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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.

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.

The spontaneous L-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-L-isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which L-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-L-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains L-isoaspartate and AdoMet binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing SOCS-box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any L-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.Abstract Figure

Background and aims: Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire 'gain-of-function' isoDGR motifs that might play a role in atherosclerotic pathology. Methods: IsoDGR-specific mAb were generated and used for ELISA-based measurement of motif levels in plasma samples from patients with coronary artery diseases (CAD) and non-CAD controls. Functional consequences of isoDGR accumulation in age-damaged fibronectin were determined by bioassay for capacity to activate monocytes, macrophages, and endothelial cells (signalling activity, pro-inflammatory cytokine expression, and recruitment/adhesion potential). Mice deficient in the isoDGR repair enzyme PCMT1 were used to assess motif distribution and macrophage localisation in vivo. Results: IsoDGR-modified fibronectin and fibrinogen levels in patient plasma were significantly enhanced in CAD and further associated with smoking status. Functional assays demonstrated that isoDGR-modified fibronectin activated both monocytes and macrophages via integrin receptor ‘outside in’ signalling, triggering an ERK:AP-1 cascade and expression of pro-inflammatory cytokines MCP-1 and TNFα to drive additional recruitment of circulating leukocytes. IsoDGR-modified fibronectin also induced endothelial cell expression of integrin β1 to further enhance cellular adhesion and matrix deposition. Analysis of murine aortic tissues confirmed accumulation of isoDGR-modified proteins co-localised with CD68+ macrophages in vivo. Conclusions: Age-damaged fibronectin features isoDGR motifs that increase binding to integrins on the surface of monocytes, macrophages, and endothelial cells. Subsequent activation of ‘outside-in’ signalling elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix. Ministry of Education (MOE) National Medical Research Council (NMRC) This work was in part supported by grants from the National Medical Research Council of Singapore (NMRC–OF–IRG-0003-2016) and Ministry of Education of Singapore (MOE2018-T1-001-078).

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

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-β1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-β1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-β1. In contrast, overexpressed wild-type PIMT and TGF-β1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-β1, while Snail stimulation by TGF-β1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-β1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-β1 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.

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.

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.

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.

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of L-isoAsp using the enzyme Protein L-isoAspartyl Methyltransferase (PIMT) and tris. The succinimide intermediate formed during reaction of L-isoAsp-containing peptides with PIMT and S-Adenosyl Methionine (SAM) is reactive with tris base and results in a tris-modified aspartic acid residue with a mass shift of +103 Daltons. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

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.

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.

Background The development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance. Methods Genome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging. Results We found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors. Conclusions Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

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.

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.

In the past decade, there have been major advances in knowledge related to mesenchymal tumors, and new genetic alterations are being delineated. We report a mesenchymal spindle cell neoplasm harboring a novel gene fusion in an infant. Histopathologically, the neoplasm shared some features with sclerosing perineurioma, but immunohistochemically, EMA was negative, whereas GLUT1, NK1-C3, and BCOR were positive. Next-generation sequencing revealed a PCMTD1-pleomorphic adenoma gene 1 (PLAG1) fusion. PLAG1 contributes to the expression of a variety of genes implicated in regulating cell proliferation, and PCMTD1 has been related to the development of certain carcinomas. Recently, other soft tissue tumors in young children associated with PLAG1 fusion variants have been reported. Perhaps, mesenchymal neoplasms presenting PLAG1 fusions with different genes would confirm a specific group (PLAG mesenchymal tumours or "plagomas") in the near future.

Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from 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 brain and kidney of PIMT-deficient mouse 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 PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In 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 expression miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expression miRNAs between brain and kidney were quantitatively assessed in brain, kidney and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating pathological hallmarks and identify potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN. This article is protected by copyright. All rights reserved.

