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Sample records for protein arginine methyltransferase

  1. Small molecule regulators of protein arginine methyltransferases.

    PubMed

    Cheng, Donghang; Yadav, Neelu; King, Randall W; Swanson, Maurice S; Weinstein, Edward J; Bedford, Mark T

    2004-06-04

    Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.

  2. Small Molecule Inhibitors of Protein Arginine Methyltransferases

    PubMed Central

    Hu, Hao; Qian, Kun; Ho, Meng-Chiao; Zheng, Y. George

    2016-01-01

    Introduction Arginine methylation is an abundant posttranslational modification occurring in mammalian cells and catalyzed by protein arginine methyltransferases (PRMTs). Misregulation and aberrant expression of PRMTs are associated with various disease states, notably cancer. PRMTs are prominent therapeutic targets in drug discovery. Areas covered The authors provide an updated review of the research on the development of chemical modulators for PRMTs. Great efforts are seen in screening and designing potent and selective PRMT inhibitors, and a number of micromolar and submicromolar inhibitors have been obtained for key PRMT enzymes such as PRMT1, CARM1, and PRMT5. The authors provide a focus on their chemical structures, mechanism of action, and pharmacological activities. Pros and cons of each type of inhibitors are also discussed. Expert opinion Several key challenging issues exist in PRMT inhibitor discovery. Structural mechanisms of many PRMT inhibitors remain unclear. There lacks consistency in potency data due to divergence of assay methods and conditions. Physiologically relevant cellular assays are warranted. Substantial engagements are needed to investigate pharmacodynamics and pharmacokinetics of the new PRMT inhibitors in pertinent disease models. Discovery and evaluation of potent, isoform-selective, cell-permeable and in vivo-active PRMT modulators will continue to be an active arena of research in years ahead. PMID:26789238

  3. Diamidine Compounds for Selective Inhibition of Protein Arginine Methyltransferase 1

    PubMed Central

    2015-01-01

    Protein arginine methylation is a posttranslational modification critical for a variety of biological processes. Misregulation of protein arginine methyltransferases (PRMTs) has been linked to many pathological conditions. Most current PRMT inhibitors display limited specificity and selectivity, indiscriminately targeting many methyltransferase enzymes that use S-adenosyl-l-methionine as a cofactor. Here we report diamidine compounds for specific inhibition of PRMT1, the primary type I enzyme. Docking, molecular dynamics, and MM/PBSA analysis together with biochemical assays were conducted to understand the binding modes of these inhibitors and the molecular basis of selective inhibition for PRMT1. Our data suggest that 2,5-bis(4-amidinophenyl)furan (1, furamidine, DB75), one leading inhibitor, targets the enzyme active site and is primarily competitive with the substrate and noncompetitive toward the cofactor. Furthermore, cellular studies revealed that 1 is cell membrane permeable and effectively inhibits intracellular PRMT1 activity and blocks cell proliferation in leukemia cell lines with different genetic lesions. PMID:24564570

  4. Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

    PubMed

    Gou, Qing; He, ShuJiao; Zhou, ZeJian

    2017-02-01

    Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

  5. PRMT11: a new Arabidopsis MBD7 protein partner with arginine methyltransferase activity.

    PubMed

    Scebba, Francesca; De Bastiani, Morena; Bernacchia, Giovanni; Andreucci, Andrea; Galli, Alvaro; Pitto, Letizia

    2007-10-01

    Plant methyl-DNA-binding proteins (MBDs), discovered by sequence homology to their animal counterparts, have not been well characterized at the physiological and functional levels. In order better to characterize the Arabidopsis AtMBD7 protein, unique in bearing three MBD domains, we used a yeast two-hybrid system to identify its partners. One of the interacting proteins we cloned is the Arabidopsis arginine methyltransferase 11 (AtPRMT11). Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that the two proteins interact with each other and can be co-isolated. Using GFP fluorescence, we show that both AtMBD7 and AtPRMT11 are present in the nucleus. Further analyses revealed that AtPRMT11 acts as an arginine methyltransferase active on both histones and proteins of cellular extracts. The analysis of a T-DNA mutant line lacking AtPRMT11 mRNA revealed reduced levels of proteins with asymmetrically dimethylated arginines, suggesting that AtPRMT11, which is highly similar to mammalian PRMT1, is indeed a type I arginine methyltransferase. Further, AtMBD7 is a substrate for AtPRMT11, which post-translationally modifies the portion of the protein-containing C-terminal methylated DNA-binding domain. These results suggest the existence of a link between DNA methylation and arginine methylation.

  6. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    SciTech Connect

    Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon; Yang, Sujeong; Choi, Seunga; Kang, Misun; Rho, Jaerang

    2010-01-01

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  7. A Potent, Selective and Cell-active Inhibitor of Human Type I Protein Arginine Methyltransferases

    PubMed Central

    Wu, Hong; Senisterra, Guillermo; Li, Fengling; Butler, Kyle V.; Kaniskan, H. Ümit; Speed, Brandon A.; dela Seña, Carlo; Dong, Aiping; Zeng, Hong; Schapira, Matthieu; Brown, Peter J.; Arrowsmith, Cheryl H.; Barsyte-Lovejoy, Dalia; Liu, Jing; Vedadi, Masoud; Jin, Jian

    2015-01-01

    Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, 3, 4, 6 and 8, but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease. PMID:26598975

  8. Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 ...

  9. A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

    PubMed Central

    Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

    2016-01-01

    Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs. PMID:26858449

  10. Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

    PubMed Central

    Moon, Eun-Kyung; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung; Kong, Hyun-Hee

    2016-01-01

    Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa. PMID:27180570

  11. Structural basis for Sfm1 functioning as a protein arginine methyltransferase

    PubMed Central

    Lv, Fengjuan; Zhang, Tianlong; Zhou, Zhen; Gao, Shuaixin; Wong, Catherine CL; Zhou, Jin-Qiu; Ding, Jianping

    2015-01-01

    SPOUT proteins constitute one class of methyltransferases, which so far are found to exert activity mainly towards RNAs. Previously, yeast Sfm1 was predicted to contain a SPOUT domain but can methylate ribosomal protein S3. Here we report the crystal structure of Sfm1, which comprises of a typical SPOUT domain and a small C-terminal domain. The active site is similar to that of protein arginine methyltransferases but different from that of RNA methyltransferases. In addition, Sfm1 exhibits a negatively charged surface surrounding the active site unsuitable for RNA binding. Our biochemical data show that Sfm1 exists as a monomer and has high activity towards ribosomal protein S3 but no activity towards RNA. It can specifically catalyze the methylation of Arg146 of S3 and the C-terminal domain is critical for substrate binding and activity. These results together provide the structural basis for Sfm1 functioning as a PRMT for ribosomal protein S3. PMID:27462434

  12. Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a

    SciTech Connect

    Miranda, Tina Branscombe; Webb, Kristofor J.; Edberg, Dale D.; Reeves, Raymond; Clarke, Steven . E-mail: clarke@mbi.ucla.edu

    2005-10-28

    The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation.

  13. Protein Arginine Methyltransferases Interact with IFT Particles and Change Location During Flagellar Growth and Resorption.

    PubMed

    Mizuno, Katsutoshi; Sloboda, Roger D

    2017-03-15

    Changes in protein activity driven by post translational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we have focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMTs 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella, and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

  14. Protein Arginine Methyltransferase 1 Interacts with and Activates p38α to Facilitate Erythroid Differentiation

    PubMed Central

    Hua, Wei-Kai; Chang, Yuan-I; Yao, Chao-Ling; Hwang, Shiaw-Min; Chang, Chung-Yi; Lin, Wey-Jinq

    2013-01-01

    Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes; however, its role in erythropoiesis is still elusive. Erythropoiesis generates circulating red blood cells which are vital for body activity. Deficiency in erythroid differentiation causes anemia which compromises the quality of life. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in protein arginine methyltransferase 1 (PRMT1) levels, via transfection or protein transduction, significantly promoted erythroid differentiation in the bipotent human K562 cell line as well as in human primary hematopoietic progenitor CD34+ cells. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated several key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient PRMT1G80R mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. Our results further showed that a specific isoform of p38 MAPK, p38α, promoted erythroid differentiation, whereas p38β did not play a role. The stimulation of erythroid differentiation by PRMT1 was diminished in p38α- but not p38β-knockdown cells. PRMT1 appeared to act upstream of p38α, since expression of p38α still promoted erythroid differentiation in PRMT1-knockdown cells, and expression of PRMT1 enhanced the activation of p38 MAPK. Importantly, we showed for the first time that PRMT1 was associated with p38α in cells by co-immunoprecipitation and that PRMT1 directly methylated p38α in in vitro methylation assays. Taken together, our findings unveil a link between PRMT1 and p38α in regulating the erythroid differentiation program and

  15. Protein arginine methyltransferase 1 is a novel regulator of MYCN in neuroblastoma

    PubMed Central

    Eberhardt, Allison; Hansen, Jeanne N.; Koster, Jan; Lotta, Louis T.; Wang, Simeng; Livingstone, Emmett; Qian, Kun; Valentijn, Linda J.; Zheng, Yujun George; Schor, Nina F.; Li, Xingguo

    2016-01-01

    Amplification or overexpression of MYCN is associated with poor prognosis of human neuroblastoma. We have recently defined a MYCN-dependent transcriptional signature, including protein arginine methyltransferase 1 (PRMT1), which identifies a subgroup of patients with high-risk disease. Here we provide several lines of evidence demonstrating PRMT1 as a novel regulator of MYCN and implicating PRMT1 as a potential therapeutic target in neuroblastoma pathogenesis. First, we observed a strong correlation between MYCN and PRMT1 protein levels in primary neuroblastoma tumors. Second, MYCN physically associates with PRMT1 by direct protein-protein interaction. Third, depletion of PRMT1 through siRNA knockdown reduced neuroblastoma cell viability and MYCN expression. Fourth, we showed that PRMT1 regulates MYCN stability and identified MYCN as a novel substrate of PRMT1. Finally, we demonstrated that mutation of putatively methylated arginine R65 to alanine decreased MYCN stability by altering phosphorylation at residues serine 62 and threonine 58. These results provide mechanistic insights into the modulation of MYCN oncoprotein by PRMT1, and suggest that targeting PRMT1 may have a therapeutic impact on MYCN-driven oncogenesis. PMID:27571165

  16. Identification and Characterization of New Molecular Partners for the Protein Arginine Methyltransferase 6 (PRMT6)

    PubMed Central

    Lo Sardo, Alessandra; Altamura, Sandro; Pegoraro, Silvia; Maurizio, Elisa; Sgarra, Riccardo; Manfioletti, Guidalberto

    2013-01-01

    PRMT6 is a protein arginine methyltransferase that has been implicated in transcriptional regulation, DNA repair, and human immunodeficiency virus pathogenesis. Only few substrates of this enzyme are known and therefore its cellular role is not well understood. To identify in an unbiased manner substrates and potential regulators of PRMT6 we have used a yeast two-hybrid approach. We identified 36 new putative partners for PRMT6 and we validated the interaction in vivo for 7 of them. In addition, using in vitro methylation assay we identified 4 new substrates for PRMT6, extending the involvement of this enzyme to other cellular processes beyond its well-established role in gene expression regulation. Holistic approaches create molecular connections that allow to test functional hypotheses. The assembly of PRMT6 protein network allowed us to formulate functional hypotheses which led to the discovery of new molecular partners for the architectural transcription factor HMGA1a, a known substrate for PRMT6, and to provide evidences for a modulatory role of HMGA1a on the methyltransferase activity of PRMT6. PMID:23326497

  17. Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation

    PubMed Central

    Alinari, Lapo; Mahasenan, Kiran V.; Yan, Fengting; Karkhanis, Vrajesh; Chung, Ji-Hyun; Smith, Emily M.; Quinion, Carl; Smith, Porsha L.; Kim, Lisa; Patton, John T.; Lapalombella, Rosa; Yu, Bo; Wu, Yun; Roy, Satavisha; De Leo, Alessandra; Pileri, Stefano; Agostinelli, Claudio; Ayers, Leona; Bradner, James E.; Chen-Kiang, Selina; Elemento, Olivier; Motiwala, Tasneem; Majumder, Sarmila; Byrd, John C.; Jacob, Samson; Sif, Said; Li, Chenglong

    2015-01-01

    Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. PMID:25742700

  18. A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)**

    PubMed Central

    Kaniskan, H. Ümit; Szewczyk, Magdalena M.; Yu, Zhengtian; Eram, Mohammad S.; Yang, Xiaobao; Schmidt, Keith; Luo, Xiao; Dai, Miao; He, Feng; Zang, Irene; Lin, Ying; Kennedy, Steven; Li, Fengling; Dobrovetsky, Elena; Dong, Aiping; Smil, David; Min, Sun-Joon; Landon, Melissa; Lin-Jones, Jennifer; Huang, Xi-Ping; Roth, Bryan L.; Schapira, Matthieu; Atadja, Peter; Barsyte-Lovejoy, Dalia; Arrowsmith, Cheryl H.; Brown, Peter J.; Zhao, Kehao; Jin, Jian; Vedadi, Masoud

    2015-01-01

    PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease. PMID:25728001

  19. Functional insights from high resolution structures of mouse protein arginine methyltransferase 6.

    PubMed

    Bonnefond, Luc; Stojko, Johann; Mailliot, Justine; Troffer-Charlier, Nathalie; Cura, Vincent; Wurtz, Jean-Marie; Cianférani, Sarah; Cavarelli, Jean

    2015-08-01

    PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7.

  20. The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex.

    PubMed

    Kafková, Lucie; Debler, Erik W; Fisk, John C; Jain, Kanishk; Clarke, Steven G; Read, Laurie K

    2017-02-10

    Prozymes are catalytically inactive enzyme paralogs that dramatically stimulate the function of weakly active enzymes through complex formation. The two prozymes described to date reside in the polyamine biosynthesis pathway of the human parasite Trypanosoma brucei, an early branching eukaryote that lacks transcriptional regulation and regulates its proteome through posttranscriptional and posttranslational means. Arginine methylation is a common posttranslational modification in eukaryotes catalyzed by protein arginine methyltransferases (PRMTs) that are typically thought to function as homodimers. We demonstrate that a major T. brucei PRMT, TbPRMT1, functions as a heterotetrameric enzyme-prozyme pair. The inactive PRMT paralog, TbPRMT1(PRO), is essential for catalytic activity of the TbPRMT1(ENZ) subunit. Mutational analysis definitively demonstrates that TbPRMT1(ENZ) is the cofactor-binding subunit and carries all catalytic activity of the complex. Our results are the first demonstration of an obligate heteromeric PRMT, and they suggest that enzyme-prozyme organization is expanded in trypanosomes as a posttranslational means of enzyme regulation.

  1. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    SciTech Connect

    Kanno, Yuichiro Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  2. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    SciTech Connect

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  3. Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression.

    PubMed

    Baldwin, R Mitchell; Haghandish, Nasim; Daneshmand, Manijeh; Amin, Shahrier; Paris, Geneviève; Falls, Theresa J; Bell, John C; Islam, Shahidul; Côté, Jocelyn

    2015-02-20

    Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer.

  4. Exploration of Cyanine Compounds as Selective Inhibitors of Protein Arginine Methyltransferases: Synthesis and Biological Evaluation

    PubMed Central

    2016-01-01

    Protein arginine methyltransferase 1 (PRMT1) is involved in many biological activities, such as gene transcription, signal transduction, and RNA processing. Overexpression of PRMT1 is related to cardiovascular diseases, kidney diseases, and cancers; therefore, selective PRMT1 inhibitors serve as chemical probes to investigate the biological function of PRMT1 and drug candidates for disease treatment. Our previous work found trimethine cyanine compounds that effectively inhibit PRMT1 activity. In our present study, we systematically investigated the structure–activity relationship of cyanine structures. A pentamethine compound, E-84 (compound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity, and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound. PMID:25559100

  5. Protein arginine methyltransferase 1 (PRMT1) represses MHC II transcription in macrophages by methylating CIITA

    PubMed Central

    Fan, Zhiwen; Li, Jianfei; Li, Ping; Ye, Qing; Xu, Huihui; Wu, Xiaoyan; Xu, Yong

    2017-01-01

    Efficient presentation of alien antigens triggers activation of T lymphocytes and robust host defense against invading pathogens. This pathophysiological process relies on the expression of major histocompatibility complex (MHC) molecules in antigen presenting cells such as macrophages. Aberrant MHC II transactivation plays a crucial role in the pathogenesis of atherosclerosis. Class II transactivator (CIITA) mediates MHC II induction by interferon gamma (IFN-γ). CIITA activity can be fine-tuned at the post-translational level, but the mechanisms are not fully appreciated. We investigated the role of protein arginine methyltransferase 1 (PRMT1) in this process. We report here that CIITA interacted with PRMT1. IFN-γ treatment down-regulated PRMT1 expression and attenuated PRMT1 binding on the MHC II promoter. Over-expression of PRMT1 repressed MHC II promoter activity while PRMT1 depletion enhanced MHC II transactivation. Mechanistically, PRMT1 methylated CIITA and promoted CIITA degradation. Therefore, our data reveal a previously unrecognized role for PRMT1 in suppressing CIITA-mediated MHC II transactivation. PMID:28094290

  6. Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1.

    PubMed

    Tibshirani, Michael; Tradewell, Miranda L; Mattina, Katie R; Minotti, Sandra; Yang, Wencheng; Zhou, Hongru; Strong, Michael J; Hayward, Lawrence J; Durham, Heather D

    2015-02-01

    Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUS(R495X) transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription.

  7. Protein Arginine Methyltransferase 6 Enhances Polyglutamine-Expanded Androgen Receptor Function and Toxicity in Spinal and Bulbar Muscular Atrophy

    PubMed Central

    Scaramuzzino, Chiara; Casci, Ian; Parodi, Sara; Lievens, Patricia M.J.; Polanco, Maria J.; Milioto, Carmelo; Chivet, Mathilde; Monaghan, John; Mishra, Ashutosh; Badders, Nisha; Aggarwal, Tanya; Grunseich, Christopher; Sambataro, Fabio; Basso, Manuela; Fackelmayer, Frank O.; Taylor, J. Paul; Pandey, Udai Bhan; Pennuto, Maria

    2015-01-01

    Summary Polyglutamine expansion in androgen receptor (AR) is responsible for spinobulbar muscular atrophy (SBMA) that leads to selective loss of lower motor neurons. Using SBMA as a model, we explored the relationship between protein structure/function and neurodegeneration in polyglutamine diseases. We show here that protein arginine methyltransferase 6 (PRMT6) is a specific co-activator of normal and mutant AR and that the interaction of PRMT6 with AR is significantly enhanced in the AR mutant. AR and PRMT6 interaction occurs through the PRMT6 steroid receptor interaction motif, LXXLL, and the AR activating function 2 surface. AR transactivation requires PRMT6 catalytic activity and involves methylation of arginine residues at Akt consensus site motifs, which is mutually exclusive with serine phosphorylation by Akt. The enhanced interaction of PRMT6 and mutant AR leads to neurodegeneration in cell and fly models of SBMA. These findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis. PMID:25569348

  8. Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2*

    PubMed Central

    Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T.; Clarke, Steven G.

    2015-01-01

    Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides. PMID:25979344

  9. Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2.

    PubMed

    Hadjikyriacou, Andrea; Yang, Yanzhong; Espejo, Alexsandra; Bedford, Mark T; Clarke, Steven G

    2015-07-03

    Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.

  10. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells.

    PubMed

    Sipos, Adrienn; Iván, Judit; Bécsi, Bálint; Darula, Zsuzsanna; Tamás, István; Horváth, Dániel; Medzihradszky, Katalin F; Erdődi, Ferenc; Lontay, Beáta

    2017-01-11

    Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1(T850) and the regulatory PRMT5(T80) residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation.

  11. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

    PubMed Central

    Sipos, Adrienn; Iván, Judit; Bécsi, Bálint; Darula, Zsuzsanna; Tamás, István; Horváth, Dániel; Medzihradszky, Katalin F.; Erdődi, Ferenc; Lontay, Beáta

    2017-01-01

    Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1T850 and the regulatory PRMT5T80 residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation. PMID:28074910

  12. The histone-binding protein COPR5 is required for nuclear functions of the protein arginine methyltransferase PRMT5

    PubMed Central

    Lacroix, Matthieu; Messaoudi, Selma El; Rodier, Geneviève; Le Cam, Aphonse; Sardet, Claude; Fabbrizio, Eric

    2008-01-01

    Protein arginine methyltransferase 5 (PRMT5) targets nuclear and cytoplasmic proteins. Here, we identified a nuclear protein, called cooperator of PRMT5 (COPR5), involved in the nuclear functions of PRMT5. COPR5 tightly binds to PRMT5, both in vitro and in living cells, but not to other members of the PRMT family. PRMT5 bound to COPR5 methylates histone H4 (R3) preferentially when compared with histone H3 (R8), suggesting that COPR5 modulates the substrate specificity of nuclear PRMT5-containing complexes, at least towards histones. Markedly, recombinant COPR5 binds to the amino terminus of histone H4 and is required to recruit PRMT5 to reconstituted nucleosomes in vitro. Consistently, COPR5 depletion in cells strongly reduces PRMT5 recruitment on chromatin at the PRMT5 target gene cyclin E1 (CCNE1) in vivo. Moreover, both COPR5 depletion and overexpression affect CCNE1 promoter expression. We propose that COPR5 is an important chromatin adaptor for PRMT5 to function on a subset of its target genes. PMID:18404153

  13. Computational Study of Symmetric Methylation on Histone Arginine Catalyzed by Protein Arginine Methyltransferase PRMT5 through QM/MM MD and Free Energy Simulations

    DOE PAGES

    Yue, Yufei; CHu, Yuzhuo; Guo, Hong

    2015-01-01

    Protein arginine methyltransferases (PRMTs) catalyze the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to arginine residues. There are three types of PRMTs (I, II and III) that produce different methylation products, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Since these different methylations can lead to different biological consequences, understanding the origin of product specificity of PRMTs is of considerable interest. In this article, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study SDMA catalyzed by the Type II PRMT5 on the basis of experimental observation that the dimethylated productmore » is generated through a distributive fashion. The simulations have identified some important interactions and proton transfers during the catalysis. Similar to the cases involving Type I PRMTs, a conserved Glu residue (Glu435) in PRMT5 is suggested to function as general base catalyst based on the result of the simulations. Moreover, our results show that PRMT5 has an energetic preference for the first methylation on N-1 followed by the second methylation on a different -guanidino nitrogen of arginine (N-2).The first and second methyl transfers are estimated to have free energy barriers of 19-20 and 18-19 kcal/mol respectively. The computer simulations suggest a distinctive catalytic mechanism of symmetric dimethylation that seems to be different from asymmetric dimethylation.« less

  14. Computational Study of Symmetric Methylation on Histone Arginine Catalyzed by Protein Arginine Methyltransferase PRMT5 through QM/MM MD and Free Energy Simulations

    SciTech Connect

    Yue, Yufei; CHu, Yuzhuo; Guo, Hong

    2015-01-01

    Protein arginine methyltransferases (PRMTs) catalyze the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to arginine residues. There are three types of PRMTs (I, II and III) that produce different methylation products, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Since these different methylations can lead to different biological consequences, understanding the origin of product specificity of PRMTs is of considerable interest. In this article, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study SDMA catalyzed by the Type II PRMT5 on the basis of experimental observation that the dimethylated product is generated through a distributive fashion. The simulations have identified some important interactions and proton transfers during the catalysis. Similar to the cases involving Type I PRMTs, a conserved Glu residue (Glu435) in PRMT5 is suggested to function as general base catalyst based on the result of the simulations. Moreover, our results show that PRMT5 has an energetic preference for the first methylation on N-1 followed by the second methylation on a different -guanidino nitrogen of arginine (N-2).The first and second methyl transfers are estimated to have free energy barriers of 19-20 and 18-19 kcal/mol respectively. The computer simulations suggest a distinctive catalytic mechanism of symmetric dimethylation that seems to be different from asymmetric dimethylation.

  15. Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque).

    PubMed

    Yeh, Hung-Yueh; Klesius, Phillip H

    2012-08-01

    Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 like. Each PRMT nucleic acid sequence has an open reading frame (ORF) and 3'-untranslated regions. Each ORF appears to encode 361, 587 and 458 amino acid residues for PRMT1, PRMT4 and variant, respectively. The partial ORF of PRMT3 and PRMT5 encode 292 and 563 amino acids, respectively. By comparison with the human counterparts, each channel catfish PRMT also has conserved domains. For expression profile, the channel catfish PRMT1 transcript was detected by RT-PCR in spleens, anterior kidneys, livers, intestines, skin and gills of fish examined. Except in liver, the PRMT3 transcript was detected in all catfish tissues examined. However, the PRMT4 cDNA was detected in livers from all three catfish and gills from two fish, but not other tissues. This information will enable us to further elucidate PRMT functions in channel catfish.

  16. Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior.

    PubMed

    Fronz, Katharina; Otto, Silke; Kölbel, Knut; Kühn, Uwe; Friedrich, Henning; Schierhorn, Angelika; Beck-Sickinger, Annette G; Ostareck-Lederer, Antje; Wahle, Elmar

    2008-07-18

    The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.

  17. Mapping of Post-translational Modifications of Transition Proteins, TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2*

    PubMed Central

    Gupta, Nikhil; Madapura, M. Pradeepa; Bhat, U. Anayat; Rao, M. R. Satyanarayana

    2015-01-01

    In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg71, Arg75, and Arg92 residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys88 and Lys91 residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome. PMID:25818198

  18. Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth

    PubMed Central

    Deng, X; Shao, G; Zhang, H-T; Li, C; Zhang, D; Cheng, L; Elzey, B D; Pili, R; Ratliff, T L; Huang, J; Hu, C-D

    2017-01-01

    Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression. PMID:27546619

  19. Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth.

    PubMed

    Deng, X; Shao, G; Zhang, H-T; Li, C; Zhang, D; Cheng, L; Elzey, B D; Pili, R; Ratliff, T L; Huang, J; Hu, C-D

    2017-03-02

    Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

  20. Protein-arginine Methyltransferase 1 Suppresses Megakaryocytic Differentiation via Modulation of the p38 MAPK Pathway in K562 Cells*

    PubMed Central

    Chang, Yuan-I; Hua, Wei-Kai; Yao, Chao-Ling; Hwang, Shiaw-Min; Hung, Yi-Chi; Kuan, Chih-Jen; Leou, Jiun-Shyang; Lin, Wey-Jinq

    2010-01-01

    Protein-arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has yet to be investigated. Human leukemia K562 cells have been used as a model to study hematopoietic differentiation. In this study, we report that ectopic expression of HA-PRMT1 in K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties, and CD41 expression, whereas knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of ERK MAPK has been shown to be essential for megakaryocytic differentiation, although the role of p38 MAPK is still poorly understood. We show that knockdown of p38α MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes activation of p38 MAPK without inhibiting activation of ERK MAPK. In p38α knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38α MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. We propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the promotion of megakaryopoiesis. PMID:20442406

  1. Caveolin1/protein arginine methyltransferase1/sirtuin1 axis as a potential target against endothelial dysfunction.

    PubMed

    Charles, Soniya; Raj, Vijay; Arokiaraj, Jesu; Mala, Kanchana

    2017-01-23

    Endothelial dysfunction (ED), an established response to cardiovascular risk factors, is characterized by increased levels of soluble molecules secreted by endothelial cells (EC). Evidence suggest that ED is an independent predictor of cardiac events and that it is associated with a deficiency in production or bioavailability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors. ED can be reversed by treating cardiovascular risk factors, hence, beyond ambiguity, ED contributes to initiation and progression of atherosclerotic disease. Majority of cardiovascular risk factors act by a common pathway, oxidative stress (OS), characterized by an imbalance in bioavailability of NO and reactive oxygen species (ROS). Enhanced ROS, through several mechanisms, alters competence of EC that leads to ED, reducing its potential to maintain homeostasis and resulting in development of cardiovascular disease (CVD). Influential mechanisms that have been implicated in the development of ED include (i) presence of elevated levels of NOS inhibitor, asymmetric dimethylarginine (ADMA) due to augmented enzyme activity of protein arginine methyl transferase-1 (PRMT1); (ii) decrease in NO generation by endothelial nitric oxide synthase (eNOS) uncoupling, or by reaction of NO with free radicals and (iii) impaired post translational modification of protein (PTM) such as eNOS, caveolin-1 (cav1) and sirtuin-1 (SIRT1). However, the inter-related mechanisms that concur to developing ED is yet to be understood. The events that possibly overlay include OS-induced sequestration of SIRT1 to caveolae facilitating cav1-SIRT1 association; potential increase in lysine acetylation of enzymes such as eNOS and PRMT1 leading to enhanced ADMA formation; imbalance in acetylation-methylation ratio (AMR); diminished NO generation and ED. Here we review current literature from research showing interdependent association between cav1-PRMT1

  2. An essential and evolutionarily conserved role of protein arginine methyltransferase 1 for adult intestinal stem cells during postembryonic development.

    PubMed

    Matsuda, Hiroki; Shi, Yun-Bo

    2010-11-01

    Organ-specific adult stem cells are critical for the homeostasis of adult organs and organ repair and regeneration. Unfortunately, it has been difficult to investigate the origins of these stem cells and the mechanisms of their development, especially in mammals. Intestinal remodeling during frog metamorphosis offers a unique opportunity for such studies. During the transition from an herbivorous tadpole to a carnivorous frog, the intestine is completely remodeled as the larval epithelial cells undergo apoptotic degeneration and are replaced by adult epithelial cells developed de novo. The entire metamorphic process is under the control of thyroid hormone, making it possible to control the development of the adult intestinal stem cells. Here, we show that the thyroid hormone receptor-coactivator protein arginine methyltransferase 1 (PRMT1) is upregulated in a small number of larval epithelial cells and that these cells dedifferentiate to become the adult stem cells. More importantly, transgenic overexpression of PRMT1 leads to increased adult stem cells in the intestine, and conversely, knocking down the expression of endogenous PRMT1 reduces the adult stem cell population. In addition, PRMT1 expression pattern during zebrafish and mouse development suggests that PRMT1 may play an evolutionally conserved role in the development of adult intestinal stem cells throughout vertebrates. These findings are not only important for the understanding of organ-specific adult stem cell development but also have important implications in regenerative medicine of the digestive tract.

  3. The Kruppel-like zinc finger protein ZNF224 recruits the arginine methyltransferase PRMT5 on the transcriptional repressor complex of the aldolase A gene.

    PubMed

    Cesaro, Elena; De Cegli, Rossella; Medugno, Lina; Florio, Francesca; Grosso, Michela; Lupo, Angelo; Izzo, Paola; Costanzo, Paola

    2009-11-20

    Gene transcription in eukaryotes is modulated by the coordinated recruitment of specific transcription factors and chromatin-modulating proteins. Indeed, gene activation and/or repression is/are regulated by histone methylation status at specific arginine or lysine residues. In this work, by co-immunoprecipitation experiments, we demonstrate that PRMT5, a type II protein arginine methyltransferase that monomethylates and symmetrically dimethylates arginine residues, is physically associated with the Kruppel-like associated box-zinc finger protein ZNF224, the aldolase A gene repressor. Moreover, chromatin immunoprecipitation assays show that PRMT5 is recruited to the L-type aldolase A promoter and that methylation of the nucleosomes that surround the L-type promoter region occurs in vivo on the arginine 3 of histone H4. Consistent with its association to the ZNF224 repressor complex, the decrease of PRMT5 expression produced by RNA interference positively affects L-type aldolase A promoter transcription. Finally, the alternating occupancy of the L-type aldolase A promoter by the ZNF224-PRMT5 repression complex in proliferating and growth-arrested cells suggests that these regulatory proteins play a significant role during the cell cycle modulation of human aldolase A gene expression. Our data represent the first experimental evidence that protein arginine methylation plays a role in ZNF224-mediated transcriptional repression and provide novel insight into the chromatin modifications required for repression of gene transcription by Kruppel-like associated box-zinc finger proteins.

  4. Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: Evidence for roles in nuclear receptor signaling and RNA metabolism

    SciTech Connect

    Mansure, Jose Joao; Furtado, Daniel Rodrigues; Bastos de Oliveira, Francisco Meirelles; Rumjanek, Franklin David; Franco, Gloria Regina; Fantappie, Marcelo Rosado . E-mail: fantappie@bioqmed.ufrj.br

    2005-10-07

    The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions.

  5. Protein arginine Methyltransferase 8 gene is expressed in pluripotent stem cells and its expression is modulated by the transcription factor Sox2.

    PubMed

    Solari, Claudia; Echegaray, Camila Vázquez; Luzzani, Carlos; Cosentino, María Soledad; Waisman, Ariel; Petrone, María Victoria; Francia, Marcos; Sassone, Alina; Canizo, Jésica; Sevlever, Gustavo; Barañao, Lino; Miriuka, Santiago; Guberman, Alejandra

    2016-04-22

    Addition of methyl groups to arginine residues is catalyzed by a group of enzymes called Protein Arginine Methyltransferases (Prmt). Although Prmt1 is essential in development, its paralogue Prmt8 has been poorly studied. This gene was reported to be expressed in nervous system and involved in neurogenesis. In this work, we found that Prmt8 is expressed in mouse embryonic stem cells (ESC) and in induced pluripotent stem cells, and modulated along differentiation to neural precursor cells. We found that Prmt8 promoter activity is induced by the pluripotency transcription factors Oct4, Sox2 and Nanog. Moreover, endogenous Prmt8 mRNA levels were reduced in ESC transfected with Sox2 shRNA vector. As a whole, our results indicate that Prmt8 is expressed in pluripotent stem cells and its transcription is modulated by pluripotency transcription factors. These findings suggest that besides its known function in nervous system, Prmt8 could play a role in pluripotent stem cells.

  6. Selective Inhibitors of Protein Methyltransferases

    PubMed Central

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  7. Protein arginine methyltransferase Prmt5-Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs.

    PubMed

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T; Hunt, Donald F; Shechter, David

    2011-12-09

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

  8. Molecular cloning, characterization and expression analysis of the protein arginine N-methyltransferase 1 gene (As-PRMT1) from Artemia sinica.

    PubMed

    Jiang, Xue; Yao, Feng; Li, Xuejie; Jia, Baolin; Zhong, Guangying; Zhang, Jianfeng; Zou, Xiangyang; Hou, Lin

    2015-07-01

    Protein arginine N-methyltransferase 1 (PRMT1) is an important epigenetic regulation factor in eukaryotic genomes. PRMT1 is involved in histone arginine loci methylation modification, changes in eukaryotic genomes' chromatin structure, and gene expression regulation. In the present paper, the full-length 1201-bp cDNA sequence of the PRMT1 homolog of Artemia sinica (As-PRMT1) was cloned for the first time. The putative As-PRMT1 protein comprises 346 amino acids with a SAM domain and a PRMT5 domain. Multiple sequence alignments revealed that the putative sequence of As-PRMT1 protein was relatively conserved across species, especially in the SAM domain. As-PRMT1 is widely expressed during embryo development of A. sinica. This is followed by a dramatic upregulation after diapause termination and then downregulation from the nauplius stage. Furthermore, As-PRMT1 transcripts are highly upregulated under conditions of high salinity and low temperature stress. These findings suggested that As-PRMT1 is a stress-related factor that might promote or inhibit the expression of certain genes, play a critical role in embryonic development and in resistance to low temperature and high salinity stress.

  9. Protein arginine methyltransferase 5 (Prmt5) promotes gene expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and its target genes during adipogenesis.

    PubMed

    LeBlanc, Scott E; Konda, Silvana; Wu, Qiong; Hu, Yu-Jie; Oslowski, Christine M; Sif, Saïd; Imbalzano, Anthony N

    2012-04-01

    Regulation of adipose tissue formation by adipogenic-regulatory proteins has long been a topic of interest given the ever-increasing health concerns of obesity and type 2 diabetes in the general population. Differentiation of precursor cells into adipocytes involves a complex network of cofactors that facilitate the functions of transcriptional regulators from the CCATT/enhancer binding protein, and the peroxisome proliferator-activated receptor (PPAR) families. Many of these cofactors are enzymes that modulate the structure of chromatin by altering histone-DNA contacts in an ATP-dependent manner or by posttranslationally modifying the histone proteins. Here we report that inhibition of protein arginine methyltransferase 5 (Prmt5) expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes. In contrast, overexpression of Prmt5 enhanced adipogenic gene expression and differentiation. Chromatin immunoprecipitation experiments indicated that Prmt5 binds to and dimethylates histones at adipogenic promoters. Furthermore, the presence of Prmt5 promoted the binding of ATP-dependent chromatin-remodeling enzymes and was required for the binding of PPARγ2 at PPARγ2-regulated promoters. The data indicate that Prmt5 acts as a coactivator for the activation of adipogenic gene expression and promotes adipogenic differentiation.

  10. Severe Hypomyelination and Developmental Defects Are Caused in Mice Lacking Protein Arginine Methyltransferase 1 (PRMT1) in the Central Nervous System*

    PubMed Central

    Hashimoto, Misuzu; Murata, Kazuya; Ishida, Junji; Kanou, Akihiko; Kasuya, Yoshitoshi; Fukamizu, Akiyoshi

    2016-01-01

    Protein arginine methyltransferase 1 (PRMT1) is involved in cell proliferation, DNA damage response, and transcriptional regulation. Although PRMT1 is extensively expressed in the CNS at embryonic and perinatal stages, the physiological role of PRMT1 has been poorly understood. Here, to investigate the primary function of PRMT1 in the CNS, we generated CNS-specific PRMT1 knock-out mice by the Cre-loxP system. These mice exhibited postnatal growth retardation with tremors, and most of them died within 2 weeks after birth. Brain histological analyses revealed prominent cell reduction in the white matter tracts of the mutant mice. Furthermore, ultrastructural analysis demonstrated that myelin sheath was almost completely ablated in the CNS of these animals. In agreement with hypomyelination, we also observed that most major myelin proteins including myelin basic protein (MBP), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG) were dramatically decreased, although neuronal and astrocytic markers were preserved in the brain of CNS-specific PRMT1 knock-out mice. These animals had a reduced number of OLIG2+ oligodendrocyte lineage cells in the white matter. We found that expressions of transcription factors essential for oligodendrocyte specification and further maturation were significantly suppressed in the brain of the mutant mice. Our findings provide evidence that PRMT1 is required for CNS development, especially for oligodendrocyte maturation processes. PMID:26637354

  11. Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors.

