L-NMMA Research Articles

Interpretable machine learning identification of arginine methylation sites

Protein methylation is one of the most prominent posttranslation modifications that essentially regulates several biological processes in eukaryotes. Therefore, identification of the arginine methylation site is crucial in deciphering its characteristics and functions in cell biology, disease mechanisms, and guided drug development. The computation methods address the long-term bottleneck together with the cost, time, and labor required in experimental methods for large-scale identification of protein arginine methylation sites. In this study, we proposed a robust machine learning-based computational tool known as iIRMethyl, employing the primary sequence and physicochemical properties of protein along with a two-step feature selection method for optimal selection of feature descriptors. Moreover, the performance of iIRMethyl was comprehensively evaluated via k-fold cross-validation on a benchmark dataset and independent test dataset. iIRMethyl demonstrated a remarkably greater performance than the state-of-the-art method and achieved an average area under the curve value of 0.99 for both k-fold cross-validation and an independent test set in the identification of protein arginine methylation sites. Furthermore, the outcomes reveal that iIRMethyl is a robust and accurate computational tool for large-scale identification of arginine methylation sites and would facilitate the understanding of their functional mechanisms and accelerating their application in drug development and clinical therapy. Additionally, the prediction mechanism of the proposed model iIRMethyl is interpreted using the SHapley Additive exPlanation algorithm.

Shotgun proteomic investigation of methyltransferase and methylation profiles in lipopolysaccharide stimulated RAW264.7 murine macrophages.

Arginine methylation is a common post-translational modification which functions as an epigenetic regulator of transcription and plays a key role in various cell signaling pathways. The methylation of arginine residues is catalyzed by protein arginine methyltransferase (PRMT). However, the expression pattern and underlying mechanism of PRMTs and protein methylation profile in lipopolysaccharide (LPS)-induced innate immune responses are poorly understood. Using a shotgun proteomic approach, we found that LPS stimulation increased arginine and proline metabolism and responses to inflammation and bacterial infections. In comparison, cysteine and methionine metabolism, the pentose phosphate pathway, purine metabolism, and protein methylation factors were also decreased in LPS stimulated murine macrophage cell lines. We revealed that LPS stimulation downregulated PRMT1, PRMT5, and protein arginine methylation profiles in RAW264.7 cells using western blot analysis. Additionally, this phenomenon occurred in parallel with nitric oxide accumulation in LPS-induced macrophages. Using inflammation models, we demonstrate for the first time that LPS stimulation decreases PRMTs, leading to the decreasing of arginine methylation in macrophages.

Abstract 3847: Tumor stem cell derived asymmetric dimethylarginine drives immune evasion via functional modulation of macrophages

Abstract Macrophages are the most abundant tumor infiltrating immune cells and perform diverse immune functions including phagocytosis and incitation of T cells to mount an immune attack against tumors. However, the macrophage plasticity can lead to different outcomes of tumor progression and the amino acid, arginine, plays a central role in macrophage dichotomy. The exact role of arginine on the management of macrophage polarization is not completely understood. We have discovered that a methylated arginine metabolite, asymmetric dimethylarginine (ADMA), serves as an important regulator of macrophage functions. Upon stimulation by ADMA, macrophages have reduced survival and show a delayed tumor-phagocytic capacity. Additionally, ADMA blocks the Nitric Oxide Synthase (NOS) but not the Arginase activity in macrophages leading to a tendency of M2-like phenotype polarization. ADMA has minor effect on tumor cell proliferation but significantly drives the epithelial to mesenchymal transition. Tumor cells, particularly the tumor stem cells, are the major sources of ADMA. Production and secretion of ADMA by tumor (stem) cells profoundly increase when co-cultured with activated T cells or treated with T cell associated inflammatory cytokine, interferon-gamma (IFN-r). IFN-r initiates autophagy of tumor (stem) cells and inhibition of autophagy reduces the output of ADMA by tumor (stem) cells. Similarly, IFN-r enhances the proteasomal degradation and inhibition of the proteasomal pathway also reduces the ADMA production induced by IFN-r. In summary, our work shows that tumor (stem) cells may escape from immune surveillance (such as their exposure to activated T lymphocytes or to cytotoxic cytokine, IFN-r) through the secretion of ADMA generated via both autophagic and proteasomal pathways. Citation Format: Yiling Chen, AKaychia T. Lowery, Andres Becerril, Zaida T. Laventure, Shareda Ferguson, Kuan-Hui Ethan Chen. Tumor stem cell derived asymmetric dimethylarginine drives immune evasion via functional modulation of macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3847.