Fibrillation of peptides and proteins is implicated in various neurodegenerative diseases and is a global concern. Aging leads to the formation of abnormal isoaspartate (isoAsp) residues from isomerization of normal aspartates in proteins, triggering fibril formation that leads to neurodegenerative diseases. Protein L-isoaspartyl methyltransferase (PIMT) is a repair enzyme which recognizes and converts altered isoAsp residues back to normal aspartate. Here we report the effect of gold nanoparticles (AuNPs) of different sizes on the structure and function of PIMT. Spherical AuNPs, viz. AuNS5, AuNS50 and AuNS100 (the number indicating the diameter in nm) stabilize PIMT, with AuNS100 exhibiting the best efficacy, as evident from various biophysical experiments. Isothermal titration calorimetry (ITC) revealed endothermic, but entropy driven mode of binding of PIMT with all the three AuNSs. Methyltransferase activity assay showed enhanced activity of PIMT in presence of all AuNSs, the maximum being with AuNS100. The efficacy of PIMT in presence of AuNS100 was further demonstrated by the reduction of fibrillation of Aβ42, the peptide that is implicated in Alzheimer’s disease. The enhancement of anti-fibrillation activity of PIMT with AuNS100 was confirmed from cell survival assay with PC12 derived neuronal cells against Aβ42 induced neurotoxicity.

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides (52)LFRTVLDSGISEVR(65) and (89)VQDDFVEIH(98), identified within this study, with the l-isoaspartate modification introduced at Asp(58) and Asp(91), respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native (52)LFRTVLDSGISEVR(65) peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin (66)SDRDKFVIFL(isoAsp)VKHF(80), results in enhanced amyloid formation in vitro. The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

Spontaneous formation of isoaspartates (isoDs) often causes protein damage. l-Isoaspartate O-methyltransferase (PIMT) repairs isoD residues by catalyzing the formation of an unstable l-isoaspartyl methyl ester that spontaneously converts to an l-aspartyl residue. PIMTs are widely distributed in all three domains of life and have been studied most intensively in connection with their role in protein repair and aging in plants and animals. Studies of bacterial PIMTs have been limited to Escherichia coli, which has one PIMT. The α-proteobacterium Rhodopseudomonas palustris has three annotated PIMT genes, one of which (rpa2580) has been found to be important for cellular longevity in a growth-arrested state. However, the biochemical activities of these three R. palustris PIMTs are unknown. Here, we expressed and characterized all three annotated PIMT proteins, finding that two of them, RPA0376 and RPA2838, had PIMT activity, whereas RPA2580 did not. RPA0376 and RPA2838 single- and double-deletion mutants did not differ in longevity from WT R. palustris and did not exhibit elevated levels of isoD residues in aged cells. Comparative sequence analyses revealed that RPA2580 belongs to a separate phylogenetic group of annotated PIMT proteins present in the α-proteobacteria. Our results suggest that this group of proteins is not involved in repair of protein isoD residues. In addition, the bona fide bacterial PIMT enzymes may play a different or subtler role in bacterial physiology than previously suggested.