    PubMed

    Heinke, Ralf; Spannhoff, Astrid; Meier, Rene; Trojer, Patrick; Bauer, Ingo; Jung, Manfred; Sippl, Wolfgang

    2009-01-01

    Lysine and arginine methyltransferases participate in the posttranslational modification of histones and regulate key cellular functions. Protein arginine methyltransferase 1 (PRMT1) has been identified as an essential component of mixed lineage leukemia (MLL) oncogenic complexes, revealing its potential as a novel therapeutic target in human cancer. The first potent arginine methyltransferase inhibitors were recently discovered by random- and target-based screening approaches. Herein we report virtual and biological screening for novel inhibitors of PRMT1. Structure-based virtual screening (VS) of the Chembridge database composed of 328 000 molecules was performed with a combination of ligand- and target-based in silico approaches. Nine inhibitors were identified from the top-scored docking solutions; these were experimentally tested using human PRMT1 and an antibody-based assay with a time-resolved fluorescence readout. Among several aromatic amines, an aliphatic amine and an amide were also found to be active in the micromolar range.

  12. Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1

    PubMed Central

    Castellano, Sabrina; Spannhoff, Astrid; Milite, Ciro; Dal Piaz, Fabrizio; Cheng, Donghang; Tosco, Alessandra; Viviano, Monica; Yamani, Abdellah; Cianciulli, Agostino; Sala, Marina; Cura, Vincent; Cavarelli, Jean; Novellino, Ettore; Mai, Antonello; Bedford, Mark T.; Sbardella, Gianluca

    2012-01-01

    Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets but their role in physiological and pathological pathways is far from being clear, due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone- (H3) or non-histone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators. PMID:23095008

  13. The protein arginine methyltransferase 5 promotes malignant phenotype of hepatocellular carcinoma cells and is associated with adverse patient outcomes after curative hepatectomy

    PubMed Central

    Shimizu, Dai; Kanda, Mitsuro; Sugimoto, Hiroyuki; Shibata, Masahiro; Tanaka, Haruyoshi; Takami, Hideki; Iwata, Naoki; Hayashi, Masamichi; Tanaka, Chie; Kobayashi, Daisuke; Yamada, Suguru; Nakayama, Goro; Koike, Masahiko; Fujiwara, Michitaka; Fujii, Tsutomu; Kodera, Yasuhiro

    2017-01-01

    The prognosis of advanced hepatocellular carcinoma (HCC) is dismal. Novel molecular targets for diagnosis and therapy is urgently required. This study evaluated expression and functions of the protein arginine methyltransferase 5 (PRMT5) in HCC. Using HCC cell lines, the expression levels of PRMT5 mRNA were determined using the quantitative real-time reverse-transcription polymerase chain reaction, and the effect of a small interfering PRMT5-siRNA on cell phenotype was evaluated. Further, PRMT5 expression was determined in 144 pairs of resected liver tissues to evaluate its clinical significance. Regardless of their differentiated phenotypes, nine HCC cell lines expressed different levels of PRMT5 mRNA. Inhibition of PRMT5 expression significantly decreased the proliferation, invasion, and migration of HCC cell lines. Although the level of PRMT5 mRNA was not influenced by patient's background liver status, it was significantly higher in HCC tissues than in the corresponding noncancerous tissues. High levels of PRMT5 mRNA in HCC tissues were significantly associated with advanced disease stage and adverse prognosis. In conclusion, our results indicate that PRMT5 may act as a putative oncogene in HCC and that the levels of PRMT5 mRNA represent a promising prognostic marker and a potential target of molecular therapy for HCC. PMID:28101581

  14. Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant

    PubMed Central

    Li, Tingting; Kong, Ah-Ng Tony; Ma, Zhiqiang; Liu, Haiyan; Liu, Pinghua; Xiao, Yu; Jiang, Xuehua; Wang, Ling

    2016-01-01

    Purpose Pregnane x receptor (PXR) - activated overexpression of the multidrug resistance 1 (MDR1) gene is an important way for tumor cells to acquire drug resistance. However, the detailed mechanism still remains unclear. In the present study, we aimed to investigate whether protein arginine methyl transferase 1(PRMT1) is involved in PXR - activated overexpression of MDR1 during acquired multidrug resistant. Experimental Design Arginine methyltransferase inhibitor 1 (AMI-1) was used to pharmacologically block PRMT1 in resistant breast cancer cells (MCF7/adr). The mRNA and protein levels of MDR1 were detected by real-time PCR and western blotting analysis. Immunofluorescence microscopy and co-immunoprecipitation were used to investigate the physical interaction between PXR and PRMT1. Then, 136 candidate compounds were screened for PRMT1 inhibitors. Lastly, luciferase reporter gene and nude mice bearing resistant breast cancer xenografts were adopted to investigate the anti-tumor effect of PRMT1 inhibitors when combined with adriamycin. Results AMI-1 significantly suppressed the expression of MDR1 in MCF7/adr cells and increased cells sensitivity of MCF7/adr to adriamycin. Physical interaction between PRMT1 and PXR exists in MCF7/adr cells, which could be disrupted by AMI-1. Those results suggest that PRMT1 may be involved in PXR-activated overexpression of MDR1 in resistant breast cancer cells, and AMI-1 may suppress MDR1 by disrupting the interaction between PRMT1 and PXR. Then, five compounds including rutin, isoquercitrin, salvianolic acid A, naproxen, and felodipline were identified to be PRMT1 inhibitors. Finally, those PRMT1 inhibitors were observed to significantly decrease MDR1 promoter activity in vitro and enhance the antitumor effect of adriamycin in nude mice that bearing resistant breast cancer xenografts. Conclusions PRMT1 may be an important co-activator of PXR in activating MDR1 gene during acquired resistance, and PRMT1 inhibitor combined with

  15. Protein arginine methylation/demethylation and cancer

    PubMed Central

    Poulard, Coralie; Corbo, Laura; Le Romancer, Muriel

    2016-01-01

    Protein arginine methylation is a common post-translational modification involved in numerous cellular processes including transcription, DNA repair, mRNA splicing and signal transduction. Currently, there are nine known members of the protein arginine methyltransferase (PRMT) family, but only one arginine demethylase has been identified, namely the Jumonji domain-containing 6 (JMJD6). Although its demethylase activity was initially challenged, its dual activity as an arginine demethylase and a lysine hydroxylase is now recognized. Interestingly, a growing number of substrates for arginine methylation and demethylation play key roles in tumorigenesis. Though alterations in the sequence of these enzymes have not been identified in cancer, their overexpression is associated with various cancers, suggesting that they could constitute targets for therapeutic strategies. In this review, we present the recent knowledge of the involvement of PRMTs and JMJD6 in tumorigenesis. PMID:27556302

  16. Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.

    PubMed

    Burgos, Emmanuel S; Wilczek, Carola; Onikubo, Takashi; Bonanno, Jeffrey B; Jansong, Janina; Reimer, Ulf; Shechter, David

    2015-04-10

    The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.

  17. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

    SciTech Connect

    Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

    2008-07-11

    Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly.

  18. Effects of a Novel Arginine Methyltransferase Inhibitor on T Helper Cell Cytokine Production

    PubMed Central

    Bonham, Kevin; Hemmers, Saskia; Lim, Yeon-Hee; Hill, Dawn M.; Finn, M.G.; Mowen, Kerri A.

    2010-01-01

    The protein arginine methyltransferase (PRMT) family of enzymes catalyzes the transfer of methyl groups from S-adenosylmethionine to the guanidino nitrogen atom of peptidylarginine to form monomethylarginine or dimethylarginine. We created several less polar analogues of the specific PRMT inhibitor AMI-1, and one such compound was found to have improved PRMT inhibitory activity over the parent molecule. The newly identified PRMT inhibitor modulated T helper cell function and thus may serve as a lead for further inhibitors useful for immune-mediated disease treatment. PMID:20345902

  19. Identification of Novel Inhibitors against Coactivator Associated Arginine Methyltransferase 1 Based on Virtual Screening and Biological Assays

    PubMed Central

    Xie, Yiqian; Luo, Cheng

    2016-01-01

    Overexpression of coactivator associated arginine methyltransferase 1 (CARM1), a protein arginine N-methyltransferase (PRMT) family enzyme, is associated with various diseases including cancers. Consequently, the development of small-molecule inhibitors targeting PRMTs has significant value for both research and therapeutic purposes. In this study, together with structure-based virtual screening with biochemical assays, two compounds DC_C11 and DC_C66 were identified as novel inhibitors of CARM1. Cellular studies revealed that the two inhibitors are cell membrane permeable and effectively blocked proliferation of cancer cells including HELA, K562, and MCF7. We further predicted the binding mode of these inhibitors through molecular docking analysis, which indicated that the inhibitors competitively occupied the binding site of the substrate and destroyed the protein-protein interactions between CARM1 and its substrates. Overall, this study has shed light on the development of small-molecule CARM1 inhibitors with novel scaffolds. PMID:27872854

  20. KAP1 is a Novel Substrate for the Arginine Methyltransferase PRMT5.

    PubMed

    di Caprio, Roberta; Ciano, Michela; Montano, Giorgia; Costanzo, Paola; Cesaro, Elena

    2015-01-09

    KRAB-associated protein 1 (KAP1), the transcriptional corepressor of Kruppel-associated box zinc finger proteins (KRAB-ZFPs), is subjected to multiple post-translational modifications that are involved in fine-tuning of the multiple biological functions of KAP1. In previous papers, we analyzed the KAP1-dependent molecular mechanism of transcriptional repression mediated by ZNF224, a member of the KRAB-ZFP family, and identified the protein arginine methyltransferase PRMT5 as a component of the ZNF224 repression complex. We demonstrated that PRMT5-mediated histone arginine methylation is required to elicit ZNF224 transcriptional repression. In this study, we show that KAP1 interacts with PRMT5 and is a novel substrate for PRMT5 methylation. Also, we present evidence that the methylation of KAP1 arginine residues regulate the KAP1-ZNF224 interaction, thus suggesting that this KAP1 post-translational modification could actively contribute to the regulation of ZNF224-mediated repression.

  1. KAP1 is a Novel Substrate for the Arginine Methyltransferase PRMT5

    PubMed Central

    di Caprio, Roberta; Ciano, Michela; Montano, Giorgia; Costanzo, Paola; Cesaro, Elena

    2015-01-01

    KRAB-associated protein 1 (KAP1), the transcriptional corepressor of Kruppel-associated box zinc finger proteins (KRAB-ZFPs), is subjected to multiple post-translational modifications that are involved in fine-tuning of the multiple biological functions of KAP1. In previous papers, we analyzed the KAP1-dependent molecular mechanism of transcriptional repression mediated by ZNF224, a member of the KRAB-ZFP family, and identified the protein arginine methyltransferase PRMT5 as a component of the ZNF224 repression complex. We demonstrated that PRMT5-mediated histone arginine methylation is required to elicit ZNF224 transcriptional repression. In this study, we show that KAP1 interacts with PRMT5 and is a novel substrate for PRMT5 methylation. Also, we present evidence that the methylation of KAP1 arginine residues regulate the KAP1-ZNF224 interaction, thus suggesting that this KAP1 post-translational modification could actively contribute to the regulation of ZNF224-mediated repression. PMID:25585209

  2. Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis

    PubMed Central

    Liu, Fan; Cheng, Guoyan; Hamard, Pierre-Jacques; Greenblatt, Sarah; Wang, Lan; Man, Na; Perna, Fabiana; Xu, Haiming; Tadi, Madhavi; Luciani, Luisa; Nimer, Stephen D.

    2015-01-01

    Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells. PMID:26258414

  3. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

    Cancer.gov

    The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

  4. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

    PubMed Central

    Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

    2016-01-01

    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

  5. Protein Methyltransferases: A Distinct, Diverse, and Dynamic Family of Enzymes.

    PubMed

    Boriack-Sjodin, P Ann; Swinger, Kerren K

    2016-03-22

    Methyltransferase proteins make up a superfamily of enzymes that add one or more methyl groups to substrates that include protein, DNA, RNA, and small molecules. The subset of proteins that act upon arginine and lysine side chains are characterized as epigenetic targets because of their activity on histone molecules and their ability to affect transcriptional regulation. However, it is now clear that these enzymes target other protein substrates, as well, greatly expanding their potential impact on normal and disease biology. Protein methyltransferases are well-characterized structurally. In addition to revealing the overall architecture of the subfamilies of enzymes, structures of complexes with substrates and ligands have permitted detailed analysis of biochemical mechanism, substrate recognition, and design of potent and selective inhibitors. This review focuses on how knowledge gained from structural studies has impacted the understanding of this large class of epigenetic enzymes.

  6. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    SciTech Connect

    Troffer-Charlier, Nathalie; Cura, Vincent; Hassenboehler, Pierre; Moras, Dino; Cavarelli, Jean

    2007-04-01

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1{sub 28–507} and two structural states of CARM1{sub 140–480} were expressed, purified and crystallized. Crystals of CARM1{sub 28–507} belong to space group P6{sub 2}22, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1{sub 28–507} was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1{sub 140–480} belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1{sub 140–480} in complex with S-adenosyl-l-homocysteine belong to space P2{sub 1}2{sub 1}2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1{sub 140–480} were solved by molecular-replacement techniques from the structure of CARM1{sub 28–507}.

  7. Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound

    PubMed Central

    2015-01-01

    A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies. PMID:26101569

  8. Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

    PubMed

    Mitchell, Lorna H; Drew, Allison E; Ribich, Scott A; Rioux, Nathalie; Swinger, Kerren K; Jacques, Suzanne L; Lingaraj, Trupti; Boriack-Sjodin, P Ann; Waters, Nigel J; Wigle, Tim J; Moradei, Oscar; Jin, Lei; Riera, Tom; Porter-Scott, Margaret; Moyer, Mikel P; Smith, Jesse J; Chesworth, Richard; Copeland, Robert A

    2015-06-11

    A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

  9. The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

    SciTech Connect

    Olsson, Ida; Berrez, Jean-Marc; Leipus, Arunas; Ostlund, Cecilia; Mutvei, Ann . E-mail: ann.mutvei@sh.se

    2007-05-15

    Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experiments with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2{delta} mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation.

  10. Regulation of arginine methyltransferase 3 by a Wolbachia-induced microRNA in Aedes aegypti and its effect on Wolbachia and dengue virus replication.

    PubMed

    Zhang, Guangmei; Hussain, Mazhar; Asgari, Sassan

    2014-10-01

    The gram-negative endosymbiotic bacteria, Wolbachia, have been found to colonize a wide range of invertebrates, including over 40% of insect species. Best known for host reproductive manipulations, some strains of Wolbachia have been shown to reduce the host life span by about 50% and inhibit replication and transmission of dengue virus (DENV) in the mosquito vector, Aedes aegypti. The molecular mechanisms underlying these effects still are not well understood. Our previous studies showed that Wolbachia uses host microRNAs (miRNAs) to manipulate host gene expression for its efficient maintenance and limiting replication of DENV in Ae. aegypti. Protein arginine methyltransferases are structurally and functionally conserved proteins from yeast to human. In mammals, it has been reported that protein arginine methyltransferases such as PRMT1, 5 and 6 could regulate replication of different viruses. Ae. aegypti contains eight members of protein arginine methyltransferases (AaArgM1-8). Here, we show that the wMelPop strain of Wolbachia introduced into Ae. aegypti significantly induces the expression of AaArgM3. Interestingly, we found that Wolbachia uses aae-miR-2940, which is highly upregulated in Wolbachia-infected mosquitoes, to upregulate the expression of AaArgM3. Silencing of AaArgM3 in a mosquito cell line led to a significant reduction in Wolbachia replication, but had no effect on the replication of DENV. These results provide further evidence that Wolbachia uses the host miRNAs to manipulate host gene expression and facilitate colonization in Ae. aegypti mosquito.

  11. Rmt1 catalyzes zinc-finger independent arginine methylation of the small ribosomal protein Rps2 in Saccharomyces cerevisiae

    PubMed Central

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-01

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells. PMID:20035717

  12. Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

    SciTech Connect

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-22

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.

  13. Expanding the yeast protein arginine methylome.

    PubMed

    Plank, Michael; Fischer, Roman; Geoghegan, Vincent; Charles, Philip D; Konietzny, Rebecca; Acuto, Oreste; Pears, Catherine; Schofield, Christopher J; Kessler, Benedikt M

    2015-09-01

    Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono-methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA-binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg-Gly-Gly motifs or Asn-rich contexts.

  14. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

  15. Discovery and mechanistic study of a class of protein arginine methylation inhibitors.

    PubMed

    Feng, You; Li, Mingyong; Wang, Binghe; Zheng, Yujun George

    2010-08-26

    Protein arginine methylation regulates multiple biological processes such as chromatin remodeling and RNA splicing. Malfunction of protein arginine methyltransferases (PRMTs) is correlated with many human diseases. Thus, small molecule inhibitors of protein arginine methylation are of great potential for therapeutic development. Herein, we report a type of compound that blocks PRMT1-mediated arginine methylation at micromolar potency through a unique mechanism. Most of the discovered compounds bear naphthalene and sulfonate groups and are structurally different from typical PRMT substrates, for example, histone H4 and glycine- and arginine-rich sequences. To elucidate the molecular basis of inhibition, we conducted a variety of kinetic and biophysical assays. The combined data reveal that this type of naphthyl-sulfo (NS) molecule directly targets the substrates but not PRMTs for the observed inhibition. We also found that suramin effectively inhibited PRMT1 activity. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation.

  16. Protein lysine methyltransferase G9a acts on non-histone targets

    PubMed Central

    Rathert, Philipp; Dhayalan, Arunkumar; Murakami, Marie; Zhang, Xing; Tamas, Raluca; Jurkowska, Renata; Komatsu, Yasuhiko; Shinkai, Yoichi; Cheng, Xiaodong; Jeltsch, Albert

    2009-01-01

    By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins. PMID:18438403

  17. Histone Arginine Methylation

    PubMed Central

    Lorenzo, Alessandra Di; Bedford, Mark T.

    2012-01-01

    Arginine methylation is a common posttranslational modification (PTM). This type of PTM occurs on both nuclear and cytoplasmic proteins, and is particularly abundant on shuttling proteins. In this review, we will focus on one aspect of this PTM: the diverse roles that arginine methylation of the core histone tails play in regulating chromatin function. A family of nine protein arginine methyltransferases (PRMTs) catalyze methylation reactions, and a subset target histones. Importantly, arginine methylation of histone tails can promote or prevent the docking of key transcriptional effector molecules, thus playing a central role in the orchestration of the histone code. PMID:21074527

  18. The Cj0588 protein is a Campylobacter jejuni RNA methyltransferase.

    PubMed

    Sałamaszyńska-Guz, Agnieszka; Taciak, Bartłomiej; Kwiatek, Agnieszka; Klimuszko, Danuta

    2014-06-06

    TlyA proteins belong to 2'-O-methyltransferases. Methylation is a common posttranscriptional RNA modification. The Campylobacter jejuni Cj0588 protein belongs to the TlyA(I) protein family and is a rRNA methyltransferase. Methylation of ribosomal RNA catalyzed by Cj0588 appears to have an impact on the biology of the cell. Presence of the cj0588 gene in bacteria appears to be important for ribosome stability and virulence properties. Absence of the Cj0588 protein causes accumulation of the 50S ribosomal subunits, reduction in the amount of functional 70S ribosomes and confers increase resistance to capreomycin.

  19. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

  20. Protein Arginine Methylation and Citrullination in Epigenetic Regulation

    PubMed Central

    2015-01-01

    The post-translational modification of arginine residues represents a key mechanism for the epigenetic control of gene expression. Aberrant levels of histone arginine modifications have been linked to the development of several diseases including cancer. In recent years, great progress has been made in understanding the physiological role of individual arginine modifications and their effects on chromatin function. The present review aims to summarize the structural and functional aspects of histone arginine modifying enzymes and their impact on gene transcription. We will discuss the potential for targeting these proteins with small molecules in a variety of disease states. PMID:26686581

  1. RmtA, a putative arginine methyltransferase, regulates secondary metabolism and development in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is found colonizing numerous oil seed crops such as corn, peanuts, sorghum, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been de...

  2. Role of arginine in the stabilization of proteins against aggregation.

    PubMed

    Baynes, Brian M; Wang, Daniel I C; Trout, Bernhardt L

    2005-03-29

    The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.

  3. Protein arginine methylation facilitates KCNQ channel-PIP2 interaction leading to seizure suppression

    PubMed Central

    Kim, Hyun-Ji; Jeong, Myong-Ho; Kim, Kyung-Ran; Jung, Chang-Yun; Lee, Seul-Yi; Kim, Hanna; Koh, Jewoo; Vuong, Tuan Anh; Jung, Seungmoon; Yang, Hyunwoo; Park, Su-Kyung; Choi, Dahee; Kim, Sung Hun; Kang, KyeongJin; Sohn, Jong-Woo; Park, Joo Min; Jeon, Daejong; Koo, Seung-Hoi; Ho, Won-Kyung; Kang, Jong-Sun; Kim, Seong-Tae; Cho, Hana

    2016-01-01

    KCNQ channels are critical determinants of neuronal excitability, thus emerging as a novel target of anti-epileptic drugs. To date, the mechanisms of KCNQ channel modulation have been mostly characterized to be inhibitory via Gq-coupled receptors, Ca2+/CaM, and protein kinase C. Here we demonstrate that methylation of KCNQ by protein arginine methyltransferase 1 (Prmt1) positively regulates KCNQ channel activity, thereby preventing neuronal hyperexcitability. Prmt1+/- mice exhibit epileptic seizures. Methylation of KCNQ2 channels at 4 arginine residues by Prmt1 enhances PIP2 binding, and Prmt1 depletion lowers PIP2 affinity of KCNQ2 channels and thereby the channel activities. Consistently, exogenous PIP2 addition to Prmt1+/- neurons restores KCNQ currents and neuronal excitability to the WT level. Collectively, we propose that Prmt1-dependent facilitation of KCNQ-PIP2 interaction underlies the positive regulation of KCNQ activity by arginine methylation, which may serve as a key target for prevention of neuronal hyperexcitability and seizures. DOI: http://dx.doi.org/10.7554/eLife.17159.001 PMID:27466704

  4. Arginine methylation in yeast proteins during stationary-phase growth and heat shock.

    PubMed

    Lakowski, Ted M; Pak, Magnolia L; Szeitz, András; Thomas, Dylan; Vhuiyan, Mynol I; Clement, Bernd; Frankel, Adam

    2015-12-01

    Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and heat shock on the formation of methylarginines on yeast proteins to determine if the conditions favor a particular type of methylation. Utilizing linear ion trap mass spectrometry, we identify methylarginines in wild-type and RMT deletion yeast strains using secondary product ion scans (MS(3)), and quantify the methylarginines using multiple reaction monitoring (MRM). Employing MS(3) and isotopic incorporation, we demonstrate for the first time that Nη1, Nη2-dimethylarginine (sDMA) is present on yeast proteins, and make a detailed structural determination of the fragment ions from the spectra. Nη-monomethylarginine (ηMMA), Nδ-monomethylarginine (δMMA), Nη1, Nη1-dimethylarginine (aDMA), and sDMA were detected in RMT deletion yeast using MS(3) and MRM with and without isotopic incorporation, suggesting that additional RMT enzymes remain to be discovered in yeast. The concentrations of ηMMA and δMMA decreased by half during heat shock and stationary phase compared to log-phase growth of wild-type yeast, whereas sDMA increased by as much as sevenfold and aDMA decreased by 11-fold. Therefore, upon entering stressful conditions like heat shock or stationary-phase growth, there is a net increase in sDMA and decreases in aDMA, ηMMA, and δMMA on yeast proteins.

  5. RmtA, a Putative Arginine Methyltransferase, Regulates Secondary Metabolism and Development in Aspergillus flavus

    PubMed Central

    Satterlee, Timothy; Cary, Jeffrey W.; Calvo, Ana M.

    2016-01-01

    Aspergillus flavus colonizes numerous oil seed crops such as corn, peanuts, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been described to be involved in epigenetics regulation through histone modification. Epigenetics regulation affects a variety of cellular processes, including morphogenesis and secondary metabolism. Our study shows that deletion of rmtA in A. flavus results in hyperconidiating colonies, indicating that rmtA is a repressor of asexual development in this fungus. The increase in conidiation in the absence of rmtA coincides with greater expression of brlA, abaA, and wetA compared to that in the wild type. Additionally, the rmtA deletion mutant presents a drastic reduction or loss of sclerotial production, while forced expression of this gene increased the ability of this fungus to generate these resistant structures, revealing rmtA as a positive regulator of sclerotial formation. Importantly, rmtA is also required for the production of aflatoxin B1 in A. flavus, affecting the expression of aflJ. Furthermore, biosynthesis of additional metabolites is also controlled by rmtA, indicating a broad regulatory output in the control of secondary metabolism. This study also revealed that rmtA positively regulates the expression of the global regulatory gene veA, which could contribute to mediate the effects of rmtA on development and secondary metabolism in this relevant opportunistic plant pathogen. PMID:27213959

  6. Peptidomimetics as protein arginine deiminase 4 (PAD4) inhibitors.

    PubMed

    Trabocchi, Andrea; Pala, Nicolino; Krimmelbein, Ilga; Menchi, Gloria; Guarna, Antonio; Sechi, Mario; Dreker, Tobias; Scozzafava, Andrea; Supuran, Claudiu T; Carta, Fabrizio

    2015-06-01

    The protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme, which catalyses the irreversible conversion of peptidyl-arginines into peptidyl-citrullines and plays an important role in several diseases such as in the rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Creutzfeldt-Jacob's disease and cancer. In this study, we report the inhibition profiles and computational docking toward the PAD4 enzyme of a series of 1,2,3-triazole peptidomimetic-based derivatives incorporating the β-phenylalanine and guanidine scaffolds. Several effective, low micromolar PAD4 inhibitors are reported in this study.

  7. Trypanosoma brucei prenylated-protein carboxyl methyltransferase prefers farnesylated substrates.

    PubMed Central

    Buckner, Frederick S; Kateete, David P; Lubega, George W; Van Voorhis, Wesley C; Yokoyama, Kohei

    2002-01-01

    Carboxyl methylation of the C-terminal prenylated cysteine, which occurs in most farnesylated and geranylgeranylated proteins, is a reversible step and is implicated in the regulation of membrane binding and cellular functions of prenylated proteins such as GTPases. The gene coding for prenylated-protein carboxyl methyltransferase (PPMT) of the protozoan parasite Trypanosoma brucei has been cloned and expressed in the baculovirus/Sf9 cell system. The protein of 245 amino acids has 24-28% sequence identity to the orthologues from other species including human and Saccharomyces cerevisiae. Methyltransferase activity was detected in the membrane fraction from Sf9 cells infected with the recombinant baculovirus using N -acetyl- S -farnesylcysteine (AFC) and S -adenosyl[ methyl -(3)H]methionine ([(3)H]AdoMet) as substrates. Recombinant T. brucei PPMT prefers AFC to N -acetyl- S -geranylgeranylcysteine (AGGC) by 10-50-fold based on the V (max)/ K (m) values. Native PPMT activity detected in the membrane fraction from T. brucei procyclics displays similar substrate specificity ( approximately 40-fold preference for AFC over AGGC). In contrast, mouse liver PPMT utilizes both AFC and AGGC as substrates with similar catalytic efficiencies. Several cellular proteins of the T. brucei bloodstream form were shown to be carboxyl methylated in a cell-free system. Incorporation of [(3)H]methyl group from [(3)H]AdoMet into most of the proteins was significantly inhibited by AFC but not AGGC at 20 microM, suggesting that T. brucei PPMT acts on farnesylated proteins in the cell. Cells of the T. brucei bloodstream form show higher sensitivity to AFC and AGGC (EC(50)=70-80 microM) compared with mouse 3T3 cells (EC(50)>150 microM). PMID:12141948

  8. Kinetic mechanism of protein N-terminal methyltransferase 1.

    PubMed

    Richardson, Stacie L; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L; Huang, Rong

    2015-05-01

    The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors.

  9. Current Chemical Biology Approaches to Interrogate Protein Methyltransferases

    PubMed Central

    Luo, Minkui

    2012-01-01

    Protein methyltransferases (PMTs) play various physiological and pathological roles through methylating histone and nonhistone targets. However, most PMTs including more than 60 human PMTs remain to be fully characterized. The current approaches to elucidate the functions of PMTs have been diversified by many emerging chemical biology technologies. This review focuses on progress in these aspects and is organized into four discussion modules (assays, substrates, cofactors and inhibitors) that are important to elucidate biological functions of PMTs. These modules are expected to provide general guidance and present emerging methods for researchers to select and combine suitable PMT-activity assays, well-defined substrates, novel SAM surrogates and PMT inhibitors to interrogate PMTs. PMID:22220966

  10. Specificity analysis of protein lysine methyltransferases using SPOT peptide arrays.

    PubMed

    Kudithipudi, Srikanth; Kusevic, Denis; Weirich, Sara; Jeltsch, Albert

    2014-11-29

    Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

  11. Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

    PubMed Central

    Kudithipudi, Srikanth; Kusevic, Denis; Weirich, Sara; Jeltsch, Albert

    2014-01-01

    Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs. PMID:25489813

  12. Coactivator-Associated Arginine Methyltransferase-1 Function in Alveolar Epithelial Senescence and Elastase-Induced Emphysema Susceptibility.

    PubMed

    Sarker, Rim S J; John-Schuster, Gerrit; Bohla, Alexander; Mutze, Kathrin; Burgstaller, Gerald; Bedford, Mark T; Königshoff, Melanie; Eickelberg, Oliver; Yildirim, Ali Ö

    2015-12-01

    Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases worldwide. A major feature of COPD is emphysema, which is the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone methylation and the transcription of genes involved in senescence, proliferation, and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type II (ATII) cells. We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and small interfering (si)RNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5 ± 9.6 μm versus 38.8 ± 5.5 μm; P < 0.01) and lung compliance (2.8 ± 0.32 μl/cm H2O versus 2.4 ± 0.4 μl/cm H2O; P < 0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased sirtuin 1 expression (0.034 ± 0.003 versus 0.022 ± 0.001; P < 0.05) but increased expression of p16 (0.27 ± 0.013 versus 0.31 ± 0.010; P < 0.5) and p21 (0.81 ± 0.088 versus 1.28 ± 0.063; P < 0.01) and higher β-galactosidase-positive senescent cells (50.57 ± 7.36% versus 2.21 ± 0.34%; P < 0.001) compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs transdifferentiation and wound healing (32.18 ± 0.9512% versus 8.769 ± 1.967%; P < 0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development

  13. Kinetic Mechanism of Protein N-terminal Methyltransferase 1*

    PubMed Central

    Richardson, Stacie L.; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L.; Huang, Rong

    2015-01-01

    The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors. PMID:25771539

  14. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    SciTech Connect

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tat pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.

  15. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins

    PubMed Central

    Mann, Daniel; Teuber, Christian; Tennigkeit, Stefan A.; Schröter, Grit; Gerwert, Klaus

    2016-01-01

    Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine “finger.” We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis. PMID:27911799

  16. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.

    PubMed

    Mann, Daniel; Teuber, Christian; Tennigkeit, Stefan A; Schröter, Grit; Gerwert, Klaus; Kötting, Carsten

    2016-12-13

    Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

  17. Effects of L-arginine on solubilization and purification of plant membrane proteins.

    PubMed

    Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

    2011-11-01

    Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins.

  18. Effects of arginine on heat-induced aggregation of concentrated protein solutions.

    PubMed

    Shah, Dhawal; Shaikh, Abdul Rajjak; Peng, Xinxia; Rajagopalan, Raj

    2011-01-01

    Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.

  19. Ribosomal Protein Methyltransferases in the Yeast Saccharomyces cerevisiae: Roles in Ribosome Biogenesis and Translation

    PubMed Central

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-01-01

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed −1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  20. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.

  1. Heterologous protein production using the twin arginine translocation pathway

    DOEpatents

    Pohlschroder, Mechtild; Kissinger, Jessica C; Rose, R. Wesley; Brueser, Thomas; Dilks, Kieran

    2008-11-04

    Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

  2. Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

    PubMed

    Miggiano, Riccardo; Perugino, Giuseppe; Ciaramella, Maria; Serpe, Mario; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, Silvia; Lahiri, Samarpita; Rizzi, Menico; Rossi, Franca

    2016-01-15

    Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.

  3. Formulating Fluorogenic Assay to Evaluate S-adenosyl-L-methionine Analogues as Protein Methyltransferase Cofactors

    PubMed Central

    Wang, Rui; Ibáñez, Glorymar; Islam, Kabirul; Zheng, Weihong; Blum, Gil; Sengelaub, Caitlin; Luo, Minkui

    2013-01-01

    Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolyase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z′ values of 0.83–0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8×5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues. PMID:21866297

  4. DNA Methyltransferase protein synthesis is reduced in CXXC finger protein 1-deficient embryonic stem cells.

    PubMed

    Butler, Jill S; Palam, Lakshmi R; Tate, Courtney M; Sanford, Jeremy R; Wek, Ronald C; Skalnik, David G

    2009-05-01

    CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life.

  5. UPF0586 Protein C9orf41 Homolog Is Anserine-producing Methyltransferase*

    PubMed Central

    Drozak, Jakub; Piecuch, Maria; Poleszak, Olga; Kozlowski, Piotr; Chrobok, Lukasz; Baelde, Hans J.; de Heer, Emile

    2015-01-01

    Anserine (β-alanyl-N(Pi)-methyl-l-histidine), a methylated derivative of carnosine (β-alanyl-l-histidine), is an abundant constituent of vertebrate skeletal muscles. Although it has been suggested to serve as a proton buffer and radical scavenger, its physiological function remains mysterious. The formation of anserine is catalyzed by carnosine N-methyltransferase, recently identified in chicken as histamine N-methyltransferase-like (HNMT-like) protein. Although the HNMT-like gene is absent in mammalian genomes, the activity of carnosine N-methyltransferase was reported in most mammalian species. In the present investigation, we purified carnosine N-methyltransferase from rat muscles about 2600-fold. Three polypeptides of ∼45, 50, and 70 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of UPF0586 protein C9orf41 homolog as the only meaningful candidate. Rat UPF0586 and its yeast, chicken, and human orthologs were expressed in COS-7 cells and purified to homogeneity. Although all recombinant proteins catalyzed the formation of anserine, as confirmed by chromatographic and mass spectrometry analysis, rat UPF0586 was more active on carnosine than other orthologs. Confocal microscopy of HeLa cells expressing recombinant UPF5086 proteins revealed their presence in both cytosol and nucleus. Carnosine and Gly-His were the best substrates for all UPF0586 orthologs studied, although the enzymes also methylated other l-histidine-containing di- and tripeptides. Finally, cotransfection of COS-7 cells with rat or human UPF0586 and carnosine synthase transformed the cells into efficient anserine producers. We conclude that UPF0586 is mammalian carnosine N-methyltransferase and hypothesize that it may also serve as a peptide or protein methyltransferase in eukaryotes. PMID:26001783

  6. DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

    PubMed

    Rowles, Daniell L; Tsai, Yuan-Chin; Greco, Todd M; Lin, Aaron E; Li, Minghao; Yeh, Justin; Cristea, Ileana M

    2015-06-01

    Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

  7. Development of a selective inhibitor of Protein Arginine Deiminase 2.

    PubMed

    Muth, Aaron; Subramanian, Venkataraman; Beaumont, Edward; Nagar, Mitesh; Kerry, Philip; McEwan, Paul; Srinath, Hema; Clancy, Kathleen Wanda; Parelkar, Sangram S; Thompson, Paul R

    2017-03-22

    Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy 30a. Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated.

  8. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  9. Isolation and properties of an arginine-binding protein from Saccharomyces cerevisiae.

    PubMed

    Opekarová, M; Kotyk, A; Horák, J; Kholodenko, V P

    1975-11-15

    Transfer of exponentially growing cells of Saccharomyces cerevisiae epsilon 1278 b to a fresh medium (or simply to distilled water) resulted in the loss of ability to transport arginine (and lysine), accompanied by the release of several proteins from the membrane surface or periplasmic space. Fractionation by ultrafiltration, Sephadex G-50 chromatography and freeze-drying yielded a homogeneous protein (55 mg per 100 g dry weight of cells) with specific binding ability for L-arginine (Kd = 3.8 X 10(-1) M) and L-lysine (Ki = 4.2 X 10(-4) M). The protein contains over 40 amino acid residues and has a molecular weight of about 5,000. In solution, it appears to aggregate as its concentration is raised, thereby decreasing the overall binding capacity for arginine. Addition of the protein to a depleted culture does not restore the transport of arginine. It is apparently the recognition protein for the specific arginine-transporting system of Saccharomyces cerevisiae but it occurs in almost identical amounts in the MG 168 mutant with impaired arginine transport.

  10. A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

    PubMed Central

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

  11. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    PubMed

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

  12. [Bioinformatics analysis and expressed level of histone methyltransferase genes in Lonicera japonica].

    PubMed

    Qi, Lin-jie; Yuan, Yuan; Huang, Lu-qi; Long, Ping; Zha, Liang-ping; Wang, Yao-long

    2015-06-01

    Twenty-three histone methyltransferase genes were obtained from transcriptome dataset of Lonicera japonica. The nucleotide and proteins characteristics, subcellular localization, senior structural domains and conservative forecasting were analyzed. The result of phylogenetic tree showed that 23 histone methyltransferases were mainly divided into two groups: lysine methyltransferase and arginine methyltransferases. The result of gene expression showed that 23 histone methyltransferases showed preference in terms of interspecies and organs. They were more expressed in buds of L. japonica than in L. japonica var. chinensis and lower in leaves of L. japonica than in L. japonica var. chinensis. Eight genes were specific expressed in flower. These results provided basis for further understanding the function of histone methyltransferase and epigenetic regulation of active ingredients of L. japonica.