Design and Synthesis of Novel PRMT1 Inhibitors and Investigation of Their Effects on the Migration of Cancer Cell.

Protein arginine methyltransferase 1 (PRMT1) can catalyze the protein arginine methylation by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of protein arginine, which influences a variety of biological processes including epithelial–mesenchymal transition (EMT) and EMT-mediated mobility of cancer cells. The upregulation of PRMT1 is involved in a diverse range of cancer, such as lung cancer, and there is an urgent need to develop novel and potent PRMT1 inhibitors. In this article, a series of 2,5-substituted furan derivatives and 2,4-substituted thiazole derivatives were designed and synthesized by targeting at the substrate arginine-binding site on PRMT1, and 10 compounds demonstrated significant inhibitory effects against PRMT1. Among them, the most potent inhibitor, compound 1r (WCJ-394), significantly affected the expression of PRMT1-related proteins in A549 cells and downregulated the expression of mesenchymal markers, by which WCJ-394 inhibited the TGF-β1-induced EMT in A549 cells and prevented the cancer cell migration. The current study demonstrated that WCJ-394 was a potent PRMT1 inhibitor, which could be used as the leading compound for further drug discovery.

Quantification of serum homoarginine, methylated arginine and inhibin-A levels in a high-risk pregnancy

The plasma levels of homoarginine (h-Arg) and methylated arginine have proven to be an independent cardiovascular risk factor. We aimed to determine the h-Arg and methyl arginine levels in serums of high-risk pregnancy causing potential complications. These participants were divided into four groups as the control group with quadruple test, the high-risk group quadruple test the control group with binary test, the high-risk group with quadruple test that have a positive result from second-trimester screening with a cut-off value of 1 in 300. The serum methyl arginine and homoarginine levels were analysed with liquid chromatography-tandem mass spectrometry. Serum h-Arg levels were found to be higher in high-risk groups compared to control groups and it was also detected higher in the groups with quadruple test than the groups with binary test (p < .05). H-Arg levels in the groups showed strong negative correlation with age and serum inhibin-A levels (r = −0.288, p < .001). Also, there was a strong negative correlation between serum asymmetric dimethylarginine (ADMA) and serum inhibin-A levels (r = −0.352, p < .001). H-Arg may be a new risk marker to detect high-risk pregnancies in early pregnancy. In addition to, methylated arginine such as ADMA has a key regulator in a physiological concentration of h-Arg. IMPACT STATEMENT What is already known on this subject? H-Arg levels decrease may be associated with preeclampsia, GDM, macrosomia, low birth weight, and preterm delivery in pregnancy. What do the results of this study add? Serum h-Arg levels were found to be higher in high-risk groups. Additionally, h-Arg levels and ADAM, one of the methylated arginines in the groups showed a strong negative correlation with serum inhibin-A levels What are the implications of these findings for clinical practice and/or further research? H-Arg may be a new risk marker to detect high-risk pregnancies

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

The lncRNA PILA promotes NF-κB signaling in osteoarthritis by stimulating the activity of the protein arginine methyltransferase PRMT1

Inflammatory cytokine-induced activation of nuclear factor κB (NF-κB) signaling plays a critical role in the pathogenesis of osteoarthritis (OA). We identified PILA as a long noncoding RNA (lncRNA) that enhances NF-κB signaling and OA. The abundance of PILA was increased in damaged cartilage from patients with OA and in human articular chondrocytes stimulated with the proinflammatory cytokine tumor necrosis factor (TNF). Knockdown of PILA inhibited TNF-induced NF-κB signaling, extracellular matrix catabolism, and apoptosis in chondrocytes, whereas ectopic expression of PILA promoted NF-κB signaling and matrix degradation. PILA promoted PRMT1-mediated arginine methylation of DExH-box helicase 9 (DHX9), leading to an increase in the transcription of the gene encoding transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of NF-κB signaling. Furthermore, intra-articular injection of an adenovirus vector encoding PILA triggered spontaneous cartilage loss and exacerbated posttraumatic OA in mice. This study provides insight into the regulation of NF-κB signaling in OA and identifies a potential therapeutic target for this disease.

Modulation of IKs channel-PIP2 interaction by PRMT1 plays a critical role in the control of cardiac repolarization.