Long-term survivability is well-known for microorganisms in nutrient-depleted environments, but the damage accrued by proteins and the associated repair processes during the starvation and recovery phase of microbial life still remain enigmatic. We focused on aspartic acid (Asp) racemization and repair in the survival of Pyrococcus furiosus and Thermococcus litoralis under starvation conditions at high temperature. Despite the dramatic decrease of viability over time, 0.002% of P. furiosus cells (2.1×103 cells/mL) and 0.23% of T. litoralis cells (2.3×105 cells/mL) remained viable after 25 and 50 days, respectively. The D/L Asp ratio in the starved cells was approximately half of those from the autoclaved cells, suggesting that the starving cells were capable of partially repairing racemized Asp. Transcriptomic analyses of the recovered cells of T. litoralis indicated that the gene encoding Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) might be involved in the repair of damaged proteins by converting D-Asp back to L-Asp during the resuscitation of starved cells. Collectively, our results provided evidence that Asp underwent racemization in the surviving hyperthermophilic cells under starved conditions and PIMT played a critical role in the repair of abnormal aspartyl residues during the initial recovery of starved, yet still viable, cells.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66–80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66–80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact β-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Human carbonic anhydrase IX (CAIX), a unique member of the &alpha, carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease. Evidence suggests that protein isoaspartate methyltransferase 1 (PCMT1) is highly expressed in brain tissue in the substantia nigra, blue plaque, and paraventricular nucleus. In this study, we investigated the effects of neural stem cell conditioned medium on the PCMT1/MST1 pathway to alleviate damage caused by amyloid β (Aβ)25-35 in SH-SY5Y cells. Results demonstrated that Aβ25-35 significantly decreased cell viability in both a time and dose-dependent manner. Neural stem cell-complete medium (NSC-CPM) or NSC-CDM had an inhibitory effect on toxicity when fibrillation of Aβ25-35 occurred, with a greater effect observed with NSC-CDM. An increase in PCMT1 expression levels was observed in the Aβ25-35 + NSC-CDM group. sh-PCMT1 significantly reduced cell viability and inhibited the protective effects of NSC-CDM through the induction of apoptosis and increased expression of p- MST1. Overexpression of PCMT1 reversed the decrease in cell viability and apoptosis induced by Aβ25-35. In summary, our findings suggest that NSC-CDM corrects the damage of Aβ25-35 to cells by increasing levels of PCMT1, which in turn reduces phosphorylation of MST.

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.

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.

To investigate whether newly identified genetic loci for primary angle-closure glaucoma (PACG) are associated with early stage angle-closure disease defined as primary angle closure suspect (PACS).Case-control study.A total of 1397 PACS patients and 943 controls of Chinese ethnicity from Singapore and 604 PACS patients and 287 controls of Indian ethnicity.The 8 PACG single nucleotide polymorphisms (SNPs; rs11024102 at PLEKHA7, rs3753841 at COL11A1, rs1015213 located between PCMTD1 and ST18 son chromosome 8q, rs3816415 at EPDR1, rs1258267 at CHAT, rs736893 at GLIS3, rs7494379 at FERMT2, and rs3739821 mapping in between DPM2 and FAM102A) were genotyped by Taqman assays. The association between SNP genotypes and PACS status was measured using logistic regression. A P value of 0.006 was set to account for the testing of 8 genetic loci using a Bonferroni correction. A meta-analysis was conducted to calculate the overall P value and accompanying per-allele odds ratios for each SNP analyzed.Association of PACG loci with PACS status.The PACS patients were significantly older in both cohorts (Chinese, P0.001; Indian, P = 0.002), and there were also more women (P0.001, both Chinese and Indian cohorts). In the Chinese cohort, significant evidence of association was noted at 3 SNPs: rs1015213 [A] in PCMTD1-ST18 (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.36-4.11; P = 0.002), rs3816415 [A] in EPDR1 (OR, 1.49; 95% CI, 1.19-1.85; P 0.001), and rs3739821 [G] in DPM2-FAM102A (OR, 1.40; 95% CI, 1.18-1.65; P0.001). Only PCMTD1-ST-18 was replicated modestly in the Indian population (P = 0.056). Meta-analysis showed significant evidence of association for PCMTD1-ST-18 (OR, 1.55; 95% CI, 1.18-2.04; P = 0.002) and DPM2-FAM102A (OR, 1.27; 95% CI, 1.12-1.45; P = 0.0002).In this study, 2 of 8 PACG-associated loci were associated significantly with PACS status, the earliest stage in the angle-closure glaucoma disease course. The association of these PACG loci with PACS status suggests that these loci may confer susceptibility to a narrow angle configuration.