  13. Effects of dietary arginine supplementation on protein turnover and tissue protein synthesis in scald-burn rats.

    PubMed

    Cui, X L; Iwasa, M; Iwasa, Y; Ohmori, Y; Yamamoto, A; Maeda, H; Kume, M; Ogoshi, S; Yokoyama, A; Sugawara, T; Funada, T

    1999-01-01

    We assessed the effects of dietary arginine supplementation on protein turnover and organ protein synthesis in burned rats. Male Wistar rats weighing about 200 g underwent catheter jejunostomy and received scald burns covering 30% of the whole-body surface area. Animals were divided into a control group (n = 9) and an arginine group (n = 9) and continuously received total enteral nutrition for 7 d (250 kcal.kg-1.d-1, 1.72 gN.kg-1.d-1). Changes in body weight, plasma total protein, plasma albumin, urinary excretion of polyamines, nitrogen balance, whole-body protein kinetics, and tissue protein synthesis rates were determined. Whole-body protein kinetics and tissue fractional protein synthetic rates (Ks, percent/d) were estimated using a 24-h constant enteral infusion of 15N glycine on the last day. The changes in body weight were not different between the control and arginine groups. The urinary excretion of polyamines was higher in the arginine group than in the control group (P < 0.01). Burned rats enterally fed arginine-supplemented diet yielded significantly greater cumulative and daily nitrogen balance on days 3 and 5 than those fed a control diet (cumulative, P < 0.05; day 3, P < 0.01; day 5, P < 0.01). Whole-body protein turnover rate was significantly elevated in the arginine group as compared to that in the control group (P < 0.05). The Ks of rectus abdominis muscles were significantly increased in the arginine group in comparison to the control group (P < 0.01). We have shown that dietary arginine supplementation improved protein anabolism and attenuated muscle protein catabolism after thermal injury.

  14. Understanding the synergistic effect of arginine and glutamic acid mixtures on protein solubility.

    PubMed

    Shukla, Diwakar; Trout, Bernhardt L

    2011-10-20

    Understanding protein solubility is a key part of physical chemistry. In particular, solution conditions can have a major effect, and the effect of multiple cosolutes is little understood. It has been shown that the simultaneous addition of L-arginine hydrochloride and L-glutamic acid enhances the maximum achievable solubility of several poorly soluble proteins up to 4-8 times (Golovanov et. al, J. Am. Chem. Soc., 2004, 126, 8933-8939) and reduces the intermolecular interactions between proteins. The observed solubility enhancement is negligible for arginine and glutamic acid solutions as compared to the equimolar mixtures. In this study, we have established the molecular mechanism behind this observed synergistic effect of arginine and glutamic acid mixtures using preferential interaction theory and molecular dynamics simulations of Drosophilia Su(dx) protein (ww34). It was found that the protein solubility enhancement is related to the relative increase in the number of arginine and glutamic acid molecules around the protein in the equimolar mixtures due to additional hydrogen bonding interactions between the excipients on the surface of the protein when both excipients are present. The presence of these additional molecules around the protein leads to enhanced crowding, which suppresses the protein association. These results highlight the role of additive-additive interaction in tuning the protein-protein interactions. Furthermore, this study reports a unique behavior of additive solutions, where the presence of one additive in solution affects the concentration of another on the protein surface.

  15. Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Guerrera, Ida Chiara; Chhuon, Cerina; Dupuis, Marion; Rigard, Mélanie; Henry, Thomas; Barel, Monique; Charbit, Alain

    2015-01-01

    Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584). PMID:25616868

  16. RamA, a protein required for reductive activation of corrinoid-dependent methylamine methyltransferase reactions in methanogenic archaea.

    PubMed

    Ferguson, Tsuneo; Soares, Jitesh A; Lienard, Tanja; Gottschalk, Gerhard; Krzycki, Joseph A

    2009-01-23

    Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.

  17. Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

    PubMed Central

    Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H.; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

    2011-01-01

    Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases. PMID:21478168

  18. Histone lysine methyltransferases as anti-cancer targets for drug discovery

    PubMed Central

    Liu, Qing; Wang, Ming-wei

    2016-01-01

    Post-translational epigenetic modification of histones is controlled by a number of histone-modifying enzymes. Such modification regulates the accessibility of DNA and the subsequent expression or silencing of a gene. Human histone methyltransferases (HMTs)constitute a large family that includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). There is increasing evidence showing a correlation between HKMTs and cancer pathogenesis. Here, we present an overview of representative HKMTs, including their biological and biochemical properties as well as the profiles of small molecule inhibitors for a comprehensive understanding of HKMTs in drug discovery. PMID:27397541

  19. Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

    PubMed Central

    Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

    2013-01-01

    Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

  20. NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein.

    PubMed

    Tooley, Christine E Schaner; Petkowski, Janusz J; Muratore-Schroeder, Tara L; Balsbaugh, Jeremy L; Shabanowitz, Jeffrey; Sabat, Michal; Minor, Wladek; Hunt, Donald F; Macara, Ian G

    2010-08-26

    The post-translational methylation of alpha-amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins L16 and L33 (refs 1, 2), but almost nothing is known about the function or enzymology of this modification. Several other bacterial and eukaryotic proteins have since been shown to be alpha-N-methylated. However, the Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of alpha-N-methylation has been identified. Methylation-defective mutants of RCC1 have reduced affinity for DNA and cause mitotic defects, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. All fungal and animal N-terminally methylated proteins contain a unique N-terminal motif, Met-(Ala/Pro/Ser)-Pro-Lys, indicating that they may be targets of the same, unknown enzyme. The initiating Met is cleaved, and the exposed alpha-amino group is mono-, di- or trimethylated. Here we report the discovery of the first alpha-N-methyltransferase, which we named N-terminal RCC1 methyltransferase (NRMT). Substrate docking and mutational analysis of RCC1 defined the NRMT recognition sequence and enabled the identification of numerous new methylation targets, including SET (also known as TAF-I or PHAPII) and the retinoblastoma protein, RB. Knockdown of NRMT recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation.

  1. Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors.

    PubMed

    Jamali, Haya; Khan, Hasan A; Tjin, Caroline C; Ellman, Jonathan A

    2016-09-08

    The protein arginine deiminases (PADs) catalyze the post-translational deimination of arginine side chains. Multiple PAD isozymes have been characterized, and abnormal PAD activity has been associated with several human disease states. PAD3 has been characterized as a modulator of cell growth via apoptosis inducing factor and has been implicated in the neurodegenerative response to spinal cord injury. Here, we describe the design, synthesis, and evaluation of conformationally constrained versions of the potent and selective PAD3 inhibitor 2. The cell activity of representative inhibitors in this series was also demonstrated for the first time by rescue of thapsigargin-induced cell death in PAD3-expressing HEK293T cells.

  2. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  3. Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase.

    PubMed

    Weirich, Sara; Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2015-09-22

    It has been reported that the Numb protein is methylated at lysine 158 and 163 and that this methylation is introduced by the SET8 protein lysine methyltransferase [Dhami et al., (2013) Molecular Cell 50, 565-576]. We studied this methylation in vitro using peptide arrays and recombinant Numb protein as substrates. Numb peptides and protein were incubated with recombinant SET8 purified after expression in E. coli or human HEK293 cells. However, no methylation of Numb by SET8 was detectable. SET8 methylation of Histone H4 and p53 peptides and proteins, which were used as positive controls, was readily observed. While SET8 methylation of Numb in cells cannot be ruled out, based on our findings, more evidence is needed to support this claim. It appears likely that another not yet identified PKMT is responsible for the reported methylation of Numb in cells.

  4. Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase

    PubMed Central

    Weirich, Sara; Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2015-01-01

    It has been reported that the Numb protein is methylated at lysine 158 and 163 and that this methylation is introduced by the SET8 protein lysine methyltransferase [Dhami et al., (2013) Molecular Cell 50, 565–576]. We studied this methylation in vitro using peptide arrays and recombinant Numb protein as substrates. Numb peptides and protein were incubated with recombinant SET8 purified after expression in E. coli or human HEK293 cells. However, no methylation of Numb by SET8 was detectable. SET8 methylation of Histone H4 and p53 peptides and proteins, which were used as positive controls, was readily observed. While SET8 methylation of Numb in cells cannot be ruled out, based on our findings, more evidence is needed to support this claim. It appears likely that another not yet identified PKMT is responsible for the reported methylation of Numb in cells. PMID:26391684

  5. The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

    PubMed Central

    Pelisch, Federico; Gerez, Juan; Druker, Jimena; Schor, Ignacio E.; Muñoz, Manuel J.; Risso, Guillermo; Petrillo, Ezequiel; Westman, Belinda J.; Lamond, Angus I.; Arzt, Eduardo; Srebrow, Anabella

    2010-01-01

    Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF. PMID:20805487

  6. The twin arginine protein transport pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies.

    PubMed

    Joshi, Madhumita V; Mann, Stefan G; Antelmann, Haike; Widdick, David A; Fyans, Joanna K; Chandra, Govind; Hutchings, Matthew I; Toth, Ian; Hecker, Michael; Loria, Rosemary; Palmer, Tracy

    2010-07-01

    Summary Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DeltatatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DeltatatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.

  7. Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk

    PubMed Central

    Zhou, Yijun; Li, Xiao-Ping; Chen, Brian Y.; Tumer, Nilgun E.

    2017-01-01

    Ricin toxin A chain (RTA) binds to stalk P-proteins to reach the α–sarcin/ricin loop (SRL) where it cleaves a conserved adenine. Arginine residues at the RTA/RTB interface are involved in this interaction. To investigate the individual contribution of each arginine, we generated single, double and triple arginine mutations in RTA. The R235A mutation reduced toxicity and depurination activity more than any other single arginine mutation in yeast. Further reduction in toxicity, depurination activity and ribosome binding was observed when R235A was combined with a mutation in a nearby arginine. RTA interacts with the ribosome via a two-step process, which involves slow and fast interactions. Single arginine mutations eliminated the fast interactions with the ribosome, indicating that they increase the binding rate of RTA. Arginine residues form a positively charged patch to bind to negatively charged residues at the C-termini of P-proteins. When electrostatic interactions conferred by the arginines are lost, hydrophobic interactions are also abolished, suggesting that the hydrophobic interactions alone are insufficient to allow binding. We propose that Arg235 serves as an anchor residue and cooperates with nearby arginines and the hydrophobic interactions to provide the binding specificity and strength in ribosome targeting of RTA. PMID:28230053

  8. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    PubMed

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.

  9. The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei.

    PubMed

    Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

    2012-06-01

    Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing ('ChIP-seq') showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.

  10. Computational prediction of methylation types of covalently modified lysine and arginine residues in proteins.

    PubMed

    Deng, Wankun; Wang, Yongbo; Ma, Lili; Zhang, Ying; Ullah, Shahid; Xue, Yu

    2016-05-30

    Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation.

  11. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  12. As(III) S-adenosylmethionine methyltransferases and other arsenic binding proteins

    PubMed Central

    Ajees, A. Abdul; Rosen, Barry P.

    2014-01-01

    Efflux is by far the most common means of arsenic detoxification is by methylation catalyzed by a family of As(III) S-adenosylmethionine (SAM) methyltransferases (MTs) enzymes designated ArsM in microbes or AS3MT in higher eukaryotes. The protein sequence of more than 5000 AS3MT/ArsM orthologues have been deposited in the NCBI database, mostly in prokaryotic and eukaryotic microbes. As(III) SAM MTs are members of a large superfamily of MTs involved in numerous physiological functions. ArsMs detoxify arsenic by conversion of inorganic trivalent arsenic (As(III)) into mono-, di- and trimethylated species that may be more toxic and carcinogenic than inorganic arsenic. The pathway of methylation remains controversial. Several hypotheses will be examined in this review. PMID:26366023

  13. De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

    PubMed

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; Ambinder, Richard F; Hayward, S Diane

    2010-11-19

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line.

  14. Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly.

    PubMed

    Sampath, Srihari C; Marazzi, Ivan; Yap, Kyoko L; Sampath, Srinath C; Krutchinsky, Andrew N; Mecklenbräuker, Ingrid; Viale, Agnes; Rudensky, Eugene; Zhou, Ming-Ming; Chait, Brian T; Tarakhovsky, Alexander

    2007-08-17

    Epigenetic gene silencing in eukaryotes is regulated in part by lysine methylation of the core histone proteins. While histone lysine methylation is known to control gene expression through the recruitment of modification-specific effector proteins, it remains unknown whether nonhistone chromatin proteins are targets for similar modification-recognition systems. Here we show that the histone H3 methyltransferase G9a contains a conserved methylation motif with marked sequence similarity to H3 itself. As with methylation of H3 lysine 9, autocatalytic G9a methylation is necessary and sufficient to mediate in vivo interaction with the epigenetic regulator heterochromatin protein 1 (HP1), and this methyl-dependent interaction can be reversed by adjacent G9a phosphorylation. NMR analysis indicates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3K9, demonstrating that the chromodomain functions as a generalized methyl-lysine binding module. These data reveal histone-like modification cassettes - or "histone mimics" - as a distinct class of nonhistone methylation targets and directly extend the principles of the histone code to the regulation of nonhistone proteins.

  15. Interaction of arginine with protein during refolding process probed by amide H/D exchange mass spectrometry and isothermal titration calorimetry.

    PubMed

    Zhao, Dawei; Liu, Yongdong; Zhang, Guifeng; Zhang, Chun; Li, Xiunan; Wang, Qingqing; Shi, Hong; Su, Zhiguo

    2015-01-01

    Arginine has been widely used as low molecular weight additive to promote protein refolding by suppressing aggregate formation. However, methods to investigate the role of arginine in protein refolding are often limited on protein's global conformational properties. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to study the effects of arginine on recombinant human granulocyte colony-stimulating factor (rhG-CSF) refolding at the scale of peptide mapping. It was found that deuteration levels of rhG-CSF refolded with arginine was higher than that without arginine during the whole refolding process, but they became almost the same when the refolding reached equilibrium. This phenomenon indicated that arginine could protect some amide deuterium atoms from being exchanged with hydrogen, but the protection diminished gradually along with refolding proceeding. Enzymatic digestion revealed six particular peptides of 16-47, 72-84, 84-93, 114-124, 145-153 and 154-162 were mainly responsible for the deuteration, and all of them dominantly located in protein's α-helix domain. Furthermore, thermodynamics analysis by isothermal titration calorimetry provided direct evidence that arginine could only react with denatured and partially refolded rhG-CSF. Taking all of the results together, we suggest that arginine suppresses protein aggregation by a reversible combination. At the initial refolding stage, arginine could combine with the denatured protein mainly through hydrogen bonding. Subsequently, arginine is gradually excluded from protein with protein's native conformation recovering.

  16. Synthesis of Mycoplasma arginine deiminase in E. coli using stress-responsive proteins.

    PubMed

    Ahn, Keum-Young; Lee, Boram; Han, Kyung-Yeon; Song, Jong-Am; Lee, Doo Sung; Lee, Jeewon

    2014-09-01

    We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

  17. Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    PubMed Central

    Jia, Qiong; Hu, Xinling; Shi, Dawei; Zhang, Yan; Sun, Meihao; Wang, Jianwei; Mi, Kaixia; Zhu, Guofeng

    2016-01-01

    The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells. PMID:27762279

  18. A plethora of plant serine/arginine-rich proteins: redundancy or evolution of novel gene functions?

    PubMed Central

    Kalyna, Maria; Barta, Andrea

    2017-01-01

    Pre-mRNA processing is an important step in gene expression and its regulation leads to the expansion of the gene product repertoire. Serine/arginine-rich (SR) proteins are key players in intron recognition and spliceosome assembly and contribute significantly to the alternative splicing process. Due to several duplication events, at least 19 SR proteins are present in the Arabidopsis genome which is almost twice as much as in humans. They fall into seven different subfamilies, three of them homologous to metazoan splicing factors whereas the other four seem to be specific for plants. The current data show that most duplicated genes have different spatio-temporal expression patterns indicating functional diversification. Interestingly, the majority of SR protein genes are alternatively spliced and in some cases this process was shown to be under developmental and/or environmental control. This might greatly influence gene expression of target genes as also exemplified by ectopic expression studies of particular SR proteins. PMID:15270675

  19. Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07.

    PubMed

    Marino, Fabio; Mommen, Geert P M; Jeko, Anita; Meiring, Hugo D; van Gaans-van den Brink, Jacqueline A M; Scheltema, Richard A; van Els, Cécile A C M; Heck, Albert J R

    2017-01-06

    Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line we detected 149 HLA class I peptides harboring mono- and/or dimethylated arginine residues by mass spectrometry. A striking preference was observed in the presentation of arginine (di)methylated peptides for HLA-B*07 molecules, likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored dimethylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra, we found most of the peptides to be asymmetrically dimethylated, most likely by CARM1. These data expand our knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and dimethylated arginine residues may therefore offer a novel target for immunotherapy.

  20. Lack of effect of acute enteral arginine infusion on whole-body and intestinal protein metabolism in humans.

    PubMed

    Claeyssens, Sophie; Lecleire, Stéphane; Leblond, Jonathan; Marion, Rachel; Hecketsweiler, Bernadette; Lavoinne, Alain; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2007-08-01

    Arginine is a conditionally essential amino acid and exerts anabolic effects. We studied the effects of enteral arginine on whole-body and duodenal protein metabolism. Eight healthy fasted volunteers received randomly a 5-hr enteral infusion of either arginine (Arg; 20 g) or an isonitrogenous amino acid mixture (AA) and an IV infusion of [13C]leucine. Duodenal biopsies were performed. Whole-body protein turnover and duodenal protein synthesis (FSR) were calculated from GC/MS-assessed enrichment. The mRNA levels for major components of proteolytic pathways, ubiquitin, cathepsin D, and m-calpain, were evaluated by RT-PCR. Results were compared using paired Wilcoxon test. Endogenous, oxidative, and nonoxidative leucine fluxes were not different after Arg and AA infusions, respectively. Duodenal mucosal protein FSR (71% +/- 26% vs 81% +/- 30%/day) and mRNA levels of ubiquitin, cathepsin D, and m-calpain were also similar after Arg and AA infusions. We conclude that in healthy subjects, arginine infusion exerts no effect on whole-body and duodenal protein metabolism. Whether arginine might specifically affect these parameters in catabolic or inflammatory situations remains to be determined.

  1. Primary structure of a human arginine-rich nuclear protein that colocalizes with spliceosome components

    SciTech Connect

    Chaudhary, N.; McMahon, C.; Blobel, G. )

    1991-09-15

    The cDNA for a 54-kDa nuclear protein (p54) has been cloned from a human hepatoma expression library. Contained within p54 is an arginine/serine-rich region similar to segments of several proteins that participate in pre-mRNA splicing including the 70-kDa component of U1 small nuclear ribonucleoprotein particle (snRNP) and the Drosophila transformer and suppressor-of-white-apricot proteins. The arginine/serine-rich region is dominated by a series of 8-amino acid imperfect repetitive motifs (consensus sequence, Arg-Arg-Ser-Arg-Ser-Arg-Ser-Arg). Antibodies raised against synthetic peptides of p54 react with an {approximately}70-kDa protein on immunoblots of HeLa cell and rat liver nuclear proteins. This apparent discrepancy in mass is also observed when p54 mRNA is translated in vitro. Indirect immunofluorescence studies in HeLa cells show that p54 is distributed throughout the nucleus in a speckled pattern, with an additional diffuse labeling of the nucleus excluding the nucleoli. Double immunofluorescence experiments indicate that these punctate regions are coincident with the speckles seen in cells stained with antibodies against several constituents of the pre-mRNA splicing machinery. Sedimentation analysis of HeLa cell extracts on sucrose gradients showed that p54 migrates at 4-6 S, indicating that the protein is not a tightly associated component of snRNPs. Although the function of p54 is not yet known, the structure and immunolocalization data suggest that this protein may have a role in pre-mRNA processing.

  2. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    SciTech Connect

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2008-01-01

    Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal a-amino group and the -amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal a-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.

  3. Trimethylation of histone H3 lysine 36 by human methyltransferase PRDM9 protein.

    PubMed

    Eram, Mohammad S; Bustos, Susan P; Lima-Fernandes, Evelyne; Siarheyeva, Alena; Senisterra, Guillermo; Hajian, Taraneh; Chau, Irene; Duan, Shili; Wu, Hong; Dombrovski, Ludmila; Schapira, Matthieu; Arrowsmith, Cheryl H; Vedadi, Masoud

    2014-04-25

    PRDM9 (PR domain-containing protein 9) is a meiosis-specific protein that trimethylates H3K4 and controls the activation of recombination hot spots. It is an essential enzyme in the progression of early meiotic prophase. Disruption of the PRDM9 gene results in sterility in mice. In human, several PRDM9 SNPs have been implicated in sterility as well. Here we report on kinetic studies of H3K4 methylation by PRDM9 in vitro indicating that PRDM9 is a highly active histone methyltransferase catalyzing mono-, di-, and trimethylation of the H3K4 mark. Screening for other potential histone marks, we identified H3K36 as a second histone residue that could also be mono-, di-, and trimethylated by PRDM9 as efficiently as H3K4. Overexpression of PRDM9 in HEK293 cells also resulted in a significant increase in trimethylated H3K36 and H3K4 further confirming our in vitro observations. Our findings indicate that PRDM9 may play critical roles through H3K36 trimethylation in cells.

  4. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    PubMed Central

    Demirci, Hasan; Gregory, Steven T.; Dahlberg, Albert E.; Jogl, Gerwald

    2008-01-01

    SUMMARY Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal α-amino group and the ε-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal α-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA. PMID:18611379

  5. Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria.

    PubMed

    Robinson, Colin; Matos, Cristina F R O; Beck, Daniel; Ren, Chao; Lawrence, Janna; Vasisht, Nishi; Mendel, Sharon

    2011-03-01

    The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler "minimalist" Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated "proofreading" activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

  6. Hydrogen bonding motifs of protein side chains: descriptions of binding of arginine and amide groups.

    PubMed Central

    Shimoni, L.; Glusker, J. P.

    1995-01-01

    The modes of hydrogen bonding of arginine, asparagine, and glutamine side chains and of urea have been examined in small-molecule crystal structures in the Cambridge Structural Database and in crystal structures of protein-nucleic acid and protein-protein complexes. Analysis of the hydrogen bonding patterns of each by graph-set theory shows three patterns of rings (R) with one or two hydrogen bond acceptors and two donors and with eight, nine, or six atoms in the ring, designated R2(2)(8), R2(2)(9), and R1(2)(6). These three patterns are found for arginine-like groups and for urea, whereas only the first two patterns R2(2)(8) and R2(2)(9) are found for asparagine- and glutamine-like groups. In each case, the entire system is planar within 0.7 A or less. On the other hand, in macromolecular crystal structures, the hydrogen bonding patterns in protein-nucleic acid complexes between the nucleic acid base and the protein are all R2(2)(9), whereas hydrogen bonding between Watson-Crick-like pairs of nucleic acid bases is R2(2)(8). These two hydrogen bonding arrangements [R2(2)(9)] and R2(2)(8)] are predetermined by the nature of the groups available for hydrogen bonding. The third motif identified, R1(2)(6), involves hydrogen bonds that are less linear than in the other two motifs and is found in proteins. PMID:7773178

  7. Insights into the Mechanism of Streptonigrin-Induced Protein Arginine Deiminase Inactivation

    PubMed Central

    Dreyton, Christina J.; Anderson, Erin D.; Subramanian, Venkataraman; Boger, Dale L.; Thompson, Paul R.

    2014-01-01

    Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the Protein Arginine Deiminases (PADs 1–4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor. PMID:24440480

  8. Insights into the mechanism of streptonigrin-induced protein arginine deiminase inactivation.

    PubMed

    Dreyton, Christina J; Anderson, Erin D; Subramanian, Venkataraman; Boger, Dale L; Thompson, Paul R

    2014-02-15

    Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the protein arginine deiminases (PADs 1-4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.

  9. RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea*S⃞

    PubMed Central

    Ferguson, Tsuneo; Soares, Jitesh A.; Lienard, Tanja; Gottschalk, Gerhard; Krzycki, Joseph A.

    2009-01-01

    Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function. PMID:19043046

  10. Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds.

    PubMed Central

    Mudgett, M B; Lowenson, J D; Clarke, S

    1997-01-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of "unrepaired" L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability. PMID:9414558

  11. Anhydrobiosis vs. aging: comparative genomics of protein repair L-isoaspartyl methyltransferases in the sleeping chironomid. .

    NASA Astrophysics Data System (ADS)

    Gusev, Oleg; Kikawada, Takahiro; Shagimardanova, Elena; Suetsugu, Yoshitaka; Ayupov, Rustam

    Origin of anhydrobiosis in the larvae of the sleeping chironomid Polypedilum vanderplanki represents unique example of set of evolutionary events in a single species, resulted in acquiring new ability allowing survival in extremely changeable environment. Complex comparative analysis of the genome of P. vanderplanki resulted in discovery of a set of features, including existence of the set of unique clusters of genes contributing in desiccation resistance. Surprisingly, in several cases, the genes mainly contributing to the formation of the molecular shield in the larvae are sleeping chironomid-specific and have no homology with genes from other insects, including P. nubifer - a chironomid from the same genus. Protein L-isoaspartyl methyltransferase (PIMT) acts on proteins that have been non-enzymatically damaged due to age, and partially restores aspartic residues, extending life of the polypeptides. PIMT a highly conserved enzyme present in nearly all eukaryotes, and microorganisms mostly in a single copy (or in a few isoforms in certain plants and some bacteria). While conducting a comparative analysis of the genomes of two chironomid midge species different in their ability to stand complete water loss, we have noticed that structure and number of PIMT-coding genes in the desiccation resistant (anhydrobiotic) midge (Polypedilum vanderplanki, Pv) is different from those of the common desiccation-sensitive midge (Polypedilum nubifer, Pn) and the rest of insects. Both species have a clear orthologous PIMT shared by all insects. At the same time, in contrast to Pn which has only one PIMT gene (PnPimt-1), the Pv genome contains 12 additional genes paralogous to Pimt1 (PvPimt-2-12) presumably coding functional PIMT proteins, which are arranged in a single cluster. Remarkably, PvPimt-1 location in the Pv is different from the rest of Pimt-like genes. PvPimt-1 gene is ubiquitously expressed during the life cycle, but expression of the PvPimt2-12 is limited to the eggs

  12. Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization

    PubMed Central

    Hong, Shangyu; Moreno-Navarrete, Jose M; Wei, Xiaojing; Kikukawa, Yusuke; Tzameli, Iphigenia; Prasad, Deepthi; Lee, Yoonjin; Asara, John M; Fernandez-Real, Jose Manuel; Maratos-Flier, Eleftheria; Pissios, Pavlos

    2015-01-01

    Nicotinamide N-methyltransferase (Nnmt) methylates nicotinamide, a form of vitamin B3, to produce N1-methylnicotinamide (MNAM). Nnmt is an emerging metabolic regulator in adipocytes but its role in the liver, a tissue with the strongest Nnmt expression, is not known. In spite of its overall high expression, here we find that hepatic expression of Nnmt is highly variable and correlates with multiple metabolic parameters in mice and in humans. Further, we find that suppression of hepatic Nnmt expression in vivo alters glucose and cholesterol metabolism and that the metabolic effects of Nnmt in the liver are mediated by its product MNAM. Supplementation of high fat diet with MNAM decreases serum and liver cholesterol and liver triglycerides levels in mice. Mechanistically, increasing Nnmt expression or MNAM levels stabilizes sirtuin 1 protein, an effect, which is required for their metabolic benefits. In summary, we describe a novel regulatory pathway for vitamin B3 that could provide a new opportunity for metabolic disease therapy. PMID:26168293

  13. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294

    SciTech Connect

    Chang, Yanqi; Zhang, Xing; Horton, John R.; Upadhyay, Anup K.; Spannhoff, Astrid; Liu, Jin; Synder, James P.; Bedford, Mark T.; Cheng, Xiaodong

    2009-03-26

    Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.

  14. Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design.

    PubMed

    Slade, Daniel J; Fang, Pengfei; Dreyton, Christina J; Zhang, Ying; Fuhrmann, Jakob; Rempel, Don; Bax, Benjamin D; Coonrod, Scott A; Lewis, Huw D; Guo, Min; Gross, Michael L; Thompson, Paul R

    2015-04-17

    Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.

  15. Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design

    PubMed Central

    2015-01-01

    Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs. PMID:25621824

  16. Protein arginine deiminase 2 binds calcium in an ordered fashion: Implications for inhibitor design

    DOE PAGES

    Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; ...

    2015-01-26

    Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

  17. Protein arginine deiminase 2 binds calcium in an ordered fashion: Implications for inhibitor design

    SciTech Connect

    Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; Zhang, Ying; Fuhrmann, Jakob; Rempel, Don; Bax, Benjamin D.; Coonrod, Scott A.; Lewis, Huw D.; Guo, Min; Gross, Michael L.; Thompson, Paul R.

    2015-01-26

    Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.

  18. Crystal Structure of the Arginine Repressor Protein in Complex With the DNA Operator From Mycobacterium Tuberculosis

    SciTech Connect

    Cherney, L.T.; Cherney, M.M.; Garen, C.R.; Lu, G.J.; James, M.N.G.

    2009-05-12

    The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 {angstrom} resolution with bound arginine and at 2.15 {angstrom} resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11{sup o} upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

  19. Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

    PubMed

    Han, Rui-Zhi; Xu, Guo-Chao; Dong, Jin-Jun; Ni, Ye

    2016-06-01

    Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

  20. Arginine metabolism in the salivary glands of protein-deficient rats and its potential association with the oral microflora.

    PubMed

    Enwonwu, C O; Ilupeju, F; Warren, R C

    1994-01-01

    Salivary glands and their secretions play key roles in the prevention of dental diseases. The antibacterial and physicochemical properties of saliva are compromised in chronic malnutrition. The present study has examined the possibility that some malnutrition-induced changes in salivary gland function are potentially capable of promoting growth and metabolic activities of pathogenic oral microorganisms. Compared to well-fed controls, rats fed a 3% protein diet for 18 days showed a significant reduction (p < 0.001) in the submandibular gland arginase (L-arginine amidinohydrolase, EC 3.5.3.1) activity. Associated with the latter finding was a marked increase (+85%) in the glandular level of free arginine, this basic amino acid accounting for 12.2% of the total essential amino acids as compared with a figure of only 4.6% for the controls. The total free amino acid pool in whole saliva was relatively unaffected by malnutrition, but the levels of the basic amino acids arginine and histidine were marginally increased. Many oral bacterial species, some of which are dominant plaque microorganisms, utilize the arginine deiminase (EC 3.5.3.6) pathway. Thus, increased availability of free arginine from salivary glands offers a plausible explanation for the frequently reported observation of differential overgrowth of several potentially pathogenic microorganisms including some mutants streptococci in protein-deficient laboratory animals and may well apply to similar findings in malnourished populations in Third World countries.

  1. Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

    PubMed Central

    Yap, Damian B.; Lewis, M.E. Suzanne; Chijiwa, Chieko; Ramos‐Arroyo, Maria A.; Tkachenko, Natália; Milano, Valentina; Fradin, Mélanie; McKinnon, Margaret L.; Townsend, Katelin N.; Xu, Jieqing; Van Allen, M.I.; Ross, Colin J.D.; Dobyns, William B.; Weaver, David D.; Gibson, William T.

    2016-01-01

    ABSTRACT Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb‐repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, whereas WS‐associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS‐associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G>C (p.Asp185His) polymorphism in EZH2. PMID:26694085

  2. Arginine methylation promotes translation repression activity of eIF4G-binding protein, Scd6

    PubMed Central

    Poornima, Gopalakrishna; Shah, Shanaya; Vignesh, Venkadasubramanian; Parker, Roy; Rajyaguru, Purusharth I.

    2016-01-01

    Regulation of translation plays a critical role in determining mRNA fate. A new role was recently reported for a subset of RGG-motif proteins in repressing translation initiation by binding eIF4G1. However the signaling mechanism(s) that leads to spatial and temporal regulation of repression activity of RGG-motif proteins remains unknown. Here we report the role of arginine methylation in regulation of repression activity of Scd6, a conserved RGG-motif protein. We demonstrate that Scd6 gets arginine methylated at its RGG-motif and Hmt1 plays an important role in its methylation. We identify specific methylated arginine residues in the Scd6 RGG-motif in vivo. We provide evidence that methylation augments Scd6 repression activity. Arginine methylation defective (AMD) mutant of Scd6 rescues the growth defect caused by overexpression of Scd6, a feature of translation repressors in general. Live-cell imaging of the AMD mutant revealed that it is defective in inducing formation of stress granules. Live-cell imaging and pull-down results indicate that it fails to bind eIF4G1 efficiently. Consistent with these results, a strain lacking Hmt1 is also defective in Scd6-eIF4G1 interaction. Our results establish that arginine methylation augments Scd6 repression activity by promoting eIF4G1-binding. We propose that arginine methylation of translation repressors with RGG-motif could be a general modulator of their repression activity. PMID:27613419

  3. The arginine finger of the Bloom syndrome protein: its structural organization and its role in energy coupling

    PubMed Central

    Ren, Hua; Dou, Shuo-Xing; Rigolet, Pascal; Yang, Ye; Wang, Peng-Ye; Amor-Gueret, Mounira; Xi, Xu Guang

    2007-01-01

    RecQ family helicases are essential in maintaining chromosomal DNA stability and integrity. Despite extensive studies, the mechanisms of these enzymes are still poorly understood. Crystal structures of many helicases reveal a highly conserved arginine residue located near the γ-phosphate of ATP. This residue is widely recognized as an arginine finger, and may sense ATP binding and hydrolysis, and transmit conformational changes. We investigated the existence and role of the arginine finger in the Bloom syndrome protein (BLM), a RecQ family helicase, in ATP hydrolysis and energy coupling. Our studies by combination of structural modelling, site-directed mutagenesis and biochemical and biophysical approaches, demonstrate that mutations of residues interacting with the γ-phosphate of ATP or surrounding the ATP-binding sites result in severe impairment in the ATPase activity of BLM. These mutations also impair BLM's DNA-unwinding activities, but do not affect its ATP and DNA-binding abilities. These data allow us to identify R982 as the residue that functions as a BLM arginine finger. Our findings further indicate how the arginine finger is precisely positioned by the conserved motifs with respect to the γ-phosphate. PMID:17766252

  4. Increased Serine-Arginine (SR) Protein Phosphorylation Changes Pre-mRNA Splicing in Hypoxia*

    PubMed Central

    Jakubauskiene, Egle; Vilys, Laurynas; Makino, Yuichi; Poellinger, Lorenz; Kanopka, Arvydas

    2015-01-01

    The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles. PMID:26023237

  5. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins

    PubMed Central

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P.; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, CT; Surjit, Milan

    2016-01-01

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66th,67th isoleucine and 101st,102nd leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV. PMID:27113483

  6. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins.

    PubMed

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, C T; Surjit, Milan

    2016-04-26

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.

  7. Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA.

    PubMed

    Brown, Jessica A; Kinzig, Charles G; DeGregorio, Suzanne J; Steitz, Joan A

    2016-12-06

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3'-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U•A-U triples interrupted by a C•G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16-MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA-protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 10(5) molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.

  8. Protein arginine deiminase 4 inhibition is sufficient for the amelioration of collagen-induced arthritis.

    PubMed

    Willis, V C; Banda, N K; Cordova, K N; Chandra, P E; Robinson, W H; Cooper, D C; Lugo, D; Mehta, G; Taylor, S; Tak, P P; Prinjha, R K; Lewis, H D; Holers, V M

    2017-01-27

    Citrullination of joint proteins by the protein arginine deiminase (PAD) family of enzymes is recognized increasingly as a key process in the pathogenesis of rheumatoid arthritis. This present study was undertaken to explore the efficacy of a novel PAD4-selective inhibitor, GSK199, in the murine collagen-induced arthritis model of rheumatoid arthritis. Mice were dosed daily from the time of collagen immunization with GSK199. Efficacy was assessed against a wide range of end-points, including clinical disease scores, joint histology and immunohistochemistry, serum and joint citrulline levels and quantification of synovial autoantibodies using a proteomic array containing joint peptides. Administration of GSK199 at 30 mg/kg led to significant effects on arthritis, assessed both by global clinical disease activity and by histological analyses of synovial inflammation, pannus formation and damage to cartilage and bone. In addition, significant decreases in complement C3 deposition in both synovium and cartilage were observed robustly with GSK199 at 10 mg/kg. Neither the total levels of citrulline measurable in joint and serum, nor levels of circulating collagen antibodies, were affected significantly by treatment with GSK199 at any dose level. In contrast, a subset of serum antibodies reactive against citrullinated and non-citrullinated joint peptides were reduced with GSK199 treatment. These data extend our previous demonstration of efficacy with the pan-PAD inhibitor Cl-amidine and demonstrate robustly that PAD4 inhibition alone is sufficient to block murine arthritis clinical and histopathological end-points.

  9. Changing Transcriptional Initiation Sites and Alternative 5'- and 3'-Splice Site Selection of the First Intron Deploys the Arabidopsis Protein Isoaspartyl Methyltransferase2 Variants to Different Subcellular Compartments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT), genes encoding an enzyme (EC 2.1.1.77) capable of converting uncoded, L-isoaspartyl residues, arising spontaneously at L-asparaginyl and L-aspartyl sites in proteins, to L-aspartate. PIMT2 produces at lea...

  10. Identification of DIM-7, a protein required to target the DIM-5 H3 methyltransferase to chromatin

    PubMed Central

    Lewis, Zachary A.; Adhvaryu, Keyur K.; Honda, Shinji; Shiver, Anthony L.; Selker, Eric U.

