Arginine Residues In Proteins Research Articles

PRMT1 arginine methyltransferase accumulates in cytoplasmic bodies that respond to selective inhibition and DNA damage.

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus, PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression, splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However, when cell nuclei were microirradiated by UV-A, the mobility of PRMT1 cytoplasmic bodies increased their, size was reduced, and they disappeared within approximately 20 min. The same response occurred after γ-irradiation of the whole cell population, but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 over-expression. Taken together, we show that PRMT1 concentrates in cytoplasmic bodies, which respond to DNA injury in the cell nucleus, and to treatment with various PRMT1 inhibitors.

Vimentin is involved in peptidylarginine deiminase 2-induced apoptosis of activated Jurkat cells.

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca2+-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.

Designing synthetic drugs against Plasmodium falciparum: a computational study of histone-lysine N-methyltransferase (PfHKMT).

Histone lysine methyltransferase (HKMT) are histone-modifying enzymes that catalyze the transfer of methyl groups to lysine and arginine residues of histone protein. HKMTs have been involved in transcriptional regulation of various proteins in organisms. Malaria parasite also has HKMT, which plays a major role in parasite development and pathogenesis and also in regulation of various biological process and pathways. Our aim is to study fundamental biology of key molecules involved in the survival of Plasmodium falciparum and use these to develop efficient synthetic peptides and chemical compounds. As a first step in this direction, we computationally predicted the three-dimensional structure of HKMT of P. falciparum (PfHKMT) by using iterative threading assembly refinement. The PfHKMT three-dimensional model was validated using PROCHECK and docked with known HKMT inhibitor Bix01294 using Autodock. Our initial results are encouraging and indicate that structural analysis of PfHKMT could be important in developing novel synthetic molecules against malaria.

β-Biguanidinium-cyclodextrin: a supramolecular mimic of mitochondrial ADP/ATP carrier protein

We reported a novel mono-β-cyclodextrin derivative, mono-6-deoxy-6-biguanidino-β-cyclodextrin (β-biGCD), which was investigated as a mimic of ADP/ATP carrier (AAC). Its affinity toward AMP, ADP, and ATP was evaluated by means of isothermal titration calorimetry (ITC). The association constants (Ka) of β-biGCD binding to AMP, ADP, and ATP were determined to be (1.07±0.04)×106, (5.86±0.02)×106, and (4.33±0.06)×106 L mol−1, respectively, which were 100-fold higher than mono-guanidino-β-cyclodextrin (ca. 104 L mol−1). UV spectroscopic titrations further confirmed the above results. The interaction between β-biGCD and nucleotides was probed by docking simulation. These results reveal that the biguanidinium moiety mimics the arginine residues of mitochondrial AAC protein.

The Link Between Periodontal Disease and Rheumatoid Arthritis: An Updated Review

Porphyromonas gingivalis is a leading pathogen in chronic periodontitis, a disease process involving progressive destruction of the tissues that support the teeth. Recently, the organism has been reported to produce a unique bacterial enzyme, P. gingivalis peptidyl-arginine deiminase (PPAD), which has the ability to convert arginine residues in proteins to citrulline. Protein citrullination alters protein structure and function; hence, PPAD may be involved in deregulation of the host’s signalling network and immune evasion. Further, accumulating evidence suggests a role for autoimmunity against citrullinated proteins in the development of rheumatoid arthritis (RA). As inflammatory conditions in the lungs of cigarette smokers contribute to the breakdown of immune tolerance to citrullinated epitopes, chronic exposure to citrullinated proteins at periodontitis sites may also predispose susceptible individuals to the development of autoantibodies and the initiation of RA. In this review, we discuss evidence that PPAD may represent a mechanistic link between periodontitis and RA, diseases that are known to be significantly associated at the epidemiological level.

