L-NMMA Research Articles

Arginine Methylation of the PGC-1α C-Terminus Is Temperature-Dependent.

We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

Association of serum ADMA, SDMA and L-NMMA concentrations with disease progression in COVID-19 patients.

This study determines and compares the concentrations of arginine and methylated arginine products ((asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), n-monomethyl-1-arginine (L-NMMA) and homoarginine (HA)) for assessment of their association with disease severity in serum samples of COVID-19 patients. Serum arginine and methylated arginine products of 57 mild-moderate and 29 severe (N = 86) COVID-19 patients and 21 controls were determined by tandem mass spectrometry. Moreover, the concentrations of some of the routine clinical laboratory parameters -neutrophil lymphocyte ratio (NLR), C-reactive protein, ferritin, D-dimer, and fibrinogen measured during COVID-19 follow-up were also taken into consideration and compared with the concentrations of arginine and methylated arginine products. Serum ADMA, SDMA and L-NMMA were found to be significantly higher in severe COVID-19 patients, than in both mild-moderate patients and the control group (P < 0.001 for each). In addition, multiple logistic regression analysis indicated L-NMMA (cut-off =120 nmol/L OR = 34, 95% confidence interval (CI) = 3.5-302.0, P= 0.002), CRP (cut-off = 32 mg/L, OR = 37, 95% CI = 4.8-287.0, P < 0.001), and NLR (cut-off = 7, OR = 22, 95% CI = 1.4-335.0, P = 0.020) as independent risk factors for identification of severe patients. The concentration of methylated arginine metabolites are significantly altered in COVID-19 disease. The results of this study indicate a significant correlation between the severity of COVID-19 disease and concentrations of CRP, NLR and L-NMMA.

Ageing and endothelium-mediated vascular dysfunction: the role of the NADPH oxidases.

The present study aimed to determine the isoform-specific role of the NADPH oxidases (NOX) in the endothelium-mediated vascular dysfunction associated with ageing. Endothelium-dependent [intraluminal flow- and acetylcholine (ACh)-induced] vasodilatation in human skeletal muscle feed arteries (SMFAs) of young (24±1 years, n=16), middle aged (45±1 years, n=18) and old (76±2 years, n=21) subjects was assessed in vitro with and without the inhibition of NOX1 (ML090), NOX2 (gp91) and NOX4 (plumbagin). To identify the role of nitric oxide (NO) bioavailability in these responses, NO synthase blockade (l-NG -monomethyl arginine citrate) was utilized. SMFA NOX1, NOX2 and NOX4 protein expression was determined by western blotting. Age related endothelium-dependent vasodilatory dysfunction was evident in response to flow (young: 69±3; middle aged: 51±3; old: 27±3%, P<0.05) and ACh (young: 89±2; middle aged: 72±3; old: 45±4%, P<0.05). NOX1 inhibition had no effect on SMFA vasodilatation, whereas NOX2 inhibition restored flow- and ACh-induced vasodilatation in the middle aged and the old SMFAs (middle aged+gp91: 69±3; 86±3, old+gp91: 65±5; 83±2%, P<0.05) and NOX4 inhibition tended to restore these vasodilatory responses in these two groups, but neither achieved statistical significance (P ≈ 0.06). l-NG -monomethyl arginine citrate negated the restorative effects of NOX2 and NOX4 blockade. Only NOX2 and NOX4 protein expression was significantly greater in the two older groups and inversely related to vascular function (r=0.48 to 0.93, P<0.05). NOX2 and, to a lesser extent, NOX4 appear to play an important, probably NO-mediated, role in age-related endothelial dysfunction. KEY POINTS: The present study aimed to determine the isoform-specific role of the NADPH oxidases (NOX) in the endothelium-mediated vascular dysfunction associated with ageing. Age related endothelium-dependent vasodilatory dysfunction was evident in skeletal muscle feed arteries in response to both flow and acetylcholine. NOX2 inhibition (gp91) restored endothelium-dependent vasodilatation in the middle aged and the old skeletal muscle feed arteries, and NOX4 inhibition (plumbagin) tended to restore these vasodilatory responses in these two groups. Nitric oxide synthase inhibition negated the restorative effects of NOX2 and NOX4 blockade. NOX2 and NOX4 protein expression was significantly greater in the two older groups and inversely related to vascular function. NOX2 and, to a lesser extent, NOX4 appear to play an important, probably nitric oxide-mediated, role in age-related endothelial dysfunction and could be important therapeutic targets to maintain vascular health with ageing.

