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.

PCMT1 knockdown attenuates malignant properties by globally regulating transcriptome profiles in triple-negative breast cancer cells.

Background: As the most frequently diagnosed cancer in women, Breast cancer has high mortality and metastasis rate, especially triple-negative breast cancer (TNBC). As an oncogene, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a prognostic biomarker in breast cancer and is highly expressed, while its underlying functions remain unknown. Methods: In this study, we silenced PCTM1 in TNBC MDA-MB-231 cells by short hairpin RNA (shPCMT1) to investigate its cellular functions using cell proliferation, apoptosis, migration, and invasion experiments. Following this, the transcriptome sequencing (RNA-seq) experiment was conducted to explore the molecular targets of PCMT1, including differentially expressed genes (DEGs) and regulated alternative splicing events (RASEs). Results: The results showed that shPCMT1 inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. We obtained 1,084 DEGs and 2,287 RASEs between shPCMT1 and negative control (NC) groups through RNA-seq. The DEGs were significantly enriched in immune or inflammation response and cell adhesion-associated pathways, pathways associated with PCMT1 cellular function in cell migration. The RASE genes were enriched in cell cycle-associated pathways and were associated with the altered cell proliferation rate. We finally validated the changed expression and splicing levels of DEGs and RASEs. We found that 34 RNA binding protein (RBP) genes were dysregulated by shPCMT1, including NQO1, S100A4, EEF1A2, and RBMS2. The dysregulated RBP genes could partially explain how PCMT1 regulates the global transcriptome profiles. Conclusion: In conclusion, our study identified the molecular targets of PCMT1 in the TNBC cell line, expands our understanding of the regulatory mechanisms of PCMT1 in cancer progression, and provides novel insights into the progression of TNBC. The identified molecular targets are potential therapeutic targets for future TNBC treatment.

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.

Adenosine dialdehyde, a methyltransferase inhibitor, induces colorectal cancer cells apoptosis by regulating PIMT:p53 interaction.

Post-translational modification (PTM) is important in controlling many biological processes by changing the structure and function of a protein. Protein methylation is an important PTM, and the role of methyltransferases has been implicated in numerous cellular functions. Protein L-isoaspartyl methyltransferase (PIMT) is ubiquitously expressed in almost all organisms and govern important cellular processes including apoptosis. Among other functions, PIMT has also been identified as a potent oncogene because it destabilizes the structure of the tumor suppressor p53 via methylation at the transactivation domain. In the present study we identified that out of the three methyltransferase inhibitors tested, namely, S-adenosyl-l-homocysteine (AdoHcy), adenosine and adenosine dialdehyde (AdOx), only AdOx augments p53 expression by destabilizing PIMT structure, as evident from far-UV CD. The effect of the inhibitors, AdOx in particular, to the structure of PIMT, and the binding of PIMT to the p53 transactivation domain have been investigated by docking and molecular dynamics simulations. AdOx significantly increases p53 accumulation and nuclear translocation in colon cancer cells, triggering the p53-mediated apoptotic pathway. To better understand the molecular mechanisms underlying p53 accumulation in colon cancer cells, we observed that the level of PIMT is considerably lower in AdOx-treated cells, reducing its association with p53, which stabilized p53. p53 then transactivated BAX, increasing the BAX: BCL-2 ratio and causing colon cancer cell death.

Involvement of protein L-isoaspartyl methyltransferase in the physiopathology of neurodegenerative diseases: Possible substrates associated with synaptic function.

Synaptic dysfunction is a typical pathophysiologic change in neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), Hintington's disease (HD) and amyotrophic lateral sclerosis (ALS), which involves protein post-translational modifications (PTMs) including L-isoaspartate (L-isoAsp) formed by isomerization of aspartate or deamidation of asparagine. The formation of L-isoAsp could be repaired by protein L-isoaspartyl methyltransferase (PIMT). Some synaptic proteins have been identified as PIMT potential substrates and play an essential role in ensuring synaptic function. In this review, we discuss the role of certain synaptic proteins as PIMT substrates in neurodegenerative disease, thus providing therapeutic synapse-centered targets for the treatment of NDs.

The Role of Protein-L-isoaspartyl Methyltransferase (PIMT) in the Suppression of Toxicity of the Oligomeric Form of Aß42, in Addition to the Inhibition of Its Fibrillization.