Special edition of Amino Acids of selected papers from the eleventh international symposium on the Maillard reaction, September 16–20, 2012

We introduce this special edition of Amino Acids with selected papers from the eleventh international symposium on the Maillard reaction held in Nancy, France, September 16–20, 2012. Glycation of proteins occurs by a complex series of sequential and parallel reactions called collectively the Maillard reaction—named after the leading pioneer of glycation research, Louis Camille Maillard (1978–1936). Glycation researchers gathered at the symposium in 2012 organised by the International Maillard Reaction Society to commemorate the centennial of Maillard reaction research and review achievements and current standing of glycation research. In this special edition there are selected papers from this meeting. Subjects of study include: the involvement of advanced glycation endproducts (AGEs) in complications of diabetes, association of AGEs with insulin secretion, enzymatic metabolism of early glycation adducts, absorption into the body of early and advanced glycation endproducts (AGEs) of dietary origin, association of AGEs in dietary protein with chronic disease, the study of the effect of AGEs on bacterial growth and inhibition of AGE formation by natural products. The Centennial Maillard Symposium began with a presentation by Nobel laureate Jean-Marie Lehn (University of Strasbourg, France) on ‘‘Towards a chemistry of complex matter’’. The Maillard reaction exemplifies how a relatively simple chemical reaction of a reducing sugar or sugar derivative with an amino group becomes highly complex through multiple parallel and sequential reactions. In recent decades there has also been increasing awareness of dicarbonyl compounds and metabolites reacting with guanidino groups in the Maillard reaction. This expands the locus of glycation adduct formation from N-terminal and lysine side chain amino groups in amino acids and proteins to guanidino groups of arginine and arginine residues in proteins. Indeed, arginine-derived AGEs are now considered to be quantitatively major AGEs in physiological systems. In 100 years the Maillard reaction has seen applications in chemistry, food, nutrition, health and disease, microbiology and clinical diagnostics and repeated, on-going development of applications in pharmacology and therapeutics. We celebrate the achievements of Louis Camille Maillard in glycation research and suggest that, if he was here today, he would marvel at the advances and development that led from his earlier studies.

Asymmetric dimethylarginine attenuates serum starvation-induced apoptosis via suppression of the Fas (APO-1/CD95)/JNK (SAPK) pathway

Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C2-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.

Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD)

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.

Biochemical Studies on Methylglyoxal-Mediated Glycated Histones: Implications for Presence of Serum Antibodies against the Glycated Histones in Patients with Type 1 Diabetes Mellitus.

Reactive carbonyl species (RCS) mainly reacts with lysine and arginine residues of proteins to form advanced glycation end products (AGEs). Histone was glycoxidated with glyoxal and methylglyoxal. It was characterized by polyacrylamide gel electrophoresis and quenching studies involving penicillamine and aminoguanidine as carbonyl scavengers. Further characterization of histone modified with methylglyoxal was done by UV, fluorescence, and IR spectrophotometry. Spectral analysis of the protein clearly demonstrates structural perturbation in the histone by methylglyoxal. Methylglyoxal-induces cross-linking in the protein leading to aggregation. Role of methylglyoxal mediated glycoxidation of histone in type 1 diabetes was also undertaken. Antibodies were detected against glycoxidated histone in sera of type 1 diabetes patients by solid-phase enzyme immunoassay. The findings indicate that as a result of structural perturbation in histone by methylglyoxal, the modified histone may be involved in production of serum antibodies in the diabetes patients.

QM/MM MD and free energy simulations of the methylation reactions catalyzed by protein arginine methyltransferase PRMT3

Protein arginine N-methyltransferases (PRMTs) catalyze the transfer of methyl group(s) from S-adenosyl-l-methionine (AdoMet) to the guanidine group of arginine residue in abundant eukaryotic proteins. Two major types of PRMTs have been identified in mammalian cells. Type I PRMTs catalyze the formation of asymmetric ω-NG, NG-dimethylarginine (ADMA), while Type II PRMTs catalyze the formation of symmetric ω-NG, N′G-dimethylarginine (SDMA). The two different methylation products (ADMA or SDMA) of the substrate could lead to different biological consequences. Although PRMTs have been the subject of extensive experimental investigations, the origin of the product specificity remains unclear. In this study, quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study the reaction mechanism for one of Type I PRMTs, PRMT3, and to gain insights into the energetic origin of its product specificity (ADMA). Our simulations have identified some important interactions and proton transfers involving the active site residues. These interactions and proton transfers seem to be responsible, at least in part, in making the Nη2 atom of the substrate arginine the target of the both 1st and 2nd methylations, leading to the asymmetric dimethylation product. The simulations also suggest that the methyl transfer and proton transfer appear to be somehow concerted processes and that Glu326 is likely to function as the general base during the catalysis.