Recent studies have shown that protein arginine methyltransferase 1 (PRMT1) is highly expressed in the human heart, and loss of PRMT1 contributes to cardiac remodeling in the heart failure. However, the functional importance of PRMT1 in cardiac ion channels remains uncertain. The slow activating delayed rectifier K+ (I Ks) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits and is a new therapeutic target for treating lethal arrhythmias in many cardiac pathologies, especially heart failure. Here, we demonstrate that PRMT1 is a critical regulator of the I Ks channel and cardiac rhythm. In the guinea pig ventricular myocytes, treatment with furamidine, a PRMT1‐specific inhibitor, prolonged the action potential duration (APD). We further show that this APD prolongation was attributable to I Ks reduction. In HEK293T cells expressing human KCNQ1 and KCNE1, inhibiting PRMT1 via furamidine reduced I Ks and concurrently decreased the arginine methylation of KCNQ1, a pore‐forming α‐subunit. Evidence presented here indicates that furamidine decreased I Ks mainly by lowering the affinity of I Ks channels for the membrane phospholipid, phosphatidylinositol 4,5‐bisphosphate (PIP2), which is crucial for pore opening. Finally, applying exogenous PIP2 to cardiomyocytes prevented the furamidine‐induced I Ks reduction and APD prolongation. Taken together, these results indicate that PRMT1 positively regulated I Ks activity through channel–PIP2 interaction, thereby restricting excessive cardiac action potential.

ProMetheusDB: An In-Depth Analysis of the High-Quality Human Methyl-proteome

Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumor development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl stable isotope labeling with amino acids in cell culture experiments analyzed with a machine learning–based tool and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein–RNA interactions and suggest a role in rewiring protein–protein interactions, for which we provide experimental evidence for a representative case (i.e., NONO [non-POU domain–containing octamer-binding protein]–paraspeckle component 1 [PSPC1]). Upon intersecting our R-methyl-sites dataset with the PhosphoSitePlus phosphorylation dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of heavy methyl stable isotope labeling with amino acids in cell culture to identify unconventional methylated residues and successfully identified novel histone methylation marks on serine 28 and threonine 32 of H3. The database generated, named ProMetheusDB, is freely accessible at https://bioserver.ieo.it/shiny/app/prometheusdb.

Endothelial PRMT5 plays a crucial role in angiogenesis after acute ischemic injury.

Arginine methylation mediated by protein arginine methyltransferases (PRMTs) has been shown to be an important posttranslational mechanism involved in various biological processes. Herein, we sought to investigate whether PRMT5, a major type II enzyme, is involved in pathological angiogenesis and, if so, to elucidate the molecular mechanism involved. Our results show that PRMT5 expression is significantly upregulated in ischemic tissues and hypoxic endothelial cells (ECs). Endothelial-specific Prmt5-KO mice were generated to define the role of PRMT5 in hindlimb ischemia–induced angiogenesis. We found that these mice exhibited impaired recovery of blood perfusion and motor function of the lower limbs, an impairment that was accompanied by decreased vascular density and increased necrosis as compared with their WT littermates. Furthermore, both pharmacological and genetic inhibition of PRMT5 significantly attenuated EC proliferation, migration, tube formation, and aortic ring sprouting. Mechanistically, we showed that inhibition of PRMT5 markedly attenuated hypoxia-induced factor 1-α (HIF-1α) protein stability and vascular endothelial growth factor–induced (VEGF-induced) signaling pathways in ECs. Our results provide compelling evidence demonstrating a crucial role of PRMT5 in hypoxia-induced angiogenesis and suggest that inhibition of PRMT5 may provide novel therapeutic strategies for the treatment of abnormal angiogenesis-related diseases, such as cancer and diabetic retinopathy.

Loss of PRMT7 reprograms glycine metabolism to selectively eradicate leukemia stem cells in CML

Our group has reported previously on the role of various members of the protein arginine methyltransferase (PRMT) family, which are involved in epigenetic regulation, in the progression of leukemia. Here, we explored the role of PRMT7, given its unique function within the PRMT family, in the maintenance of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Genetic loss of Prmt7, and the development and testing of a small-molecule specific inhibitor of PRMT7, showed that targeting PRMT7 delayed leukemia development and impaired self-renewal of LSCs in a CML mouse model and in primary CML CD34+ cells from humans without affecting normal hematopoiesis. Mechanistically, loss of PRMT7 resulted in reduced expressions of glycine decarboxylase, leading to the reprograming of glycine metabolism to generate methylglyoxal, which is detrimental to LSCs. These findings link histone arginine methylation with glycine metabolism, while suggesting PRMT7 as a potential therapeutic target for the eradication of LSCs in CML.