Arginine methylation of BRD4 by PRMT2/4 governs transcription and DNA repair.

BRD4 functions as an epigenetic reader and plays a crucial role in regulating transcription and genome stability. Dysregulation of BRD4 is frequently observed in various human cancers. However, the molecular details of BRD4 regulation remain largely unknown. Here, we report that PRMT2- and PRMT4-mediated arginine methylation is pivotal for BRD4 functions on transcription, DNA repair, and tumor growth. Specifically, PRMT2/4 interacts with and methylates BRD4 at R179, R181, and R183. This arginine methylation selectively controls a transcriptional program by promoting BRD4 recruitment to acetylated histones/chromatin. Moreover, BRD4 arginine methylation is induced by DNA damage and thereby promotes its binding to chromatin for DNA repair. Deficiency in BRD4 arginine methylation significantly suppresses tumor growth and sensitizes cells to BET inhibitors and DNA damaging agents. Therefore, our findings reveal an arginine methylation-dependent regulatory mechanism of BRD4 and highlight targeting PRMT2/4 for better antitumor effect of BET inhibitors and DNA damaging agents.

PRMT1 and PRMT5: on the road of homologous recombination and non-homologous end joining.

DNA double-strand breaks (DSBs) are widely accepted to be the most deleterious form of DNA lesions that pose a severe threat to genome integrity. Two predominant pathways are responsible for repair of DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR relies on a template to faithfully repair breaks, while NHEJ is a template-independent and error-prone repair mechanism. Multiple layers of regulation have been documented to dictate the balance between HR and NHEJ, such as cell cycle and post-translational modifications (PTMs). Arginine methylation is one of the most common PTMs, which is catalyzed by protein arginine methyltransferases (PRMTs). PRMT1 and PRMT5 are the predominate PRMTs that promote asymmetric dimethylarginine and symmetric dimethylarginine, respectively. They have emerged to be crucial regulators of DNA damage repair. In this review, we summarize current understanding and unaddressed questions of PRMT1 and PRMT5 in regulation of HR and NHEJ, providing insights into their roles in DSB repair pathway choice and the potential of targeting them for cancer therapy.

Arginine methylation sites on SepIVA help balance elongation and septation in Mycobacterium smegmatis.

The growth of mycobacterial cells requires successful coordination between elongation and septation. However, it is not clear which factors mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of its methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG and that this regulation may impact polar elongation. Finally, we map SepIVA's two regulatory functions to different ends of the protein: the N-terminus regulates elongation while the C-terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.

Do arginine metabolites have a role in periodontitis due to smoking? A new perspective.

Cigarette consumption is common around the world and besides its negative effects on health, and its effects on periodontitis draw attention. Arginine metabolites are involved in the pathogenesis of several systemic inflammatory diseases' including cardiovascular diseases. Our aim was to determine periodontitis and healthy individuals' arginine metabolites and IL-6 levels in saliva and serum and to evaluate those according to smoking status. The study consisted of four groups: healthy individuals (control [C]; n=20), smokers with healthy periodontium (S-C; n=20), nonsmokers with Stage-III Grade-B generalized periodontitis (P; n=20) and smokers with Stage-III Grade-C generalized periodontitis (S-P; n=18). Periodontal parameters were measured. Analysis of methylated arginine metabolites was performed by LC-MS/MS, and IL-6 levels were determined by ELISA kits. In nonsmokers, salivary concentrations of asymmetric dimethylarginine (ADMA) and symmetrical dimethylarginine (SDMA) were higher in the periodontitis than control (p < 0.001, p=0.010). Smokers with periodontitis exhibited higher ADMA (p=0.033, p < 0.001) and arginine (p=0.030, p=0.001) saliva concentrations than smoking and nonsmoking controls. Our results demonstrated that salivary concentrations of ADMA and SDMA were associated with periodontitis. Smoking increased ADMA, SDMA and NG -monomethyl L-arginine (L-NMMA) levels in serum only in periodontitis patients.