    2010-01-01

    Functionally distinct chromatin domains are delineated by distinct posttranslational modifications of histones, and in some organisms by differences in DNA methylation. Proper establishment and maintenance of chromatin domains is critical but not well understood. We previously demonstrated that heterochromatin in the filamentous fungus Neurospora crassa is marked by cytosine methylation directed by trimethylated Lysine 9 on histone H3 (H3K9me3). H3K9me3 is the product of the DIM-5 Lysine methyltransferase and is recognized by a protein complex containing heterochromatin protein-1 and the DIM-2 DNA methyltransferase. To identify additional components that control the establishment and function of DNA methylation and heterochromatin, we built a strain harboring two selectable reporter genes that are silenced by DNA methylation and employed this strain to select for mutants that are defective in DNA methylation (dim). We report a previously unidentified gene (dim-7) that is essential for H3K9me3 and DNA methylation. DIM-7 homologs are found only in fungi and are highly divergent. We found that DIM-7 interacts with DIM-5 in vivo and demonstrated that a conserved domain near the N terminus of DIM-7 is required for its stability. In addition, we found that DIM-7 is essential for recruitment of DIM-5 to form heterochromatin. PMID:20404183

  11. L-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway.

    PubMed

    Ma, Xi; Han, Meng; Li, Defa; Hu, Shengdi; Gilbreath, Kyler R; Bazer, Fuller W; Wu, Guoyao

    2017-03-04

    L-Arginine has been reported to enhance brown adipose tissue developments in fetal lambs of obese ewes, but the underlying mechanism is unknown. The present study tested the hypothesis that L-arginine stimulates growth and development of brown adipocyte precursor cells (BAPCs) through activation of mammalian target of rapamycin cell signaling. BAPCs isolated from fetal lambs at day 90 of gestation were incubated   for 6 h in arginine-free DMEM, and then cultured in DMEM with concentrations of 50, 100, 200, 500 or 1000 μmol L-arginine/L for 24-96 h. Cell proliferation, protein turnover, the mammalian target of rapamycin (mTOR) signaling pathway and pre-adipocyte differentiation markers were determined. L-arginine treatment enhanced (P < 0.05) BAPC growth and protein synthesis, while inhibiting proteolysis in a dose-dependent manner. Compared with 50 and 100 μmol/L (the concentrations of arginine in the maternal plasma of obese ewes), 200 μmol L-arginine/L (the concentrations of arginine in the maternal plasma of obese ewes receiving arginine supplementation) increased (P < 0.05) the abundances of phosphorylated mTOR, P70(S6K) and 4EBP1, as well as the abundances of PGC1α, UCP1, BMP7 and PRDM16. These novel findings indicate that increasing extra-cellular arginine concentration from 50 to 200 µmol/L activates mTOR cell signaling in BAPCs and enhances their growth and development in a dose-dependent manner. Our results provide a mechanism for arginine supplementation to enhance the development of brown adipose tissue in fetal lambs.

  12. DNA methyltransferase DNMT3b protein overexpression as a prognostic factor in patients with diffuse large B-cell lymphomas.

    PubMed

    Amara, Khaled; Ziadi, Sonia; Hachana, Mohamed; Soltani, Nabil; Korbi, Sadok; Trimeche, Mounir

    2010-07-01

    Diffuse large B-cell lymphomas (DLBCL) are the most common type of aggressive lymphomas, with considerable heterogeneity in clinical presentation, molecular characteristics, and outcome. Previous studies have showed significant correlations between DNA methyltransferase (DNMT) overexpression and unfavorable prognosis in human cancers. Therefore, we investigated in this study the biological and prognostic significance of DNMT1, DNMT3a, and DNMT3b protein expression in DLBCL. DNA methyltransferase (DNMT) expression was analyzed by immunohistochemistry in 81 DLBCL cases and correlated with clinicopathological parameters. Kaplan-Meier curves were used to estimate survival rates, and the Cox proportional hazard regression model was used to evaluate the prognostic impact of DNMT expression. Our results showed that overexpression of DNMT1, DNMT3a, and DNMT3b were detected in 48%, 13%, and 45% of investigated cases, respectively. DNA methyltransferase 1 (DNMT1) and DNMT3b overexpression was significantly correlated with advanced clinical stages (P = 0.028 and P = 0.016, respectively). Moreover, concomitant expression of DNMT1 and DNMT3b was significantly correlated with resistance to treatment (P = 0.015). With regard to survival rates, although data was available only for 40 patients, DNMT3b overexpression was significantly correlated with shorter overall survival (P = 0.006) and progression-free survival (P = 0.016). Interestingly, multivariate analysis demonstrated that DNMT3b overexpression was an independent prognostic factor for predicting shortened overall survival (P = 0.004) and progression-free survival (P = 0.024). In conclusion, DNMT3b overexpression was identified as an independent prognostic factor for predicting shortened survival of patients with DLBCL and could be, therefore, useful in identifying patients who would benefit from aggressive therapy.

  13. LLY-507, a cell-active, potent, and selective inhibitor of protein-lysine methyltransferase SMYD2

    SciTech Connect

    Nguyen, Hannah; Allali-Hassani, Abdellah; Antonysamy, Stephen; Chang, Shawn; Chen, Lisa Hong; Curtis, Carmen; Emtage, Spencer; Fan, Li; Gheyi, Tarun; Li, Fengling; Liu, Shichong; Martin, Joseph R.; Mendel, David; Olsen, Jonathan B.; Pelletier, Laura; Shatseva, Tatiana; Wu, Song; Zhang, Feiyu Fred; Arrowsmith, Cheryl H.; Brown, Peter J.; Campbell, Robert M.; Garcia, Benjamin A.; Barsyte-Lovejoy, Dalia; Mader, Mary; Vedadi, Masoud

    2015-03-30

    SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. As a result, these findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.

  14. LLY-507, a cell-active, potent, and selective inhibitor of protein-lysine methyltransferase SMYD2

    DOE PAGES

    Nguyen, Hannah; Allali-Hassani, Abdellah; Antonysamy, Stephen; ...

    2015-03-30

    SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex withmore » LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. As a result, these findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.« less

  15. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

    PubMed

    Ouchi, Takuya; Tomita, Takeo; Horie, Akira; Yoshida, Ayako; Takahashi, Kento; Nishida, Hiromi; Lassak, Kerstin; Taka, Hikari; Mineki, Reiko; Fujimura, Tsutomu; Kosono, Saori; Nishiyama, Chiharu; Masui, Ryoji; Kuramitsu, Seiki; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2013-04-01

    LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

  16. Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism.

    PubMed

    Dreyton, Christina J; Knuckley, Bryan; Jones, Justin E; Lewallen, Daniel M; Thompson, Paul R

    2014-07-15

    Citrullination, which is catalyzed by protein arginine deiminases (PADs 1-4 and 6), is a post-translational modification (PTM) that effectively neutralizes the positive charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homologue of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at R26, and this PTM leads to the increased transcription of more than 200 genes under the control of the estrogen receptor. Given that our understanding of PAD2 biology remains incomplete, we initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Herein, we report that the substrate specificity and calcium dependence of PAD2 are similar to those of PADs 1, 3, and 4. However, unlike those isozymes, PAD2 appears to use a substrate-assisted mechanism of catalysis in which the positively charged substrate guanidinium depresses the pKa of the nucleophilic cysteine. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isozyme-specific inhibitors.

  17. Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites

    PubMed Central

    Chen, Patty B.; Ding, Shuai; Zanghì, Gigliola; Soulard, Valérie; DiMaggio, Peter A.; Fuchter, Matthew J.; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A.

    2016-01-01

    Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

  18. A feedback regulatory loop between methyltransferase PRMT1 and orphan receptor TR3

    PubMed Central

    Lei, Na-zi; Zhang, Xiao-yan; Chen, Hang-zi; Wang, Yuan; Zhan, Yan-yan; Zheng, Zhong-hui; Shen, Yue-mao; Wu, Qiao

    2009-01-01

    PRMT1, an arginine methyltransferase, plays an important role in numerous cellular processes. In this study, we demonstrate a feedback regulatory loop between PRMT1 and the orphan receptor TR3. Unlike another orphan receptor HNF4, TR3 is not methylated by PRMT1 although they physically interact with each other. By delaying the TR3 protein degradation, PRMT1 binding leads to the elevation of TR3 cellular protein level, thereby enhances the DNA binding and transactivation activity of TR3 in a non-methyltransferase manner. Another coactivator SRC-2 acts synergistically with PRMT1 to regulate TR3 functions. In turn, TR3 binding to the catalytic domain of PRMT1 causes an inhibition of the PRMT1 methyltransferase activity. This repression results in the functional changes in some of PRMT1 substrates, including STAT3 and Sam68. The negative regulation of PRMT1 by TR3 was further confirmed in both TR3-knockdown cells and TR3-knockout mice with the use of an agonist for TR3. Taken together, our study not only identifies a regulatory role of PRMT1, independent on methyltransferase activity, in TR3 transactivation, but also characterizes a novel function of TR3 in the repression of PRMT1 methyltransferase activity. PMID:19095693

  19. The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase.

    PubMed

    Bachmann, Julie; Bauer, Brigitte; Zwicker, Klaus; Ludwig, Bernd; Anderka, Oliver

    2006-11-01

    The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.

  20. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α.

    PubMed

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement T Y; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-03-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours.

  1. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α

    PubMed Central

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement TY; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-01-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours. PMID:23381944

  2. O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients.

    PubMed

    Spiegl-Kreinecker, Sabine; Pirker, Christine; Filipits, Martin; Lötsch, Daniela; Buchroithner, Johanna; Pichler, Josef; Silye, Rene; Weis, Serge; Micksche, Michael; Fischer, Johannes; Berger, Walter

    2010-01-01

    O(6)-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (chi(2) test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76-17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45-2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.

  3. Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity

    PubMed Central

    Ishimoto, Kenji; Kawamata, Natsuko; Uchihara, Yoshie; Okubo, Moeka; Fujimoto, Reiko; Gotoh, Eiko; Kakinouchi, Keisuke; Mizohata, Eiichi; Hino, Nobumasa; Okada, Yoshiaki; Mochizuki, Yasuhiro; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Inoue, Tsuyoshi; Tachibana, Keisuke; Doi, Takefumi

    2016-01-01

    Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin peptidase inhibitor, clade E, member 1 (SERPINE1) by methylating H3K9. These results suggest that the ubiquitination of SETDB1 at lysine 867 controls the expression of its target gene by activating its H3K9 methyltransferase activity. PMID:27798683

  4. Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase*

    PubMed Central

    Owings, Joshua P.; Kuiper, Emily G.; Prezioso, Samantha M.; Meisner, Jeffrey; Varga, John J.; Zelinskaya, Natalia; Dammer, Eric B.; Duong, Duc M.; Seyfried, Nicholas T.; Albertí, Sebastián; Conn, Graeme L.; Goldberg, Joanna B.

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa. PMID:26677219

  5. Protein repair L-isoaspartyl methyltransferase 1 is involved in both seed longevity and germination vigor in Arabidopsis.

    PubMed

    Ogé, Laurent; Bourdais, Gildas; Bove, Jérôme; Collet, Boris; Godin, Béatrice; Granier, Fabienne; Boutin, Jean-Pierre; Job, Dominique; Jullien, Marc; Grappin, Philippe

    2008-11-01

    The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments.

  6. Protein repair L-isoaspartyl methyltransferase 1 (PIMT1) in rice improves seed longevity by preserving embryo vigor and viability.

    PubMed

    Wei, Yidong; Xu, Huibin; Diao, Lirong; Zhu, Yongsheng; Xie, Hongguang; Cai, Qiuhua; Wu, Fangxi; Wang, Zonghua; Zhang, Jianfu; Xie, Huaan

    2015-11-01

    Damaged proteins containing abnormal isoaspartyl (isoAsp) accumulate as seeds age and the abnormality is thought to undermine seed vigor. Protein-L-isoaspartyl methyltransferase (PIMT) is involved in isoAsp-containing protein repair. Two PIMT genes from rice (Oryza sativa L.), designated as OsPIMT1 and OsPIMT2, were isolated and investigated for their roles. The results indicated that OsPIMT2 was mainly present in green tissues, but OsPIMT1 largely accumulated in embryos. Confocal visualization of the transient expression of OsPIMTs showed that OsPIMT2 was localized in the chloroplast and nucleus, whereas OsPIMT1 was predominately found in the cytosol. Artificial aging results highlighted the sensitivity of the seeds of OsPIMT1 mutant line when subjected to accelerated aging. Overexpression of OsPIMT1 in transgenic seeds reduced the accumulation of isoAsp-containing protein in embryos, and increased embryo viability. The germination percentage of transgenic seeds overexpressing OsPIMT1 increased 9-15% compared to the WT seeds after 21-day of artificial aging, whereas seeds from the OsPIMT1 RNAi lines overaccumulated isoAsp in embryos and experienced rapid loss of seed germinability. Taken together, these data strongly indicated that OsPIMT1-related seed longevity improvement is probably due to the repair of detrimental isoAsp-containing proteins that over accumulate in embryos when subjected to accelerated aging.

  7. A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism.

    PubMed

    Klingel, U; Miller, C M; North, A K; Stockley, P G; Baumberg, S

    1995-08-21

    In Bacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducible beta-galactosidase activity. One such isolate was obtained. The region upstream of lacZ was subcloned in Escherichia coli in such a way that it could be replaced in the B. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3' end of an ORF homologous to gdh genes encoding glutamate dehydrogenase, with highest homology to the homologue from Clostridium difficile; the 5' end of an ORF homologous to a Saccharomyces cerevisiae gene encoding delta 1-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosynthetic argCJBD-cpa-F cluster. The same region has also been sequenced by others as part of the B. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termed rocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 bp AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to bp -16/-22 to +1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

  8. Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2

    PubMed Central

    2016-01-01

    Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents. PMID:27075367

  9. Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: interaction with cofactors and effect of osmolytes on unfolding.

    PubMed

    Chatterjee, Tanaya; Pal, Aritrika; Chakravarty, Devlina; Dey, Sucharita; Saha, Rudra P; Chakrabarti, Pinak

    2013-04-01

    Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.

  10. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-01

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  11. Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

    PubMed

    Lazar, G; Schaal, T; Maniatis, T; Goodman, H M

    1995-08-15

    We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

  12. Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

    PubMed

    Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

    2012-08-01

    Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV.

  13. Plant serine/arginine-rich proteins: roles in precursor messenger RNA splicing, plant development, and stress responses.

    PubMed

    Reddy, Anireddy S N; Shad Ali, Gul

    2011-01-01

    Global analyses of splicing of precursor messenger RNAs (pre-mRNAs) have revealed that alternative splicing (AS) is highly pervasive in plants. Despite the widespread occurrence of AS in plants, the mechanisms that control splicing and the roles of splice variants generated from a gene are poorly understood. Studies on plant serine/arginine-rich (SR) proteins, a family of highly conserved proteins, suggest their role in both constitutive splicing and AS of pre-mRNAs. SR proteins have a characteristic domain structure consisting of one or two RNA recognition motifs at the N-terminus and a C-terminal RS domain rich in arginine/serine dipeptides. Plants have many more SR proteins compared to animals including several plant-specific subfamilies. Pre-mRNAs of plant SR proteins are extensively alternatively spliced to increase the transcript complexity by about six-fold. Some of this AS is controlled in a tissue- and development-specific manner. Furthermore, AS of SR pre-mRNAs is altered by various stresses, raising the possibility of rapid reprogramming of the whole transcriptome by external signals through regulation of the splicing of these master regulators of splicing. Most SR splice variants contain a premature termination codon and are degraded by up-frameshift 3 (UPF3)-mediated nonsense-mediated decay (NMD), suggesting a link between NMD and regulation of expression of the functional transcripts of SR proteins. Limited functional studies with plant SRs suggest key roles in growth and development and plant responses to the environment. Here, we discuss the current status of research on plant SRs and some promising approaches to address many unanswered questions about plant SRs.

  14. A second protein L-isoaspartyl methyltransferase gene in Arabidopsis produces two transcripts whose products are sequestered in the nucleus.

    PubMed

    Xu, Qilong; Belcastro, Marisa P; Villa, Sarah T; Dinkins, Randy D; Clarke, Steven G; Downie, A Bruce

    2004-09-01

    The spontaneous and deleterious conversion of l-asparaginyl and l-aspartyl protein residues to l-iso-Asp or d-Asp occurs as proteins age and is accelerated under stressful conditions. Arabidopsis (Arabidopsis L. Heynh.) contains two genes (At3g48330 and At5g50240) encoding protein-l-isoaspartate methyltransferase (EC 2.1.1.77; PIMT), an enzyme capable of correcting this damage. The gene located on chromosome 5 (PIMT2) produces two proteins differing by three amino acids through alternative 3' splice site selection in the first intron. Recombinant protein from both splicing variants has PIMT activity. Subcellular localization using cell fractionation followed by immunoblot detection, as well as confocal visualization of PIMT:GFP fusions, demonstrated that PIMT1 is cytosolic while a canonical nuclear localization signal, present in PIMT2psi and the shorter PIMT2omega, is functional. Multiplex reverse transcription-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to beta-TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous abscisic acid (ABA). Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA and applied stress presumably through an ABA-dependent pathway. Furthermore, cleaved amplified polymorphic sequences from PIMT2 amplicons determined that ABA preferentially enhances the production of PIMT2omega transcript in leaves and possibly in tissues other than germinating seeds.

  15. Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases

    PubMed Central

    Kanwal, Rajnee; Datt, Manish; Liu, Xiaoqi; Gupta, Sanjay

    2016-01-01

    Methylation of DNA and histone proteins are mutually involved in the epigenetic regulation of gene expression mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). DNMTs methylate cytosine residues within gene promoters, whereas HMTs catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins, thus causing chromatin condensation and transcriptional repression, which play an important role in the pathogenesis of cancer. The potential reversibility of epigenetic alterations has encouraged the development of dual pharmacologic inhibitors of DNA and histone methylation as anticancer therapeutics. Dietary flavones can affect epigenetic modifications that accumulate over time and have shown anticancer properties, which are undefined. Through DNA binding and in silico protein-ligand docking studies with plant flavones viz. Apigenin, Chrysin and Luteolin, the effect of flavones on DNA and histone methylation was assessed. Spectroscopic analysis of flavones with calf-thymus DNA revealed intercalation as the dominant binding mode, with specific binding to a GC-rich sequence in the DNA duplex. A virtual screening approach using a model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones are tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Epigenetic studies performed with flavones exhibited a decrease in DNMT enzyme activity and a reversal of the hypermethylation of cytosine bases in the DNA and prevented cytosine methylation in the GC-rich promoter sequence incubated with the M.SssI enzyme. Furthermore, a marked decrease in HMT activity and a decrease in EZH2 protein expression and trimethylation of H3K27 were noted in histones isolated from cancer cells treated with plant flavones. Our results suggest that dietary flavones can alter DNMT and HMT activities and the methylation of DNA and histone proteins that regulate epigenetic modifications, thus

  16. AMP-activated protein kinase regulates L-arginine mediated cellular responses

    PubMed Central

    2013-01-01

    Background Our prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown. Method Human umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO2–/NO3–), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. 13C6 glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O2•–) was identified by EPR-spin-trap, and peroxynitrite (ONOO–) was measured by flow-cytometer using aminophenyl fluorescein dye. Results Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-NG-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO2–/NO3– after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O2•– and ONOO–. Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased

  17. Malignant progression in O6-methylguanine-DNA methyltransferase-deficient esophageal cancer cells is associated with Ezrin protein.

    PubMed

    Su, Yaoyao; Liu, Ran; Sheng, JingYi; Liu, Hui; Wang, Yi; Pan, Enchun; Guo, Wei; Pu, Yuepu; Zhang, Juan; Liang, Geyu; Tang, Derong; Yin, Lihong

    2012-05-01

    The abnormal function of O(6)-methylguanine-DNA methyltransferase (MGMT) is reported to be associated with the occurrence of various tumors and malignant tumor progression. However, little evidence is available to describe its role in esophageal carcinogenesis. To address this issue, we constructed a stable MGMT-silenced esophageal cancer cell line by RNA interference, and exposed the cells to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) to investigate the role that MGMT plays in toxicity. During this time, we also observed the malignant behavior of cells in vitro and in vivo. In addition, two-dimensional electrophoresis and mass spectrometry were used to detect and confirm the proteins that were differentially expressed in the MGMT-deficient and MGMT-proficient cells, which might be responsible for the malignant alteration of cells. Results showed that the IC(50) of MGMT-deficient and MGMT-proficient cells exposed to MNNG was 30 μM and 65 μM, respectively, and MGMT-deficient cells had more aggressive motility and invasive abilities compared with MGMT-proficient cells. Nineteen differentially expressed proteins were detected between the MGMT-deficient and MGMT-proficient cells, 14 of which were identified, including the membrane-cytoskeleton linker protein, Ezrin, which was confirmed by both mass spectrometry and western blot analysis. The correlation between MGMT, Ezrin expression, and the malignant behavior of one normal epithelial esophageal cell line and seven esophageal cancer lines is discussed. In conclusion, loss of MGMT expression leads EC109 esophageal cancer cells to have increased malignant behavior, which may correlate with its high Ezrin protein expression.

  18. Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

    PubMed Central

    Fisher, Adam C.; Kim, Jae‐Young; Perez‐Rodriguez, Ritsdeliz; Tullman‐Ercek, Danielle; Fish, Wallace R.; Henderson, Lee A.; DeLisa, Matthew P.

    2008-01-01

    Summary Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step. PMID:21261860

  19. PRMT1-mediated arginine methylation controls ATXN2L localization

    SciTech Connect

    Kaehler, Christian; Guenther, Anika; Uhlich, Anja; Krobitsch, Sylvia

    2015-05-15

    Arginine methylation is a posttranslational modification that is of importance in diverse cellular processes. Recent proteomic mass spectrometry studies reported arginine methylation of ataxin-2-like (ATXN2L), the paralog of ataxin-2, a protein that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. Here, we investigated the methylation state of ATXN2L and its significance for ATXN2L localization. We first confirmed that ATXN2L is asymmetrically dimethylated in vivo, and observed that the nuclear localization of ATXN2L is altered under methylation inhibition. We further discovered that ATXN2L associates with the protein arginine-N-methyltransferase 1 (PRMT1). Finally, we showed that neither mutation of the arginine–glycine-rich motifs of ATXN2L nor methylation inhibition alters ATXN2L localization to stress granules, suggesting that methylation of ATXN2L is probably not mandatory. - Highlights: • ATXN2L is asymmetrically dimethylated in vivo. • ATXN2L interacts with PRMT1 under normal and stress conditions. • PRMT1-mediated dimethylation of ATXN2L controls its nuclear localization. • ATXN2L localization to stress granules appears independent of its methylation state.

  20. The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin

    PubMed Central

    Giri, Sumanprava; Aggarwal, Vasudha; Pontis, Julien; Shen, Zhen; Chakraborty, Arindam; Khan, Abid; Mizzen, Craig; Prasanth, Kannanganattu V; Ait-Si-Ali, Slimane; Ha, Taekjip; Prasanth, Supriya G

    2015-01-01

    Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure. DOI: http://dx.doi.org/10.7554/eLife.06496.001 PMID:25922909

  1. Folding forms of Escherichia coli DmsD, a twin-arginine leader binding protein.

    PubMed

    Sarfo, Kwabena J; Winstone, Tara L; Papish, Andriyka L; Howell, Jenika M; Kadir, Hakan; Vogel, Hans J; Turner, Raymond J

    2004-03-05

    Escherichia coli DmsD interacts with the twin-arginine leader sequence of the catalytic sub-unit (DmsA) of DMSO reductase. DmsD was purified as a mixture of a number of different folding forms including: dimer (A); monomer (B); a minor thiol oxidized form; a heterogeneously folded or multi-conformational monomer form which displayed a ladder of bands on native-PAGE (D); and proteolytically degraded and aggregated forms. Polyacrylamide gel electrophoresis (PAGE), under denaturing and non-denaturing conditions, was used to examine the folding and stability of DmsD. Additionally, the biophysical methods of dynamic light scattering, circular dichroism, fluorescence, and mass spectroscopy were also used. Form D could be converted to form B by treatment with 4M urea, which is the concentration at which form B begins to denature. Forms A/B could be converted to D by incubation at pH 5.0. Forms A/B and D all had twin-arginine leader binding activity.

  2. The human hnRNP M proteins: identification of a methionine/arginine-rich repeat motif in ribonucleoproteins.

    PubMed Central

    Datar, K V; Dreyfuss, G; Swanson, M S

    1993-01-01

    Recent reports indicate that proteins which directly bind to nascent RNA polymerase II transcripts, the heterogeneous nuclear ribonucleoproteins (hnRNPs), play an important role in both transcript-specific packaging and alternative splicing of pre-mRNAs. Here we describe the isolation and characterization of a group of abundant hnRNPs, the M1-M4 proteins, which appear as a cluster of four proteins of 64,000-68,000 daltons by two-dimensional electrophoresis. The M proteins are pre-mRNA binding proteins in vivo, and they bind avidly to poly(G) and poly(U) RNA homopolymers in vitro. Covalently associated polyadenylated RNA-protein complexes, generated by irradiating living HeLa cells with UV light, were purified and used to elicit antibodies in mice. The resulting antisera were then employed to isolate cDNA clones for the largest M protein, M4, by immunological screening. The deduced amino acid sequence of M4 indicates that the M proteins are members of the ribonucleoprotein consensus sequence family of RNA-binding proteins with greatest similarity to a hypothetical RNA-binding protein from Saccharomyces cerevisiae. The M proteins also possess an unusual hexapeptide-repeat region rich in methionine and arginine residues (MR repeat motif) that resembles a repeat in the 64,000 dalton subunit of cleavage stimulation factor, which is involved in 3'-end maturation of pre-mRNAs. Proteins immunologically related to M exist in divergent eukaryotes ranging from human to yeast. Images PMID:8441656

  3. Serine phosphorylation and arginine methylation at the crossroads to neurodegeneration.

    PubMed

    Basso, Manuela; Pennuto, Maria

    2015-09-01

    Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, polyglutamine diseases and motor neuron diseases, are late-onset and progressive disorders characterized by the accumulation of misfolded proteins inside and outside neurons. No effective therapies exist to delay the onset or arrest the progression of these diseases. One novel and promising therapeutic approach consists of targeting disease-causing proteins at the post-translational level. Here we illustrate this concept using the example of spinal and bulbar muscular atrophy, a neurodegenerative disease caused by polyglutamine expansion in the androgen receptor. Emerging evidence suggests that two key post-translational modifications of polyglutamine-expanded androgen receptor, namely serine phosphorylation by protein kinase B/Akt and arginine methylation by protein arginine methyltransferases, occur at the same consensus site, are mutually exclusive, and have opposing effects on neurotoxicity. Because several proteins linked to neurodegenerative diseases have canonical Akt consensus site motifs, these findings may have a broad impact in the field of neurological diseases caused by misfolded proteins.

  4. Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

    SciTech Connect

    Lu, G.J.; Garen, C.R.; Cherney, M.M.; Cherney, L.T.; Lee, C.; James, M.N.J.

    2009-06-03

    The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.

  5. Integrated proteomic analysis of major isoaspartyl-containing proteins in the urine of wild type and protein L-isoaspartate O-methyltransferase-deficient mice.

    PubMed

    Dai, Shujia; Ni, Wenqin; Patananan, Alexander N; Clarke, Steven G; Karger, Barry L; Zhou, Zhaohui Sunny

    2013-02-19

    The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.

  6. An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function

    PubMed Central

    Orlando, Joseph S.; Ornelles, David A.

    1999-01-01

    A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection. PMID:10233919

  7. Arginine Methylation by PRMT1 Regulates Muscle Stem Cell Fate.

    PubMed

    Blanc, Roméo Sébastien; Vogel, Gillian; Li, Xing; Yu, Zhenbao; Li, Shawn; Richard, Stéphane

    2017-02-01

    Quiescent muscle stem cells (MSCs) become activated in response to skeletal muscle injury to initiate regeneration. Activated MSCs proliferate and differentiate to repair damaged fibers or self-renew to maintain the pool and ensure future regeneration. The balance between self-renewal, proliferation, and differentiation is a tightly regulated process controlled by a genetic cascade involving determinant transcription factors such as Pax7, Myf5, MyoD, and MyoG. Recently, there have been several reports about the role of arginine methylation as a requirement for epigenetically mediated control of muscle regeneration. Here we report that the protein arginine methyltransferase 1 (PRMT1) is expressed in MSCs and that conditional ablation of PRMT1 in MSCs using Pax7(CreERT2) causes impairment of muscle regeneration. Importantly, PRMT1-deficient MSCs have enhanced cell proliferation after injury but are unable to terminate the myogenic differentiation program, leading to regeneration failure. We identify the coactivator of Six1, Eya1, as a substrate of PRMT1. We show that PRMT1 methylates Eya1 in vitro and that loss of PRMT1 function in vivo prevents Eya1 methylation. Moreover, we observe that PRMT1-deficient MSCs have reduced expression of Eya1/Six1 target MyoD due to disruption of Eya1 recruitment at the MyoD promoter and subsequent Eya1-mediated coactivation. These findings suggest that arginine methylation by PRMT1 regulates muscle stem cell fate through the Eya1/Six1/MyoD axis.

  8. Control of arginine utilization in Neurospora.

    PubMed Central

    Weiss, R L; Davis, R H

    1977-01-01

    The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora. PMID:838690

  9. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

    PubMed

    Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C; Majee, Manoj

    2013-03-01

    PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

  10. Recruitment of Histone Methyltransferase G9a Mediates Transcriptional Repression of Fgf21 Gene by E4BP4 Protein*

    PubMed Central

    Tong, Xin; Zhang, Deqiang; Buelow, Katie; Guha, Anirvan; Arthurs, Blake; Brady, Hugh J. M.; Yin, Lei

    2013-01-01

    The liver responds to fasting-refeeding cycles by reprogramming expression of metabolic genes. Fasting potently induces one of the key hepatic hormones, fibroblast growth factor 21 (FGF21), to promote lipolysis, fatty acid oxidation, and ketogenesis, whereas refeeding suppresses its expression. We previously reported that the basic leucine zipper transcription factor E4BP4 (E4 binding protein 4) represses Fgf21 expression and disrupts its circadian oscillations in cultured hepatocytes. However, the epigenetic mechanism for E4BP4-dependent suppression of Fgf21 has not yet been addressed. Here we present evidence that histone methyltransferase G9a mediates E4BP4-dependent repression of Fgf21 during refeeding by promoting repressive histone modification. We find that Fgf21 expression is up-regulated in E4bp4 knock-out mouse liver. We demonstrate that the G9a-specific inhibitor BIX01294 abolishes suppression of the Fgf21 promoter activity by E4BP4, whereas overexpression of E4bp4 leads to increased levels of dimethylation of histone 3 lysine 9 (H3K9me2) around the Fgf21 promoter region. Furthermore, we also show that E4BP4 interacts with G9a, and knockdown of G9a blocks repression of Fgf21 promoter activity and expression in cells overexpressing E4bp4. A G9a mutant lacking catalytic activity, due to deletion of the SET domain, fails to inhibit the Fgf21 promoter activity. Importantly, acute hepatic knockdown by adenoviral shRNA targeting G9a abolishes Fgf21 repression by refeeding, concomitant with decreased levels of H3K9me2 around the Fgf21 promoter region. In summary, we show that G9a mediates E4BP4-dependent suppression of hepatic Fgf21 by enhancing histone methylation (H3K9me2) of the Fgf21 promoter. PMID:23283977

  11. Structural Biology of Human H3K9 Methyltransferases

    SciTech Connect

    Wu, H.; Min, J; Lunin, V; Antoshenko, T; Dombrovsk, L; Zeng, H; Allali-Hassani, A; Campagna-Slater, V; Vedadi, M; et. al.

    2010-01-01

    SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. Post-translational modifications of histone proteins regulate chromatin compaction, mediate epigenetic regulation of transcription, and control cellular differentiation in health and disease. Methylation of histone tails is one of the fundamental events of epigenetic signaling. Tri-methylation of lysine 9 of histone 3 (H3K9) mediates chromatin recruitment of HP1, heterochromatin condensation and gene silencing. Similarly, methylation of H3K27 and H4K20 are associated with a repressed state of chromatin, whereas expressed genes are methylated at H3K4, H3K36 and H3K79. Histone methyltransferases are divided into protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs). HKMTs catalyze the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) to a substrate lysine and, with the exception of DOT1L, are all organized around a canonical SET domain. The structures of a number of HKMTs have been reported, including ternary complexes of human orthologs with co-factor and substrate peptides (SETD7-H3K4, SETD8-H4K20 and MLL1-H3K4), as well

  12. Impaired Homocysteine Transmethylation and Protein-Methyltransferase Activity Reduce Expression of Selenoprotein P: Implications for Obesity and Metabolic Syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity causes Metabolic Syndrome and Type-II Diabetes, disrupting hepatic function, methionine (Met)/homocysteine (Hcy) transmethylation and methyltransferase (PRMT) activities. Selenoprotein P (SEPP1), exported from the liver, is the predominate form of plasma selenium (Se) and the physiological S...

  13. Activity and specificity of the human SUV39H2 protein lysine methyltransferase.

    PubMed

    Schuhmacher, Maren Kirstin; Kudithipudi, Srikanth; Kusevic, Denis; Weirich, Sara; Jeltsch, Albert

    2015-01-01

    The SUV39H1 and SUV39H2 enzymes introduce H3K9me3, which is essential for the viability of mammalian cells. It was the aim of the present work to investigate the substrate specificity and product pattern of SUV39H2. Methylation of peptide SPOT arrays showed that SUV39H2 recognizes a long motif on H3 comprising T6-K14, with highly specific readout of R8, S10, T11 and G12 and partial specificity at T6, A7, G13 and K14. Modification of R8 and phosphorylation of S10 or T11 lead to a reduction or loss of SUV39H2 activity towards H3K9. The specificity of SUV39H2 differs from other H3K9 PKMTs, like Dim-5 or G9a, and these biochemical differences can be explained by the structures of the corresponding enzymes. Based on the specificity profile we identified additional non-histone candidate substrates in human proteins, but all of them were only weakly methylated by SUV39H2 at the peptide level. We conclude that SUV39H2 displays a high preference for the methylation of H3. Using the catalytic SET domain we show here that the enzyme prefers H3K9me0 as a substrate over H3K9me1 and H3K9me2 and it introduces the first two methyl groups into H3K9me0 in a processive reaction. SUV39H2 can transfer up to three methyl groups to lysine 9 of histone H3 but the last methylation reaction is much slower than the first two steps. We also demonstrate that the N324K mutant in the SET domain of SUV39H2 that has been shown to cause an inherited nasal skin disease in Labrador Retrievers renders SUV39H2 inactive. Differences in the circular dichroism spectra of wild type and mutant proteins indicated that the mutation causes slight structural changes.

  14. The Influence of Arginine on the Response of Enamel Matrix Derivative (EMD) Proteins to Thermal Stress: Towards Improving the Stability of EMD-Based Products

    PubMed Central

    Bolisetty, Sreenath; Marascio, Matteo; Gemperli Graf, Anja; Garamszegi, Laszlo; Mezzenga, Raffaele; Fischer, Peter; Månson, Jan-Anders

    2015-01-01

    In a current procedure for periodontal tissue regeneration, enamel matrix derivative (EMD), which is the active component, is mixed with a propylene glycol alginate (PGA) gel carrier and applied directly to the periodontal defect. Exposure of EMD to physiological conditions then causes it to precipitate. However, environmental changes during manufacture and storage may result in modifications to the conformation of the EMD proteins, and eventually premature phase separation of the gel and a loss in therapeutic effectiveness. The present work relates to efforts to improve the stability of EMD-based formulations such as Emdogain™ through the incorporation of arginine, a well-known protein stabilizer, but one that to our knowledge has not so far been considered for this purpose. Representative EMD-buffer solutions with and without arginine were analyzed by 3D-dynamic light scattering, UV-Vis spectroscopy, transmission electron microscopy and Fourier transform infrared spectroscopy at different acidic pH and temperatures, T, in order to simulate the effect of pH variations and thermal stress during manufacture and storage. The results provided evidence that arginine may indeed stabilize EMD against irreversible aggregation with respect to variations in pH and T under these conditions. Moreover, stopped-flow transmittance measurements indicated arginine addition not to suppress precipitation of EMD from either the buffers or the PGA gel carrier when the pH was raised to 7, a fundamental requirement for dental applications. PMID:26670810

  15. Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism1[W

    PubMed Central

    Cao, Feng-Qiu; Werner, Andrea K.; Dahncke, Kathleen; Romeis, Tina; Liu, Lai-Hua; Witte, Claus-Peter

    2010-01-01

    Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (Km = 67 mm, kcat = 490 s−1). The activity depended on the presence of manganese (Kd = 1.3 μm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution. PMID:20631318

  16. Identification and characterization of proteins involved in rice urea and arginine catabolism.

    PubMed

    Cao, Feng-Qiu; Werner, Andrea K; Dahncke, Kathleen; Romeis, Tina; Liu, Lai-Hua; Witte, Claus-Peter

    2010-09-01

    Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.

  17. Beneficial effects of cod protein on inflammatory cell accumulation in rat skeletal muscle after injury are driven by its high levels of arginine, glycine, taurine and lysine.

    PubMed

    Dort, Junio; Leblanc, Nadine; Maltais-Giguère, Julie; Liaset, Bjørn; Côté, Claude H; Jacques, Hélène

    2013-01-01

    We have shown that feeding cod protein, which is rich in anti-inflammatory arginine, glycine, and taurine, may beneficially modulate the inflammatory response during recovery following skeletal muscle injury; however it is unknown if these amino acids are responsible for this effect. This study was designed to assess whether supplementing casein with an amino acid mixture composed of arginine, glycine, taurine and lysine, matching their respective levels in cod protein, may account for the anti-inflammatory effect of cod protein. Male Wistar rats were fed isoenergetic diets containing either casein, cod protein, or casein supplemented with L-arginine (0.45%), glycine (0.43%), L-taurine (0.17%) and L-lysine (0.44%) (casein+). After 21 days of ad libitum feeding, one tibialis anterior muscle was injured with 200 µl bupivacaine while the saline-injected contra-lateral tibialis anterior was served as sham. Cod protein and casein+ similarly modulated the inflammation as they decreased COX-2 level at day 2 post-injury (cod protein, p=0.014; casein+, p=0.029) and ED1(+) macrophage density at days 2 (cod protein, p=0.012; casein+, p<0.0001), 5 (cod protein, p=0.001; casein+, p<0.0001) and 14 (cod protein, p<0.0001; casein+, p<0.0001) post-injury, and increased ED2(+) macrophage density at days 5 (cod protein, p<0.0001; casein+, p=0.006), 14 (cod protein, p=0.001; casein+, p<0.002) and 28 (cod protein, p<0.009; casein+, p<0.005) post-injury compared with casein. Furthermore, cod protein up-regulated (p=0.037) whereas casein+ tended to up-regulate (p=0.062) myogenin expression at day 5 post-injury compared with casein. In the cod protein-fed group, these changes resulted in greater muscle mass at days 14 (p=0.002), and 28 (p=0.001) post-injury and larger myofiber cross-sectional area at day 28 post-injury compared with casein (p=0.012). No such effects were observed with casein+. These data indicate that anti-inflammatory actions of cod protein, contrary to its effect on

  18. mraW, an essential gene at the dcw cluster of Escherichia coli codes for a cytoplasmic protein with methyltransferase activity.