Characterization of CARM1 Inhibition in Skeletal Muscle

Coactivator‐associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues of target proteins critical for health and disease. CARM1 has recently emerged as an important regulator of skeletal muscle biology. The purpose of this study was to further investigate CARM1 biology in skeletal muscle. For our first experiment, we utilized 3‐month‐old male and female mice (n = 3/sex) that were housed and cared for in accordance with Canadian Council for Animal Care guidelines. Mice were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; Epizyme, Inc.) or vehicle via oral gavage BID for 2, 4, or 8 days. Tissues were collected 6 hours following the final dose and muscle mass was recorded. Western blotting was performed to evaluate protein expression in the tibialis anterior (TA) muscle and liver. To assess CARM1 activity, we investigated the arginine methylation status (i.e., the methylated form of the protein relative to its total amount) of known CARM1 substrates BAF155 and PABP1, as well as the myocellular level of arginine methylated proteins preferentially marked by CARM1. We observed similar outcomes between males and females, so the data were collapsed by sex (n = 6). CARM1 inhibition significantly reduced BAF155 methylation status in the TA muscle by 70‐80% between 2‐8 days of treatment. Muscle PABP1 methylation status was also attenuated (p &lt; 0.05) by 60‐65% after 4 and 8 days of exposure. Similarly, arginine methylation of CARM1‐specific substrates across all muscle proteins was reduced by ~50% (p &lt; 0.05) between 2‐8 days of CARM1 inhibitor administration. The attenuation of BAF155 and PABP1 methylation status, as well as arginine methylated CARM1‐specific substrates, was greater in liver as compared to muscle. Next, we completed additional experiments to assess the effect of pharmacological CARM1 inhibition on skeletal muscle function. After 11 days of CARM1 inhibitor treatment, we observed a sex‐specific reduction (p &lt; 0.05) in exercise capacity and muscular endurance such that muscle function in female mice was unaffected by CARM1 suppression. Collectively, these results suggest that global, pharmacological CARM1 inhibition preferentially impacts liver enzyme activity over muscle. Furthermore, CARM1 inhibitor treatment may affect muscle performance with males being more sensitive to the functional outcomes of CARM1 suppression than females. Future work in our laboratory will examine the mechanisms and functional consequences of sex‐based differences in CARM1 biology within skeletal muscle.

The Influence of Arginine Methylation in Immunity and Inflammation.

Exploration in the field of epigenetics has revealed that protein arginine methyltransferases (PRMTs) contribute to disease, and this has given way to the development of specific small molecule compounds that inhibit arginine methylation. Protein arginine methylation is known to regulate fundamental cellular processes, such as transcription; pre-mRNA splicing and other RNA processing mechanisms; signal transduction, including the anti-viral response; and cellular metabolism. PRMTs are also implicated in the regulation of physiological processes, including embryonic development, myogenesis, and the immune system. Finally, the dysregulation of PRMTs is apparent in cancer, neurodegeneration, muscular disorders, and during inflammation. Herein, we review the functions of PRMTs in immunity and inflammation. We also discuss recent progress with PRMTs regarding the modulation of gene expression related to T and B lymphocyte differentiation, germinal center dynamics, and anti-viral signaling responses, as well as the clinical relevance of using PRMT inhibitors alone or in combination with other drugs to treat cancer, immune, and inflammatory-related diseases.

PRMT5 regulates lipid metabolism in skeletal muscle via epigenetic suppression of Pnpla2

Skeletal muscle plays a vital role in regulating systemic energy metabolism. Defects in skeletal muscle energy production exacerbate chronic metabolic disorders. Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation and regulates a plethora of cellular processes in many tissues, but its function in skeletal muscle is poorly defined. Using a skeletal muscle specific Prmt5 knockout mouse model (Prmt5MKO), we observed that Prmt5 KO led to a reduction of myofiber size, compromised contractile function, and exercise performance. In addition, Prmt5KO muscles had fewer oxidative fiber type and correspondingly more glycolytic myofibers. Prmt5MKO mice also exhibit systemic metabolic defects and abnormal lipid droplet biogenesis within skeletal muscle, resulting in reduced fat mass in response to high‐fat‐diet. Mechanistic studies demonstrated that PRMT5 mediates dimethylation of H4R3 in the promoter region of Pnpla2 (encoding ATGL, the rate‐limiting enzyme in lipolysis), and loss of Prmt5 results in elevated level of ATGL. Accordingly, constitutive deletion of Pnpla2in skeletal muscle rescued muscle mass and function of Prmt5MKO mice. Taken together, our findings delineate a novel function of Prmt5 as a regulator of lipid metabolism in myocytes through repressing Pnpla2 expression.