Histone arginine methylation in the freeze-tolerant wood frog, Rana sylvatica

Histone arginine methylation in the freeze-tolerant wood frog, Rana sylvatica

The Role of L-Arginine-NO System in Female Reproduction: A Narrative Review.

Accumulating evidence are available on the involvement of l-arginine-nitric oxide (NO) system in complex biological processes and numerous clinical conditions. Particular attention was made to reveal the association of l-arginine and methylarginines to outcome measures of women undergoing in vitro fertilization (IVF). This review attempts to summarize the expression and function of the essential elements of this system with particular reference to the different stages of female reproduction. A literature search was performed on the PubMed and Google Scholar systems. Publications were selected for evaluation according to the results presented in the Abstract. The regulatory role of NO during the period of folliculogenesis, oocyte maturation, fertilization, embryogenesis, implantation, placentation, pregnancy, and delivery was surveyed. The major aspects of cellular l-arginine uptake via cationic amino acid transporters (CATs), arginine catabolism by nitric oxide synthases (NOSs) to NO and l-citrulline and by arginase to ornithine, and polyamines are presented. The importance of NOS inhibition by methylated arginines and the redox-sensitive elements of the process of NO generation are also shown. The l-arginine-NO system plays a crucial role in all stages of female reproduction. Insufficiently low or excessively high rates of NO generation may have adverse influences on IVF outcome.

Hsa_circ_0094606 promotes malignant progression of prostate cancer by inducing M2 polarization of macrophages through PRMT1-mediated arginine methylation of ILF3.

Circular RNA (circRNA), a type of noncoding RNAs, has been demonstrated to act vital roles in tumorigenesis and cancer deterioration. Although tumor-associated macrophages are involved in tumor malignancy, the interactions between circRNAs and tumor-associated macrophages in prostate cancer (PCa) remain unclear. In the present study, we found that hsa_circ_0094606 (subsequently named circ_0094606) could promote proliferation, epithelial-mesenchymal transition (EMT) as well as migration of PCa cells through cell viability and migration assays and the determination of EMT markers. Mass spectrometry analysis after RNA pull-down experiment identified that circ_0094606 bound to protein arginine methyltransferase 1 (PRMT1) in PCa cells, and further functional assays revealed that circ_0094606 promoted the malignant progression of PCa by binding to PRMT1. Moreover, co-immunoprecipitation (Co-IP), glutathione-S-transferase (GST) pull-down and immunofluorescence showed that PRMT1 mediated arginine methylation of ILF3 to stabilize the protein. Bioinformatics analysis combined with data from RNA-binding protein immunoprecipitation and RNA pull-down suggested that ILF3 could stabilize IL-8 mRNA, which promoted the M2 polarization in coculture study. Finally, in vivo experiments showed that circ_0094606 subserve PCa growth and promoted the M2 polarization of macrophages through the PRMT1/ILF3/IL-8 regulation pathway, supporting circ_0094606 as a potential novel effective target for PCa treatment.

Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization

BackgroundAsymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known.MethodsThree mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system.ResultsFor macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor.ConclusionOur results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

Protein Arginine Methyltransferases as Therapeutic Targets in Hematological Malignancies.