    PubMed

    Carrión, M; Gómez, M J; Merchante-Schubert, R; Dongarrá, S; Ayala, J A

    1999-01-01

    Three new open reading frames, mraZ, mraW and mraR (also called ftsL), were revealed by DNA sequencing immediately upstream of gene pbpB in the dcw cluster of Escherichia coli. We have found that mraW and mraZ are active genes, coding for two proteins with relative molecular masses of 34 800 and 17 300, respectively. MraW is a cytoplasmic protein that under overproduction condition is also loosely bound to the membrane. Soluble MraW was purified up to 90% by a single high performance electrophoresis (HPEC) step from an extract of an overproducing strain. The protein exhibits a S-adenosyl-dependent methyltransferase activity on membrane-located substrates.

  19. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity.

    PubMed

    Hoskisson, Paul A; Sumby, Paul; Smith, Margaret C M

    2015-03-01

    The phage growth limitation system of Streptomyces coelicolor A3(2) is an unusual bacteriophage defence mechanism. Progeny ϕC31 phage from an initial infection are thought to be modified such that subsequent infections are attenuated in a Pgl(+) host but normal in a Pgl(-) strain. Earlier work identified four genes required for phage resistance by Pgl. Here we demonstrate that Pgl is an elaborate and novel phage restriction system that, in part, comprises a toxin/antitoxin system where PglX, a DNA methyltransferase is toxic in the absence of a functional PglZ. In addition, the ATPase activity of PglY and a protein kinase activity in PglW are shown to be essential for phage resistance by Pgl. We conclude that on infection of a Pgl(+) cell by bacteriophage ϕC31, PglW transduces a signal, probably via phosphorylation, to other Pgl proteins resulting in the activation of the DNA methyltransferase, PglX and this leads to phage restriction.

  20. Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

    PubMed

    Spotts, R O; Chakerian, A E; Matthews, K S

    1991-12-05

    Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the

  1. Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD).

    PubMed

    Chen, Heng-Li; Su, Pei-Yi; Chang, Ya-Shu; Wu, Szu-Yao; Liao, You-Di; Yu, Hui-Ming; Lauderdale, Tsai-Ling; Chang, Kaichih; Shih, Chiaho

    2013-01-01

    The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176) and ARD I-III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I-IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD

  2. Identification of a Novel Antimicrobial Peptide from Human Hepatitis B Virus Core Protein Arginine-Rich Domain (ARD)

    PubMed Central

    Chen, Heng-Li; Su, Pei-Yi; Chang, Ya-Shu; Wu, Szu-Yao; Liao, You-Di; Yu, Hui-Ming; Lauderdale, Tsai-Ling; Chang, Kaichih; Shih, Chiaho

    2013-01-01

    The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I–IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II–IV (HBc153-176) and ARD I–III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I–IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that

  3. The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.

    PubMed

    Batut, Julie; Vandel, Laurence; Leclerc, Catherine; Daguzan, Christiane; Moreau, Marc; Néant, Isabelle

    2005-10-18

    We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.

  4. Mass Spectrometric Identification of the Arginine and Lysine deficient Proline Rich Glutamine Rich Wheat Storage Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tandem mass spectrometry (MS/MS) of enzymatic digest has made possible identification of a wide variety of proteins and complex samples prepared by such techniques as RP-HPLC or 2-D gel electrophoresis. Success requires peptide fragmentation to be indicative of the peptide amino acid sequence. The f...

  5. Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

    PubMed Central

    Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

    2013-01-01

    Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

  6. Purification and characterization of moschins, arginine-glutamate-rich proteins with translation-inhibiting activity from brown pumpkin (Cucurbita moschata) seeds.

    PubMed

    Ng, T B; Parkash, A; Tso, W W

    2002-10-01

    From fresh brown pumpkin seeds, two proteins with a molecular mass of 12kDa and an N-terminal sequence rich in arginine and glutamate residues were obtained. The protein designated alpha-moschin closely resembled the fruitfly programmed-cell death gene product and the protein designated beta-moschin demonstrated striking similarity to prepro 2S albumin in N-terminal sequence. alpha- and beta-moschins inhibited translation in the rabbit reticulocyte lysate system with an IC(50) of 17 microM and 300nM, respectively.

  7. Immunological responses as affected by dietary protein and arginine concentrations in starting broiler chicks.

    PubMed

    Jahanian, R

    2009-09-01

    The study presented here aimed to investigate the effect of dietary protein content on Arg needs and immunological responses of broiler chicks during the starter period. A total of 715 one-day-old male Ross broiler chicks were randomly assigned to 5 replicate pens for each of 11 experimental diets during a 21-d feeding trial. The dietary treatments included a corn-soybean meal control diet or experimental diets (corn-soybean meal-corn gluten meal) containing 5 dietary Arg levels of 80, 90, 100, 110, or 120% of NRC recommendations and 2 dietary protein levels of 19 and 22.35% of diet. Increasing dietary CP content significantly (P<0.001) increased daily feed consumption and weight gain. Also, feeding diets deficient in Arg to the chicks led to a noticeable decline in feed intake, and dietary Arg supplementation overcame decreased feed consumption and weight gain observed in Arg-deficient chicks. Feed efficiency was affected only by dietary Arg concentration so that chicks on Arg-deficient diets markedly (P<0.001) increased feed conversion ratio. Contrast comparisons showed that the highly variable responses of chicks to dietary Arg level were mainly attributed to dietary protein concentration: more dietary protein content and higher Arg demands. Among lymphoid organs, thymus (P<0.001) and spleen (P<0.05) were affected by dietary Arg deficiency, whereas diets low in CP content decreased (P<0.001) relative weights of thymus and bursa of Fabricius. Increase in dietary CP level from 19 to 22.35% caused an increase (P<0.001) in the proportion of lymphocytes and consequently lower (P<0.05) heterophil-to-lymphocyte ratio. Broiler chicks on Arg-deficient diets decreased the proportion of heterophils in peripheral blood. Furthermore, skin reaction to phytohemagglutinin P was impaired when the diets were low in CP and Arg contents. Similarly, a decrease in dietary CP and Arg levels diminished the antibody production response to Newcastle disease virus. The broken

  8. Implementing a Rational and Consistent Nomenclature for Serine/Arginine-Rich Protein Splicing Factors (SR Proteins) in Plants

    PubMed Central

    Barta, Andrea; Kalyna, Maria; Reddy, Anireddy S.N.

    2010-01-01

    Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species. PMID:20884799

  9. Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants.

    PubMed

    Barta, Andrea; Kalyna, Maria; Reddy, Anireddy S N

    2010-09-01

    Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species.

  10. Theoretical study of the mechanism of protein arginine deiminase 4 (PAD4) inhibition by F-amidine.

    PubMed

    Li, Dongmei; Liu, Cui; Lin, Jianping

    2015-02-01

    Protein arginine deiminase 4 (PAD4) catalyzes the hydrolysis of a peptidylarginine residue to form a citrulline residue and ammonia during posttranslational modification. This process plays a pivotal role in rheumatoid arthritis (RA) and gene regulation. F-amidine belongs to a series of haloacetamidine compounds that are the most potent PAD4 inhibitors described to date. F-amidine acts as a mechanism-based inhibitor of PAD4, inactivating PAD4 by the covalent modification of the active site Cys645. In this manuscript, the fundamental mechanism of PAD4 inhibition by F-amidine is investigated using a QM/MM approach. Our simulations show that in the PAD4-F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. This is consistent with the reverse protonation mechanism wherein the active site nucleophile, Cys645, in PAD4 exists as a thiolate in the active form of the enzyme. Inhibition of PAD4 by F-amidine is initiated by the nucleophilic addition of Sγ to the Cζ of F-amidine, leading to the formation of a tetrahedral intermediate. His471 serves as a proton donor, helping F to leave the fluoroacetamidine moiety of F-amidine; meanwhile, Sγ forms a three-membered ring with Cζ and Cη of F-amidine. Subsequently, the three-membered sulfonium ring collapses and rearranges to the final thioether product. His471 acts as a proton donor in the transition state and facilitates the inhibition reaction of PAD4.

  11. Homocysteine homeostasis in the rat is maintained by compensatory changes in cystathionine β-synthase, betaine-homocysteine methyltransferase, and phosphatidylethanolamine N-methyltransferase gene transcription occurring in response to maternal protein and folic acid intake during pregnancy and fat intake after weaning.

    PubMed

    Chmurzynska, Agata; Malinowska, Anna M

    2011-07-01

    The reactions of the methionine/homocysteine pathway are mediated by several enzymes, including phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase. Homocysteine homeostasis is regulated by these enzymes. We hypothesized here that the protein and folic acid content in the maternal diet affects methionine/homocysteine metabolism in the progeny. To test this hypothesis, pregnant rats were fed a diet with normal protein and normal folic acid levels (a modified casein-based AIN-93G diet), a protein-restricted and normal folic acid diet, a protein-restricted and folic acid-supplemented diet, or a normal protein and folic acid-supplemented diet. The progeny were fed either the modified AIN-93G diet or a high-fat lard-based diet. Progeny were analyzed for expression of the phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase genes in the liver and for serum homocysteine concentration. Interactions between prenatal and postnatal nutrition were also determined. The progeny of the dams fed the diets supplemented with folic acid showed decreased expression of all 3 genes (P < .001). An interaction effect between the protein and folic acid content in the maternal diet contributed to this down-regulation (P < .001), and the postweaning diet modified these effects. Serum homocysteine concentrations were approximately 15% higher in the male rats (P < .01), but neither prenatal nutrition nor the postweaning diet affected it significantly. We conclude that maternal diet during gestation has an important effect on the transcription level of these 3 genes, but changes in gene expression were not associated with significant changes in progeny homocysteine concentrations.

  12. Arginine methylation initiates BMP-induced Smad signaling

    PubMed Central

    Xu, Jian; Wang, A. Hongjun; Oses-Prieto, Juan; Makhijani, Kalpana; Katsuno, Yoko; Pei, Ming; Yan, Leilei; Zheng, Y. George; Burlingame, Alma; Brückner, Katja; Derynck, Rik

    2014-01-01

    Summary Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-β family ligands, induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases. However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in the initiation of BMP signaling. We now show that arginine methylation, which is known to regulate gene expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling. BMP-induced receptor complex formation promotes interaction of the methyltransferase PRMT1 with the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the receptor, leading to activation of effector Smads through phosphorylation. PRMT1 is required for BMP-induced biological responses across species, as evidenced by the role of its ortholog Dart1 in BMP signaling during Drosophila wing development. Activation of signaling by arginine methylation may also apply to other signaling pathways. PMID:23747011

  13. An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE[C][W

    PubMed Central

    Nayak, Nihar R.; Putnam, Andrea A.; Addepalli, Balasubrahmanyam; Lowenson, Jonathan D.; Chen, Tingsu; Jankowsky, Eckhard; Perry, Sharyn E.; Dinkins, Randy D.; Limbach, Patrick A.; Clarke, Steven G.; Downie, A. Bruce

    2013-01-01

    Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75’s complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination. PMID:23903319

  14. FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System

    PubMed Central

    Willenborg, Jörg; Koczula, Anna; Fulde, Marcus; de Greeff, Astrid; Beineke, Andreas; Eisenreich, Wolfgang; Huber, Claudia; Seitz, Maren; Valentin-Weigand, Peter; Goethe, Ralph

    2016-01-01

    Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system. PMID:27455333

  15. FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System.

    PubMed

    Willenborg, Jörg; Koczula, Anna; Fulde, Marcus; de Greeff, Astrid; Beineke, Andreas; Eisenreich, Wolfgang; Huber, Claudia; Seitz, Maren; Valentin-Weigand, Peter; Goethe, Ralph

    2016-07-21

    Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system.

  16. Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding.

    PubMed

    Chatterjee, Tanaya; Mukherjee, Debadrita; Banerjee, Mousumi; Chatterjee, Barun K; Chakrabarti, Pinak

    2015-10-01

    The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39-58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 10(6) M(-1), quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.

  17. Protein-L-Isoaspartyl Methyltransferase (PIMT) Is Required for Survival of Salmonella Typhimurium at 42°C and Contributes to the Virulence in Poultry

    PubMed Central

    Pesingi, Pavan K.; Kumawat, Manoj; Behera, Pranatee; Dixit, Sunil K.; Agarwal, Rajesh K.; Goswami, Tapas K.; Mahawar, Manish

    2017-01-01

    Poultry birds are asymptomatic reservoir of Salmonella Typhimurium (S. Typhimurium) but act as source of human infection for this bacterium. Inside the poultry, S. Typhimurium experiences several stresses, 42°C body temperature of birds is one of them. Proteins are highly susceptible to temperature mediated damage. Conversion of protein bound aspartate (Asp) residues to iso-aspartate (iso-Asp) is one of such modifications that occur at elevated temperature. Iso-Asp formation has been linked to protein inactivation and compromised cellular survival. Protein-L-isoaspartyl methyltransferase (PIMT) can repair iso-Asp back to Asp, thus enhances the cellular survival at elevated temperature. Here, we show that the pimt gene deletion strain of S. Typhimurium (Δpimt mutant strain) is hypersensitive to 42°C in vitro. The hypersusceptibility of Δpimt strain is partially reversed by plasmid based complementation (trans-complementation) of Δpimt strain. Following oral inoculation, Δpimt strain showed defective colonization in poultry caecum, and compromised dissemination to spleen and liver. Interestingly, we have observed three and half folds induction of the PIMT protein following exposure of S. Typhimurium to 42°C. Our data suggest a novel role of pimt gene in the survival of S. Typhimurium at elevated temperature and virulence. PMID:28326072

  18. Protein-L-Isoaspartyl Methyltransferase (PIMT) Is Required for Survival of Salmonella Typhimurium at 42°C and Contributes to the Virulence in Poultry.

    PubMed

    Pesingi, Pavan K; Kumawat, Manoj; Behera, Pranatee; Dixit, Sunil K; Agarwal, Rajesh K; Goswami, Tapas K; Mahawar, Manish

    2017-01-01

    Poultry birds are asymptomatic reservoir of Salmonella Typhimurium (S. Typhimurium) but act as source of human infection for this bacterium. Inside the poultry, S. Typhimurium experiences several stresses, 42°C body temperature of birds is one of them. Proteins are highly susceptible to temperature mediated damage. Conversion of protein bound aspartate (Asp) residues to iso-aspartate (iso-Asp) is one of such modifications that occur at elevated temperature. Iso-Asp formation has been linked to protein inactivation and compromised cellular survival. Protein-L-isoaspartyl methyltransferase (PIMT) can repair iso-Asp back to Asp, thus enhances the cellular survival at elevated temperature. Here, we show that the pimt gene deletion strain of S. Typhimurium (Δpimt mutant strain) is hypersensitive to 42°C in vitro. The hypersusceptibility of Δpimt strain is partially reversed by plasmid based complementation (trans-complementation) of Δpimt strain. Following oral inoculation, Δpimt strain showed defective colonization in poultry caecum, and compromised dissemination to spleen and liver. Interestingly, we have observed three and half folds induction of the PIMT protein following exposure of S. Typhimurium to 42°C. Our data suggest a novel role of pimt gene in the survival of S. Typhimurium at elevated temperature and virulence.

  19. Arginine Methylation by PRMT1 Regulates Muscle Stem Cell Fate

    PubMed Central

    Blanc, Roméo Sébastien; Vogel, Gillian; Li, Xing; Yu, Zhenbao; Li, Shawn

    2016-01-01

    ABSTRACT Quiescent muscle stem cells (MSCs) become activated in response to skeletal muscle injury to initiate regeneration. Activated MSCs proliferate and differentiate to repair damaged fibers or self-renew to maintain the pool and ensure future regeneration. The balance between self-renewal, proliferation, and differentiation is a tightly regulated process controlled by a genetic cascade involving determinant transcription factors such as Pax7, Myf5, MyoD, and MyoG. Recently, there have been several reports about the role of arginine methylation as a requirement for epigenetically mediated control of muscle regeneration. Here we report that the protein arginine methyltransferase 1 (PRMT1) is expressed in MSCs and that conditional ablation of PRMT1 in MSCs using Pax7CreERT2 causes impairment of muscle regeneration. Importantly, PRMT1-deficient MSCs have enhanced cell proliferation after injury but are unable to terminate the myogenic differentiation program, leading to regeneration failure. We identify the coactivator of Six1, Eya1, as a substrate of PRMT1. We show that PRMT1 methylates Eya1 in vitro and that loss of PRMT1 function in vivo prevents Eya1 methylation. Moreover, we observe that PRMT1-deficient MSCs have reduced expression of Eya1/Six1 target MyoD due to disruption of Eya1 recruitment at the MyoD promoter and subsequent Eya1-mediated coactivation. These findings suggest that arginine methylation by PRMT1 regulates muscle stem cell fate through the Eya1/Six1/MyoD axis. PMID:27849571

  20. Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER

    PubMed Central

    2009-01-01

    Background In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails. Results Here, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport. Conclusion Altogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type

  1. Arginine Metabolism in Developing Soybean Cotyledons 1

    PubMed Central

    Micallef, Barry J.; Shelp, Barry J.

    1989-01-01

    The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-14C]glutamate and [U-14C]glutamine were fed to excised cotyledons. After 4 hours, 14C was incorporated into protein and released as 14CO2, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ. PMID:16666818

  2. Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

    SciTech Connect

    Luka, Zigmund; Pakhomova, Svetlana; Loukachevitch, Lioudmila V; Newcomer, Marcia E; Wagner, Conrad

    2012-06-27

    Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

  3. Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl CpG-/CpG domain-binding proteins in embryonic orofacial cells.

    PubMed

    Mukhopadhyay, Partha; Greene, Robert M; Pisano, M Michele

    2015-12-01

    Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80μg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting.

  4. CIGARETTE SMOKE INDUCES PROTEASOMAL-MEDIATED DEGRADATION OF DNA METHYLTRANSFERASES AND METHYL CpG-/CpG DOMAIN-BINDING PROTEINS IN EMBRYONIC OROFACIAL CELLS

    PubMed Central

    Mukhopadhyay, Partha; Greene, Robert M.; Pisano, M. Michele

    2015-01-01

    Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80 μg/ml cigarette smoke extract (CSE) for 24 hrs, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1, and - 3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting. PMID:26482727

  5. Arginine metabolism in developing soybean cotyledons

    SciTech Connect

    Micallef, B.J.; Shelp, B.J. )

    1989-09-01

    Tracerkinetic experiments were performed using L-(guanidino-{sup 14}C)arginine, L-(U-{sup 14}C)arginine, L-(ureido-{sup 14}C)citrulline, and L-(1-{sup 14}C)ornithine to investigate arginine utilization in developing cotyledons of Gycine max (L.) Merrill. Excised cotyledons were injected with carrier-free {sup 14}C compounds and incubated in sealed vials containing a CO{sub 2} trap. The free and protein amino acids were analyzed using higher performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the {sup 14}C metabolized from (guanidino-{sup 14}C)arginine and (U-{sup 14}C)arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours {sup 14}CO{sub 2} was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra {sup 14}CO{sub 2} was evolved during the catabolism of ornithine-derived glutamate; {sup 14}C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.

  6. Physiological implications of arginine metabolism in plants

    PubMed Central

    Winter, Gudrun; Todd, Christopher D.; Trovato, Maurizio; Forlani, Giuseppe; Funck, Dietmar

    2015-01-01

    Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions. PMID:26284079

  7. Enzymology of Mammalian DNA Methyltransferases.

    PubMed

    Jurkowska, Renata Z; Jeltsch, Albert

    2016-01-01

    DNA methylation is currently one of the hottest topics in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA nucleotide methyltransferases (DNMTs), principles of their regulation in cells have only begun to be uncovered. In mammals, DNA methylation is introduced by the DNMT1, DNMT3A, and DNMT3B enzymes, which are all large multi-domain proteins. These enzymes contain a catalytic C-terminal domain with a characteristic cytosine-C5 methyltransferase fold and an N-terminal part with different domains that interacts with other proteins and chromatin and is involved in targeting and regulation of the DNMTs. The subnuclear localization of the DNMT enzymes plays an important role in their biological function: DNMT1 is localized to replicating DNA via interaction with PCNA and UHRF1. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD and PWWP domains. Recently, a novel regulatory mechanism has been discovered in DNMTs, as latest structural and functional data demonstrated that the catalytic activities of all three enzymes are under tight allosteric control of their N-terminal domains having autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of autoinhibitory domains by protein factors, noncoding RNAs, or by posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, including their specificity and processivity, and afterward we focus on the regulation of their activity and targeting via allosteric processes, protein interactors, and posttranslational modifications.

  8. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

    PubMed

    Jarmalavicius, Saulius; Trefzer, Uwe; Walden, Peter

    2010-03-01

    The aim of the study was to identify as potential therapeutic targets specific molecular alterations in tumor cells recognized by the immune system. To identify such targets, we analyzed the human leukocyte antigen (HLA) peptidomes of human melanoma cells by 2-dimensional nano-HPLC/mass spectrometry and tested the immunological significance of the peptides by ex vivo ELISpot assays with lymphocytes from melanoma patients. The peptide SQNPRFYHK was identified as derived from the regulator of the nuclear corepressor complex (NCoR) G-protein pathway suppressor 2 (GPS-2) and to be differentially unmethylated, monomethylated or asymmetrically dimethylated at the arginine. The methylation state was specifically recognized by the immune system in that only the monomethylated variant induced T-cell responses and significantly stronger responses in patients than in healthy controls. The methylations were confirmed with synthetic analogues and in vitro radiolabeling assays with recombinant GPS-2 and synthetic peptides. The immunity of the 3 variants of GPS-2 was tested in T-cell assays with T lymphocytes of melanoma patients compared with healthy donors. The results show for the first time that GPS-2 is differentially methylated at a site that lacks known methylation motifs and that the methylation state is detected by the immune system.-Jarmalavicius, S., Trefzer, U., Walden, P. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

  9. Caffeine synthase and related methyltransferases in plants.

    PubMed

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  10. The BioC O-Methyltransferase Catalyzes Methyl Esterification of Malonyl-Acyl Carrier Protein, an Essential Step in Biotin Synthesis*

    PubMed Central

    Lin, Steven; Cronan, John E.

    2012-01-01

    Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682–688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-l-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-l-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis. PMID:22965231

  11. A chimera carrying the functional domain of the orphan protein SLC7A14 in the backbone of SLC7A2 mediates trans-stimulated arginine transport.

    PubMed

    Jaenecke, Isabel; Boissel, Jean-Paul; Lemke, Matthias; Rupp, Johanna; Gasnier, Bruno; Closs, Ellen I

    2012-08-31

    In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker(®). To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called "functional domain" of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-L-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.

  12. Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

    PubMed

    Li, Xiao-Ping; Kahn, Peter C; Kahn, Jennifer Nielsen; Grela, Przemyslaw; Tumer, Nilgun E

    2013-10-18

    Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K(m) values and catalytic rates (k(cat)) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K(m) and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

  13. Depletion of Arabidopsis SC35 and SC35-like serine/arginine-rich proteins affects the transcription and splicing of a subset of genes

    PubMed Central

    Xia, Xi; Sun, Zhenfei

    2017-01-01

    Serine/arginine-rich (SR) proteins are important splicing factors which play significant roles in spliceosome assembly and splicing regulation. However, little is known regarding their biological functions in plants. Here, we analyzed the phenotypes of mutants upon depleting different subfamilies of Arabidopsis SR proteins. We found that loss of the functions of SC35 and SC35-like (SCL) proteins cause pleiotropic changes in plant morphology and development, including serrated leaves, late flowering, shorter roots and abnormal silique phyllotaxy. Using RNA-seq, we found that SC35 and SCL proteins play roles in the pre-mRNA splicing. Motif analysis revealed that SC35 and SCL proteins preferentially bind to a specific RNA sequence containing the AGAAGA motif. In addition, the transcriptions of a subset of genes are affected by the deletion of SC35 and SCL proteins which interact with NRPB4, a specific subunit of RNA polymerase II. The splicing of FLOWERING LOCUS C (FLC) intron1 and transcription of FLC were significantly regulated by SC35 and SCL proteins to control Arabidopsis flowering. Therefore, our findings provide mechanistic insight into the functions of plant SC35 and SCL proteins in the regulation of splicing and transcription in a direct or indirect manner to maintain the proper expression of genes and development. PMID:28273088

  14. Conjugation to octa-arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization.

    PubMed

    Kuwada, Eri; Tadaki, Toshimasa; Kambara, Kaori; Egawa, Kohji; Noguchi, Katsuo

    2011-01-01

    Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.

  15. Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

    PubMed

    Snijders, Ambrosius P; Hautbergue, Guillaume M; Bloom, Alex; Williamson, James C; Minshull, Thomas C; Phillips, Helen L; Mihaylov, Simeon R; Gjerde, Douglas T; Hornby, David P; Wilson, Stuart A; Hurd, Paul J; Dickman, Mark J

    2015-03-01

    Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

  16. A putative transport protein is involved in citrulline excretion and re-uptake during arginine deiminase pathway activity by Lactobacillus sakei.

    PubMed

    Rimaux, Tom; Rivière, Audrey; Hebert, Elvira María; Mozzi, Fernanda; Weckx, Stefan; De Vuyst, Luc; Leroy, Frédéric

    2013-04-01

    Arginine conversion through the arginine deiminase (ADI) pathway is a common metabolic trait of Lactobacillus sakei which is ascribed to an arc operon and which inquisitively involves citrulline excretion and re-uptake. The aim of this study was to verify whether a putative transport protein (encoded by the PTP gene) plays a role in citrulline-into-ornithine conversion by L. sakei strains. This was achieved through a combination of fermentation experiments, gene expression analysis via quantitative real-time reverse transcription PCR (RT-qPCR) and construction of a PTP knock-out mutant. Expression of the PTP gene was modulated by environmental pH and was highest in the end-exponential or mid-exponential growth phase for L. sakei strains CTC 494 and 23K, respectively. In contrast to known genes of the arc operon, the PTP gene showed low expression at pH 7.0, in agreement with the finding that citrulline-into-ornithine conversion is inhibited at this pH. The presence of additional energy sources also influenced ADI pathway activity, in particular by decreasing citrulline-into-ornithine conversion. Further insight into the functionality of the PTP gene was obtained with a knock-out mutant of L. sakei CTC 494 impaired in the PTP gene, which displayed inhibition in its ability to convert extracellular citrulline into ornithine. In conclusion, results indicated that the PTP gene may putatively encode a citrulline/ornithine antiporter.

  17. The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis

    PubMed Central

    Otsuka, Taketo; Kirkham, Charmaine; Brauer, Aimee; Koszelak-Rosenblum, Mary; Malkowski, Michael G.

    2015-01-01

    Moraxella catarrhalis is an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to prevent M. catarrhalis infections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface of M. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement of M. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth of M. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen. PMID:26597985

  18. Protein Lysine Methyltransferase G9a Inhibitors: Design, Synthesis, and Structure Activity Relationships of 2,4-Diamino-7-aminoalkoxy-quinazolines.†

    PubMed Central

    Liu, Feng; Chen, Xin; Allali-Hassani, Abdellah; Quinn, Amy M.; Wigle, Tim J.; Wasney, Gregory A.; Dong, Aiping; Senisterra, Guillermo; Chau, Irene; Siarheyeva, Alena; Norris, Jacqueline L.; Kireev, Dmitri B.; Jadhav, Ajit; Herold, J. Martin; Janzen, William P.; Arrowsmith, Cheryl H.; Frye, Stephen V.; Brown, Peter J.; Simeonov, Anton; Vedadi, Masoud; Jin, Jian

    2010-01-01

    Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is over expressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth and the di-methylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first co-crystal structure of G9a with a small molecule inhibitor, was obtained. Based on the structural insights revealed by this co-crystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison Ki = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date. PMID:20614940

  19. Protein lysine methyltransferase G9a inhibitors: design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines.

    PubMed

    Liu, Feng; Chen, Xin; Allali-Hassani, Abdellah; Quinn, Amy M; Wigle, Tim J; Wasney, Gregory A; Dong, Aiping; Senisterra, Guillermo; Chau, Irene; Siarheyeva, Alena; Norris, Jacqueline L; Kireev, Dmitri B; Jadhav, Ajit; Herold, J Martin; Janzen, William P; Arrowsmith, Cheryl H; Frye, Stephen V; Brown, Peter J; Simeonov, Anton; Vedadi, Masoud; Jin, Jian

    2010-08-12

    Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is overexpressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth, and the dimethylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. On the basis of the structural insights revealed by this cocrystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison K(i) = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date.

  20. The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

    PubMed Central

    Senthong, Pattama; Millington, Christopher L.; Wilkinson, Oliver J.; Marriott, Andrew S.; Watson, Amanda J.; Reamtong, Onrapak; Eyers, Claire E.; Williams, David M.; Margison, Geoffrey P.; Povey, Andrew C.

    2013-01-01

    The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O6-carboxymethylguanine (O6-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O6-CMG is not a substrate for the human version of the DNA damage reversal protein O6-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O6-alkylguanine lesions by removing alkyl groups from the O6-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O6-methylguanine (O6-MeG) or O6-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O6-alkyl group and its transfer to the active-site cysteine residue of MGMT. O6-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O6-CMG is a potential causative agent. PMID:23335782

  1. Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

    PubMed

    Göder, Anja; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-08-01

    Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.

  2. A novel role for protein arginine deiminase 4 in pluripotency: the emerging role of citrullinated histone H1 in cellular programming.

    PubMed

    Slade, Daniel J; Horibata, Sachi; Coonrod, Scott A; Thompson, Paul R

    2014-08-01

    Histone post-translational modifications (PTMs) alter the chromatin architecture, generating "open" and "closed" states, and these structural changes can modulate gene expression under specific cellular conditions. While methylation and acetylation are the best-characterized histone PTMs, citrullination by the protein arginine deiminases (PADs) represents another important player in this process. In addition to "fine tuning" chromatin structure at specific loci, histone citrullination can also promote rapid global chromatin decondensation during the formation of extracellular traps (ETs) in immune cells. Recent studies now show that PAD4-mediated citrullination of histone H1 at promoter elements can also promote localized chromatin decondensation in stem cells, thus regulating the pluripotent state. These observations suggest that PAD-mediated histone deimination profoundly affects chromatin structure, possibly above and beyond that of other PTMs. Additionally, these recent findings further enhance our understanding of PAD biology and the important contributions that these enzymes play in development, health, and disease.

  3. Arginine requirement of starting broiler chicks.

    PubMed

    Cuca, M; Jensen, L S

    1990-08-01

    Three experiments were conducted to estimate the arginine requirement of male broiler chicks from 0 to 3 wk of age. The experiments were conducted in battery brooders with wires floors, and the birds received water and feed ad libitum. In the first experiment, chicks were fed a diet based on corn, soybean meal, casein, and corn-gluten meal containing 3,200 kcal ME per kg and either 20 or 23% crude protein. Regression analysis indicated an arginine requirement of 1.22% for maximum growth rate and feed efficiency with the 20% protein diet. For chicks fed the 23% protein diet, neither growth rate nor feed efficiency was significantly different among the diets containing arginine ranging from 1.13 to 1.43%. In the second experiment, a basal diet was used containing 17.5% casein and 22.5% protein with arginine ranging from 1.03 to 1.43%. An arginine requirement of 1.18% for maximum body weight gain was estimated by regression analysis, but no significant response to arginine above the basal level was observed for feed efficiency. Performance of chicks fed the basal diet was somewhat reduced because of a difficulty with adherence of feed to the beaks. In a third experiment, three basal diets containing 21, 22, or 23% protein were formulated from practical ingredients without use of casein. The requirement for maximum growth rate and feed efficiency was estimated to be 1.24 to 1.28% for the three diets. The results of these investigations indicate that the arginine requirement for starting chicks suggested by the National Research Council in 1984 of 1.44% in diets containing 3,200 kcal ME per kg is too high for practical diets. The data presented here support an arginine requirement of 1.25%.

  4. Functional Analysis of Genetic Variation in Catechol-O-Methyltransferase (COMT): Effects on mRNA, Protein, and Enzyme Activity in Postmortem Human Brain

    PubMed Central

    Chen, Jingshan; Lipska, Barbara K.; Halim, Nader; Ma, Quang D.; Matsumoto, Mitsuyuki; Melhem, Samer; Kolachana, Bhaskar S.; Hyde, Thomas M.; Herman, Mary M.; Apud, Jose; Egan, Michael F.; Kleinman, Joel E.; Weinberger, Daniel R.

    2004-01-01

    Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3′ flanking region (rs165599)—both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val—had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3′ SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a

  5. The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

    PubMed Central

    Kheddo, Priscilla; Golovanov, Alexander P.; Mellody, Kieran T.; Uddin, Shahid; van der Walle, Christopher F.; Dearman, Rebecca J.

    2016-01-01

    The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~ 400 mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo. PMID:26873863

  6. Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome.

    PubMed

    Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

    2015-08-14

    PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome.

  7. Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome*

    PubMed Central

    Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

    2015-01-01

    PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. PMID:26149688

  8. Supplemental effects and metabolic fate of crystalline arginine in juvenile shrimp Marsupenaeus japonicus.

    PubMed

    Teshima, Shin-ichi; Ishikawa, Manabu; Shah Alam, Md; Koshio, Shunsuke; Michael, Fady Raafat

    2004-02-01

    Feeding experiments with juvenile kuruma shrimp (Marsupenaeus japonicus) were conducted to understand the effects of supplemental levels of crystalline arginine hydrochloride on the growth and assimilation of arginine. In experiment 1 the juvenile shrimp were maintained on diets with and without arginine supplements. The addition of 3.0% arginine to a casein-based diet was slightly effective in improving the growth of the juveniles. In experiment 2, tracer experiments using [14C] arginine were conducted to clarify the ingestion and assimilation of arginine, 9 and 24 h after feeding, at different levels of supplemental arginine. Tracer experiments showed that the leaching rate of arginine 1 h after feeding ranged from 16 to 26% in the diets with different levels (0, 0.1, 1.5, 3.0 and 6.0%) of supplemental arginine; that is, 74-84% of the given arginine was actually ingested by the shrimp fed the diets. However, with increasing levels of supplemental arginine the incorporation rate of arginine into the whole body decreased and the utilization of absorbed arginine for body protein synthesis was reduced, whereas the excretion of absorbed arginine was increased. Thus, the absorbed arginine was not effectively utilized for body protein synthesis when large amounts of arginine were supplemented to the diets.

  9. Chemical modification of arginine residues in the lactose repressor

    SciTech Connect

    Whitson, P.A.; Matthews, K.S.

    1987-10-06

    The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino aced analysis or incorporation of /sup 14/C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of approx. 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.

