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BACKGROUND AIMS: Nitric oxide, a major inhibitory nonadrenergic, noncholinergic neurotransmitter that relaxes smooth muscle, may be implicated in the pathophysiology of visceral hypersensitivity in irritable bowel syndrome (IBS). Impaired bioavailability of the nitric oxide precursor molecule L-arginine and higher concentrations of methylarginines (endogenous inhibitors of nitric oxide synthesis) are known to impair nitric oxide synthesis in numerous gastrointestinal cell types. We therefore examined serum concentrations of L-arginine and the methylarginines in a nested case-control study, to assess whether these factors are associated with adult IBS.Data on clinical characteristics, methylarginines, and L-arginine (measured using LC-MS/MS) were collected from a random population-based cohort of Australian adults (median age = 64 years; IQR = 60-70). Cases of IBS, defined according to Rome III criteria (N = 156), and controls (N = 332) were identified from within the cohort at the 5-year follow-up.In adjusted logistic regression analyses, L-arginine, asymmetric dimethylarginine, symmetric dimethylarginine, L-arginine/asymmetric dimethylarginine ratio, and Kessler-10 psychological distress scores were significantly associated with IBS (p 0.05). [Correction added on 18 September 2021, after first online publication: In the preceding sentence, the value (p 0.05) has been changed to (p 0.05)]. Similar results were found for IBS subtypes. Higher serum L-arginine concentration had the strongest association with IBS diagnosis, with an odds ratio of 9.03 for those with serum L-arginine at the 75th (84 μmol/L) versus 25th (46 μmol/L) percentile (95% CI: 5.99-13.62). L-arginine had the best discriminative ability with a bias-adjusted area under the receiver operator characteristic curve of 0.859.Higher serum concentrations of L-arginine and endogenous methylarginines are strongly associated with IBS in adults.

Our aim in this study was to measure serum levels of methylarginines and related metabolites in patients with ankylosing spondylitis (AS), moreover, to investigate the relationship between these parameters and various clinical and laboratory parameters of patients with AS. The study included 60 patients with AS and 60 healthy volunteers. Serum asymmetric dimethylarginine (ADMA), L–N monomethylarginine (L-NMMA), symmetric dimethylarginine (SDMA), arginine (Arg), homoarginine (hArg), ornithine, and citrulline concentrations were measured with tandem mass spectrometry. In addition, participants were divided into three groups according to the treatment regimen: TNF-α inhibitor group (n = 25), conventional therapy group (n = 35), and control group (n = 60). These groups were compared in terms of serum levels of methylarginine pathway metabolites and various biochemical parameters. It was found that total methylated arginine load significantly increased in patients with AS (p

Thousands of proteins undergo arginine methylation, a widespread post-translational modification catalyzed by several protein arginine methyltransferases (PRMTs). However, global understanding of their biological functions is limited due to the lack of a complete picture of the catalytic network for each PRMT. Here, we systematically identified interacting proteins for all human PRMTs and demonstrated their functional importance in mRNA splicing and translation. We demonstrated significant overlapping of interactomes of human PRMTs with the known methylarginine-containing proteins. Different PRMTs are functionally redundant with a high degree of overlap in their substrates and high similarities between their putative methylation motifs. Importantly, RNA-binding proteins involved in regulating RNA splicing and translation contain highly enriched arginine methylation regions. Moreover, inhibition of PRMTs globally alternates alternative splicing (AS) and suppresses translation. In particular, ribosomal proteins are extensively modified with methylarginine, and mutations in their methylation sites suppress ribosome assembly, translation, and eventually cell growth. Collectively, our study provides a global view of different PRMT networks and uncovers critical functions of arginine methylation in regulating mRNA splicing and translation.