Exploring the Role of Arginine Methylation in the RNA Helicase, Ded1

Ded1p is an essential ATP‐dependent RNA helicase that promotes nuclear export (1), stress granule accumulation, and translation initiation through association with translation factors, such as eIF4A and eIF4G (2, 3). Ded1 is methylated by Hmt1 at five arginine residues in its intrinsically disordered domains (4, 5). Methylation strengthens the interaction between Ded1 and Npl3, a protein involved in nuclear export (4), but it is unclear if methylation impacts translation.We find that Ded1 methylation is stably detected under a range of growth conditions and stresses, suggesting that this modification is common within in vivo stores of Ded1. To test the function of Ded1 methylation in vivo, we mutated four of the five methylated arginines to either alanine (ded1‐quadA) or lysine (ded1‐quadK). We are currently testing the effects of a recently discovered fifth methylation site (5). The ded1‐quadA and quadK mutants show no Ded1 methylation detectable by western blot, indicating that methylation is significantly decreased. As these mutants complement a null, Ded1 methylation is not required for the essential function of Ded1 and is, therefore, unlikely to be required for global translation.Ded1 methylation may be important for a subset of mRNAs that are most dependent on Ded1 for translation (6). Consistent with this idea, the methylation deficient mutants do show a range of phenotypes distinct from wild type yeast, which were identified via phenotypic microarray screen. These phenotypes suggest that the lack of methylation becomes important under certain growth conditions. Additionally, the methylation defective ded1 mutant fails to localize to stress granules, but whether that affects stress granule formation or RNA accumulation within stress granules is unknown.Ded1 is important for the translation of many mRNAs, especially those with highly structured 5’ UTRs (6). We have screened several Ded1‐dependent translation reporters for their translation efficiency in cells with methylation defective ded1 mutants. We found one reporter that is sensitive to the methylation defective mutant and have assessed the entire transcriptome for mRNAs that are similarly affected by Ded1 methylation. We are currently analyzing these results to identify any similarities in GO terms, structure potential, length, etc. among the RNAs whose translation is sensitive to the methylation defective ded1 mutant.We have successfully purified recombinant methylated versus non‐methylated Ded1. Recombinant methylated Ded1 is as efficient as non‐methylated Ded1 in repressing translation in vitro when Ded1 is in excess, consistent with retaining aspects of Ded1 function. We are currently testing whether methylation affects other biochemical functions of Ded1, such as its ability to interact with eIF4A or eIF4G (2, 3).(1) Hauk and Bowman. 2015. PloS One. 10: e0131690.(2) Gao et al. 2016. eLife 5:e16408.(3) Hilliker et al. 2011. Molecular Cell 43: 962‐972.(4) Erce et al. 2013. Molecular &amp; Cellular Proteomics 12: 3184‐3198.(5) Hamey et al. 2021. J. Proteome Res. 20(5): 2420–2434.(6) Sen et al. 2015. Genome Res 25: 1196‐205.

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction–reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Novel Chemicals Derived from Tadalafil Exhibit PRMT5 Inhibition and Promising Activities against Breast Cancer.

Breast cancer seriously endangers women’s health worldwide. Protein arginine methyltransferase 5 (PRMT5) is highly expressed in breast cancer and represents a potential druggable target for breast cancer treatment. However, because the currently available clinical PRMT5 inhibitors are relatively limited, there is an urgent need to develop new PRMT5 inhibitors. Our team previously found that the FDA-approved drug tadalafil can act as a PRMT5 inhibitor and enhance the sensitivity of breast cancer patients to doxorubicin treatment. To further improve the binding specificity of tadalafil to PRMT5, we chemically modified tadalafil, and designed three compounds, A, B, and C, based on the PRMT5 protein structure. These three compounds could bind to PRMT5 through different binding modes and inhibit histone arginine methylation. They arrested the proliferation and triggered the apoptosis of breast cancer cells in vitro and also promoted the antitumor effects of the chemotherapy drugs cisplatin, doxorubicin, and olaparib in combination regimens. Among them, compound A possessed the highest potency. Finally, the anti-breast cancer effects of PRMT5 inhibitor A and its ability to enhance chemosensitivity were further verified in a xenograft mouse model. These results indicate that the new PRMT5 inhibitors A, B, and C may be potential candidates for breast cancer treatment.