  10. Arginine metabolism: nitric oxide and beyond.

    PubMed Central

    Wu, G; Morris, S M

    1998-01-01

    Arginine is one of the most versatile amino acids in animal cells, serving as a precursor for the synthesis not only of proteins but also of nitric oxide, urea, polyamines, proline, glutamate, creatine and agmatine. Of the enzymes that catalyse rate-controlling steps in arginine synthesis and catabolism, argininosuccinate synthase, the two arginase isoenzymes, the three nitric oxide synthase isoenzymes and arginine decarboxylase have been recognized in recent years as key factors in regulating newly identified aspects of arginine metabolism. In particular, changes in the activities of argininosuccinate synthase, the arginases, the inducible isoenzyme of nitric oxide synthase and also cationic amino acid transporters play major roles in determining the metabolic fates of arginine in health and disease, and recent studies have identified complex patterns of interaction among these enzymes. There is growing interest in the potential roles of the arginase isoenzymes as regulators of the synthesis of nitric oxide, polyamines, proline and glutamate. Physiological roles and relationships between the pathways of arginine synthesis and catabolism in vivo are complex and difficult to analyse, owing to compartmentalized expression of various enzymes at both organ (e.g. liver, small intestine and kidney) and subcellular (cytosol and mitochondria) levels, as well as to changes in expression during development and in response to diet, hormones and cytokines. The ongoing development of new cell lines and animal models using cDNA clones and genes for key arginine metabolic enzymes will provide new approaches more clearly elucidating the physiological roles of these enzymes. PMID:9806879

  11. The Fusobacterium nucleatum Outer Membrane Protein RadD Is an Arginine-Inhibitable Adhesin Required for Inter-Species Adherence and the Structured Architecture of Multi-Species Biofilm

    PubMed Central

    Kaplan, Christopher W.; Lux, Renate; Haake, Susan Kinder; Shi, Wenyuan

    2009-01-01

    Summary A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation. PMID:19007407

  12. Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

    PubMed

    Madeira, Marta S; Rolo, Eva S; Alfaia, Cristina M; Pires, Virgínia R; Luxton, Richard; Doran, Olena; Bessa, Rui J B; Prates, José A M

    2016-03-28

    The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

  13. Regulation of macrophage functions by L-arginine

    PubMed Central

    1989-01-01

    Sites of inflammation with prominent macrophage infiltration, such as wounds and certain tumors, are uniquely deficient in free arginine. The effects of arginine availability on macrophage physiology were investigated. When cultured in media containing less than 0.1 mM L- arginine, rat resident peritoneal macrophages exhibited enhanced spreading, tumor cytotoxicity, superoxide production, phagocytosis, and protein synthesis. Thus, arginine concentrations similar to those found in sites of inflammation can augment macrophage functions, while those found in plasma (approximately 0.1 mM) and in commonly used culture media (0.4 to 1.2 mM) are inhibitory. Culture in homoarginine, but not D-arginine, ornithine, citrulline, urea, histidine, or lysine also inhibited macrophage tumor cytotoxicity, indicating the specificity of the effect. In contrast to resident macrophages, the tumor cytotoxicity of peritoneal macrophages obtained after C. parvum injection was suppressed by culture in arginine-deficient media. However, L-arginine- deficient media enhanced all other activation-associated functions in C. parvum-elicited macrophages as in resident cells. Arginine-free wound fluid promoted resident macrophage tumoricidal activity when compared with rat serum, and again, the addition of L-arginine was inhibitory. The marked effects of L-arginine availability on macrophage functions, together with the knowledge that these cells modify the extracellular arginine concentration in sites of inflammation through arginase, provide evidence for an autoregulatory mechanism of macrophage activation. PMID:2538541

  14. Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006

    PubMed Central

    Ouellette, Matthew; Jackson, Laura; Chimileski, Scott; Papke, R. Thane

    2015-01-01

    Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: Cm4TAG and GCAm6BN6VTGC. Analysis of the ΔHVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006. PMID:25904898

  15. SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth1[W

    PubMed Central

    Bhatia, Shikha; Gangappa, Sreeramaiah N.; Kuswaha, Ritu; Kundu, Snehangshu; Chattopadhyay, Sudip

    2008-01-01

    Light is an important factor for plant growth and development. We have identified and functionally characterized a regulatory gene SHORT HYPOCOTYL IN WHITE LIGHT1 (SHW1) involved in Arabidopsis (Arabidopsis thaliana) seedling development. SHW1 encodes a unique serine-arginine-aspartate-rich protein, which is constitutively localized in the nucleus of hypocotyl cells. Transgenic analyses have revealed that the expression of SHW1 is developmentally regulated and is closely associated with the photosynthetically active tissues. Genetic and molecular analyses suggest that SHW1 acts as a negative regulator of light-mediated inhibition of hypocotyl elongation, however, plays a positive regulatory role in light-regulated gene expression. The shw1 mutants also display shorter hypocotyl in dark, and analyses of shw1 cop1 double mutants reveal that SHW1 acts nonredundantly with COP1 to control hypocotyl elongation in the darkness. Taken together, this study provides evidences that SHW1 is a regulatory protein that is functionally interrelated to COP1 and plays dual but opposite regulatory roles in photomorphogenesis. PMID:18375596

  16. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

    SciTech Connect

    Yu, Wenyu; Chory, Emma J.; Wernimont, Amy K.; Tempel, Wolfram; Scopton, Alex; Federation, Alexander; Marineau, Jason J.; Qi, Jun; Barsyte-Lovejoy, Dalia; Yi, Joanna; Marcellus, Richard; Iacob, Roxana E.; Engen, John R.; Griffin, Carly; Aman, Ahmed; Wienholds, Erno; Li, Fengling; Pineda, Javier; Estiu, Guillermina; Shatseva, Tatiana; Hajian, Taraneh; Al-awar, Rima; Dick, John E.; Vedadi, Masoud; Brown, Peter J.; Arrowsmith, Cheryl H.; Bradner, James E.; Schapira, Matthieu

    2012-12-18

    Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound toEPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

  17. Serine-arginine protein kinase 1 (SRPK1), a determinant of angiogenesis, is upregulated in prostate cancer and correlates with disease stage and invasion.

    PubMed

    Bullock, Nicholas; Potts, Jonathan; Simpkin, Andrew J; Koupparis, Anthony; Harper, Steve J; Oxley, Jon; Oltean, Sebastian

    2016-02-01

    Vascular endothelial growth factor (VEGF) undergoes alternative splicing to produce both proangiogenic and antiangiogenic isoforms. Preferential splicing of proangiogenic VEGF is determined by serine-arginine protein kinase 1 (SRPK1), which is upregulated in a number of cancers. In the present study, we aimed to investigate SRPK1 expression in prostate cancer (PCa) and its association with cancer progression. SRPK1 expression was assessed using immunohistochemistry of PCa tissue extracted from radical prostatectomy specimens of 110 patients. SRPK1 expression was significantly higher in tumour compared with benign tissue (p<0.00001) and correlated with higher pT stage (p=0.004), extracapsular extension (p=0.003) and extracapsular perineural invasion (p=0.008). Interestingly, the expression did not correlate with Gleason grade (p=0.21), suggesting that SRPK1 facilitates the development of a tumour microenvironment that favours growth and invasion (possibly through stimulating angiogenesis) while having little bearing on the morphology or function of the tumour cells themselves.

  18. An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.

    PubMed

    Pattanayek, Rekha; Xu, Yao; Lamichhane, Aashish; Johnson, Carl H; Egli, Martin

    2014-05-01

    A post-translational oscillator (PTO) composed of the proteins KaiA, KaiB and KaiC is at the heart of the cyanobacterial circadian clock. KaiC interacts with KaiA and KaiB over the daily cycle, and CII domains undergo rhythmic phosphorylation/dephosphorylation with a 24 h period. Both the N-terminal (CI) and C-terminal (CII) rings of KaiC exhibit ATPase activity. The CI ATPase proceeds in an input-independent fashion, but the CII ATPase is subject to metabolic input signals. The crystal structure of KaiC from Thermosynechococcus elongatus allows insight into the different anatomies of the CI and CII ATPases. Four consecutive arginines in CI (Arg linker) that connect the P-loop, CI subunits and CI and CII at the ring interface are primary candidates for the coordination of the CI and CII activities. The mutation of linker residues alters the period or triggers arhythmic behavior. Comparison between the CI and CII structures also reveals differences in loop regions that are key to KaiA and KaiB binding and activation of CII ATPase and kinase. Common packing features in KaiC crystals shed light on the KaiB-KaiC interaction.

  19. Effects of arginine on multimodal anion exchange chromatography.

    PubMed

    Hirano, Atsushi; Arakawa, Tsutomu; Kameda, Tomoshi

    2015-12-01

    The effects of arginine on binding and elution properties of a multimodal anion exchanger, Capto adhere, were examined using bovine serum albumin (BSA) and a monoclonal antibody against interleukin-8 (mAb-IL8). Negatively charged BSA was bound to the positively charged Capto adhere and was readily eluted from the column with a stepwise or gradient elution using 1M NaCl at pH 7.0. For heat-treated BSA, small oligomers and remaining monomers were also eluted using a NaCl gradient, whereas larger oligomers required arginine for effective elution. The positively charged mAb-IL8 was bound to Capto adhere at pH 7.0. Arginine was also more effective for elution of the bound mAb-IL8 than was NaCl. The results imply that arginine interacts with the positively charged Capto adhere. The mechanism underlying the interactions of arginine with Capto adhere was examined by calculating the binding free energy between an arginine molecule and a Capto adhere ligand in water through molecular dynamics simulations. The overall affinity of arginine for Capto adhere is attributed to the hydrophobic and π-π interactions between an arginine side chain and the aromatic moiety of the ligand as well as hydrogen bonding between arginine and the ligand hydroxyl group, which may account for the characteristics of protein elution using arginine.

  20. Proline/arginine-rich end leucine-rich repeat protein N-terminus is a novel osteoclast antagonist that counteracts bone loss.

    PubMed

    Rucci, Nadia; Capulli, Mattia; Ventura, Luca; Angelucci, Adriano; Peruzzi, Barbara; Tillgren, Viveka; Muraca, Maurizio; Heinegård, Dick; Teti, Anna

    2013-09-01

    (hbd) PRELP is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (hbd) PRELP inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-κB transcription factor activity. In this work, we hypothesized that (hbd) PRELP could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (hbd) PRELP counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (hbd) PRELP reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (hbd) PRELP also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (hbd) PRELP was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (hbd) PRELP could be employed as a

  1. Arginine Vasopressin Potentiates the Stimulatory Action of CRH on Pituitary Corticotropes via a Protein Kinase C-Dependent Reduction of the Background TREK-1 Current.

    PubMed

    Lee, Andy K; Tse, Frederick W; Tse, Amy

    2015-10-01

    The hypothalamic hormone arginine vasopressin (AVP) potentiates the stimulatory action of CRH on ACTH secretion from pituitary corticotropes, but the underlying mechanism is elusive. Using the perforated patch-clamp technique to monitor membrane potentials in mouse corticotropes, we found that AVP triggered a transient hyperpolarization that was followed by a sustained depolarization. The hyperpolarization was caused by intracellular Ca(2+) release that in turn activated the small conductance Ca(2+)-activated K(+) (SK) channels. The depolarization was due to the suppression of background TWIK-related K(+) (TREK)-1 channels. Direct activation of protein kinase C (PKC) reduced the TREK-1 current, whereas PKC inhibition attenuated the AVP-mediated reduction of the TREK-1 current, implicating the involvement of PKC. The addition of CRH (which stimulates the protein kinase A pathway) in the presence of AVP, or vice versa, resulted in further suppression of the TREK-1 current. In corticotropes with buffered cytosolic Ca(2+) concentration ([Ca(2+)]i), AVP evoked a sustained depolarization, and the coapplication of AVP and CRH caused a larger depolarization than that evoked by AVP or CRH alone. In cells with minimal perturbation of [Ca(2+)]i and background TREK-1 channels, CRH evoked a sustained depolarization that was superimposed with action potentials, and the subsequent coapplication of AVP and CRH triggered a transient hyperpolarization that was followed by a larger depolarization. In summary, AVP and CRH have additive effects on the suppression of the TREK-1 current, resulting in a more robust depolarization in corticotropes. We suggest that this mechanism contributes to the potentiating action of AVP on CRH-evoked ACTH secretion.

  2. Stabilizing and destabilizing effects of arginine on deoxyribonucleic acid.

    PubMed

    Arakawa, Tsutomu; Hirano, Atsushi; Shiraki, Kentaro; Kita, Yoshiko; Koyama, A Hajime

    2010-03-01

    Aqueous arginine solution now finds a wide range of applications in biotechnology fields, including protein refolding, chromatography and virus inactivation. While progress has been made for mechanistic understanding of the effects of arginine on proteins, we have little understanding on how arginine inactivates viruses. One of the viral components is nucleic acid. We have examined the effects of arginine on the structure and thermal stability of calf thymus deoxyribonucleic acid (DNA) using circular dichroism (CD). Both NaCl and arginine reduced CD intensity. At low concentrations, arginine showed a stronger effect on CD intensity than NaCl. Both NaCl and arginine sharply increased the melting temperature at low concentrations (below 0.25 M). However, they had an opposite effect at higher concentrations. Above this concentration, NaCl gradually increased the melting temperature, leading to the onset melting temperature above 90 degrees C. On the other hand, the thermal stability in the presence of arginine reached a maximum at 0.2-0.5 M, after which further addition of arginine caused decreased melting temperature. It is most likely that the increased melting temperature at low concentration is due to electrostatic stabilization of DNA structure by both NaCl and arginine and that the opposite effects at higher salt concentration are due to salt-specific effects, i.e., stabilizing (salting-out) effects of NaCl and destabilizing (salting-in) effects of arginine. Solubility measurements of nucleic acid bases showed that arginine, but not NaCl, increases the solubilities of the bases, supporting their effects on DNA stability at higher concentration.

  3. Arginine: Its pKa value revisited

    PubMed Central

    Fitch, Carolyn A; Platzer, Gerald; Okon, Mark; Garcia-Moreno E, Bertrand; McIntosh, Lawrence P

    2015-01-01

    Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group. PMID:25808204

  4. Arginine: Its pKa value revisited.

    PubMed

    Fitch, Carolyn A; Platzer, Gerald; Okon, Mark; Garcia-Moreno, Bertrand E; McIntosh, Lawrence P

    2015-05-01

    Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼ 12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.

  5. The role of arginine and arginine-metabolizing enzymes during Giardia – host cell interactions in vitro

    PubMed Central

    2013-01-01

    Background Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections. Results RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline. Conclusions Giardia affects the host’s arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy. PMID:24228819

  6. Single amino acid substitution in the methyltransferase domain of Paprika mild mottle virus replicase proteins confers the ability to overcome the high temperature-dependent Hk gene-mediated resistance in Capsicum plants.

    PubMed

    Matsumoto, Katsutoshi; Johnishi, Kousuke; Hamada, Hiroyuki; Sawada, Hiromasa; Takeuchi, Shigeharu; Kobayashi, Kappei; Suzuki, Kazumi; Kiba, Akinori; Hikichi, Yasufumi

    2009-03-01

    Capsicum plants harboring the Hk gene (Hk) show resistance to Paprika mild mottle virus (PaMMV) at 32 degrees C but not 24 degrees C. To identify the viral elicitor that activates the Hk-mediated resistance, several chimeric viral genomes were constructed between PaMMV and Tobacco mosaic virus-L. Infection patterns of these chimeric viruses in Hk-harboring plants revealed responsibility of PaMMV replicase genes for activation of the Hk-mediated resistance. The comparison of nucleotide sequence of replicase genes between PaMMV and PaHk1, an Hk-resistance-breaking strain of PaMMV, revealed that the adenine-to-uracil substitution at the nucleotide position 721 causes an amino acid change from threonine to serine at the 241st residue in the methyltransferase domain. Introduction of the A721U mutation into the replicase genes of parental PaMMV overcame the Hk resistance at 32 degrees C. The results indicate that Hk-mediated resistance is induced by PaMMV replicase proteins and that methyltransferase domain has a role in this elicitation.

  7. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development.

    PubMed

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael; Kück, Ulrich

    2016-01-01

    Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the

  8. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development

    PubMed Central

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael

    2016-01-01

    ABSTRACT Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at

  9. Two distinct O-methyltransferases in aflatoxin biosynthesis.

    PubMed Central

    Yabe, K; Ando, Y; Hashimoto, J; Hamasaki, T

    1989-01-01

    The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium. Images PMID:2802602

  10. The Fanconi anemia pathway controls oncogenic response in hematopoietic stem and progenitor cells by regulating PRMT5-mediated p53 arginine methylation

    PubMed Central

    Du, Wei; Amarachintha, Surya; Erden, Ozlem; Wilson, Andrew; Pang, Qishen

    2016-01-01

    The Fanconi anemia (FA) pathway is involved in DNA damage and other cellular stress responses. We have investigated the role of the FA pathway in oncogenic stress response by employing an in vivo stress-response model expressing the Gadd45β-luciferase transgene. Using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we show that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA core complex components Fanca or Fancc exhibit aberrant short-lived response to oncogenic insults. Mechanistic studies reveal that FA deficiency in HSPCs impairs oncogenic stress-induced G1 cell-cycle checkpoint, resulting from a compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Furthermore, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca−/− mice prolongs oncogenic response and delays leukemia development in recipient mice. Our study defines an arginine methylation-dependent FA-p53 interplay that controls oncogenic stress response. PMID:27507053

  11. The Selective Estrogen Receptor Modulator Raloxifene Regulates Arginine-Vasopressin Gene Expression in Human Female Neuroblastoma Cells Through G Protein-Coupled Estrogen Receptor and ERK Signaling.

    PubMed

    Grassi, Daniela; Ghorbanpoor, Samar; Acaz-Fonseca, Estefania; Ruiz-Palmero, Isabel; Garcia-Segura, Luis M

    2015-10-01

    The selective estrogen receptor modulator raloxifene reduces blood pressure in hypertensive postmenopausal women. In the present study we have explored whether raloxifene regulates gene expression of arginine vasopressin (AVP), which is involved in the pathogenesis of hypertension. The effect of raloxifene was assessed in human female SH-SY5Y neuroblastoma cells, which have been recently identified as a suitable cellular model to study the estrogenic regulation of AVP. Raloxifene, within a concentration ranging from 10(-10) M to 10(-6) M, decreased the mRNA levels of AVP in SH-SY5Y cells with maximal effect at 10(-7) M. This effect of raloxifene was imitated by an agonist (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone of G protein-coupled estrogen receptor-1 (GPER) and blocked by an antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline of GPER and by GPER silencing. Raloxifene induced a time-dependent increase in the level of phosphorylated ERK1 and ERK2, by a mechanism blocked by the GPER antagonist. The treatment of SH-SY5Y cells with either a MAPK/ERK kinase 1/2-specific inhibitor (1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadine) or a protein kinase C inhibitor (sotrastaurin) blocked the effects of raloxifene on the phosphorylation of ERK1/2 and the regulation of AVP mRNA levels. These results reveal a mechanism mediating the regulation of AVP expression by raloxifene, involving the activation of GPER, which in turn activates protein kinase C, MAPK/ERK kinase, and ERK. The regulation of AVP by raloxifene and GPER may have implications for the treatment of blood hypertension(.).

  12. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

    SciTech Connect

    Ticak, Tomislav; Kountz, D. J.; Girosky, K. E.; Krzycki, J. A.; Ferguson, D. J.

    2014-10-13

    COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. Additionally, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. Lastly, they are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.

  13. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

    DOE PAGES

    Ticak, Tomislav; Kountz, D. J.; Girosky, K. E.; ...

    2014-10-13

    COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptorsmore » such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. Additionally, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. Lastly, they are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.« less

  14. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.

    PubMed

    Ticak, Tomislav; Kountz, Duncan J; Girosky, Kimberly E; Krzycki, Joseph A; Ferguson, Donald J

    2014-10-28

    COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. In addition, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. They are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.

  15. Selective Interactions between Vertebrate Polycomb Homologs and the SUV39H1 Histone Lysine Methyltransferase Suggest that Histone H3-K9 Methylation Contributes to Chromosomal Targeting of Polycomb Group Proteins

    PubMed Central

    Sewalt, Richard G. A. B.; Lachner, Monika; Vargas, Mark; Hamer, Karien M.; den Blaauwen, Jan L.; Hendrix, Thijs; Melcher, Martin; Schweizer, Dieter; Jenuwein, Thomas; Otte, Arie P.

    2002-01-01

    Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures. PMID:12101246

  16. Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins.

    PubMed

    Sewalt, Richard G A B; Lachner, Monika; Vargas, Mark; Hamer, Karien M; den Blaauwen, Jan L; Hendrix, Thijs; Melcher, Martin; Schweizer, Dieter; Jenuwein, Thomas; Otte, Arie P

    2002-08-01

    Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.

  17. Purification of free arginine from chickpea (Cicer arietinum) seeds.

    PubMed

    Cortés-Giraldo, Isabel; Megías, Cristina; Alaiz, Manuel; Girón-Calle, Julio; Vioque, Javier

    2016-02-01

    Chickpea is a grain legume widely consumed in the Mediterranean region and other parts of the world. Chickpea seeds are rich in proteins but they also contain a substantial amount of free amino acids, especially arginine. Hence chickpea may represent a useful source of free amino acids for nutritional or pharmaceutical purposes. Arginine is receiving great attention in recent years because it is the substrate for the synthesis of nitric oxide, an important signaling molecule involved in numerous physiological and pathological processes in mammals. In this work we describe a simple procedure for the purification of arginine from chickpea seeds, using nanofiltration technology and an ion-exchange resin, Amberlite IR-120. Arginine was finally purified by precipitation or crystallization, yielding preparations with purities of 91% and 100%, respectively. Chickpea may represent an affordable green source of arginine, and a useful alternative to production by fermentation or protein hydrolysis.

  18. Arginine-induced stimulation of protein synthesis and survival in IPEC-J2 cells is mediated by mTOR but not nitric oxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arginine (ARG) is an indispensable amino acid in neonates and required for growth. Neonatal intestinal epithelial cells (IEC) are capable of ARG transport, catabolism, and synthesis, and express nitric oxide synthase (NOS) to produce NO from ARG. Our aim was to determine whether ARG directly stimul...

  19. Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

    PubMed Central

    Finnegan, E J; Dennis, E S

    1993-01-01

    A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana. Images PMID:8389441

  20. Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response

    PubMed Central

    Reintjes, Anja; Fuchs, Julian E.; Kremser, Leopold; Lindner, Herbert H.; Liedl, Klaus R.; Huber, Lukas A.; Valovka, Taras

    2016-01-01

    Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB–activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA–NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling. PMID:27051065

  1. Arginine metabolism in wounds

    SciTech Connect

    Albina, J.E.; Mills, C.D.; Barbul, A.; Thirkill, C.E.; Henry, W.L. Jr.; Mastrofrancesco, B.; Caldwell, M.D.

    1988-04-01

    Arginine metabolism in wounds was investigated in the rat in 1) lambda-carrageenan-wounded skeletal muscle, 2) Schilling chambers, and 3) subcutaneous polyvinyl alcohol sponges. All showed decreased arginine and elevated ornithine contents and high arginase activity. Arginase could be brought to the wound by macrophages, which were found to contain arginase activity. However, arginase was expressed by macrophages only after cell lysis and no arginase was released by viable macrophages in vitro. Thus the extracellular arginase of wounds may derive from dead macrophages within the injured tissue. Wound and peritoneal macrophages exhibited arginase deiminase activity as demonstrated by the conversion of (guanido-/sup 14/C)arginine to radiolabeled citrulline during culture, the inhibition of this reaction by formamidinium acetate, and the lack of prokaryotic contamination of the cultures. These findings and the known metabolic fates of the products of arginase and arginine deiminase in the cellular populations of the wound suggest the possibility of cooperativity among cells for the production of substrates for collagen synthesis.

  2. Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

    PubMed Central

    Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C.; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S.; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

    2017-01-01

    For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei. PMID:28045943

  3. Effects of arginine on hair damage via oxidative coloring process.

    PubMed

    Oshimura, Eiko; Ino, Masahiro

    2004-01-01

    The purpose of this study was to measure the protective effects of arginine in oxidative coloring or bleaching process. Contact angle measurement, tensile measurement and amino acid analysis were employed. As the first step, it was shown that oxidative coloring or bleaching process decreases hair surface hydrophobicity and tensile strength in wet condition. Next the study has been conducted with coloring agents in which part of the ammonia was replaced with arginine, to find that arginine reduced the oxidative change in contact angle and tensile strength. These results suggest that arginine prevents the undesirable attack by hydrogen peroxide on hair proteins and hair surface lipids. Furthermore, it is also suggested from amino acid analysis that a considerable amount of arginine is deposited on, or in hair fibers from coloring agents.

  4. Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

    PubMed

    Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj

    2016-07-01

    PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.

  5. Altered brain arginine metabolism in schizophrenia

    PubMed Central

    Liu, P; Jing, Y; Collie, N D; Dean, B; Bilkey, D K; Zhang, H

    2016-01-01

    Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease. PMID:27529679

  6. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1.

    PubMed

    Zheng, Shunsheng; Moehlenbrink, Jutta; Lu, Yi-Chien; Zalmas, Lykourgos-Panagiotis; Sagum, Cari A; Carr, Simon; McGouran, Joanna F; Alexander, Leila; Fedorov, Oleg; Munro, Shonagh; Kessler, Benedikt; Bedford, Mark T; Yu, Qiang; La Thangue, Nicholas B

    2013-10-10

    The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.

  7. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1

    PubMed Central

    Zheng, Shunsheng; Moehlenbrink, Jutta; Lu, Yi-Chien; Zalmas, Lykourgos-Panagiotis; Sagum, Cari A.; Carr, Simon; McGouran, Joanna F.; Alexander, Leila; Fedorov, Oleg; Munro, Shonagh; Kessler, Benedikt; Bedford, Mark T.; Yu, Qiang; La Thangue, Nicholas B.

    2014-01-01

    Summary The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. PMID:24076217

  8. Homology modeling, docking and structure-based pharmacophore of inhibitors of DNA methyltransferase

    NASA Astrophysics Data System (ADS)

    Yoo, Jakyung; Medina-Franco, José L.

    2011-06-01

    DNA methyltransferase 1 (DNMT1) is an emerging epigenetic target for the treatment of cancer and other diseases. To date, several inhibitors from different structural classes have been published. In this work, we report a comprehensive molecular modeling study of 14 established DNTM1 inhibitors with a herein developed homology model of the catalytic domain of human DNTM1. The geometry of the homology model was in agreement with the proposed mechanism of DNA methylation. Docking results revealed that all inhibitors studied in this work have hydrogen bond interactions with a glutamic acid and arginine residues that play a central role in the mechanism of cytosine DNA methylation. The binding models of compounds such as curcumin and parthenolide suggest that these natural products are covalent blockers of the catalytic site. A pharmacophore model was also developed for all DNMT1 inhibitors considered in this work using the most favorable binding conformations and energetic terms of the docked poses. To the best of our knowledge, this is the first pharmacophore model proposed for compounds with inhibitory activity of DNMT1. The results presented in this work represent a conceptual advance for understanding the protein-ligand interactions and mechanism of action of DNMT1 inhibitors. The insights obtained in this work can be used for the structure-based design and virtual screening for novel inhibitors targeting DNMT1.

  9. Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia

    PubMed Central

    Du, Hui; Wu, Jie; Ji, Kui-Xian; Zeng, Qing-Yin; Bhuiya, Mohammad-Wadud; Su, Shang; Shu, Qing-Yan; Ren, Hong-Xu; Liu, Zheng-An; Wang, Liang-Sheng

    2015-01-01

    Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp. PMID:26208646

  10. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

    EPA Science Inventory

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  11. Inhibition of lytic infection of pseudorabies virus by arginine depletion

    SciTech Connect

    Wang, H.-C.; Kao, Y.-C.; Chang, T-J.; Wong, M.-L. . E-mail: mlwong@dragon.nchu.edu.tw

    2005-08-26

    Pseudorabies virus (PRV) is a member of Alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines (such as spermine and spermidine) are ubiquitous in animal cells and participate in cellular proliferation and differentiation. Previous results of our laboratory showed that the PRV can accomplish lytic infection either in the presence of exogenous spermine (or spermidine) or depletion of cellular polyamines. The amino acid arginine is a precursor of polyamine biosynthesis. In this work, we investigated the role of arginine in PRV infection. It was found that the plaque formation of PRV was inhibited by arginase (enzyme catalyzing the conversion of arginine into ornithine and urea) treatment whereas this inhibition can be reversed by exogenous arginine, suggesting that arginine is essential for PRV proliferation. Western blotting was conducted to study the effect of arginine depletion on the levels of structural proteins of PRV in virus-infected cells. Four PRV structural proteins (gB, gE, UL47, and UL48) were chosen for examination, and results revealed that the levels of viral proteins were obviously reduced in long time arginase treatment. However, the overall protein synthesis machinery was apparently not influenced by arginase treatment either in mock or PRV-infected cells. Analyzing with native gel, we found that arginase treatment affected the mobility of PRV structural proteins, suggesting the conformational change of viral proteins by arginine depletion. Heat shock proteins, acting as molecular chaperons, participate in protein folding and translocation. Our results demonstrated that long time arginase treatment could reduce the expression of cellular heat shock proteins 70 (hsc70 and hsp70), and transcriptional suppression of heat shock protein 70 gene promoter was one of the mechanisms involved in this reduced expression.

  12. A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

    PubMed Central

    Chen, Hongshan; Lorton, Benjamin; Gupta, Varun; Shechter, David

    2016-01-01

    Protein arginine methyltransferase 5 (PRMT5) complexed with MEP50/WDR77 catalyzes arginine methylation on histones and other proteins. PRMT5-MEP50 activity is elevated in cancer cells and its expression is highly correlated with poor prognosis in many human tumors. We demonstrate that PRMT5-MEP50 is essential for transcriptional regulation promoting cancer cell invasive phenotypes in lung adenocarcinoma, lung squamous cell carcinoma and breast carcinoma cancer cells. RNA-Seq transcriptome analysis demonstrated that PRMT5 and MEP50 are required to maintain expression of metastasis and Epithelial-to-mesenchymal transition (EMT) markers and to potentiate an epigenetic mechanism of the TGFβ response. We show that PRMT5-MEP50 activity both positively and negatively regulates expression of a wide range of genes. Exogenous TGFβ promotes EMT in a unique pathway of PRMT5-MEP50 catalyzed histone mono- and dimethylation of chromatin at key metastasis suppressor and EMT genes, defining a new mechanism regulating cancer invasivity. PRMT5 methylation of histone H3R2me1 induced transcriptional activation by recruitment of WDR5 and concomitant H3K4 methylation at targeted genes. In parallel, PRMT5 methylation of histone H4R3me2s suppressed transcription at distinct genomic loci. Our decoding of histone methylarginine at key genes supports a critical role for complementary PRMT5-MEP50 transcriptional activation and repression in cancer invasion pathways and in response to TGFβ stimulation and therefore and orients future chemotherapeutic opportunities. PMID:27270440

  13. DNA methyltransferase 3a and mitogen-activated protein kinase signaling regulate the expression of fibroblast growth factor-inducible 14 (Fn14) during denervation-induced skeletal muscle atrophy.

    PubMed

    Tajrishi, Marjan M; Shin, Jonghyun; Hetman, Michal; Kumar, Ashok

    2014-07-18

    The TWEAK-fibroblast growth factor-inducible 14 (Fn14) system is a critical regulator of denervation-induced skeletal muscle atrophy. Although the expression of Fn14 is a rate-limiting step in muscle atrophy on denervation, mechanisms regulating gene expression of Fn14 remain unknown. Methylation of CpG sites within promoter region is an important epigenetic mechanism for gene silencing. Our study demonstrates that Fn14 promoter contains a CpG island close to transcription start site. Fn14 promoter also contains multiple consensus DNA sequence for transcription factors activator protein 1 (AP1) and specificity protein 1 (SP1). Denervation diminishes overall genomic DNA methylation and causes hypomethylation at specific CpG sites in Fn14 promoter leading to the increased gene expression of Fn14 in skeletal muscle. Abundance of DNA methyltransferase 3a (Dnmt3a) and its interaction with Fn14 promoter are repressed in denervated skeletal muscle of mice. Overexpression of Dnmt3a inhibits the gene expression of Fn14 and attenuates skeletal muscle atrophy upon denervation. Denervation also causes the activation of ERK1/2, JNK1/2, and ERK5 MAPKs and AP1 and SP1, which stimulate the expression of Fn14 in skeletal muscle. Collectively, our study provides novel evidence that Dnmt3a and MAPK signaling regulate the levels of Fn14 in skeletal muscle on denervation.

  14. Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus

    PubMed Central

    Moncorgé, Olivier; Panthu, Baptiste; Prchal, Jan; Décimo, Didier; Ohlmann, Théophile; Lina, Bruno; Favard, Cyril; Decroly, Etienne; Ottmann, Michèle; Roingeard, Philippe; Muriaux, Delphine

    2016-01-01

    The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular

  15. Arginine deiminase inhibits Porphyromonas gingivalis surface attachment.

    PubMed

    Cugini, Carla; Stephens, Danielle N; Nguyen, Daniel; Kantarci, Alpdogan; Davey, Mary E

    2013-02-01

    The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity.

  16. FcWRKY70, a WRKY protein of Fortunella crassifolia, functions in drought tolerance and modulates putrescine synthesis by regulating arginine decarboxylase gene.

    PubMed

    Gong, Xiaoqing; Zhang, Jingyan; Hu, Jianbing; Wang, Wei; Wu, Hao; Zhang, Qinghua; Liu, Ji-Hong

    2015-11-01

    WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report functional characterization of a Group III WRKY gene (FcWRKY70) from Fortunella crassifolia. FcWRKY70 was greatly induced by drought and abscisic acid, but slightly or negligibly by salt and cold. Overexpression of FcWRKY70 in tobacco (Nicotiana nudicaulis) and lemon (Citrus lemon) conferred enhanced tolerance to dehydration and drought stresses. Transgenic tobacco and lemon exhibited higher expression levels of ADC (arginine decarboxylase), and accumulated larger amount of putrescine in comparison with wild type (WT). Treatment with D-arginine, an inhibitor of ADC, caused transgenic tobacco plants more sensitive to dehydration. Knock-down of FcWRKY70 in kumquat down-regulated ADC abundance and decreased putrescine level, accompanied by compromised dehydration tolerance. The promoter region of FcADC contained two W-box elements, which were shown to be interacted with FcWRKY70. Taken together, our data demonstrated that FcWRKY70 functions in drought tolerance by, at least partly, promoting production of putrescine via regulating ADC expression.

  17. A second DNA methyltransferase repair enzyme in Escherichia coli.

    PubMed Central

    Rebeck, G W; Coons, S; Carroll, P; Samson, L

    1988-01-01

    The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains. Images PMID:3283737

  18. Convergent evolution of the arginine deiminase pathway: the ArcD and ArcE arginine/ornithine exchangers.

    PubMed

    Noens, Elke E E; Lolkema, Juke S

    2017-02-01

    The arginine deiminase (ADI) pathway converts L-arginine into L-ornithine and yields 1 mol of ATP per mol of L-arginine consumed. The L-arginine/L-ornithine exchanger in the pathway takes up L-arginine and excretes L-ornithine from the cytoplasm. Analysis of the genomes of 1281 bacterial species revealed the presence of 124 arc gene clusters encoding the pathway. About half of the clusters contained the gene encoding the well-studied L-arginine/L-ornithine exchanger ArcD, while the other half contained a gene, termed here arcE, encoding a membrane protein that is not a homolog of ArcD. The arcE gene product of Streptococcus pneumoniae was shown to take up L-arginine and L-ornithine with affinities of 0.6 and 1 μmol/L, respectively, and to catalyze metabolic energy-independent, electroneutral exchange. ArcE of S. pneumoniae could replace ArcD in the ADI pathway of Lactococcus lactis and provided the cells with a growth advantage. In contrast to ArcD, ArcE catalyzed translocation of the pathway intermediate L-citrulline with high efficiency. A short version of the ADI pathway is proposed for L-citrulline catabolism and the presence of the evolutionary unrelated arcD and arcE genes in different organisms is discussed in the context of the evolution of the ADI pathway.

  19. Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes

    PubMed Central

    Balasubramaniyan, Natarajan; Ananthanarayanan, Meena

    2012-01-01

    The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes. PMID:22345554

  20. Arginine production in the neonate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endogenous arginine synthesis in adults is a complex multiorgan process, in which citrulline is synthesized in the gut, enters the general circulation, and is converted into arginine in the kidney, by what is known as the intestinal-renal axis. In neonates, the enzymes required to convert citrulline...

  1. Arabidopsis MET1 cytosine methyltransferase mutants.

    PubMed Central

    Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

    2003-01-01

    We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

  2. The Trimethylamine Methyltransferase Gene and Multiple Dimethylamine Methyltransferase Genes of Methanosarcina barkeri Contain In-Frame and Read-Through Amber Codons†

    PubMed Central

    Paul, Ligi; Ferguson, Donald J.; Krzycki, Joseph A.

    2000-01-01

    Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1. However, two other nearly identical copies of mtbB1, designated mtbB2 and mtbB3, were also found in the genome. A 6.8-kb transcript was detected with probes to mttB and mtbB1, as well as to mtbC and mttC, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP, encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order mtbC, mttB, mttC, mttP, and mtbB1, form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC. The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1, mtbB2, or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M. barkeri mttB gene. The UAG codon is also present in mttB transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis

  3. New insights into estrogenic regulation of O(6)-methylguanine DNA-methyltransferase (MGMT) in human breast cancer cells: Co-degradation of ER-α and MGMT proteins by fulvestrant or O(6)-benzylguanine indicates fresh avenues for therapy.

    PubMed

    Paranjpe, Ameya; Bailey, Nathan I; Konduri, Santhi; Bobustuc, George C; Ali-Osman, Francis; Yusuf, Mohd A; Punganuru, Surendra R; Madala, Hanumantha Rao; Basak, Debasish; Mostofa, Agm; Srivenugopal, Kalkunte S

    2016-09-01

    Endocrine therapy using estrogen receptor-α (ER-α) antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O(6)-methylguanine DNA methyltransferase (MGMT), a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines (MCF-7, T47D) and ER- negative cell lines (MDAMB-468, MDAMB-231), and established inhibitors of ER-α and MGMT, namely, ICI-182,780 (Faslodex) and O(6)-benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements (EREs) and two antioxidant-responsive elements (AREs). MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 (nuclear factor-erythroid 2-related factor-2) increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this drug-resistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O(6)-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O(6)-benzylguanine also induced a specific loss of ER-α and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-α and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-α proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated

  4. The role of water and K + ion in the charge transfer between PO4- groups of DNA and the lysine + and arginine + side chains of histone proteins

    NASA Astrophysics Data System (ADS)

    Bende, A.; Bogár, F.; Ladik, J.

    2008-09-01

    We have calculated the charge transfer (CT) between the PO4- group of DNA and the lysine (Lys) and arginine (Arg) positive side chains of histones in presence of water and K + ions. The calculations were performed at the HF + MP2 level, using the TZVP basis set. The calculations were corrected for basis set superposition error and besides Mulliken's population analysis we have introduced the - for charged systems more reliable - natural population analysis. The results show that the bare PO4--Lys and the PO4--Arg interactions become weaker, mainly, due to the presence of the K + ion. We have found 0.067 e CT for Lys and 0.050 e for Arg.

  5. Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells.

    PubMed

    Oliveira, Andressa P; Simabuco, Fernando M; Tamura, Rodrigo E; Guerrero, Manuel C; Ribeiro, Paulo G G; Libermann, Towia A; Zerbini, Luiz F; Ventura, Armando M

    2013-10-01

    Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

  6. Molecular basis for the regulation of the H3K4 methyltransferase activity of PRDM9.

    PubMed

    Wu, Hong; Mathioudakis, Nikolas; Diagouraga, Boubou; Dong, Aiping; Dombrovski, Ludmila; Baudat, Frédéric; Cusack, Stephen; de Massy, Bernard; Kadlec, Jan

    2013-10-17

    PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

  7. Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs.

    PubMed Central

    Xu, S; Xiao, J; Posfai, J; Maunus, R; Benner, J

    1997-01-01

    BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector. PMID:9321648

  8. Nucleolar Methyltransferase Fibrillarin: Evolution of Structure and Functions.

    PubMed

    Shubina, M Y; Musinova, Y R; Sheval, E V

    2016-09-01

    Fibrillarin is one of the most studied nucleolar proteins. Its main functions are methylation and processing of pre-rRNA. Fibrillarin is a highly conserved protein; however, in the course of evolution from archaea to eukaryotes, it acquired an additional N-terminal glycine and arginine-rich (GAR) domain. In this review, we discuss the evolution of fibrillarin structure and its relation to the functions of the protein in prokaryotes and eukaryotes.