The oligomeric form of amyloid-ß peptide (Aß42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD) and is responsible for cognitive deficits. The soluble oligomers are believed to be more toxic compared to the fibril form. Protein-L-isoaspartyl methyltransferase (PIMT) is a repair enzyme that converts aberrant isoAsp residues, formed spontaneously on isomerization of normal Asp and Asn residues, back to typical Asp. It was shown to inhibit the fibrillization of Aß42 (containing three Asp residues), and here, we investigate its effect on the size, conformation, and toxicity of Aß42 oligomers (AßO). Far-UV CD indicated a shift in the conformational feature of AßOs from the random coil to ß-sheet in the presence of PIMT. Binding of bis-ANS to different AßOs (obtained using different concentrations of Aß42 monomer) indicated the correlation of size of oligomers to hydrophobicity: the smallest AßO having the highest hydrophobicity is the most toxic. Dynamic light scattering showed an increase in size of AßO with the addition of PIMT, a contrasting role to that on Aß fibril. Assays using PC12-derived neurons showed the neuroprotective role of PIMT against AßO-induced toxicity. Furthermore, we have elaborated on the molecular mechanism of the antifibrillar action of PIMT and how this function is correlated with its enzymatic activity. PIMT has a more pronounced effect on AßO as compared to a small heat shock protein, pointing to its importance for the amelioration of the adverse effect of both Aß42 oligomers and fibrils.

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.

In vitro Anti-malignant Property of PCMT1 Silencing and Identification of the SNHG16/miR-195/PCMT1 Regulatory Axis in Breast Cancer Cells.

BACKGROUND: Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a highly conserved protein repair enzyme that participates in regulating the progression of human cancers. We therefore studied the function and the related mechanisms of PCMT1 in breast cancer cells. METHODS: Expression profile and prognostic analysis of PCMT1 in breast cancer patients were analyzed using online databases. PCMT1 expression in breast cancer cells was detected by western blot analysis. Cell proliferation was determined by CCK-8 and colony formation assays. Apoptosis was evaluated using flow cytometry analysis and caspase-3/7 activity assay. Cell invasion was assessed by Transwell invasion assay. The small nucleolar RNA host gene 16 (SNHG16)/miR-195/PCMT1 regulatory axis was identified using bioinformatics analysis. RESULTS: PCMT1 expression was increased in breast cancer tissues and cells. High PCMT1 expression was correlated with poor prognosis in breast cancer patients. PCMT1 knockdown suppressed cell proliferation and colony formation ability in breast cancer cells. Moreover, PCMT1 knockdown induced apoptosis and restrained the invasive ability in breast cancer cells. PCMT1 overexpression increased the proliferative and invasive abilities of breast cancer cells. miR-195 was identified as the unique upstream miRNA of PCMT1. SNHG16 was identified as the unique upstream lncRNA of miR-195. SNHG16 knockdown downregulated PCMT1 by increasing miR-195 expression. Breast cancer cell proliferation was regulated by the SNHG16/miR-195/PCMT1 axis. CONCLUSION: PCMT1 silencing inhibited cell proliferation and invasion and induced apoptosis in breast cancer cells and the SNHG16/miR-195/PCMT1 regulatory axis might serve as a potential therapeutic target for breast cancer.

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.

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin.

Aspartimides are notorious as undesired side products in solid-phase peptide synthesis and in pharmaceutical formulations. However, we have discovered several ribosomally synthesized and post-translationally modified peptides (RiPPs) in which aspartimide is installed intentionally via enzymatic activity of protein l-isoaspartyl methyltransferase (PIMT) homologues. In the case of the lasso peptide lihuanodin, the methyltransferase LihM recognizes the lassoed substrate pre-lihuanodin, specifically methylating the side chain of an l-Asp residue in the ring portion of the lasso peptide. The subsequent nucleophilic attack from the adjacent amide leads to the formation of an aspartimide. The resulting aspartimide hydrolyzes regioselectively to l-Asp in buffers above pH 7. Here we report the first Michaelis-Menten kinetic measurements of such a RiPP-associated PIMT homologue, LihM, acting on its cognate substrate pre-lihuanodin. Additionally, we measured the rate of aspartimide hydrolysis, which allowed us to deduce the kinetics of the entire reaction network. The relative magnitudes of these rates explain the accumulation and relative stability of aspartimide-containing lihuanodin. We also demonstrate that the residue C-terminal to the aspartimide controls the regioselectivity of hydrolysis and thus the threadedness of the peptide.

[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.

Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa).

Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of methionine sulfoxide reductase (MSR): MSRA and MSRB. Though the role of MSR enzymes has been elucidated in various physiological processes, the regulation and role of MSR in seeds remains poorly understood. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that an age-induced reduction in the vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and is predominantly localized to the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase and PROTEIN l-ISOASPARTYL METHYLTRANSFERASE undergo MetSO modification in seeds that affects their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds.

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.