Protein Arginine Methylation: An Emerging Modification in Cancer Immunity and Immunotherapy.

In recent years, protein arginine methyltransferases (PRMTs) have emerged as new members of a gene expression regulator family in eukaryotes, and are associated with cancer pathogenesis and progression. Cancer immunotherapy has significantly improved cancer treatment in terms of overall survival and quality of life. Protein arginine methylation is an epigenetic modification function not only in transcription, RNA processing, and signal transduction cascades, but also in many cancer-immunity cycle processes. Arginine methylation is involved in the activation of anti-cancer immunity and the regulation of immunotherapy efficacy. In this review, we summarize the most up-to-date information on regulatory molecular mechanisms and different underlying arginine methylation signaling pathways in innate and adaptive immune responses during cancer. We also outline the potential of PRMT-inhibitors as effective combinatorial treatments with immunotherapy.

Evaluation of salivary and serum methylated arginine metabolites and nitric oxide synthase in advanced periodontitis patients.

Methylated arginine metabolites and nitric oxide synthase (NOS) play a critical role in regulating endothelial function. The aim of this study was to determine levels of NOS, and methylated arginine metabolites (ADMA, SDMA, homoarginine, arginine, and L-NMMA) and IL-6 in serum and saliva in patients with advanced periodontal diseases and identify their association with clinical parameters. The study consisted of two groups: healthy individuals (control: n = 24), and generalized Stage III Grade B periodontitis (P: n = 21). Clinical periodontal parameters (probing pocket depth, bleeding on probing, clinical attachment level) were recorded. IL 6 and NOS levels in saliva and serum were analyzed by enzyme-linked immunosorbent assay (ELISA). ADMA, SDMA, homoArg, arginine, and L-NMMA in saliva and serum were analyzed by liquid chromatography-mass spectrometry (LC MS/MS). Clinical parameters were significantly higher in the periodontitis group (p < 0.001). In periodontitis group, NOS, ADMA, and arginine levels in saliva were statistically significantly higher than control group (p < 0.05). Serum levels of SDMA were statistically significantly lower, and IL-6 was statistically significantly higher in P group than C group (p < 0.05). ADMA, NOS, and arginine levels were significantly positive correlated with all clinical periodontal parameters (p < 0.05). These findings suggest that there is a relationship between severity of periodontal disease and endothelial dysfunction by means of ADMA. Salivary ADMA may be related with periodontal inflammation. ADMA levels in periodontal inflammation are associated with endothelial dysfunction. According to the results of our study, periodontal inflammation is effective on both local and systemic methylated arginine metabolites and nitric oxide synthase levels. This may shed light on the relationship between periodontal disease and systemic status.

Pan-methylarginine antibody generation using PEG linked GAR motifs as antigens.

Arginine methylation is a prevalent posttranslational modification which is deposited by a family of protein arginine methyltransferases (PRMTs), and is found in three different forms in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Pan-methylarginine antibodies are critical for identifying proteins that are methylated on arginine residues, and are also used for evaluating signaling pathways that modulate this methyltransferase activity. Although good pan-MMA, -ADMA and -SDMA antibodies have been developed over the years, there is still room for improvement. Here we use a novel antigen approach, which involves the separation of short methylated motifs with inert polyethylene glycol (PEG) linkers, to generate a set of pan antibodies to the full range of methylarginine marks. Using these antibodies, we observed substrate scavenging by PRMT1, when PRMT5 activity is blocked. Specifically, we find that the splicing factor SmD1 displays increased ADMA levels upon PRMT5 inhibitor treatment. Furthermore, when the catalysis of both SDMA and ADMA is blocked with small molecule inhibitors, we demonstrate that SmD1 and SMN no longer interact. This could partially explain the synergistic effect of PRMT5 and type I PRMT inhibition on RNA splicing and cancer cell growth.