Background Nitric oxide (NO) plays an important role in endothelial homeostasis. Asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA) and symmetric dimethyl arginine (SDMA), which are derivatives of methylarginine, directly or indirectly reduce NO production. Therefore, these metabolites are an important risk factor for various diseases, including cardiovascular diseases. Numerous methods have been developed for the measurement of methylarginine derivatives, but various difficulties have been encountered. This study aimed to develop a reliable, fast and cost-effective method for the analysis and measurement of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, citrulline, homoarginine, ornithine), and to validate this method according to Clinical and Laboratory Standards Institute (CLSI) protocols. Methods For the analysis of ADMA, SDMA, L-NMMA, arginine, homoarginine, citrulline, ornithine, 200 µl of serum were precipitated with methanol, and subsequently derivatized with a butanol solution containing 5% acetyl chloride. Butyl derivatives were separated using a C18 reverse phase column with a 5 min run time. Detection of analytes was achieved by utilising the specific fragmentation patterns identified through tandem mass spectrometry. Results The method was linear for ADMA, SDMA, L-NMMA, ornithine, arginine, homoarginine and citrulline in the ranges of 0.023–6.0, 0.021–5.5, 0.019–5.0, 0.015–250, 0.015–250, 0.019–5 and 0.015–250 µM, respectively. The inter-assay CV% values for all analytes was less than 9.8%. Conclusions Data obtained from method validation studies shows that the developed method is highly sensitive, precise and accurate. Short analysis time, cost-effectiveness, and multiplexed analysis of these metabolites, with the same pretreatment steps, are the main advantages of the method.

Objectives Thyroid disorders are important risk factor for cardiovascular diseases. Levels of methylarginines such as asymmetric dimethyl arginine (ADMA), L-monomethyl arginine (L-NMMA), symmetric dimethyl arginine (SDMA) are increase in cardiovascular diseases. Multinodular goiter (MNG) is the most common type of goiter in adults. To date, no study has been conducted to determine the levels of methylarginine in euthyroid MNG patients. Our aim in this study is to compare levels of methylarginines and related metabolites in the preoperative, postoperative MNG patients and controls. Methods Serum ADMA, SDMA, L-NMMA, homoarginine (hArg), arginine and citrulline concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results ADMA (pl-arginine (p=0.006) and citrulline (p Conclusions The levels of methylarginines and related metabolites altered in the euthyroid MNG patients compared to the control group, and more importantly, there were significant differences between the preop and postop groups. Therefore, these metabolites can be useful in the diagnosis and prognosis of thyroid disorders, even if thyroid hormone levels are normal.

Objectives Infertility is defined as the absence of pregnancy within the reproductive period despite regular sexual intercourse. Methylarginines are formed as a result of methylation of arginine residues in proteins and formed in three forms as asymmetric dimethyl arginine (ADMA), symmetrical dimethyl arginine (SDMA) and monomethylarginine (L-NMMA). So, here, we aimed to evaluate arginine and their derivatives levels in fertile and infertile individuals. Methods Present study were consist of 30 oligozoospermia patients (proven by spermiogram analysis) and 30 healthy individuals with normozoospermia group who were applied to the urology department. With blood samples taken from individuals, serum methylarginine and its derivatives levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clinic data and demographic characteristics of individuals were also recorded at the same time. Results The serum ADMA level (0.38 ± 0.07) of the oligozoospermia group was found to be significantly higher than the normozoospermia group (0.35 ± 0.05) (p=0.046). A positive correlation were observed between ADMA and SDMA (r=0.686, p=0.000), HArg and SDMA (r=0.611, p=0.001), citrulline and L-NMMA (r=0.595, p=0.001) in patients with oligosospermia. The increase in SDMA, arginine and HArg levels and a decrease in L-NMMA and citrulline levels were not significant as statistically. Also, the ADMA level was found to be high in individuals with low sperm concentration. Conclusions Consequently, serum ADMA levels of individuals with oligozoospermia were statistically significantly higher than those with normozoospermia. As proposal, determination of ADMA levels may be a potential biomarker parameter in terms of early diagnosis of fertility and infertility.

Arginine methylation is one of the most essential protein post-translational modifications. Identifying the site of arginine methylation is a critical problem in biology research. Unfortunately, biological experiments such as mass spectrometry are expensive and time-consuming. Hence, predicting arginine methylation by machine learning is an alternative fast and efficient way. In this paper, we focus on the systematic characterization of arginine methylation with composition-transition-distribution (CTD) features. The presented framework consists of three stages. In the first stage, we extract CTD features from 1750 samples and exploit decision tree to generate accurate prediction. The accuracy of prediction can reach 96%. In the second stage, the support vector machine can predict the number of arginine methylation sites with 0.36 R-squared. In the third stage, experiments carried out with the updated arginine methylation site data set show that utilizing CTD features and adopting random forest as the classifier outperform previous methods. The accuracy of identification can reach 82.1 and 82.5% in single methylarginine and double methylarginine data sets, respectively. The discovery presented in this paper can be helpful for future research on arginine methylation.