The role of protein l-isoaspartate (d-aspartate) O-methyltransferase (PCMT1) in human cancer was generally cognized. The clinical significance and biological function of PCMT1 in bladder cancer is still unknown. PCMT1 mRNA and protein expression levels in bladder cancer tissues and cell lines were detected by qRT-PCR, immunohistochemistry, or western blot. The correlation between PCMT1 expression and clinicopathological factors was analyzed through immunohistochemistry in 108 bladder cancer patients. Loss-of-function and gain-of-function studies were conducted to explore the biological function of PCMT1 in bladder cancer cell lines in regulating cell proliferation, migration, and invasion. In our results, we found that PCMT1 was overexpressed in bladder cancer tissues compared with normal urothelium tissues in microarray datasets (GSE3167). Then, we confirmed PCMT1 mRNA and protein expression were increased in bladder cancer tissues and cell lines compared with paired normal urothelium tissues and normal uroepithelial cell line. PCMT1 protein expression was obviously correlated with clinical stage, muscularis invasion, lymph node metastasis, and distant metastasis. Survival analysis showed that PCMT1 protein high-expression was an independent unfavorable prognostic factor for bladder cancer patients. The in vitro experiments showed PCMT1 regulated bladder cancer cells migration and invasion through modulating epithelial-mesenchymal transition (EMT)-associated genes expression including E-cadherin, vimentin, Snail and Slug, but had no effect on proliferation. In conclusion, PCMT1 is an unfavorable prognostic biomarker and involves in cells migration and invasion through regulating EMT-associated genes. © 2018 IUBMB Life, 70(4):291-299, 2018.

Neuronal apoptosis chiefly contributes to the cell loss following traumatic brain injury (TBI). CGP3466B is a compound related to the anti-Parkinsonism drug R-(−)-deprenyl. Previous studies have illuminated anti-apoptosis effects of CGP3466B in different cell lines, but the underlying mechanisms have not been fully elucidated. Mammalian sterile 20 (STE20)-like kinase1 (Mst1) is a core component of the Hippo signaling pathway. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is an enzyme that repairs damaged L-isoaspartyl residues in proteins. The present study was performed to investigate the neuroprotective effects of CGP3466B and to determine a potential PCMT1/Mst1 neuronal anti-apoptotic pathway after TBI. Double immunofluorescence staining demonstrated that PCMT1 and Mst1 are co-located in neurons. Administration of CGP3466B improved neurological function, downregulated the ROS level and alleviated brain edema at 24 h after TBI. CGP3466B alleviates neuronal apoptosis by increasing PCMT1 expression and subsequently inhibiting MST1 activation, resulting in changing the expression levels of Bax, Bcl-2 and active-caspase3. The TUNEL staining results also support the anti-apoptosis effects of CGP3466B. The anti-apoptotic effects of CGP3466B were abolished by chelerythrine, an Mst1 activator, without changing PCMT1 levels. In conclusion, our findings suggest CGP3466B may have a promising therapeutic potential by modulating PCMT1/Mst1 signaling pathway after TBI injury.

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.

Amino acids undergo many covalent modifications, but only few amino acid repair enzymes have been identified. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), also known as L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), methylates covalently modified isoaspartate (isoAsp) residues accumulated in proteins via Asn deamidation and Asp hydrolysis. This cytoplasmic reaction occurs through the formation of succinimide cyclical intermediate and generates either isoAsp or Asp from succinimide. Succinimide conversion into Asp is spontaneous, while isoAsp is restored by PIMT using S-adenosylmethionine as a methyl donor. PIMT transforms isoAsp into succinimide, thereby creating an opportunity for the later to be converted into Asp. Apart from normal cell physiology, formation of isoAsp in proteins is promoted by various stress conditions. The resulting isoAsp can form a kink or bend in the protein backbone thus making the protein conformationally and functionally distorted. Many PIMT-interacting proteins (proteins with isoAsp residues) have been reported in eukaryotes, but only few of them have been found in prokaryotes. Extensive studies in mice have shown the importance of PIMT in neurodegeneration. Detail elucidation of PIMT function can create a platform for addressing various disorders such as Alzheimer's disease and cancer.