Arginine methylation is a common post-translational modification affecting protein activity and the transcription of target genes when methylation occurs on histone tails. There are nine protein arginine methyltransferases (PRMTs) in mammals, divided into subgroups depending on the methylation they form on a molecule of arginine. During the formation and maturation of the different types of blood cells, PRMTs play a central role by controlling cell differentiation at the transcriptional level. PRMT enzymatic activity is necessary for many cellular processes in hematological malignancies, such as the activation of cell cycle and proliferation, inhibition of apoptosis, DNA repair processes, RNA splicing, and transcription by methylating histone tails' arginine. Chemical tools have been developed to inhibit the activity of PRMTs and have been tested in several models of hematological malignancies, including primary samples from patients, xenografts into immunodeficient mice, mouse models, and human cell lines. They show a significant effect by reducing cell viability and increasing the overall survival of mice. PRMT5 inhibitors have a strong therapeutic potential, as phase I clinical trials in hematological malignancies that use these molecules show promising results, thus, underlining PRMT inhibitors as useful therapeutic tools for cancer treatment in the future.

Multifactorial feature extraction and site prognosis model for protein methylation data.

Integrated studies (multi-omics studies) comprising genetic, proteomic and epigenetic data analyses have become an emerging topic in biomedical research. Protein methylation is a posttranslational modification that plays an essential role in various cellular activities. The prediction of methylation sites (arginine and lysine) is vital to understand the molecular processes of protein methylation. However, current experimental techniques used for methylation site predictions are tedious and expensive. Hence, computational techniques for predicting methylation sites in proteins are necessary. For predicting methylation sites, various computational methods have been proposed in recent years. Most existing methods require structural and evolutionary information for retrieving features, acquiring this information is not always convenient. Thus, we proposed a novel method, called multi-factorial feature extraction and site prognosis model (MufeSPM), for the prediction of protein methylation sites based on information theory features (Renyi, Shannon, Havrda-Charvat and Arimoto entropy), amino acid composition and physicochemical properties acquired from protein methylation data. A random forest algorithm was used to predict methylation sites in protein sequences. This paper also studied the impact of different features and classifiers on arginine and lysine methylation data sets. For the R methylation data set, MufeSPM yielded 82.45%($\pm $ 3.47) accuracy, and for the K methylation data set, it provided an average accuracy of 71.94%($\pm $ 2.12). Additionally, the area under the receiver operating characteristic curve for different classifiers in predicting methylation site was provided. The experimental results signify that MufeSPM performs better than the state-of-the-art predictors.

Modulation of the activity of histone lysine methyltransferases and demethylases by curcumin analog in leukaemia cells.

Curcumin is a known epigenetic modifier that demonstrated antitumor effect in different types of cancer. The poor solubility and metabolic stability are major drawbacks that limit its development as an antitumor agent. Dimethoxycurcumin (DMC) is a more soluble and stable curcumin analog. In this study, we compared the effect of both drugs on a variety of histone posttranslational modifications and on the activity of histone lysine methyltransferase (HKMTs) and demethylase (HKDMTs) enzymes that target the H3K4, H3K9 and H3K27 epigenetic marks. Mass spectrometry was used to quantitate the changes in 95 histone posttranslational modifications induced by curcumin or DMC. The effect of both drugs on the enzymatic activity of HKMTs and HKDMs was measured using an antibody-based assay. Mass spectrometry analysis showed that curcumin and DMC modulated several histone modifications. Histone changes were not limited to lysine methylation and acetylation but included arginine and glutamine methylation. Only few histone modifications were similarly changed by both drugs. On the contrary, the effect of both drugs on the activity of HKMTs and HKDMs was very similar. Curcumin and DMC inhibited the HKMTs enzymes that target the H3K4, H3K9 and H3K27 marks and increased the activity of the HKDMs enzymes LSD1, JARID and JMJD2. In conclusion, we identified novel enzymatic targets for both curcumin and DMC that support their use and development as epigenetic modifiers in cancer treatment. The multiple targets modulated by both drugs could provide a therapeutic advantage by overcoming drug resistance development.