Histone H3 arginine 2 (H3R2) is post-translationally modified in three different states by “writers” of the protein arginine methyltransferase (PRMT) family. H3R2 methylarginine isoforms include PRMT5-catalyzed mono- and symmetric di-methylation (me1, me2s), and PRMT6-catalyzed me1 and asymmetric dimethylation (me2a). WD-40 repeat-containing protein 5 (WDR5) is an epigenetic “reader” protein that interacts with H3R2 and is a subunit of numerous chromatin-modifying complexes, such as theMixedLineageLeukemia (MLL) H3 lysine 4 methyltransferase complex. Previous studies suggested that MLL recruitment to chromatin was specified by the high-affinity interaction between WDR5 and H3R2me2s. However, our prior biological data prompted the hypothesis that WDR5 may also interact with H3R2me1 to recruit MLL activity. Here, using highly accurate quantitative binding analysis combined with high-resolution crystal structures of WDR5 in complex with unmodified (me0) and me1/me2s L-Arginine amino acids and in complex with H3R2me1 peptide, we provide a rigorous biochemical study of this important biological interaction. Despite modest structural differences at the binding interface, our study supports an interaction model regulated by a binary arginine methylation switch: H3R2me2a prevents interaction with WDR5, whereas H3R2me0/me1/me2s are equally permissive.

Arginine methylation is a ubiquitous post-translational modification. Three predominant types of arginine-guanidino methylation occur in Eukarya: mono (Rme1/MMA); symmetric (Rme2s/SDMA); and asymmetric (Rme2a/ADMA). Arginine methylation frequently occurs at sites of protein-protein and protein-nucleic acid interactions, providing specificity for binding partners and stabilization of important biological interactions in diverse cellular processes. Each methylarginine isoform—catalyzed by members of the protein arginine methyltransferase family, Type I (PRMT1–4,6,8) and Type II (PRMT5,9)—has unique downstream consequences. Methylarginines are found in ordered domains, domains of low-complexity, and in intrinsically-disordered regions of proteins—the latter two of which are intimately connected with biological liquid-liquid phase separation. This review highlights discoveries illuminating how arginine methylation affects genome integrity; gene transcription; mRNA splicing and mRNP biology; protein translation and stability; and phase separation. As more proteins and processes are found to be regulated by arginine methylation, its importance for understanding cellular physiology will continue to grow.

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5’-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

A striking increase in homoarginine concentrations, about more than 100-fold that observed in humans, was recently reported during pregnancy in a nutritionally induced model of intra-uterine growth restriction in ewes. To determine whether this phenomenon is at least partially related to the nutritional regimen, estrus synchronization, or analytical method, thirty-four one-year-old primiparous, non-synchronized, and well-fed Sarda breed ewes were exposed to fertile rams allowing those who came into estrus to naturally mate. Plasma arginine, homoarginine, asymmetric dimethylarginine, symmetric dimethylarginine, mono methylarginine, and citrulline concentrations were measured in each sample using LC-MS/MS. Homoarginine concentrations showed a 44-fold variation between the highest and the lowest values while the fluctuations of arginine and its analogues and metabolites were much smaller, between 1.1 and 1.6-fold. Repeated-measures correlation analysis showed a significant negative correlation between homoarginine/arginine and arginine/asymmetric dimethylarginine ratios (Rm = -0.40; P

Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (∼0.1% of total arginine), Rme2a was highly abundant (∼1.1%), and Rme2s was intermediate (∼0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis—a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

SUMMARY The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC) development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B−/− mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis., In Brief Li et al. identify the arginine demethylase (RDM) activity of JMJD1B, a known lysine demethylase (KDM). They reveal that JMJD1B actively mediates demethylation of histone markers H4R3me2s and H3K9me2 in hematopoietic stem/progenitor cells (HSPCs).