The discovery of active microbial life deeply buried beneath the seafloor has opened important questions: how do microorganisms cope with extreme energy limitation, what is their metabolic activity, and how do they repair damages to essential biomolecules? We used a D:L-amino acid model to calculate microbial biomass turnover times. We used a metagenome and metatranscriptome analysis to investigate the distribution of the gene that encodes Protein-L-iso aspartate(D-aspartate) O-methyltransferase (PCMT), an enzyme which recognizes damaged L-isoapartyl and D-aspartyl residues in proteins and catalyzes their repair. Sediment was retrieved during the Integrated Ocean Drilling Program (IODP) Expedition 347 from Landsort Deep and the Little Belt in the Baltic Sea. The study covers the period from the Baltic Ice Lake ca. 13 000 years ago to the present. Our results provide new knowledge on microbial biomass turnover times and protein repair in relation to different regimes of organic matter input. For the first time, we show that the PCMT gene was widely distributed and expressed among phylogenetically diverse groups of microorganisms. Our findings suggest that microbial communities are capable of repairing D-amino acids within proteins using energy obtained from the degradation of a mixture of labile compounds in microbial necromass and more recalcitrant organic matter.

Asparaginyl deamidation, a spontaneous protein post-biosynthetic modification, determines isoaspartyl formation and structure-function impairment. The isoaspartyl protein carboxyl-O-methyltransferase (PCMT1; EC 2.1.1.77) catalyzes the repair of the isopeptide bonds at isoaspartyl sites, preventing deamidation-related functional impairment. Protein deamidation affects key apoptosis mediators, such as BclxL, thus increasing susceptibility to apoptosis, whereas PCMT1 activity may effectively counteract such alterations. The aim of this work was to establish the role of RNAi as a potential mechanism for regulating PCMT1 expression and its possible implications in apoptosis. We investigated the regulatory properties of the microRNA 15a/16-1 cluster on PCMT1 expression on HepG2 cells. MicroRNA 15a or microRNA 16-1 transfection, as well as their relevant antagonists, showed that PCMT1 is effectively regulated by this microRNA cluster. The direct interaction of these two microRNAs with the seed sequence at the 3' UTR of PCMT1 transcripts was demonstrated by the luciferase assay system. The role of PCMT1 down-regulation in conditioning the susceptibility to apoptosis was investigated using various specific siRNA or shRNA approaches, to prevent non-PCMT1-specific pleiotropic effects to take place. We found that PCMT1 silencing is associated with an increase of the BclxL isoform reported to be inactivated by deamidation, thus making cells more susceptible to apoptosis induced by cisplatinum. We conclude that PCMT1 is effectively regulated by the microRNA 15a/16-1 cluster and is involved in apoptosis by preserving the structural stability and biological function of BclxL from deamidation. Control of PCMT1 expression by microRNA 15a/16-1 may thus represent a late checkpoint in apoptosis regulation.

The l -isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δ pcm mutant produced persisters for an extended period in stationary phase, and a Δ glpD mutation drastically increased persisters in a Δ pcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the Δ pcm Δ glpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δ pcm mutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage. IMPORTANCE Protein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population in Escherichia coli bacteria increased, especially if the metabolic gene glpD was also mutated. High levels of persisters in pcm glpD double mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p

The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells.We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1.We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content.The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

Endoplasmic reticulum stress and chaperone dysfunction have recently been associated with poor prognoses in various cancers. The newly discovered chaperone protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) regulates the viability of cancer cells in various cancers, although no clinical information regarding the relationship between lung cancer and PIMT expression has been reported. In this study, we aimed to elucidate the relationship between PIMT expression and the prognosis of lung adenocarcinoma. Paraffin-embedded lung tissues obtained from 208 patients with surgically resected lung adenocarcinoma were subjected to immunohistochemical analyses using primary antibodies against PIMT. Kaplan-Meier curves, log-rank tests, and the Cox proportional hazards model were used to analyze the association between PIMT expression and patient survival. Strong PIMT expression was detected in 106 (50.9%) patients, being particularly observed in patients with advanced stages of lung adenocarcinoma. Strong PIMT expression was associated with that of 78-kDa glucose-regulated protein, a marker of endoplasmic reticulum stress. Patients with strong PIMT expression had a shorter survival time (Kaplan-Meier analysis, P