  9. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    PubMed

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods.

  10. L-Arginine modulates CXC chemokines in the human intestinal epithelial cell line HCT-8 by the NO pathway.

    PubMed

    Marion, Rachel; Coëffier, Moïse; Lemoulan, Sabrina; Gargala, Gilles; Ducrotté, Philippe; Déchelotte, Pierre

    2005-12-01

    Arginine has immunomodulating properties in different animal models but its effects in human intestine remain unknown. This study examined whether arginine modulates inflammatory mediators as chemokines and nitric oxide (NO) in the human intestinal epithelial cell line HCT-8 induced by cytokines. Under basal conditions, arginine did not influence iNOS protein expression, NO and chemokine production and mRNA levels (P>0.05 for all). Stimulation with cytokines-induced a significant increase of NO and chemokine production, iNOS and chemokine mRNA level and iNOS protein expression. Under inflammatory conditions, arginine increased 30% NO production (P<0.05) but did not influence iNOS mRNA level or iNOS protein expression. Under stimulated conditions, arginine decreased IL-8 and Mig mRNA level (57% and 39%, for 0.1 vs. 2 mmol/l l-arginine, P<0.05, respectively), and production (respectively, 28 and 23%, both P<0.05). IP-10 and I-TAC mRNA level and production were not significantly influenced by arginine. Under inflammatory conditions, l-arginine as well as a NO donor (sodium nitroprusside (SNP)) increased NO production, which was inversely correlated with IL-8 production (r'=-0.66, P=0.007 for arginine; r'=-0.79, P<0.0001 for SNP). Use of NG-Methyl-l-arginine acetate, a NOS inhibitor which prevents arginine-induced NO production, suppressed the arginine-induced IL-8 inhibition (P<0.05). In HCT-8 cells, arginine enhanced cytokine-induced NO production, reduced IL-8 and Mig production and mRNA level and had no effects on other assessed chemokines. In conclusion, arginine-induced IL-8 inhibition in HCT-8 cells involves NO pathway under inflammatory conditions. These data suggest that arginine-enriched enteral nutrition may have significant influence on inflammatory response in human intestine.

  11. Structure and Function of Flavivirus NS5 Methyltransferase

    SciTech Connect

    Zhou,Y.; Ray, D.; Zhao, Y.; Dong, H.; Ren, S.; Li, Z.; Guo, Y.; Bernard, K.; Shi, P.; Li, H.

    2007-01-01

    The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

  12. Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.

    PubMed

    Lu, Zhengchun; Sarkar, Sanjay; Zhang, Jianqiang; Balasuriya, Udeni B R

    2016-04-01

    Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation.

  13. Specificity of the SUV4-20H1 and SUV4-20H2 protein lysine methyltransferases and methylation of novel substrates.

    PubMed

    Weirich, Sara; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-06-05

    The SUV4-20H1 and SUV4-20H2 enzymes methylate histone H4 at K20, and they have overlapping and distinct biological effects. Here, by in vitro methylation studies we confirmed that both the murine SUV4-20H enzymes strongly favor the monomethylated H4K20 peptide substrate. We also show that both enzymes only generate dimethylated H4K20 products. We determined the substrate sequence recognition motif of both enzymes using SPOT peptide arrays showing that SUV4-20H1 recognizes an (RY)-Kme1-(IVLM)-(LFI)-X-D sequence. In contrast, SUV4-20H2 shows less specificity and recognizes an X-Kme1-(IVLMK)-(LVFI)-X-(DEV) sequence, which is partially overlapping with SUV4-20H1 but has relaxed specificity at the -1 and +4 positions (if the target H4K20me1 is positon 0). Based on our data, we identify novel peptide substrates for SUV4-20H1 (K1423 of Zinc finger protein castor homolog 1) and SUV4-20H2 (K1423 of Zinc finger protein castor homolog 1, K215 of Protein Mis18-beta and K308 of Centromere protein U). All these lysine residues were already identified to be methylated in human cells, but the responsible PKMT was not known. In addition, we also tested the activity of SUV4-20H enzymes on ERK1, which was recently reported to be methylated by SUV4-20H1 at K302 and K361. However the sequences surrounding both methylation sites do not fit to the specificity profile of SUV4-20H1 and we could not detect methylation of ERK1 by any of the SUV4-20H enzymes. The possible reasons of this discrepancy and its consequences are discussed.

  14. Listeria monocytogenes 10403S Arginine Repressor ArgR Finely Tunes Arginine Metabolism Regulation under Acidic Conditions

    PubMed Central

    Cheng, Changyong; Dong, Zhimei; Han, Xiao; Sun, Jing; Wang, Hang; Jiang, Li; Yang, Yongchun; Ma, Tiantian; Chen, Zhongwei; Yu, Jing; Fang, Weihuan; Song, Houhui

    2017-01-01

    Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti

  15. Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells.

    PubMed

    Joshi, Mahesh S; Ferguson, T Bruce; Johnson, Fruzsina K; Johnson, Robert A; Parthasarathy, Sampath; Lancaster, Jack R

    2007-06-12

    Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and alpha-2 adrenoceptors (alpha-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and alpha-2 adrenoceptors, partly inhibited L-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of alpha-2 AR, at very low concentrations completely inhibited NO formation. Like L-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of alpha-2 AR was very potent in activating cellular NO, thus indicating a possible role for alpha-2 AR in L-arginine-mediated NO synthesis. D-arginine also activated NO production and could be inhibited by imidazoline and alpha-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated L-arginine-mediated NO synthesis, thus indicating mediation via G proteins. L-type Ca(2+) channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the L-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of L-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development.

  16. Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells

    PubMed Central

    Joshi, Mahesh S.; Ferguson, T. Bruce; Johnson, Fruzsina K.; Johnson, Robert A.; Parthasarathy, Sampath; Lancaster, Jack R.

    2007-01-01

    Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and α-2 adrenoceptors (α-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and α-2 adrenoceptors, partly inhibited l-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of α-2 AR, at very low concentrations completely inhibited NO formation. Like l-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of α-2 AR was very potent in activating cellular NO, thus indicating a possible role for α-2 AR in l-arginine-mediated NO synthesis. d-arginine also activated NO production and could be inhibited by imidazoline and α-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated l-arginine-mediated NO synthesis, thus indicating mediation via G proteins. l-type Ca2+ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the l-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of l-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development. PMID:17535904

  17. Convergent Mechanistic Features between the Structurally Diverse N- and O-Methyltransferases: Glycine N-Methyltransferase and Catechol O-Methyltransferase.

    PubMed

    Zhang, Jianyu; Klinman, Judith P

    2016-07-27

    Although an enormous and still growing number of biologically diverse methyltransferases have been reported and identified, a comprehensive understanding of the enzymatic methyl transfer mechanism is still lacking. Glycine N-methyltransferase (GNMT), a member of the family that acts on small metabolites as the substrate, catalyzes methyl transfer from S-adenosyl-l-methionine (AdoMet) to glycine to form S-adenosyl-l-homocysteine and sarcosine. We report primary carbon ((12)C/(14)C) and secondary ((1)H3/(3)H3) kinetic isotope effects at the transferred methyl group, together with (1)H3/(3)H3 binding isotope effects for wild-type GNMT and a series of Tyr21 mutants. The data implicate a compaction effect in the methyl transfer step that is conferred by the protein structure. Furthermore, a remarkable similarity of properties is observed between GNMT and catechol O-methyltransferase, despite significant differences between these enzymes with regard to their active site structures and catalyzed reactions. We attribute these results to a catalytically relevant reduction in the methyl donor-acceptor distance that is dependent on a tyrosine side chain positioned behind the methyl-bearing sulfur of AdoMet.

  18. The crystal structure of a novel SAM-dependent methyltransferase PH1915 from Pyrococcus horikoshii.

    SciTech Connect

    Sun, W.; Xu, X.; Pavlova, M.; Edwards, A.; Joachimiak, A.; Savchenko, A.; Christendat, D.; Biosciences Division; Univ. of Toronto; Univ. Health Network

    2005-01-01

    The S-adenosyl-L-methionine (SAM)-dependent methyltransferases represent a diverse and biologically important class of enzymes. These enzymes utilize the ubiquitous methyl donor SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. Here we present the crystal structure of PH1915 from Pyrococcus horikoshii OT3, a predicted SAM-dependent methyltransferase. This protein belongs to the Cluster of Orthologous Group 1092, and the presented crystal structure is the first representative structure of this protein family. Based on sequence and 3D structure analysis, we have made valuable functional insights that will facilitate further studies for characterizing this group of proteins. Specifically, we propose that PH1915 and its orthologs are rRNA- or tRNA-specific methyltransferases.

  19. Ameliorative Effects of Curcumin on Fibrinogen-Like Protein-2 Gene Expression, Some Oxido-Inflammatory and Apoptotic Markers in a Rat Model of l-Arginine-Induced Acute Pancreatitis.

    PubMed

    Shafik, Noha M; Abou-Fard, Ghada M

    2016-06-01

    The aim of the study was to investigate the ameliorative effects of curcumin on fibrinogen like protein-2 (fgl-2), some oxido-inflammatory and apoptotic markers in rat-induced acute pancreatitis (AP). Seventy-five albino rats were divided into control group, l-arginine (l-Arg)-induced AP group, curcumin pre-treated group before AP induction, curcumin post-treated group after AP induction, and curcumin injected group only. AP group showed severe necrotizing pancreatitis confirmed by histopathological changes and elevations in serum amylase and lipase activities, levels of epithelial neutrophil-activating peptide 78, tissue content of protein carbonyls, levels of tumor necrosis factor α, and caspase-3 as well as myeloperoxidase activity. Significant elevation in pancreatic fgl-2 mRNA expression was detected in AP group. Improvement of all parameters was detected with increase of caspase-3 in both curcumin-treated groups that confirmed curcumin ameliorative effects against AP through induction of apoptosis and inhibition of micro-thrombosis, inflammation, and oxidative stress.

  20. Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana).

    PubMed

    Brown, Ashli E; France, Richard M; Grossman, Steven H

    2004-06-01

    The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.

  1. Spinocerebellar ataxia-13 Kv3.3 potassium channels: arginine-to-histidine mutations affect both functional and protein expression on the cell surface.

    PubMed

    Zhao, Jian; Zhu, Jing; Thornhill, William B

    2013-09-01

    The voltage-gated potassium channel Kv3.3 is the causative gene of SCA13 (spinocerebellar ataxia type 13), an autosomal dominant neurological disorder. The four dominant mutations identified to date cause Kv3.3 channels to be non-functional or have altered gating properties in Xenopus oocytes. In the present paper, we report that SCA13 mutations affect functional as well as protein expression of Kv3.3 channels in a mammalian cell line. The reduced protein level of SCA13 mutants is caused by a shorter protein half-life, and blocking the ubiquitin-proteasome pathway increases the total protein of SCA13 mutants more than wild-type. SCA13 mutated amino acids are highly conserved, and the side chains of these residues play a critical role in the stable expression of Kv3.3 proteins. In addition, we show that mutant Kv3.3 protein levels could be partially rescued by treatment with the chemical chaperone TMAO (trimethylamine N-oxide) and to a lesser extent with co-expression of Kv3.1b. Thus our results suggest that amino acid side chains of SCA13 positions affect the protein half-life and/or function of Kv3.3, and the adverse effect on protein expression cannot be fully rescued.

  2. PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b

    PubMed Central

    Du, Jin; Zhao, Tao-Lan; Wang, Peng-Fei; Zhao, Ping-Xia; Xie, Qi; Cao, Xiao-Feng; Xiang, Cheng-Bin

    2016-01-01

    Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance. PMID:27676073

  3. An Arabidopsis thaliana methyltransferase Capable of Methylating Farnesoic Acid

    SciTech Connect

    Yang,Y.; Yuan, J.; Ross, J.; Noel, J.; Pichersky, E.

    2006-01-01

    We previously reported the identification of a new family of plant methyltransferases (MTs), named the SABATH family, that use S-adenosyl-l-methionine (SAM) to methylate a carboxyl moiety or a nitrogen-containing functional group on a diverse array of plant compounds. The Arabidopsis genome alone contains 24 distinct SABATH genes. To identify the catalytic specificities of members of this protein family in Arabidopsis, we screened recombinantly expressed and purified enzymes with a large number of potential substrates. Here, we report that the Arabidopsis thaliana gene At3g44860 encodes a protein with high catalytic specificity towards farnesoic acid (FA). Under steady-state conditions, this farnesoic acid carboxyl methyltransferase (FAMT) exhibits K{sub M} values of 41 and 71 {mu}M for FA and SAM, respectively. A three-dimensional model of FAMT constructed based upon similarity to the experimentally determined structure of Clarkia breweri salicylic acid methyltransferase (SAMT) suggests a reasonable model for FA recognition in the FAMT active site. In plants, the mRNA levels of At3g44860 increase in response to the exogenous addition of several compounds previously shown to induce plant defense responses at the transcriptional level. Although methyl farnesoate (MeFA) has not yet been detected in Arabidopsis, the presence of a FA-specific carboxyl methyltransferase in Arabidopsis capable of producing MeFA, an insect juvenile hormone made by some plants as a presumed defense against insect herbivory, suggests that MeFA or chemically similar compounds are likely to serve as new specialized metabolites in Arabidopsis.

  4. Crystal structure of dengue virus methyltransferase without S-adenosyl-L-methionine.

    PubMed

    Noble, Christian G; Li, Shi-Hua; Dong, Hongping; Chew, Sock Hui; Shi, Pei-Yong

    2014-11-01

    Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2'-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket.

  5. Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine.

    PubMed Central

    Patterson-Curtis, S I; Johnson, E A

    1989-01-01

    Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A. Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine. Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine. Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation. In contrast, certain other nutritional conditions stimulated protease and toxin formation in C. botulinum and counteracted the repression by arginine. Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers. Casein supplementation of a medium containing high levels of arginine prevented protease inactivation. High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine. These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C. botulinum. Images PMID:2669631

  6. The role of the arginine metabolome in pain: implications for sickle cell disease

    PubMed Central

    Bakshi, Nitya; Morris, Claudia R

    2016-01-01

    Sickle cell disease (SCD) is the most common hemoglobinopathy in the US, affecting approximately 100,000 individuals in the US and millions worldwide. Pain is the hallmark of SCD, and a subset of patients experience pain virtually all of the time. Of interest, the arginine metabolome is associated with several pain mechanisms highlighted in this review. Since SCD is an arginine deficiency syndrome, the contribution of the arginine metabolome to acute and chronic pain in SCD is a topic in need of further attention. Normal arginine metabolism is impaired in SCD through various mechanisms that contribute to endothelial dysfunction, vaso-occlusion, pulmonary complications, risk of leg ulcers, and early mortality. Arginine is a semiessential amino acid that serves as a substrate for protein synthesis and is the precursor to nitric oxide (NO), polyamines, proline, glutamate, creatine, and agmatine. Since arginine is involved in multiple metabolic processes, a deficiency of this amino acid has the potential to disrupt many cellular and organ functions. NO is a potent vasodilator that is depleted in SCD and may contribute to vaso-occlusive pain. As the obligate substrate for NO production, arginine also plays a mechanistic role in SCD-related pain, although its contribution to pain pathways likely extends beyond NO. Low global arginine bioavailability is associated with pain severity in both adults and children with SCD as well as other non-SCD pain syndromes. Preliminary clinical studies of arginine therapy in SCD demonstrate efficacy in treating acute vaso-occlusive pain, as well as leg ulcers and pulmonary hypertension. Restoration of arginine bioavailability through exogenous supplementation of arginine is, therefore, a promising therapeutic target. Phase II clinical trials of arginine therapy for sickle-related pain are underway and a Phase III randomized controlled trial is anticipated in the near future. PMID:27099528

  7. Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives.

    PubMed

    Coutard, Bruno; Barral, Karine; Lichière, Julie; Selisko, Barbara; Martin, Baptiste; Aouadi, Wahiba; Lombardia, Miguel Ortiz; Debart, Françoise; Vasseur, Jean-Jacques; Guillemot, Jean Claude; Canard, Bruno; Decroly, Etienne

    2017-03-01

    The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2'-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2'-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-l-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the αX α-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs.IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns, and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune

  8. Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas.

    PubMed

    Cheng, Feng; Zhu, Leilei; Lue, Hongqi; Bernhagen, Jürgen; Schwaneberg, Ulrich

    2015-02-01

    Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, ADI's application as anticancer drug is hampered by its low activity for arginine under physiological conditions mainly due to its high "K M" (S₀.₅) values which are often 1 magnitude higher than the arginine concentration in blood (0.10-0.12 mM arginine in human plasma). Previous evolution campaigns were directed by us with the aim of boosting activity of PpADI (ADI from Pseudomonas plecoglossicida, k cat = 0.18 s(-1); S₀.₅ = 1.30 mM), and yielded variant M6 with slightly reduced S₀.₅ values and enhanced k cat (S₀.₅ = 0.81 mM; k cat = 11.64 s(-1)). In order to further reduce the S₀.₅ value and to increase the activity of PpADI at physiological arginine concentration, a more sensitive screening system based on ammonia detection in 96-well microtiter plate to reliably detect ≥0.005 mM ammonia was developed. After screening ~5,500 clones with the ammonia detection system (ADS) in two rounds of random mutagenesis and site-directed mutagenesis, variant M19 with increased k cat value (21.1 s(-1); 105.5-fold higher compared to WT) and reduced S₀.₅ value (0.35 mM compared to 0.81 mM (M6) and 1.30 mM (WT)) was identified. Improved performance of M19 was validated by determining IC₅₀ values for two melanoma cell lines. The IC₅₀ value for SK-MEL-28 dropped from 8.67 (WT) to 0.10 (M6) to 0.04 μg/mL (M19); the IC₅₀ values for G361 dropped from 4.85 (WT) to 0.12 (M6) to 0.05 μg/mL (M19).

  9. Arginine Decarboxylase Is Localized in Chloroplasts.

    PubMed Central

    Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

    1995-01-01

    Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

  10. DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA).

    PubMed

    Erova, Tatiana E; Kosykh, Valeri G; Sha, Jian; Chopra, Ashok K

    2012-05-01

    Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam(+)) and GM33 (∆dam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA(+) strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ∆gidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation

  11. An Arginine-rich Motif of Ring Finger Protein 4 (RNF4) Oversees the Recruitment and Degradation of the Phosphorylated and SUMOylated Krüppel-associated Box Domain-associated Protein 1 (KAP1)/TRIM28 Protein during Genotoxic Stress*

    PubMed Central

    Kuo, Ching-Ying; Li, Xu; Kong, Xiang-Qian; Luo, Cheng; Chang, Che-Chang; Chung, Yiyin; Shih, Hsiu-Ming; Li, Keqin Kathy; Ann, David K.

    2014-01-01

    Krüppel-associated box domain-associated protein 1 (KAP1) is a universal transcriptional corepressor that undergoes multiple posttranslational modifications (PTMs), including SUMOylation and Ser-824 phosphorylation. However, the functional interplay of KAP1 PTMs in regulating KAP1 turnover during DNA damage response remains unclear. To decipher the role and cross-talk of multiple KAP1 PTMs, we show here that DNA double strand break-induced KAP1 Ser-824 phosphorylation promoted the recruitment of small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, ring finger protein 4 (RNF4), and subsequent RNF4-mediated, SUMO-dependent degradation. Besides the SUMO interacting motif (SIM), a previously unrecognized, but evolutionarily conserved, arginine-rich motif (ARM) in RNF4 acts as a novel recognition motif for selective target recruitment. Results from combined mutagenesis and computational modeling studies suggest that RNF4 utilizes concerted bimodular recognition, namely SIM for Lys-676 SUMOylation and ARM for Ser(P)-824 of simultaneously phosphorylated and SUMOylated KAP1 (Ser(P)-824-SUMO-KAP1). Furthermore, we proved that arginines 73 and 74 within the ARM of RNF4 are required for efficient recruitment to KAP1 or accelerated degradation of promyelocytic leukemia protein (PML) under stress. In parallel, results of bimolecular fluorescence complementation assays validated the role of the ARM in recognizing Ser(P)-824 in living cells. Taken together, we establish that the ARM is required for RNF4 to efficiently target Ser(P)-824-SUMO-KAP1, conferring ubiquitin Lys-48-mediated proteasomal degradation in the context of double strand breaks. The conservation of such a motif may possibly explain the requirement for timely substrate selectivity determination among a myriad of SUMOylated proteins under stress conditions. Thus, the ARM dynamically regulates the SIM-dependent recruitment of targets to RNF4, which could be critical to dynamically fine-tune the

  12. The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase.

    PubMed Central

    Kanipes, M I; Hill, J E; Henry, S A

    1998-01-01

    The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms. PMID:9755189

  13. Production of l-Arginine by Arginine Hydroxamate-Resistant Mutants of Bacillus subtilis

    PubMed Central

    Kisumi, Masahiko; Kato, Jyoji; Sugiura, Masaki; Chibata, Ichiro

    1971-01-01

    l-Arginine hydroxamate inhibited the growth of various bacteria, and the inhibition was readily reversed by arginine. l-Arginine hydroxamate (10−3m) completely inhibited the growth of Bacillus subtilis. This inhibitory effect was prevented by 2.5 × 10−4ml-arginine, which was the most effective of all the natural amino acids in reversing the inhibition. l-Arginine hydroxamate-resistant mutants of Bacillus subtilis were isolated and found to excrete l-arginine in relatively high yields. One of the mutants, strain AHr-5, produced 4.5 mg of l-arginine per ml in shaken culture in 3 days. PMID:5002904

  14. Purification and characterization of arginine kinase from locust.

    PubMed

    Li, Miao; Wang, Xiao-Yun; Bai, Ji-Gang

    2006-01-01

    L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30 degrees C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k(cat)/K(m)(Arg) 174. The activity of AK required divalent cations such as Mg(2+) and Mn(2+). In the presence of Cu(2+) and Zn(2+), AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.

  15. Dietary arginine requirement of juvenile red drum (Sciaenops ocellatus) based on weight gain and feed efficiency.

    PubMed

    Barziza, D E; Buentello, J A; Gatlin, D M

    2000-07-01

    Increasing aquacultural production of red drum (Sciaenops ocellatus) has prompted the determination of many of this species' nutritional requirements. However, limited information is available concerning its amino acid requirements, especially for arginine. Therefore, a feeding trial was conducted with juvenile red drum to determine their quantitative dietary requirement for arginine. Experimental diets contained 35 g crude protein/100 g from red drum muscle and crystalline amino acids. Incremental levels of arginine were added to the diets in place of a mixture of glycine and aspartic acid to maintain all diets isonitrogenous. All diets were fed in triplicate to juvenile red drum for 7 wk. Graded levels of arginine significantly (P < 0.05) affected weight gain, feed efficiency, protein efficiency ratio (PER) and plasma arginine levels of the fish. Based on least-squares regression of feed efficiency and PER data, the minimum requirement (+/- SEM) of red drum for arginine was estimated at 1.44 (+/- 0.15) and 1.48 (+/- 0.12) g/100 g diet (4.11 and 4.23 g/100 g dietary protein), respectively. The arginine requirements estimated from weight gain data were 1.75 (+/- 0.18) g/100 g diet or 5.0 g/100 g dietary protein. These values are similar to those reported for other carnivorous fish species.

  16. Arginine methylation of SmB is required for Drosophila germ cell development.

    PubMed

    Anne, Joël

    2010-09-01

    Sm proteins constitute the common core of spliceosomal small nuclear ribonucleoproteins. Although Sm proteins are known to be methylated at specific arginine residues within the C-terminal arginine-glycine dipeptide (RG) repeats, the biological relevance of these modifications remains unknown. In this study, a tissue-specific function of arginine methylation of the SmB protein was identified in Drosophila. Analysis of the distribution of SmB during oogenesis revealed that this protein accumulates at the posterior pole of the oocyte, a cytoplasmic region containing the polar granules, which are necessary for the formation of primordial germ cells. The pole plasm localisation of SmB requires the methylation of arginine residues in its RG repeats by the Capsuléen-Valois methylosome complex. Functional studies showed that the methylation of these arginine residues is essential for distinct processes of the germline life cycle, including germ cell formation, migration and differentiation. In particular, the methylation of a subset of these arginine residues appears essential for the anchoring of the polar granules at the posterior cortex of the oocyte, whereas the methylation of another subset controls germ cell migration during embryogenesis. These results demonstrate a crucial role of arginine methylation in directing the subcellular localisation of SmB and that this modification contributes specifically to the establishment and development of germ cells.

  17. Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity

    PubMed Central

    Jacques-Fricke, Bridget T.; Gammill, Laura S.

    2014-01-01

    Neural crest precursors express genes that cause them to become migratory, multipotent cells, distinguishing them from adjacent stationary neural progenitors in the neurepithelium. Histone methylation spatiotemporally regulates neural crest gene expression; however, the protein methyltransferases active in neural crest precursors are unknown. Moreover, the regulation of methylation during the dynamic process of neural crest migration is unclear. Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells. NSD3 expression commences before up-regulation of neural crest genes, and NSD3 is necessary for expression of the neural plate border gene Msx1, as well as the key neural crest transcription factors Sox10, Snail2, Sox9, and FoxD3, but not gene expression generally. Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation. In addition, by temporally limiting expression of a dominant negative to migratory stages, we identify a novel, direct requirement for NSD3-related methyltransferase activity in neural crest migration. These results identify NSD3 as the first protein methyltransferase essential for neural crest gene expression during specification and show that NSD3-related methyltransferase activity independently regulates migration. PMID:25318671

  18. Fidelity Index Determination of DNA Methyltransferases

    PubMed Central

    Borgaro, Janine G.; Benner, Nicole; Zhu, Zhenyu

    2013-01-01

    DNA methylation is the most frequent form of epigenetic modification in the cell, which involves gene regulation in eukaryotes and protection against restriction enzymes in prokaryotes. Even though many methyltransferases exclusively modify their cognate sites, there have been reports of those that exhibit promiscuity. Previous experimental approaches used to characterize these methyltransferases do not provide the exact concentration at which off-target methylation occurs. Here, we present the first reported fidelity index (FI) for a number of DNA methyltransferases. We define the FI as the ratio of the highest amount of methyltransferase that exhibits no star activity (off-target effects) to the lowest amount that exhibits complete modification of the cognate site. Of the methyltransferases assayed, M.MspI and M.AluI exhibited the highest fidelity of ≥250 and ≥500, respectively, and do not show star activity even at very high concentrations. In contrast, M.HaeIII, M.EcoKDam and M.BamHI have the lowest fidelity of 4, 4 and 2, respectively, and exhibit star activity at concentrations close to complete methylation of the cognate site. The fidelity indexes provide vital information on the usage of methyltransferases and are especially important in applications where site specific methylation is required. PMID:23671703

  19. Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

    PubMed

    Bentur, Ohad S; Schwartz, Doron; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Chernin, Gil; Schwartz, Idit F

    2015-08-15

    Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.

  20. Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

    PubMed

    Andersen, Synne M; Holen, Elisabeth; Aksnes, Anders; Rønnestad, Ivar; Zerrahn, Jens-Erik; Espe, Marit

    2013-12-14

    In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for β-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and β-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

  1. Comparative Analysis of Serine/Arginine-Rich Proteins across 27 Eukaryotes: Insights into Sub-Family Classification and Extent of Alternative Splicing

    PubMed Central

    Richardson, Dale N.; Rogers, Mark F.; Labadorf, Adam; Ben-Hur, Asa; Guo, Hui; Paterson, Andrew H.; Reddy, Anireddy S. N.

    2011-01-01

    Alternative splicing (AS) of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs) at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and “basal” eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs) as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes). AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3′ or 5′ splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%–88% of their SR genes experiencing some type of AS compared to the 40%–54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms. PMID:21935421

  2. Putrescine N-methyltransferase--the start for alkaloids.

    PubMed

    Biastoff, Stefan; Brandt, Wolfgang; Dräger, Birgit

    2009-01-01

    Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.

  3. Glucagon-like peptide-1 receptor agonist inhibits asymmetric dimethylarginine generation in the kidney of streptozotocin-induced diabetic rats by blocking advanced glycation end product-induced protein arginine methyltranferase-1 expression.

    PubMed

    Ojima, Ayako; Ishibashi, Yuji; Matsui, Takanori; Maeda, Sayaka; Nishino, Yuri; Takeuchi, Masayoshi; Fukami, Kei; Yamagishi, Sho-ichi

    2013-01-01

    Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, contributes to diabetic nephropathy. We have found that glucagon-like peptide-1 (GLP-1) inhibits the AGE-induced inflammatory reactions in endothelial cells. However, effects of GLP-1 on the AGE-RAGE-ADMA axis are unknown. This study examined the effects of GLP-1 on reactive oxygen species (ROS) generation, gene expression of protein arginine methyltransfetase-1 (PRMT-1), an enzyme that mainly generates ADMA, and ADMA levels in human proximal tubular cells. Streptozotocin-induced diabetic rats received continuous i.p. infusion of 0.3 μg of vehicle or 1.5 μg of the GLP-1 analog exendin-4 per kilogram of body weight for 2 weeks. We further investigated whether and how exendin-4 treatment reduced ADMA levels and renal damage in streptozotocin-induced diabetic rats. GLP-1 inhibited the AGE-induced RAGE and PRMT-1 gene expression, ROS, and ADMA generation in tubular cells, which were blocked by small-interfering RNAs raised against GLP-1 receptor. Exendin-4 treatment decreased gene expression of Rage, Prmt-1, Icam-1, and Mcp-1 and ADMA level; reduced urinary excretions of 8-hydroxy-2'-deoxyguanosine and albumin; and improved histopathologic changes of the kidney in diabetic rats. Our present study suggests that GLP-1 receptor agonist may inhibit the AGE-RAGE-mediated ADMA generation by suppressing PRMT-1 expression via inhibition of ROS generation, thereby protecting against the development and progression of diabetic nephropathy.

  4. Downregulation of a barley (Hordeum vulgare) leucine-rich repeat, non-arginine-aspartate receptor-like protein kinase reduces expression of numerous genes involved in plant pathogen defense.

    PubMed

    Parrott, David L; Huang, Li; Fischer, Andreas M

    2016-03-01

    Pattern recognition receptors represent a first line of plant defense against pathogens. Comparing the flag leaf transcriptomes of barley (Hordeum vulgare L.) near-isogenic lines varying in the allelic state of a locus controlling senescence, we have previously identified a leucine-rich repeat receptor-like protein kinase gene (LRR-RLK; GenBank accession: AK249842), which was strongly upregulated in leaves of early-as compared to late-senescing germplasm. Bioinformatic analysis indicated that this gene codes for a subfamily XII, non-arginine-aspartate (non-RD) LRR-RLK. Virus-induced gene silencing resulted in a two-fold reduction of transcript levels as compared to controls. Transcriptomic comparison of leaves from untreated plants, from plants treated with virus only without any plant sequences (referred to as 'empty virus' control), and from plants in which AK249842 expression was knocked down identified numerous genes involved in pathogen defense. These genes were strongly induced in 'empty virus' as compared to untreated controls, but their expression was significantly reduced (again compared to 'empty virus' controls) when AK249842 was knocked down, indicating that their expression partially depends on the LRR-RLK investigated here. Expression analysis, using datasets from BarleyBase/PLEXdb, demonstrated that AK249842 transcript levels are heavily influenced by the allelic state of the well-characterized mildew resistance a (Mla) locus, and that the gene is induced after powdery mildew and stem rust infection. Together, our data suggest that AK249842 is a barley pattern recognition receptor with a tentative role in defense against fungal pathogens, setting the stage for its full functional characterization.

  5. Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.

    PubMed

    Hoff, R H; Hengge, A C; Wu, L; Keng, Y F; Zhang, Z Y

    2000-01-11

    General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

  6. HNA-3a–specific antibodies recognize choline transporter–like protein-2 peptides containing arginine, but not glutamine at Position 154

    PubMed Central

    Curtis, Brian R.; Sullivan, Mia J.; Holyst, M. Trudy; Szabo, Aniko; Bougie, Daniel W.; Aster, Richard H.

    2013-01-01

    BACKGROUND Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter–like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope. STUDY DESIGN AND METHODS Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a–specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154. RESULTS Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2 peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide. CONCLUSION The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a–specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood. PMID:21517890

  7. Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.

    PubMed

    Afolayan, Adeleye J; Alexander, Maxwell; Holme, Rebecca L; Michalkiewicz, Teresa; Rana, Ujala; Teng, Ru-Jeng; Zemanovic, Sara; Sahoo, Daisy; Pritchard, Kirkwood A; Konduri, Girija G

    2017-02-10

    Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at (445)GERAMT(450) Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu(446) and Arg(447) cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

  8. Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco.

    PubMed

    Ying, Z; Mulligan, R M; Janney, N; Houtz, R L

    1999-12-17

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.

  9. Localization of arginine decarboxylase in tobacco plants.

    PubMed

    Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa

    2004-01-01

    The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

  10. Effects of arginine on intestinal epithelial cell integrity and nutrient uptake.

    PubMed

    Xia, Mi; Ye, Lulu; Hou, Qihang; Yu, Qinghua

    2016-11-14

    Arginine is a multifaceted amino acid that is critical to the normal physiology of the gastrointestinal tract. Oral arginine administration has been shown to improve mucosal recovery following intestinal injury. The present study investigated the influence of extracellular arginine concentrations on epithelial cell barrier regulation and nutrition uptake by porcine small intestinal epithelial cell line (IPEC-J2). The results show that reducing arginine concentration from 0·7 to 0·2 mm did not affect the transepithelial electrical resistance value, tight-junction proteins (claudin-1, occludin, E-cadherin), phosphorylated extracellular signal-regulated protein kinases (p-ERK) and mucin-1 expression. Furthermore, reducing arginine concentration stimulated greater expression of cationic amino acid transporter (CAT1), excitatory amino acid transporter (EAAT3) and alanine/serine/cysteine transporter (ASCT1) mRNA by IPEC-J2 cells, which was verified by elevated efficiency of amino acid uptake. Glucose consumption by IPEC-J2 cells treated with 0·2 mm-arginine remained at the same physiological level to guarantee energy supply and to maintain the cell barrier. This experiment implied that reducing arginine concentration is feasible in IPEC-J2 cells guaranteed by nutrient uptake and cell barrier function.

  11. Purification of Arsenic (+3 Oxidation State) Methyltransferase from Rat Liver Cytosol

    PubMed Central

    Drobna, Zuzana; Styblo, Miroslav; Thomas, David J.

    2015-01-01

    Demonstrating the enzymatic basis of arsenic methylation is critical to further studies of the pathway for the conversion of inorganic arsenic into a variety of methylated metabolites. This protocol describes a procedure for the purification of an arsenic methyltransferase from rat liver cytosol. Purification of this enzyme and subsequent cloning of its gene has permitted studies of enzyme structure and function and has lead to the identification of orthologous genes in genomes of organisms ranging in complexity from sea urchins to humans. These proteins are referred to as arsenic (+3 oxidation state) methyltransferases. PMID:20949431

  12. Multimethylation of Rickettsia OmpB Catalyzed by Lysine Methyltransferases*

    PubMed Central

    Abeykoon, Amila; Wang, Guanghui; Chao, Chien-Chung; Chock, P. Boon; Gucek, Marjan; Ching, Wei-Mei; Yang, David C. H.

    2014-01-01

    Methylation of rickettsial OmpB (outer membrane protein B) has been implicated in bacterial virulence. Rickettsial methyltransferases RP789 and RP027-028 are the first biochemically characterized methyltransferases to catalyze methylation of outer membrane protein (OMP). Methylation in OMP remains poorly understood. Using semiquantitative integrated liquid chromatography-tandem mass spectroscopy, we characterize methylation of (i) recombinantly expressed fragments of Rickettsia typhi OmpB exposed in vitro to trimethyltransferases of Rickettsia prowazekii RP027-028 and of R. typhi RT0101 and to monomethyltransferases of R. prowazekii RP789 and of R. typhi RT0776, and (ii) native OmpBs purified from R. typhi and R. prowazekii strains Breinl, RP22, and Madrid E. We found that in vitro trimethylation occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), whereas monomethylation is pervasive throughout OmpB. Native OmpB from virulent R. typhi contains mono- and trimethyllysines at locations well correlated with methylation in recombinant OmpB catalyzed by methyltransferases in vitro. Native OmpBs from highly virulent R. prowazekii strains Breinl and RP22 contain multiple clusters of trimethyllysine in contrast to a single cluster in OmpB from mildly virulent R. typhi. Furthermore, OmpB from the avirulent strain Madrid E contains mostly monomethyllysine and no trimethyllysine. The native OmpB from Madrid E was minimally trimethylated by RT0101 or RP027-028, consistent with a processive mechanism of trimethylation. This study provides the first in-depth characterization of methylation of an OMP at the molecular level and may lead to uncovering the link between OmpB methylation and rickettsial virulence. PMID:24497633

  13. Metabolic and immune effects of dietary arginine supplementation after burn.

    PubMed

    Saito, H; Trocki, O; Wang, S L; Gonce, S J; Joffe, S N; Alexander, J W

    1987-07-01

    The effect of supplemental dietary arginine on metabolism and immunity was studied in 36 burned guinea pigs (30% of total body surface area) with previously placed catheter gastrostomies. The animals were randomized into four groups. After an initial three-day adaptation period, all groups received continuous isonitrogenous, isocaloric (175 kcal [735 kJ]/kg/d), and isovolemic intragastric tube feedings until postburn day (PBD) 14. Groups A, B, C, and D received 0%, 1%, 2%, and 4%, respectively, of total energy intake as arginine given in the form of crystalline arginine hydrochloride with 22%, 21%, 20%, and 18%, respectively, of total energy as whey protein. The average body weight after burn decreased equally in all groups. Resting metabolic expenditure on PBD 6 was higher in groups B (151% +/- 6% of preburn) and C (156% +/- 7%) than in groups A (131% +/- 4%) and D (136% +/- 3%). Ear-thickness response to dinitrofluorobenzene challenge on PBD 12 showed the best response in group C. The mortality rates of groups A, B, C, and D were 56%, 29%, 22%, and 56%, respectively. This study suggests that oral dietary arginine supplementation up to 2% of energy intake may be beneficial after burn injury.

