant of hydrolysis for each peptide. The rate constant of succinimidyl hydrolysis in the peptide bearing a Ser residue at the carboxyl side (0.50 ± 0.02 /h) was 3.0 times higher than that for the peptide bearing an Ala residue (0.17 ± 0.01 /h), whereas it was just 1.2 times higher for the peptide bearing a Gly residue (0.20 ± 0.01 /h). The rate constant of succinimidyl formation in the peptide bearing a Ser residue [(2.44 ± 0.11) × 10-3 /d] was only 1.2 times higher than that for the peptide bearing an Ala residue ([1.87 ± 0.09) × 10-3 /d], whereas 5.5 times higher for the peptide bearing a Gly residue [(10.2 ± 0.2) × 10-3 /d]. These results show that the Gly and Ser residues at the carboxyl end of the succinimidyl residue have opposing roles in succinimidyl formation and hydrolysis. Catalysis of Ser residue's hydroxyl group plays a crucial role in succinimidyl hydrolysis.

Protein Kinase C Theta Modulates PCMT1 through hnRNPL to Regulate FOXP3 Stability in Regulatory T Cells.

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.

Isolation and Identification of Protein L-Isoaspartate-O-Methyltransferase (PIMT) Interacting Proteins in Salmonella Typhimurium.

Protein L-isoaspartate-O-methyltransferase (PIMT) plays an important role in restoration of covalently damaged Asn/Asp residues. It repairs the racemized forms of these amino acids in protein by forming a labile isoAsp methyl ester which readily converts back to the succinimide intermediate. Spontaneous hydrolysis of the intermediate further restores a minor portion to the normal Asp residues. While significant numbers of PIMT targets have been identified in eukaryotes, very few are documented from prokaryotes. Temperature (42 °C) induced elevation in PIMT expression level has been recently shown in a poultry isolate of Salmonella Typhimurium (ST). The enzyme was also found to be crucial for survival, virulence and colonization of ST in poultry. In the present study, co-immunoprecipitation (Co-IP) approach was used (for isolation) followed by LC-MS analysis to identify the PIMT interacting proteins of ST. Four different proteins were identified among which cytochrome C biogenesis protein A (CcmA) was further expressed in recombinant form and analysed for interaction with recombinant PIMT (rPIMT) by microtiter plate assay. Additionally, the findings were supported by alterations in secondary structure of the proteins upon co-incubation.