Global profiling of arginine dimethylation in regulating protein phase separation by a steric effect–based chemical-enrichment method

Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid-liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect-based chemical-enrichment method (SECEM) coupled with liquid chromatography-tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG-rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both invitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG-rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics.

CNNArginineMe: A CNN structure for training models for predicting arginine methylation sites based on the One-Hot encoding of peptide sequence.

Protein arginine methylation (PRme), as one post-translational modification, plays a critical role in numerous cellular processes and regulates critical cellular functions. Though several in silico models for predicting PRme sites have been reported, new models may be required to develop due to the significant increase of identified PRme sites. In this study, we constructed multiple machine-learning and deep-learning models. The deep-learning model CNN combined with the One-Hot coding showed the best performance, dubbed CNNArginineMe. CNNArginineMe performed best in AUC scoring metrics in comparisons with several reported predictors. Additionally, we employed CNNArginineMe to predict arginine methylation proteome and performed functional analysis. The arginine methylated proteome is significantly enriched in the amyotrophic lateral sclerosis (ALS) pathway. CNNArginineMe is freely available at https://github.com/guoyangzou/CNNArginineMe.

Flavokawain A is a natural inhibitor of PRMT5 in bladder cancer

BackgroundProtein arginine methyltransferases (PRMTs) regulate protein biological activity by modulating arginine methylation in cancer and are increasingly recognized as potential drug targets. Inhibitors targeting PRMTs are currently in the early phases of clinical trials and more candidate drugs are needed. Flavokawain A (FKA), extracted from kava plant, has been recognized as a potential chemotherapy drug in bladder cancer (BC), but its action mechanism remains unclear.MethodsWe first determined the role of a type II PRMT, PRMT5, in BC tissue samples and performed cytological experiments. We then utilized bioinformatics tools, including computational simulation, virtual screening, molecular docking, and energy analysis, to identify the potential use of PRMT5 inhibitors for BC treatment. In vitro and in vivo co-IP and mutation assays were performed to elucidate the molecular mechanism of PRMT5 inhibitor. Pharmacology experiments like bio-layer interferometry, CETSA, and pull-down assays were further used to provide direct evidence of the complex binding process.ResultsAmong PRMTs, PRMT5 was identified as a therapeutic target for BC. PRMT5 expression in BC was correlated with poor prognosis and manipulating its expression could affect cancer cell growth. Through screening and extensive experimental validation, we recognized that a natural product, FKA, was a small new inhibitor molecule for PRMT5. We noticed that the product could inhibit the action of BC, in vitro and in vivo, by inhibiting PRMT5. We further demonstrated that FKA blocks the symmetric arginine dimethylation of histone H2A and H4 by binding to Y304 and F580 of PRMT5.ConclusionsIn summary, our research strongly suggests that PRMT5 is a potential epigenetic therapeutic target in bladder cancer, and that FKA can be used as a targeted inhibitor of PRMT5 for the treatment of bladder cancer.

Endometrial stromal PRMT5 plays a crucial role in decidualization by regulating NF-κB signaling in endometriosis

Decidualization is a prerequisite for successful embryo implantation, in which elongated fibroblast-like endometrial stromal cells differentiate into more rounded decidual cells. Accumulating evidence has stressed the important role of the defective eutopic endometrium in infertility in endometriosis patients. However, the role of arginine methylation in the process of physiological decidualization and pathological decidualization defects is not clear. Here, we observed that the expression level of PRMT5, the main type II PRMT, was decreased in the endometrium of endometriosis patients, predominantly in stromal cells. Compared with the undecidualized state, PRMT5 was increased in the stromal cells of normal secretory endometrium in humans and in the decidua of normal pregnant mice or mice with artificially induced decidualization. The inhibition of PRMT5 resulted in a significant decrease in uterine weight and decidualization-related regulator expression, including FOXO1, HOXA10 and WNT4, in mice and IGFBP1 and prolactin levels in human endometrial stromal cells. Transcriptome analysis showed that decreased PRMT5 activity led to NF-κB signaling activation by inducing p65 translocation to the nucleus, which was also observed in endometriosis patients. Finally, overexpression of PRMT5 rescued the defective expression of IGFBP1 and prolactin in primary endometrial stromal cells from endometriosis patients. Our results indicate that promotion of PRMT5 may provide novel therapeutic strategies for the treatment of decidualization defects in infertile women, such as those with endometriosis.