Impact of GLO1 Knock Down on GLUT4 Trafficking and Glucose Uptake in L6 Myoblasts

Methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.

Drugging the human methylome: an emerging modality for reversible control of aberrant gene transcription

Protein and DNA methylation have emerged as critical mechanisms for the control of regulated gene transcription. In humans, the addition, recognition and removal of methyl groups are orchestrated by at least 344 proteins that we collectively refer to as the 'methylome'. The large size of the methylome likely reflects the importance of precise control over this small covalent modification. An increasing number of reports implicating the misregulation of methylation in disease make the proteins governing this modification attractive target for small molecule drug discovery. In light of the emerging opportunities for the development of therapeutics that modulate methylation-dependent pathways, this review examines the protein families that constitute the methylome, with emphasis on the methylation of arginine and lysine residues of proteins. Genetic aberrations that give rise to disease are highlighted, in addition to recent proof-of-concept successes in the development of small molecule modulators of methylome constituents.

Abstract 1128: Preclinical and pharmacological activities of protein arginine methyltransferase 5(PRMT5) enzyme inhibition in acute myeloid leukemia .

Abstract With standard cytotoxic chemotherapy the majority of patients with Acute Myeloid Leukemia (AML) fail to achieve long term remission. Protein Arginine Methyltransferase 5 (PRMT5) can catalyze methylation of arginine residues in target proteins like histones and P53. The epigenetic marks of PRMT5 on histones H3 (S2Me-H3R8) and H4 (S2Me-H4R3), directly lead to transcriptional silencing of genes. Overexpression of PRMT5 has been reported in hematologic and solid malignancies and represents a promising therapeutic target. Thus, we hypothesized that inhibition of PRMT5 enzymatic activity using a first in class, selective small molecule inhibitor “BLL1” would result in antileukemic activity and novel methods to treat AML. PRMT5 was upregulated in AML-derived cell lines and primary patient samples. PRMT5 in complex with co-repressors like histone deacetylase (HDAC)s and DNA methyltrasferase (DNMT)s is involved in epigenetic silencing of tumor suppressor genes. Using Co-IP assay we captured PRMT5 in association with DNMT3a and HDAC2 in AML cells, in which treatment with BLL1 (25μM) resulted in disruption of this interaction. We confirmed the ability and specificity of BLL1 by detecting the loss of unique epigenetic mark of PRMT5, S2Me-H3R8. We previously reported that pharmacologic inhibition of DNMTs (by decitabine) and HDACs (by AR42) or the disruption of their repressor complexes (by bortezomib) can inhibit critical oncogenes in AML, KIT and FLT3, by upregulating miR-29b and consequently lead to better clinical response in AML. Similar to these epigenetic-targeting compounds, BLL1 upregulated miR-29b (>7 fold; MV411, FLT3 ITD+ cells) and resulted in downregulation of FLT3 RNA (>4 fold) and protein (3.8 fold). We examined the in vitro antileukemic activity of BLL1 in AML and demonstrated a dose-dependent inhibition of cell proliferation (MTS assay) in AML cell lines (MV411; IC50=6μM & Kasumi1, KITmut, IC50=11μM) and primary patient blasts (#1, FLT3 ITD+, NPM1+, CD34−; IC50=8μM & #2, FLT3 ITD+, CD34+; IC50=7.5μM). Colony forming ability was significantly decreased (p<0.001) in BLL1 (2μM) treated AML cells compared to controls. We observed a BLL1-dependent increase in apoptosis measured by AnnexinV staining (25 & 50μM, MV411; 8.6 fold & Kasumi1; 4 fold) within 48 hrs. BLL1 treatment promoted cell maturation measured by increased monocyte differentiation marker CD11b in AML cells (50μM, MV411; 11.5 fold & Kasumi1; 6.8 fold) and primary patient blasts (25μM, #1; 4 fold). In conclusion, our data suggest that inhibiting PRMT5 activity interferes with the production or activity of oncoproteins and, subsequently, disrupts leukemogenic and proliferation pathways in AML. Given the substantial antileukemic potential of PRMT5 inhibitor compound on AML cells and primary patient blasts, an assessment of therapeutic implications of this novel drug in vivo is highly warranted. Citation Format: Somayeh S. Tarighat, Kiran Mahasenan, Danilo Perrotti, Ramiro Garzon, Michael Caligiuri, Chenglong Li, William Blum, Guido Marcucci, Robert A. Baiocchi. Preclinical and pharmacological activities of protein arginine methyltransferase 5(PRMT5) enzyme inhibition in acute myeloid leukemia . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1128. doi:10.1158/1538-7445.AM2013-1128