A key step in the biosynthesis of the polyene polyketide ECO-0501 by Amycolatopsis orientalis ATCC 43491 is thought to involve oxidative decarboxylation of arginine or Nδ-methylarginine to the corresponding primary amide. This reaction is the centerpiece of a recently identified biosynthetic cassette that generates 4-guanidinobutyryl thioesters to serve as starter units for polyketide synthesis. We examined the reaction of ORF7, the predicted ECO-0501 biosynthetic decarboxylase, with arginine, and saw no evidence of decarboxylation. Instead, we observed exclusive amine oxidation to generate 2-oxoarginine, with a kcat/KM,Arg of 5.6 × 106 M− 1 s− 1, typical of values measured for physiological amino acid decarboxylases. In contrast, when ORF7 was incubated with Nδ-methylarginine, we observed exclusive decarboxylation to generate 4-(N1-methylguanidino)butyramide. These differing reactive pathways provide insight into the biosyntheses of guanidinobutyryl-derived polyketides and demonstrate the biosynthetic versatility of arginine-processing decarboxylases. In addition, it suggests that ORF7 may be an incisive model system for dissecting the determinants of flavoprotein-catalyzed oxidase and monooxygenase modes of reactivity.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a stress granule-associated RNA-binding protein that plays a role in apoptosis and cellular stress recovery. HnRNP A1 is a major non-histone target of protein arginine methyltransferase 1, which asymmetrically dimethylates hnRNP A1 at several key arginine residues within its arginine-glycine-glycine (RGG)-motif region. Although arginine methylation is known to regulate general RNA binding of hnRNP A1 in vitro, the functional role of arginine methylation in hnRNP A1 cytoplasmic activity is unknown. To test the impact of key methylarginine residues on hnRNP A1 cytoplasmic activity and stress granule association, cytoplasmically restricted Flag-tagged mutants of hnRNP A1 were generated in which key methylarginine residues within the RGG-motif region were changed to either lysine or alanine. Lysine substitution, which mimics unmethylated arginine, resulted in a 40% increase in internal ribosome entry site trans-acting factor (ITAF) activity and the protein readily associates with stress granules. Alanine substitution resulted in a loss of ITAF activity and reduced mRNA binding. The alanine mutant also displays reduced stress granule association and suppresses stress granule formation. Our data suggest that arginine residues within the RGG-motif region are critical for hnRNP A1 cytoplasmic activities and that endogenous asymmetric dimethylation of the RGG-motif region suppresses hnRNP A1 ITAF activity in cells. Our findings indicate that methylarginine residues within the RGG-motif region of hnRNP A1 are important for its cytoplasmic activities and that hypomethylation and/or mutation of the RGG-motif region may contribute to the role of hnRNP A1 in diseases such as cancer.

Arginine methylation, catalyzed by various protein arginine methyltransferases (PRMTs), is increasingly recognized as a widespread post-translational modification in eukaryotes. Thousands of proteins undergo arginine methylation, however, a full picture of the catalytic network for each PRMT is lacking, limiting the global understanding of their biological roles. In this study, we reported a systematic identification of interacting proteins for all human PRMTs, and the resulting interactomes are significantly overlapped with the known proteins containing methylarginine. The conserved motifs for arginine methylation by each PRMT were further determined, with several novel motifs being validated. Among different PRMTs, we found a high degree of overlap in their substrates and high similarities between their putative methylation motifs, suggesting possible functional complementation. We demonstrated that arginine methylation is significantly enriched in RNA binding proteins involved in regulating RNA splicing and translation. Consistently, inhibition of PRMTs leads to global alteration of alternative splicing and suppression of translation. In particular, the ribosomal proteins are pervasively modified with methylarginine, and the mutations on methylation sites inhibit ribosome assembly and translation. Collectively, this study provides a global network of different PRMTs and putative substrates, revealing critical functions of arginine methylation in the regulation of mRNA splicing and translation.

Multiple sclerosis (MS) is an autoinflammatory, chronic central nervous system disease. In the pathogenesis of the disease increased nitric oxide (NO) levels play an important role. Nitric oxide (NO) has neuroprotective effects in physiological conditions, however, it is thought that excessive NO formation in MS may lead to demyelination and axonal damage. Derivatives of methylarginine including asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA), symmetric dimethyl arginine (SDMA) directly or indirectly reduce NO production. Our aim was to measure the levels of methylarginine derivatives and citrulline, ornithine, arginine, homoarginine levels, which are metabolites associated with NO metabolism, in MS subgroups.

It was shown that dopamine amides of arachidonic, oleic, and docosahexaenoic acids exhibit toxicity with respect to PC12 pheochromocytoma cell line. The mechanism of realization of the cytotoxic effect of acyl dopamines is the induction of oxidative stress. This event is preceded by triggering the synthesis of nitric oxide.