ROS inhibits RORα degradation by decreasing its arginine methylation in liver cancer.

Retinoic acid receptor-related orphan receptor α (RORα) is a transcription factor involved in nuclear gene expression and a known tumor suppressor. RORα was the first identified substrate of lysine methylation-dependent degradation. However, the mechanisms of other post-translational modifications (PTMs) that occur in RORα remain largely unknown, especially in liver cancer. Arginine methylation is a common PTM in arginine residues of nonhistone and histone proteins and affects substrate protein function and fate. We found an analogous amino acid disposition containing R37 at the ROR N-terminus compared to histone H3 residue, which is arginine methylated. Here, we provide evidence that R37 methylation-dependent degradation is carried out by protein arginine methyltransferase 5 (PRMT5). Further, we discovered that PRMT5 regulated the interaction between the E3 ubiquitin ligase ITCH and RORα through RORα arginine methylation. Arginine methylation-dependent ubiquitination-mediated RORα degradation reduced downstream target gene activation. H2 O2 -induced reactive oxygen species (ROS) decreased PRMT5 protein levels, consequently increasing RORα protein levels in HepG2 liver cancer cells. In addition, ROS inhibited liver cancer progression by inducing apoptosis via PRMT5-mediated RORα methylation and the ITCH axis. Our results potentiate PRMT5 as an elimination target in cancer therapy, and this additional regulatory level within ROS signaling may help identify new targets for therapeutic intervention in liver cancer.

Evaluation of a Panel of 105 Cytokines from Human Glioblastoma (GBM) Tissue Maintained, and Treated, on a Unique Microfluidic Platform

Abstract AIMS The first aim was to measure the changes in the levels of 105 cytokines potentially released by GBM tissue maintained on-chip. The second aim was to compare the cytokine profiles from control GBM tissue and tissue treated with anti-GBM drugs {temozolomide (TMZ) at 1 and 10μM both with 1μM arginine methylation inhibitors}. METHOD Intra-operative tissue biopsies were taken from the tumour and transferred immediately from the operating theatre to Hull University laboratories (&amp;lt; 1 hour). Tissue samples were maintained in a viable state in a bespoke chip device for eight days. The biopsy was perfused (3μl/min) with culture medium, with or without drug treatment. Effluents from the biopsies were collected at 24h intervals. Cytokines levels were measured using the Proteome Profiler Human XL Cytokine Array Kit (R&amp;D Systems), each membrane is spotted with 105 different cytokine antibodies in duplicate. RESULTS Six GBM diagnosed patients (five negative for MGMT) were recruited and studied. Eleven cytokines showed consistent, high level, release from GBM tissue either in control or treated samples: Chitinase3-like1, IL8(CXCL8) ,MCP-1(CCL2 ),MIF(Macrophage migration inhibitory factor), MMP-9(Matrix metallopeptidase 9), Osteopontin(OPN), Serpin E1, VEGF(Vascular endothelial growth factor) ,Apolipoprotein A-I, Angiogenin and Emmprin. Multivariate analysis (PERMANOVA) did not show evidence for differential cytokine release profiles with time (P=0.113), treatment (P=0.629), , nor the combined interaction (P=0.936). CONCLUSION GBM biopsies remained viable on the microfluidic platform, shown by sustained cytokine production cytokines. We have also shown that treatment with TMZ plus arginine methylation inhibitors does not significantly affect cytokine release over an eight day period.