Abstract 1297: SETD6-mediated deregulation of NF- κB signaling in 4-ABP-induced bladder carcinogenesis.

Abstract Epidemiological data support an etiological role for chemicals from occupational exposure and cigarette smoke with increased risk of bladder cancer. However, epigenetic effects of these chemicals remain to be elucidated. Protein methyltransferases (PMTs) methylate protein lysine and arginine residues and play an important role in regulating gene transcription. Deregulation of SET-domain-containing PMTs has a crucial role in carcinogenesis. SETD6 has been shown to monomethylate RelA (a principal subunit of NF-κB at Lys310). Monomethylated RelA recruits GLP (G9A-like protein), a histone dimethyl transferase to the NF-κB target genes, which results in transcriptional repression. The role of SETD6 in bladder cancer is unknown. We used qPCR and immunoblotting to determine changes in SETD6 at the mRNA and protein level in bladder cancer cells relative to primary urothelial cells. We also found that knockdown of SETD6 significantly decreases the viability and proliferation of bladder cancer cells suggesting that SETD6 may function as an oncogene in bladder cancer. Furthermore, we also found that message and protein levels of SETD6 are induced in primary urothelial cells following treatment of 4-aminobiphenyl (4-ABP), an important constituent of cigarette smoke. Preliminary results indicate that induction may be mediated by 4-ABP-induced ROS. RelAK310Me1 level also increased with 4-ABP treatment showing the increase in SETD6 functional activity with 4-ABP exposure. We also found the decrease in expression of RelB, one of the target genes of RelA. Overall our results show that SETD6 may function as an oncogene in 4-ABP induced bladder cancer by deregulating transactivation property of RelA. These results show for the first time the relevance of PKMTs and epigenetic regulation of NF-κB in carcinogen induced bladder cancer. Supported by RO3 CA136058 (RG). Citation Format: Neelam Mukherjee, Rita Ghosh. SETD6-mediated deregulation of NF- κB signaling in 4-ABP-induced bladder carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1297. doi:10.1158/1538-7445.AM2013-1297

Assay Development for Histone Methyltransferases

Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-L-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpot(SM) platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC₅₀ values at a low μM range.

PRMT4 Is a Novel Coactivator of c-Myb-Dependent Transcription in Haematopoietic Cell Lines

Protein arginine methyltransferase 4 (PRMT4)–dependent methylation of arginine residues in histones and other chromatin-associated proteins plays an important role in the regulation of gene expression. However, the exact mechanism of how PRMT4 activates transcription remains elusive. Here, we identify the chromatin remodeller Mi2α as a novel interaction partner of PRMT4. PRMT4 binds Mi2α and its close relative Mi2β, but not the other components of the repressive Mi2-containing NuRD complex. In the search for the biological role of this interaction, we find that PRMT4 and Mi2α/β interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. This coactivation requires the methyltransferase and ATPase activity of PRMT4 and Mi2, respectively. Chromatin immunoprecipitation analysis shows that c-Myb target genes are direct transcriptional targets of PRMT4 and Mi2. Knockdown of PRMT4 or Mi2α/β in haematopoietic cells of the erythroid lineage results in diminished transcriptional induction of c-Myb target genes, attenuated cell growth and survival, and deregulated differentiation resembling the effects caused by c-Myb depletion. These findings reveal an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling.