Atherosclerosis, in turn preceded by endothelial dysfunction, underlies a series of important cardiovascular diseases. Reduced bioavailability of endothelial nitric oxide, by increasing vascular tone and promoting platelet aggregation, leukocyte adhesion, and smooth muscle cell proliferation, plays a key role in the onset of the majority of cardiovascular diseases. In addition, high blood levels of asymmetric dimethylarginine, a potent inhibitor of nitric oxide synthesis, are associated with future development of adverse cardiovascular events and cardiac death. Recent reports have demonstrated that another methylarginine, i.e., symmetric dimethylarginine, is also involved in the onset of endothelial dysfunction and hypertension. Almost a decade ago, prematurity at birth and intrauterine growth retardation were first associated with a potential negative influence on the cardiovascular apparatus, thus constituting risk factors or leading to early onset of cardiovascular diseases. This condition is referred to as cardiovascular perinatal programming. Accordingly, cardiovascular morbidity and mortality are higher among former preterm adults than in those born at term. The aim of this paper was to undertake a comprehensive literature review focusing on cellular and biochemical mechanisms resulting in both reduced nitric oxide bioavailability and increased methylarginine levels in subjects born preterm. Evidence of the involvement of these compounds in the perinatal programming of cardiovascular risk are also discussed.

Frontotemporal lobar degeneration (FTLD) is heterogeneous in clinical presentation, neuropathological characteristics and genetics. An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of both FTLD and motor neuron disease (MND). Dipeptide repeat polymers (DPR) are generated through repeat-associated non-ATG translation, and they aggregate in neuronal inclusions with a distribution distinct from that of TDP-43 pathology. Recent studies from animal and cell culture models suggest that DPR might be toxic, but that toxicity may differ for specific DPR. Arginine containing DPR (poly-GR and poly-PR) have the greatest toxicity and are less frequent than other DPR (poly-GP, poly-GA). A unique feature of arginine-containing DPR is their potential for post-translational modification by methyl-transferases, which produces methylarginine DPR. In this report, we explored the relationship of DPR and methylarginine to markers of neurodegeneration using quantitative digital microscopic methods in 40 patients with C9ORF72 mutations and one of three different clinicopathologic phenotypes, FTLD, FTLD-MND or MND. We find that density and distribution of poly-GR inclusions are different from poly-GA and poly-GP inclusions. We also demonstrate colocalization of poly-GR with asymmetrical dimethylarginine (aDMA) immunoreactivity in regions with neurodegeneration. Differences in aDMA were also noted by clinical phenotype. FTLD-MND had the highest burden of poly-GR pathology compared to FTLD and MND, while FTLD-MND had higher burden of aDMA than FTLD. The results suggest that poly-GR pathology is associated with toxicity and neurodegeneration. It remains to be determined if dimethylarginine modification of poly-GR could contribute to its toxicity. Electronic supplementary material The online version of this article (10.1186/s40478-018-0564-7) contains supplementary material, which is available to authorized users.

Endogenously produced asymmetrically methylated forms of L-arginine (ADMA) competitively inhibit all three isoforms of nitric oxide synthase enzymes and therefore have the potential to exert significant cardiovascular effects. Elevated circulating ADMA concentrations have been reported in a number of cardiovascular disease states, including preeclampsia, suggesting that impaired ADMA metabolism may contribute to pathology. In order to test the causal relationship between ADMA concentrations and hemodynamics in pregnancy we employed radiotelemetric measurement of hemodynamic function in mice with either global or fetal-specific deletion of the predominant ADMA metabolising enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1). Global deletion of DDAH1 in non-pregnant female mice caused an elevation in circulating ADMA concentrations (2.851±0.289 vs 1.216±0.072 μM p-/- mice showed no further elevation in ADMA and normal pregnancy related changes in blood pressure were observed. However, during the third trimester ADMA levels in DDAH1 -/- mice increased further and this was associated with increased concentrations of sEng and sFlt1 (2288±301.58 vs 1181.35 ± 161.83 pg/ml p-/- mice. Our data indicate that elevated ADMA is sufficient to increase levels of sFLlt1 and sEng and disrupt maternal hemodynamics. The fetus is a significant source of ADMA in late pregnancy suggesting that dysfunctional fetal ADMA metabolism is sufficient to impair maternal hemodynamic function.

Amine quantification is an important strategy in patient stratification and personalised medicine. This is because amines, including amino acids and methylarginines impact on many homeostatic processes. One important pathway regulated by amine levels is nitric oxide synthase (NOS). NOS is regulated by levels of (i) the substrate, arginine, (ii) amino acids which cycle with arginine and (iii) methylarginine inhibitors of NOS. However, biomarker research in this area is hindered by the lack of a unified analytical platform. Thus, the development of a common metabolomics platform, where a wide range of amino acids and methylarginines can be measured constitutes an important unmet need. Here we report a novel high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) platform where ≈40 amine analytes, including arginine and methylarginines can be detected and quantified on a molar basis, in a single sample of human plasma. To validate the platform and to generate biomarkers, human plasma from a well-defined cohort of patients before and after coronary artery bypass surgery, who developed systemic inflammatory response syndrome (SIRS), were analysed. Bypass surgery with SIRS significantly altered 26 amine analytes, including arginine and ADMA. Consequently, pathway analysis revealed significant changes in a range of pathways including those associated with NOS.

Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, PRMT biology during muscle development is complex and not completely understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C2C12 muscle cell maturation, assessed during the myoblast (MB) stage, and during days 1, 3, 5, and 7 of differentiation, was employed as an in vitro model of myogenesis. We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines (MMAs) and symmetric dimethylarginines (SDMAs), indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines (ADMAs) increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, the present study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially mediated mechanism, in driving the muscle differentiation program.