The methylarginines NMMA, ADMA, and SDMA are ubiquitous constituents of the main vegetables of human nutrition

Endogenous methylarginines, NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA), NG-N′G-dimethyl-l-arginine (symmetric dimethylarginine; SDMA), and NG-monomethyl-l-arginine (monomethylarginine; NMMA) are supposed to be produced in human body through the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and released during proteolysis of the methylated proteins. Micromolar concentration of ADMA and NMMA can compete with arginine for nitric oxide synthase (NOS) reducing nitric oxide (NO) formation, whereas SDMA does not. Indeed, increased ADMA and SDMA plasma levels or a decreased arginine/ADMA ratio is related with risk factors for chronic kidney disease and cardiovascular disease. To the best of our knowledge the exogenous presence of methylarginines, like that in fruits and vegetables, has never been described so far. Here, we report the finding that methylarginines are ubiquitous in vegetables which represent an important part of human daily diet. Some of these vegetables contain discrete amounts of ADMA, SDMA, and NMMA. Specifically, among the vegetables examined, soybean, rye, sweet pepper, broad bean, and potato contain the highest ADMA and NMMA mean levels. Our results establish that the three methylarginines, in addition to being produced endogenously, can also be taken daily through the diet in conspicuous amounts. We propose that the contribution of the methylarginines contained in the vegetables of daily diet should be taken into account when the association between vegetable assumption and their levels is evaluated in clinical studies. Furthermore, a comprehensive understanding on the role of the digestive breakdown process and intestinal absorption grade of the methylarginines contained in vegetables is now needed.

Suppression of PRMT6-mediated arginine methylation of p16 protein potentiates its ability to arrest A549 cell proliferation

The tumor suppressor p16INK4A (p16) blocks the cell cycle progression by inhibiting phosphorylation of the retinoblastoma protein. We describe here a novel aspect of the posttranslational control that has an important functional consequence on p16 protein. We first discovered that the p16 protein was methylated in various cell lineages. We then determined that the arginine 22, 131 and 138 of p16 were the main methylation sites. Western blotting and TUNEL analyses revealed that the p16 protein bearing these point mutations induced a higher apoptosis ratio than wild-type p16 in A549 cells. Furthermore, co-immunoprecipitation assays suggested that decrease of p16 arginine methylation level promoted the association of p16 with CDK4. Additionally, we determined that the protein arginine methyltransferase 6 (PRMT6) was responsible for the p16 arginine methylation. Results from flow cytometric analysis demonstrated that PRMT6 overexpression counteracted the cell cycle arrest at G1 phase induced by wild-type p16 in A549 cells. We also provided evidence that PRMT6 was able to interact with p16, and that the intensity of p16-CDK4 association was reduced upon PRMT6 overexpression. Together, data presented in this report establish that methylation at specific arginine residues of p16 protein by PRMT6 may be critical for the activity of p16.

Protein arginine methyltransferase 5 is essential for growth of lung cancer cells

PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.

New insights into the genetics of immune responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is a common autoimmune disease with a strong genetic component. Numerous aberrant immune responses have been described during the evolution of the disease. In later years, the appearance of anti-citrullinated protein antibodies (ACPAs) has become a hallmark for the diagnosis and prognosis of RA. The post-translational transformation of arginine residues of proteins and peptides into citrulline (citrullination) is a natural process in the body, but for unknown reasons autoreactivity towards citrullinated residues may develop in disposed individuals. ACPAs are often found years before clinical manifestations. ACPAs are present in about 70% of RA patients and constitute an important disease marker, distinguishing patient groups with different prognoses and different responses to various treatments. Inside the human leukocyte antigen (HLA) region, some HLA-DRB1 alleles are strongly associated with their production. Genome-wide association studies in large patient cohorts have defined a great number of single nucleotide polymorphisms (SNPs) outside of the HLA region that are associated with ACPA positive (ACPA+) RA. The SNPs are generally located close to or within genes involved in the immune response or signal transduction in immune cells. Some environmental factors such as tobacco smoking are also positively correlated with ACPA production. In this review, we will describe the genes and loci associated with ACPA+ RA or ACPA- RA and attempt to clarify their potential role in the development of the disease.