Asimetrik dimetil arjinin (ADMA) klinik tanıda değerlendirilmek maksadıyla üzerinde. araştırmaların devam ettiği, metillenmiş bir arjinin türevidir. ADMA, Nitrik Oksit (NO) sentezini bloke ederek vazospazma ve endotel disfonksiyonuna sebebiyet vermekte ve gebelik döneminde preeklampsi oluşumu ile ilgili risk faktörü olarak kabul edilmektedir Homoarjinin, arjininin homoloğudur ve arjininle yarışarak NOS'ın subsratı olarak görev yapar. Fakat NO üretiminde arjininden daha az etkilidir. Gebelikte erken dönemde ADMA düzeyi yüksek olan kadınlarda ADMA seviyeleri ile endotelyal disfonksiyon arasında ilişki vardır. Gebelik döneminde ADMA seviyesinin ve diğer metilarjinin seviyelerinin nasıl değişkenlik gösterdiği, hastalıkların patogenezini anlamak ve bunları tedavi ile ilişkilendirmek, yeni tedavi protokollerinin geliştirilmesi ve hatta hastalıklar oluşmadan sağlıklı kişilerin risk faktörlerinin elimine edilmesi açısından son derece önemlidir. Bu sebeple çalışmamızda ikili ve dörtlü test istemi yapılan gebe katılımcıların serumlarında metilarjinin seviyelerini belirleyebilmeyi amaçladık. Çalışmaya, toplam 200 gebe kadın dahil edildi. Bu katılımcılar 4'lü test istemi yapılan kontrol grubu (n=50), 4'lü test istemi yapılan riskli grup (n=50), 2'li test istemi yapılan kontrol grubu(n=50) ve 2'li test istemi yapılan riskli grup(n=50) olacak şekilde dört gruba ayrıldı. Her gruba ait serum metilarjinin ve bazı biyokimya testlerinin seviyeleri ölçüldü. Dörtlü ve ikili test istemi yapılan riskli gruplardaki bireylerde serum homoarjinin seviyeleri bu grupların kontrollerine göre anlamlı derecede düşüktü. (sırasıyla p=0.001, p= 0.001) ve en düşük homoarjinin seviyelerinin ikili test istemi yapılan riskli gruba [0.91(0.45- 2.71)] ait olduğu görüldü. Gruplarda HArg seviyeleri yaş parametresi (r= -0.295**, p=0.000) ve serum İnhibin-A seviyeleri ile( r= -0.288**, p=0.005) güçlü negatif bir korelasyon gösterirken ,serum ADMA seviyeleri (r= 0.309**, p=0.000) ile güçlü pozitif bir korelasyon gösterdi. Ayrıca arjinin (r= 0.471**, p=0.000) ve total metilarjininler (r= 0.476**, p=0.000) ile de arasında güçlü pozitif bir korelasyon söz konusuydu. Dörtlü test istemi yapılan kontrol grubunda serum ADMA seviyeleri [0.13(0.02- 0.44)], dörtlü test istemi yapılan riskli gruba [0.06(0.02- 0.17)] ve ikili test istemi yapılan riskli gruba [0.09(0.03-0.24)] göre sırasıyla anlamlı derecede yüksek bulundu (p= 0.000, p= 0.038). İkili test istemi yapılan kontrol grubunda riskli gruptan daha yüksek serum ADMA düzeyleri bulunmasına rağmen aradaki fark istatiksel olarak anlamlı değildi (p= 0.080). Tüm gruplar arasında en yüksek ADMA seviyelerinin dörtlü test istemi yapılan kontrol grubuna ait olduğu görüldü [0.13(0.02- 0.44)]. Serum ADMA seviyeleri serum İnhibin-A seviyeleri (r= -0.352**, p=0.001) ile güçlü negatif bir korelasyon gösterirken, serum HCG seviyeleri (r= -0.261**, p=0.012) zayıf negatif bir korelasyon gösterdi. Normal gebelik sırasında düşük serum maternal ADMA ve L-arjinin konsantrasyonu gibi gebelikle ilgili metabolik değişikliklerin endotel fonksiyonu düzenleyemeyebileceği düşünmekteyiz. Preeklampsi dışında serum ADMA düzeyi iyi bir belirteç olarak kullanılamayabilir. Homoarjinin serum seviyelerinin gebe kadınlardaki düzeyleri açısından literatürde bir boşluk olduğu görülmektedir. Gebelikte yüksek risk altındaki kadınların erken dönemde belirlenmesinde homoarjinin yeni bir risk markırı olarak ortaya çıkabilir., Asymmetric dimethyl arginine (ADMA) is a derivative of a methylated arginine that continues to be studied for use in clinical diagnosis. Nitric oxide synthase synthesizes NO from L-arginine. ADMA inhibits nitric oxide (NO) synthesis and causes vasospasm and endothelial .dysfunction and it is considered as a risk factor for the occurrence of preeclampsia during pregnancy. Homo arginine is the homologous of arginine and serves as a subsrat of NOS, competing with arginine. But it is less effective than arginine in NO production. There is a relationship between ADMA level and endothelial dysfunction in women with high ADMA levels in early stage in pregnancy. How the levels of ADMA and other methlyarginines vary during pregnancy and related diseases, to understand pathogenesis of diseases and to associate them with treatment are extremely important in terms of the development of new treatment protocols and even the elimination of risk factors for healthy persons and as a subject that needs to be investigated. Due to thıs reason we aimed to determine the levels of the methlyarginine levels in the serum of pregnant participants who were subjected to binary- quad test in our study In this study, a total of 200 pregnancy woman were enrolled. These individuals were divided into four groups as the control group with quadruple test (n=50), high risk group with quadruple test (n=50), control group with binary test (n=50) and high risk group with binary test (n=50). In each group, serum methylarginine levels and some of biochemical tests were measured. Serum homoarginine levels were significantly lower (p = 0.001, p = 0.001, respectively) in the subjects high risk groups with in the quadruple and binary test than in the controls of these groups and the lowest homoarginine levels were found to belong to the high risk group [0.91 (0.45-2.71)] with binary test. While serum homorgininne levels showed a strong negative relationship with parameter of age (r= -0.295**, p=0.000) and serum inhibin A levels ( r= -0.288**, p=0.005) there was a strong positive correlation with serum ADMA levels (r= 0.309**, p=0.000). There was also a strong positive correlation between serum homoarginine and arginine levels (r= -0.352**, p=0.001) and total methylarginines levels (r= 0.476** , p= 0.000). In the control group with quadruple test serum ADMA values [0.13(0.02- 0.44)], were significantly higher than ( p= 0.000, p= 0.038, respectively) high risk group with quadruple test [0.06(0.02- 0.17)] and high risk group with binary test [0.09(0.03-0.24)]. Although there was a higher serum ADMA levels in the control group with binary test than high risk group the difference was not statistically significant (p = 0.080). The highest ADMA levels among all groups were found to belong to the control group with quadruple test [0.13 (0.02-0.44)]. While serum ADMA levels showed a strong negative correlation with serum inhibin A (r= -0.352**, p=0.001) there was weak negative correlation with serum HCG levels (r= -0.261**, p=0.012). We think that pregnancy-related metabolic changes such as low serum maternal ADMA and L-arginine concentration during normal pregnancy may not regulate endothelial function. Serum ADMA levels other than preeclampsia may not be a good indicator. It seems that there is a gap in the literatüre regarding the serum levels of homoarginine in pregnant women. Homoarginine may emerge as a new risk marker when women at high risk are identified early stage in pregnancy., Bu araştırma Selçuk Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü tarafından 16202040 proje numarası ile desteklenmiştir.

NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.