  14. Citrulline a More Suitable Substrate than Arginine to Restore NO Production and the Microcirculation during Endotoxemia

    PubMed Central

    Wijnands, Karolina A. P.; Vink, Hans; Briedé, Jacob J.; van Faassen, Ernst E.; Lamers, Wouter H.; Buurman, Wim A.; Poeze, Martijn

    2012-01-01

    Background Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. Methodology/Principal Findings To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg•g bodyweight−1•h−1), combined with either L-Citrulline (6.25 mg•h-1), L-Arginine (6.25 mg•h−1), or L-Alanine (isonitrogenous control; 12.5 mg•h−1) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. Conclusion/Significance L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the

  15. L-arginine independent macrophage tumor cytotoxicity

    SciTech Connect

    Klostergaard, J.; Leroux, M.E. )

    1989-12-29

    We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, NG-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.

  16. Interethnic difference in thiopurine methyltransferase activity.

    PubMed

    Klemetsdal, B; Tollefsen, E; Loennechen, T; Johnsen, K; Utsi, E; Gisholt, K; Wist, E; Aarbakke, J

    1992-01-01

    A number of metabolic pathways are subject to both genetic polymorphism and interethnic differences. A catabolic pathway of 6-mercaptopurine, red blood cell (RBC) thiopurine methyltransferase (TPMT) activity showed genetic polymorphism in Caucasians, but variation according to ethnicity has not been studied. We investigated if red blood cell thiopurine methyltransferase was subject to interethnic variation in a Saami (Lappish; n = 36) and a Caucasian population (n = 50). The Saami population sample had 29% higher thiopurine methyltransferase activity, 17.0 +/- 3.3 U/ml red blood cell compared with the Caucasian population sample, 13.1 +/- 2.9 U/ml red blood cell (p much less than 0.001). Probit plots and frequency distribution histograms supported bimodality consistent with genetic polymorphism in both study populations. Differences in chronic diseases, drug consumption, age, or gender could not explain the interethnic difference in red blood cell thiopurine methyltransferase activity. The higher red blood cell thiopurine methyltransferase activity in the Saami population group indicates that these subjects may require higher dosages of thiopurine drugs than Caucasians.

  17. Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia coli.

    PubMed Central

    Madduri, K; Torti, F; Colombo, A L; Hutchinson, C R

    1993-01-01

    Sequence analysis of a portion of the Streptomyces peucetius daunorubicin biosynthetic gene cluster revealed a complete open reading frame (dnrK) that showed DNA and protein sequence homology to several O-methyltransferases. Expression of dnrK in Streptomyces lividans and Escherichia coli was done to show that this gene codes for carminomycin 4-O-methyltransferase. The deduced carminomycin 4-O-methyltransferase protein shows a conserved nucleotide binding site for its S-adenosyl-L-methionine cofactor. Images PMID:8509343

  18. Enzymes of creatine biosynthesis, arginine and methionine metabolism in normal and malignant cells.

    PubMed

    Bera, Soumen; Wallimann, Theo; Ray, Subhankar; Ray, Manju

    2008-12-01

    The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

  19. Critical Role for Arginine Methylation in Adenovirus-Infected Cells▿

    PubMed Central

    Iacovides, Demetris C.; O'Shea, Clodagh C.; Oses-Prieto, Juan; Burlingame, Alma; McCormick, Frank

    2007-01-01

    During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection. PMID:17686851

  20. Identification and purification of arginine deiminase that originated from Mycoplasma arginini.

    PubMed Central

    Sugimura, K; Fukuda, S; Wada, Y; Taniai, M; Suzuki, M; Kimura, T; Ohno, T; Yamamoto, K; Azuma, I

    1990-01-01

    A lymphocyte blastogenesis inhibitory factor, (LBIF), was purified from the culture supernatant of human histiocytic lymphoma U937 by fast protein liquid chromatography. In this study, we demonstrated, first, that LBIF originated from a mycoplasma, Mycoplasma arginini, infecting U937 cells, and second, that LBIF bore the arginine deiminase activity. The implication of in vivo immunosuppression induced by arginine-utilizing mycoplasma species is discussed. Images PMID:2370103

  1. Epigenetic Regulation of Autophagy by the Methyltransferase G9a

    PubMed Central

    Artal-Martinez de Narvajas, Amaia; Gomez, Timothy S.; Zhang, Jin-San; Mann, Alexander O.; Taoda, Yoshiyuki; Gorman, Jacquelyn A.; Herreros-Villanueva, Marta; Gress, Thomas M.; Ellenrieder, Volker; Bujanda, Luis; Kim, Do-Hyung; Kozikowski, Alan P.

    2013-01-01

    Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy. PMID:23918802

  2. Depletion of arginine in yeast cells decreases the resistance to hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Nomura, Kazuki; Iwahashi, Hitoshi; Iguchi, Akinori; Shigematsu, Toru

    2015-07-01

    High hydrostatic pressure (HP) inhibits growth and inactivates microorganisms by destabilizing non-covalent molecular interactions. Arginine contributes to stress resistance because it has a guanidine side chain, which assists in the refolding of aggregated proteins. We attempted to analyze the contribution of arginine to high HP stress using a pressure-sensitive mutant strain of Saccharomyces cerevisiae and a metabolomics approach. Our results showed that the content of 136 out of 250 detected metabolites differed in the mutant and parent strains. Decreased metabolites were involved in the tricarboxylic acid cycle and arginine biosynthesis. The expression of genes contributing to arginine biosynthesis was significantly lower in the mutant strain than in the parent strain. When arginine was supplemented to the medium, the mutant strain showed more tolerance to pressure. These results suggest that yeast cells survived due to the contribution of arginine to high pressure resistance. This indicates that depletion of arginine caused by decreased activity of the biosynthesis pathway confers sensitivity to HP.

  3. Geometry of guanidinium groups in arginines.

    PubMed

    Malinska, Maura; Dauter, Miroslawa; Dauter, Zbigniew

    2016-09-01

    The restraints in common usage today have been obtained based on small molecule X-ray crystal structures available 25 years ago and recent reports have shown that the values of bond lengths and valence angles can be, in fact, significantly different from those stored in libraries, for example for the peptide bond or the histidine ring geometry. We showed that almost 50% of outliers found in protein validation reports released in the Protein Data Bank on 23 March 2016 come from geometry of guanidine groups in arginines. Therefore, structures of small molecules and atomic resolution protein crystal structures have been used to derive new target values for the geometry of this group. The most significant difference was found for NE-CZ-NH1 and NE-CZ-NH2 angles, showing that the guanidinium group is not symmetric. The NE-CZ-NH1 angle is larger, 121.5(10)˚, than NE-CZ-NH2, 119.2(10)˚, due to the repulsive interaction between NH1 and CD1 atom.

  4. Histone Arginine Methylation by PRMT7 Controls Germinal Center Formation via Regulating Bcl6 Transcription.

    PubMed

    Ying, Zhengzhou; Mei, Mei; Zhang, Peizhun; Liu, Chunyi; He, Huacheng; Gao, Fei; Bao, Shilai

    2015-08-15

    B cells are the center of humoral immunity and produce Abs to protect against foreign Ags. B cell defects lead to diseases such as leukemia and lymphomas. Histone arginine methylation is important for regulating gene activation and silencing in cells. Although the process commonly exists in mammalian cells, its roles in B cells are unknown. To explore the effects of aberrant histone arginine methylation on B cells, we generated mice with a B cell-specific knockout of PRMT7, a member of the methyltransferases that mediate arginine methylation of histones. In this article, we showed that the loss of PRMT7 led to decreased mature marginal zone B cells and increased follicular B cells and promoted germinal center formation after immunization. Furthermore, mice lacking PRMT7 expression in B cells secreted low levels of IgG1 and IgA. Abnormal expression of germinal center genes (i.e., Bcl6, Prdm1, and Irf4) was detected in conditional knockout mice. By overexpressing PRMT7 in the Raji and A20 cell lines derived from B cell lymphomas, we validated the fact that PRMT7 negatively regulated Bcl6 expression. Using chromatin immunoprecipitation-PCR, we found that PRMT7 could recruit H4R3me1 and symmetric H4R3me2 to the Bcl6 promoter. These results provide evidence for the important roles played by PRMT7 in germinal center formation.

  5. Combined glutamine and arginine decrease proinflammatory cytokine production by biopsies from Crohn's patients in association with changes in nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways.

    PubMed

    Lecleire, Stéphane; Hassan, Aktham; Marion-Letellier, Rachel; Antonietti, Michel; Savoye, Guillaume; Bôle-Feysot, Christine; Lerebours, Eric; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2008-12-01

    Glutamine (Gln) and arginine (Arg) are conditionally essential amino acids with immunomodulatory properties. The aim of the study was to assess the effects of Gln and Arg alone or in combination on cytokine release by cultured colonic biopsies from patients with active Crohn's disease (CD). Ten consecutive patients [mean (range) age 26 (18-39) y] with active colonic CD (mean CD activity index: 383.7 +/- 129.8) were prospectively included in the study. Eight colonic biopsies were obtained via a colonoscopy and incubated during 18 h with low (physiological) or high (pharmacological) doses of Arg (0.1 or 2 mmol/L designated as Arg(low) or Arg(high), respectively) and Gln (0.6 or 10 mmol/L designated as Gln(low) or Gln(high), respectively). The concentrations of cytokines [interleukin (IL)-4, IL-10, IL-8, IL-6, tumor necrosis factor-alpha (TNFalpha), IL-1beta, interferon-gamma) were assessed by ELISA, and nitric oxide (NO) production was evaluated by Griess assay. Nuclear factor (NF)-kappaB p65 subunit, inhibitor of NFkappaB-alpha, and p38 mitogen-activated protein kinase (MAPK) were assessed by immunoblotting. Arg(high)/Gln(high) decreased the production of TNFalpha, IL-1beta, IL-8, and IL-6 (each P < 0.01). Arg(low)/Gln(high) decreased IL-6 and IL-8 production (both P < 0.01), whereas Arg(high)/Gln(low) did not affect cytokine and NO production. Arg(low)/Gln(high) and Arg(high)/Gln(high) decreased NF-kappaB p65 subunit expression, whereas p38 MAPK was decreased only by Arg(high)/Gln(high). Combined pharmacological doses of Arg and Gln decreased TNFalpha and the main proinflammatory cytokines release in active colonic CD biopsies via NF-kappaB and p38 MAPK pathways. These results could be the basis of prospective studies evaluating the effects of enteral supply of combined Arg and Gln during active CD.

  6. Evaluation of two year treatment outcome and limited impact of arginine restriction in a patient with GAMT deficiency.

    PubMed

    Mercimek-Mahmutoglu, Saadet; Dunbar, Mary; Friesen, Andrea; Garret, Susan; Hartnett, Carol; Huh, Linda; Sinclair, Graham; Stockler, Sylvia; Wellington, Stephen; Pouwels, Petra J W; Salomons, Gajja S; Jakobs, Cornelis

    2012-01-01

    A 4-year-old female with history of developmental regression and autistic features was diagnosed with guanidinoacetate methyltransferase deficiency at age 21 months. Upon treatment, she showed improvements in her developmental milestones, sensorial-neural hearing loss and brain atrophy on cranial-MRI. The creatine/choline ratio increased 82% in basal ganglia and 88% in white matter on cranial MR-spectroscopy. The CSF guanidinoacetate decreased 80% after six months of ornithine and creatine supplementation and an additional 8% after 18 months of additional arginine restricted diet. We report the most favorable clinical and biochemical outcome on treatment in our patient.

  7. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    PubMed

    Garg, Rohini; Kumari, Romika; Tiwari, Sneha; Goyal, Shweta

    2014-01-01

    DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases), namely Methyltransferase (MET), Chromomethylase (CMT) and Domains Rearranged Methyltransferase (DRM), which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2) subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA) MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  8. Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

    PubMed

    Wang, Maoliang; Basu, Amartya; Palm, Thomas; Hua, Jack; Youngster, Stephen; Hwang, Lisa; Liu, Hsien-Ching; Li, Xiguang; Peng, Ping; Zhang, Yue; Zhao, Hong; Zhang, Zhihua; Longley, Clifford; Mehlig, Mary; Borowski, Virna; Sai, Prakash; Viswanathan, Manickam; Jang, Eun; Petti, Gerald; Liu, Sam; Yang, Karen; Filpula, David

    2006-01-01

    Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics

  9. Expression of DNA methyltransferases is influenced by growth hormone in the long-living Ames dwarf mouse in vivo and in vitro.

    PubMed

    Armstrong, Vanessa L; Rakoczy, Sharlene; Rojanathammanee, Lalida; Brown-Borg, Holly M

    2014-08-01

    Methyltransferase expression and DNA methylation are linked to aging and age-related disease. We utilized 3-, 12-, and 24-month-old Ames dwarf and their wild-type siblings to examine the genotype and age-related differences in the expression of methyltransferase enzymes related to DNA methylation in the liver, glycine-N-methyltransferase and DNA methyltransferase (DNMT). We found that DNMT proteins and transcripts are differentially expressed in dwarf mice compared with wild-type siblings that can be attributed to age and/or genotype. However, DNMT1 protein expression is drastically reduced compared with wild-type controls at every age. DNMT3a protein levels coincide with differences observed in DNMT activity. Growth hormone appears to modulate expression of DNMT1 and 3a in dwarf liver tissue and primary hepatocytes. Therefore, growth hormone may contribute to age-related processes, DNA methylation, and, ultimately, longevity.

  10. PSEUDOMONAS PYOCYANEA AND THE ARGININE DIHYDROLASE SYSTEM.

    PubMed

    TAYLOR, J J; WHITBY, J L

    1964-03-01

    Non-pigmented strains of Pseudomonas pyocyanea occur frequently and this organism has only limited activity in conventional biochemical tests; 50 strains were tested for the presence of arginine dihydrolase and found positive whereas only Salmonella sp. and Enterobacter sp. among other Gram-negative species were positive. The test for arginine dihydrolase is rapid and simple and suitable for routine use.

  11. Novel CARM1-Interacting Protein, DZIP3, Is a Transcriptional Coactivator of Estrogen Receptor-α

    PubMed Central

    Purcell, Daniel J.; Chauhan, Swati; Jimenez-Stinson, Diane; Elliott, Kathleen R.; Tsewang, Tenzin D.; Lee, Young-Ho; Marples, Brian

    2015-01-01

    Coactivator-associated arginine methyltransferase 1 (CARM1) is known to promote estrogen receptor (ER)α-mediated transcription in breast cancer cells. To further characterize the regulation of ERα-mediated transcription by CARM1, we screened CARM1-interacting proteins by yeast two-hybrid. Here, we have identified an E3 ubiquitin ligase, DAZ (deleted in azoospermia)-interacting protein 3 (DZIP3), as a novel CARM1-binding protein. DZIP3-dependent ubiquitination of histone H2A has been associated with repression of transcription. However, ERα reporter gene assays demonstrated that DZIP3 enhanced ERα-mediated transcription and cooperated synergistically with CARM1. Interaction with CARM1 was observed with the E3 ligase RING domain of DZIP3. The methyltransferase activity of CARM1 partially contributed to the synergy with DZIP3 for transcription activation, but the E3 ubiquitin ligase activity of DZIP3 was dispensable. DZIP3 also interacted with the C-terminal activation domain 2 of glucocorticoid receptor-interacting protein 1 (GRIP1) and enhanced the interaction between GRIP1 and CARM1. Depletion of DZIP3 by small interfering RNA in MCF7 cells reduced estradiol-induced gene expression of ERα target genes, GREB1 and pS2, and DZIP3 was recruited to the estrogen response elements of the same ERα target genes. These results indicate that DZIP3 is a novel coactivator of ERα target gene expression. PMID:26505218

  12. A cytochrome c methyltransferase from Crithidia oncopelti.

    PubMed Central

    Valentine, J; Pettigrew, G W

    1982-01-01

    The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c. PMID:6282265

  13. Transcriptional regulation by the Set7 lysine methyltransferase.

    PubMed

    Keating, Samuel T; El-Osta, Assam

    2013-04-01

    Posttranslational histone modifications define chromatin structure and function. In recent years, a number of studies have characterized many of the enzymatic activities and diverse regulatory components required for monomethylation of histone H3 lysine 4 (H3K4me1) and the expression of specific genes. The challenge now is to understand how this specific chemical modification is written and the Set7 methyltransferase has emerged as a key regulatory enzyme mediating methylation of lysine residues of histone and non-histone proteins. In this review, we comprehensively explore the regulatory proteins modified by Set7 and highlight mechanisms of specific co-recruitment of the enzyme to activating promoters. With a focus on signaling and transcriptional control in disease we discuss recent experimental data emphasizing specific components of diverse regulatory complexes that mediate chromatin modification and reinterpretation of Set7-mediated gene expression.

  14. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  15. New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

    PubMed Central

    Nield, Blair S.; Willows, Robert D.; Torda, Andrew E.; Gillings, Michael R.; Holmes, Andrew J.; Nevalainen, K.M. Helena; Stokes, H.W.; Mabbutt, Bridget C.

    2004-01-01

    By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%–28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins. PMID:15152095

  16. Impacts of arginine nutrition on embryonic and fetal development in mammals.

    PubMed

    Wu, Guoyao; Bazer, Fuller W; Satterfield, M Carey; Li, Xilong; Wang, Xiaoqiu; Johnson, Gregory A; Burghardt, Robert C; Dai, Zhaolai; Wang, Junjun; Wu, Zhenlong

    2013-08-01

    Embryonic loss and intrauterine growth restriction (IUGR) are significant problems in humans and other animals. Results from studies involving pigs and sheep have indicated that limited uterine capacity and placental insufficiency are major factors contributing to suboptimal reproduction in mammals. Our discovery of the unusual abundance of the arginine family of amino acids in porcine and ovine allantoic fluids during early gestation led to the novel hypothesis that arginine plays an important role in conceptus (embryo and extra-embryonic membranes) development. Arginine is metabolized to ornithine, proline, and nitric oxide, with each having important physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas ornithine and proline are substrates for uterine and placental synthesis of polyamines that are key regulators of gene expression, protein synthesis, and angiogenesis. Additionally, arginine activates the mechanistic (mammalian) target of rapamycin cell signaling pathway to stimulate protein synthesis in the placenta, uterus, and fetus. Thus, dietary supplementation with 0.83 % L-arginine to gilts consuming 2 kg of a typical gestation diet between either days 14 and 28 or between days 30 and 114 of pregnancy increases the number of live-born piglets and litter birth weight. Similar results have been reported for gestating rats and ewes. In sheep, arginine also stimulates development of fetal brown adipose tissue. Furthermore, oral administration of arginine to women with IUGR has been reported to enhance fetal growth. Collectively, enhancement of uterine as well as placental growth and function through dietary arginine supplementation provides an effective solution to improving embryonic and fetal survival and growth.

  17. Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity

    PubMed Central

    Carroll, Bernadette; Maetzel, Dorothea; Maddocks, Oliver DK; Otten, Gisela; Ratcliff, Matthew; Smith, Graham R; Dunlop, Elaine A; Passos, João F; Davies, Owen R; Jaenisch, Rudolf; Tee, Andrew R; Sarkar, Sovan; Korolchuk, Viktor I

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling. DOI: http://dx.doi.org/10.7554/eLife.11058.001 PMID:26742086

  18. Characterization of the arginine deiminase of Streptococcus equi subsp. zooepidemicus.

    PubMed

    Hong, Kyongsu

    2006-09-01

    Streptococcus equi subsp. zooepidemicus is an important cause of infectious diseases in horses and rarely humans. Little is known about the virulence factors or protective antigens of S. equi subsp. zooepidemicus. In the present study, I designed original primers based on an alignment of the gene sagp(arcA) from Streptococcus pyogenes encoding streptococcal acid glycoprotein-arginine deiminase (SAGP/AD) to amplify the S. equi subsp. zooepidemicus counterpart sequence by polymerase chain reaction, and I analyzed the sagp(arcA) gene of the organism. Using chromosomal walking steps, I identified a contiguous eight-gene locus involved in SAGP/AD production. Their open reading frames were found to share significant homologies and to correspond closely in molecular mass to previously sequenced arc genes of S. pyogenes, thus they were designated ahrC.2 (arginine repressor), arcR (CRP/FNR transcription regulator), sagp(arcA) (streptococcal acid glycoprotein-arginine deiminase), putative acetyltransferase gene, arcB (ornithine carbamyl transferase), arcD (arginine-ornithine antiporter), arcT (Xaa-His peptidase), and arcC (carbamate kinase). The SAGP homologue of S. equi subsp. zooepidemicus (SzSAGP), encoded by arcA gene of the bacteria (arcA(SZ)), was successfully expressed in Escherichia coli and purified to homogeneity. When in vitro growth inhibitory activity of the recombinant SzSAGP was tested against MOLT-3 cells, it inhibited the growth of the cells during the 3 days of culture in a dose-dependent manner, accompanied by the induction of apoptotic cell death. The recombinant protein also possessed AD activity. By immunoblot analysis using both anti-SzSAGP-SfbI(H8) and anti-SfbI(H8) sera, I was able to demonstrate that the SzSAGP protein is expressed on the streptococcal surface.

  19. Structural characterization of CalO1: a putative orsellinic acid methyltransferase in the calicheamicin-biosynthetic pathway

    SciTech Connect

    Chang, Aram; Singh, Shanteri; Bingman, Craig A.; Thorson, Jon S.; Phillips, Jr, George N.

    2011-11-07

    The X-ray structure determination at 2.4 {angstrom} resolution of the putative orsellinic acid C3 O-methyltransferase (CalO1) involved in calicheamicin biosynthesis is reported. Comparison of CalO1 with a homology model of the functionally related calicheamicin orsellinic acid C2 O-methyltransferase (CalO6) implicates several residues that are likely to contribute to the regiospecificity of alkylation. Consistent with the proposed requirement of an acyl-carrier-protein-bound substrate, this structural study also reveals structural determinants within CalO1 that are anticipated to accommodate an association with an acyl carrier protein.

  20. Occurrence of Arginine Deiminase Pathway Enzymes in Arginine Catabolism by Wine Lactic Acid Bacteria

    PubMed Central

    Liu, S.; Pritchard, G. G.; Hardman, M. J.; Pilone, G. J.

    1995-01-01

    l-Arginine, an amino acid found in significant quantities in grape juice and wine, is known to be catabolized by some wine lactic acid bacteria. The correlation between the occurrence of arginine deiminase pathway enzymes and the ability to catabolize arginine was examined in this study. The activities of the three arginine deiminase pathway enzymes, arginine deiminase, ornithine transcarbamylase, and carbamate kinase, were measured in cell extracts of 35 strains of wine lactic acid bacteria. These enzymes were present in all heterofermentative lactobacilli and most leuconostocs but were absent in all the homofermentative lactobacilli and pediococci examined. There was a good correlation among arginine degradation, formation of ammonia and citrulline, and the occurrence of arginine deiminase pathway enzymes. Urea was not detected during arginine degradation, suggesting that the catabolism of arginine did not proceed via the arginase-catalyzed reaction, as has been suggested in some earlier studies. Detection of ammonia with Nessler's reagent was shown to be a simple, rapid test to assess the ability of wine lactic acid bacteria to degrade arginine, although in media containing relatively high concentrations (>0.5%) of fructose, ammonia formation is inhibited. PMID:16534912

  1. The RNA-binding protein SERBP1 interacts selectively with the signaling protein RACK1.

    PubMed

    Bolger, Graeme B

    2017-03-04

    The RACK1 protein interacts with numerous proteins involved in signal transduction, the cytoskeleton, and mRNA splicing and translation. We used the 2-hybrid system to identify additional proteins interacting with RACK1 and isolated the RNA-binding protein SERBP1. SERPB1 shares amino acid sequence homology with HABP4 (also known as Ki-1/57), a component of the RNA spicing machinery that has been shown previously to interact with RACK1. Several different isoforms of SERBP1, generated by alternative mRNA splicing, interacted with RACK1 with indistinguishable interaction strength, as determined by a 2-hybrid beta-galactosidase assay. Analysis of deletion constructs of SERBP1 showed that the C-terminal third of the SERBP1 protein, which contains one of its two substrate sites for protein arginine N-methyltransferase 1 (PRMT1), is necessary and sufficient for it to interact with RACK1. Analysis of single amino acid substitutions in RACK1, identified in a reverse 2-hybrid screen, showed very substantial overlap with those implicated in the interaction of RACK1 with the cAMP-selective phosphodiesterase PDE4D5. These data are consistent with SERBP1 interacting selectively with RACK1, mediated by an extensive interaction surface on both proteins.

  2. Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration

    PubMed Central

    Alkaitis, Matthew S.; Nardone, Glenn; Chertow, Jessica H.

    2015-01-01

    Abstract NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd. PMID:26130049

  3. Keeping them all together: β-propeller domains in histone methyltransferase complexes.

    PubMed

    Bergamin, Elisa; Blais, Alexandre; Couture, Jean-François

    2014-10-09

    Histone methyltransferases (HKMTs) residing in multi-subunit protein complexes frequently require the presence of β-propeller proteins to achieve their biological functions. Recent biochemical studies have highlighted the functional diversity of these scaffolding proteins in maintaining the integrity of the complexes, allosterically regulating HKMT enzymatic activity and acting as "histone tethering devices" to facilitate the interaction between HKMTs and their substrates. Structural studies have revealed that, while β-propeller domain proteins share structural similarity, they employ divergent mechanisms to achieve their functions. This review focuses on the progress made in the last decade to identify the biochemical determinants underlying the functions of these important proteins.

  4. Synthesis of PET probe O(6)-[(3-[(11)C]methyl)benzyl]guanine by Pd(0)-mediated rapid C-[(11)C]methylation toward imaging DNA repair protein O(6)-methylguanine-DNA methyltransferase in glioblastoma.

    PubMed

    Koyama, Hiroko; Ikenuma, Hiroshi; Toda, Hiroshi; Kondo, Goro; Hirano, Masaki; Kato, Masaya; Abe, Junichiro; Yamada, Takashi; Wakabayashi, Toshihiko; Ito, Kengo; Natsume, Atsushi; Suzuki, Masaaki

    2017-03-18

    O(6)-Benzylguanine (O(6)-BG) is a substrate of O(6)-methylguanine-DNA methyltransferase (MGMT), which is involved in drug resistance of chemotherapy in the majority of glioblastoma multiform. For clinical diagnosis, it is hoped that the MGMT expression level could be determined by a noninvasive method to understand the detailed biological properties of MGMT-specific tumors. We synthesized (11)C-labeled O(6)-[(3-methyl)benzyl]guanine ([(11)C]mMeBG) as a positron emission tomography probe. Thus, a mixed amine-protected stannyl precursor, N(9)-(tert-butoxycarbonyl)-O(6)-[3-(tributylstannyl)benzyl]-N(2)-(trifluoroacetyl)guanine, was subjected to rapid C-[(11)C]methylation under [(11)C]CH3I/[Pd2(dba)3]/P(o-CH3C6H4)3/CuCl/K2CO3 in NMP, followed by quick deprotection with LiOH/H2O, giving [(11)C]mMeBG with total radioactivity of 1.34GBq and ≥99% radiochemical and chemical purities.

  5. Differential role of arginine mutations on the structure and functions of α-crystallin☆

    PubMed Central

    Panda, Alok Kumar; Nandi, Sandip Kumar; Chakraborty, Ayon; Nagaraj, Ram H.; Biswas, Ashis

    2016-01-01

    Background α-Crystallin is a major protein of the eye lens in vertebrates. It is composed of two subunits, αA- and αB-crystallin. α-Crystallin is an oligomeric protein having these two subunits in 3:1 ratio. It belongs to small heat shock protein family and exhibits molecular chaperone function, which plays an important role in maintaining the lens transparency. Apart from chaperone function, both subunits also exhibit anti-apoptotic property. Comparison of their primary sequences reveals that αA- and αB-crystallin posses 13 and 14 arginine residues, respectively. Several of them undergo mutations which eventually lead to various eye diseases such as congenital cataract, juvenile cataract, and retinal degeneration. Interestingly, many arginine residues of these subunits are modified during glycation and even some are truncated during aging. All these facts indicate the importance of arginine residues in α-crystallin. Scope of review In this review, we will emphasize the recent in vitro and in vivo findings related to congenital cataract causing arginine mutations in α-crystallin. Major conclusions Congenital cataract causing arginine mutations alters the structure and decreases the chaperone function of α-crystallin. These mutations also affect the lens morphology and phenotypes. Interestingly, non-natural arginine mutations (generated for mimicking the glycation and truncation environment) improve the chaperone function of α-crystallin which may play an important role in maintaining the eye lens transparency during aging. General significance The neutralization of positive charge on the guanidino group of arginine residues is not always detrimental to the functionality of α-crystallin. PMID:26080000

  6. Attenuated glomerular arginine transport prevents hyperfiltration and induces HIF-1α in the pregnant uremic rat.

    PubMed

    Schwartz, Idit F; Grupper, Ayelet; Soetendorp, Hila; Hillel, Oren; Laron, Ido; Chernichovski, Tamara; Ingbir, Merav; Shtabski, Allexander; Weinstein, Talia; Chernin, Gil; Shashar, Moshe; Hershkoviz, Rami; Schwartz, Doron

    2012-08-01

    Pregnancy worsens renal function in females with chronic renal failure (CRF) through an unknown mechanism. Reduced nitric oxide (NO) generation induces renal injury. Arginine transport by cationic amino acid transporter-1 (CAT-1), which governs endothelial NO generation, is reduced in both renal failure and pregnancy. We hypothesize that attenuated maternal glomerular arginine transport promotes renal damage in CRF pregnant rats. In uremic rats, pregnancy induced a significant decrease in glomerular arginine transport and cGMP generation (a measure of NO production) compared with CRF or pregnancy alone and these effects were prevented by l-arginine. While CAT-1 abundance was unchanged in all experimental groups, protein kinase C (PKC)-α, phosphorylated PKC-α (CAT-1 inhibitor), and phosphorylated CAT-1 were significantly augmented in CRF, pregnant, and pregnant CRF animals; phenomena that were prevented by coadministrating l-arginine. α-Tocopherol (PKC inhibitor) significantly increased arginine transport in both pregnant and CRF pregnant rats, effects that were attenuated by ex vivo incubation of glomeruli with PMA (a PKC stimulant). Renal histology revealed no differences between all experimental groups. Inulin and p-aminohippurate clearances failed to augment and renal cortical expression of hypoxia inducible factor-1α (HIF-1α) significantly increased in CRF pregnant rat, findings that were prevented by arginine. These studies suggest that in CRF rats, pregnancy induces a profound decrease in glomerular arginine transport, through posttranslational regulation of CAT-1 by PKC-α, resulting in attenuated NO generation. These events provoke renal damage manifested by upregulation of renal HIF-1α and loss of the ability to increase glomerular filtration rate during gestation.

  7. Genetic and biochemical characterization of arginine biosynthesis in Sinorhizobium meliloti 1021.

    PubMed

    Hernández, Victor M; Girard, Lourdes; Hernández-Lucas, Ismael; Vázquez, Alejandra; Ortíz-Ortíz, Catalina; Díaz, Rafael; Dunn, Michael F

    2015-08-01

    L-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates l-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of argB in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. argE and argJ single mutants, and an argEJ double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate ama genes (smc00682 (hipO1), smc02256 (hipO2) and smb21279) all possessed NAO deacetylase activity. hipO1 and hipO2, but not smb21279, expressed in trans functionally complemented an Escherichia coli ΔargE : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the argEJ mutant. Transcriptional assays of argB, argE and argJ, fused to a promoterless gusA gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.

  8. Effect of arginine on oligomerization and stability of N-acetylglutamate synthase

    PubMed Central

    Haskins, N.; Mumo, A.; Brown, P. H.; Tuchman, M.; Morizono, H.; Caldovic, L.

    2016-01-01

    N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-acetylglutamate synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS. PMID:27934952

  9. COBALAMIN- AND COBAMIDE-DEPENDENT METHYLTRANSFERASES

    PubMed Central

    Matthews, Rowena G.; Koutmos, Markos; Datta, Supratim

    2008-01-01

    Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple SN2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co+1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled. PMID:19059104

  10. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia

    PubMed Central

    He, Yupeng; Ferguson, Debra; Jagadeeswaran, Sujatha; Osterling, Donald J.; Gao, Wenqing; Spence, Julie K.; Pliushchev, Marina; Sweis, Ramzi F.; Buchanan, Fritz G.; Michaelides, Michael R.; Shoemaker, Alexander R.; Tse, Chris; Chiang, Gary G.

    2015-01-01

    Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2) is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells. PMID:26147105

  11. Preparation, Biochemical Analysis, and Structure Determination of SET Domain Histone Methyltransferases.

    PubMed

    Bergamin, E; Couture, J F

    2016-01-01

    In eukaryotes, several lysine residues on histone proteins are methylated. This posttranslational modification is linked to a myriad of nuclear-based transactions such as epigenetic inheritance of heterochromatin, regulation of gene expression, DNA damage repair, and DNA replication. The majority of the enzymes responsible for writing these marks onto chromatin belong to the SET domain family of histone lysine methyltransferases. Although they often share important structural features, including a conserved catalytic domain, SET domain enzymes use different mechanisms to achieve substrate recognition, mono-, di-, or trimethylate lysine residues and some require other proteins to achieve maximal methyltransferase activity. In this chapter, we summarize our efforts to purify, crystallize, and enzymatically characterize SET domain enzymes with a specific focus on the histone H3K27 monomethyltransferase ATXR5.

  12. Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

    SciTech Connect

    Som, S.; Friedman, S. )

    1990-03-15

    Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either (methyl-3H)AdoMet or (35S)AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of (3H)AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported. The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.

  13. Glycine N-methyltransferase tumor susceptibility gene in the benzo(a)pyrene-detoxification pathway.

    PubMed

    Chen, Shih-Yin; Lin, Jane-Ru Vivan; Darbha, Ramalakshmi; Lin, Pinpin; Liu, Tsung-Yun; Chen, Yi-Ming Arthur

    2004-05-15

    Glycine N-methyltransferase (GNMT) affects genetic stability by (a) regulating the ratio of S-adenosylmethionine to S-adenosylhomocystine and (b) binding to folate. Based on the identification of GNMT as a 4 S polyaromatic hydrocarbon-binding protein, we used liver cancer cell lines that expressed GNMT either transiently or stably in cDNA transfections to analyze the role of GNMT in the benzo(a)pyrene (BaP) detoxification pathway. Results from an indirect immunofluorescent antibody assay showed that GNMT was expressed in cell cytoplasm before BaP treatment and translocated to cell nuclei after BaP treatment. Compared with cells transfected with the vector plasmid, the number of BaP-7,8-diol 9,10-epoxide-DNA adducts that formed in GNMT-expressing cells was significantly reduced. Furthermore, the dose-dependent inhibition of BaP-7,8-diol 9,10-epoxide-DNA adduct formation by GNMT was observed in HepG2 cells infected with different multiplicities of infection of recombinant adenoviruses carrying GNMT cDNA. According to an aryl hydrocarbon hydroxylase enzyme activity assay, GNMT inhibited BaP-induced cytochrome P450 1A1 enzyme activity. Automated BaP docking using a Lamarckian genetic algorithm with GNMT X-ray crystallography revealed a BaP preference for the S-adenosylmethionine-binding domain of the dimeric form of GNMT, a novel finding of a cellular defense against potentially damaging exposures. In addition to GNMT, results from docking experiments showed that BaP binds readily with other DNA methyltransferases, including HhaI, HaeIII, PvuII methyltransferases and human DNA methyltransferase 2. We therefore hypothesized that BaP-DNA methyltransferase and BaP-GNMT interactions may contribute to carcinogenesis.

  14. Arginine Enhances Osteoblastogenesis and Inhibits Adipogenesis through the Regulation of Wnt and NFATc Signaling in Human Mesenchymal Stem Cells

    PubMed Central

    Huh, Jeong-Eun; Choi, Jun-Young; Shin, Ye-Ok; Park, Dong-Suk; Kang, Jung Won; Nam, Dongwoo; Choi, Do-Young; Lee, Jae-Dong

    2014-01-01

    Arginine, an α-amino acid, has been reported to exert beneficial effects that ameliorate health problems and prevent excessive fat deposition. In this study, we investigated whether the activation of cell signaling by arginine can induce osteogenic differentiation and modulate excessive adipogenic differentiation in human mesenchymal stem cells (MSCs). Arginine potently induced the expression of type Iα1 collagen, osteocalcin, and ALP in a dose-dependent manner without causing cytotoxicity. Arginine significantly increased the mRNA expression of the osteogenic transcription factors runt-related transcription factor 2 (Runx2), DIx5, and osterix. Furthermore, arginine demonstrated its antiadipogenicity by decreasing adipocyte formation and triglyceride (TG) content in MSCs and inhibiting the mRNA expression of the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (Fabp4). This effect was associated with increased expression of Wnt5a, and nuclear factor of activated T-cells (NFATc), and was abrogated by antagonists of Wnt and NFATc, which indicated a role of Wnt and NFATc signaling in the switch from adipogenesis to osteoblastogenesis induced by arginine. In conclusion, this is the first report of the dual action of arginine in promoting osteogenesis and inhibiting adipocyte formation through involving Wnt5a and NFATc signaling pathway. PMID:25054323

  15. Association of Single Nucleotide Polymorphisms in Catechol-O-Methyltransferase and Serine-Threonine Protein Kinase Genes in the Pakistani Schizophrenic Population: A Study with Special Emphasis on Cannabis and Smokeless Tobacco.

    PubMed

    Nawaz, Rukhsana; Siddiqui, Sonia

    2015-01-01

    Schizophrenia is a neuropsychiatric disorder in which abnormalities in the prefrontal cortex lead to impaired synthesis of dopamine. It is associated with hallucination, psychosis and hearing impairments. Many susceptible genes have been identified in schizophrenia such as catechol-O-methyltransferase (COMT) and serine/threonine kinase (AKT1). Single nucleotide polymorphisms (SNPs) in these genes have not been identified in Pakistan. Therefore, we investigated the allelic and genotypic frequencies in COMT and AKT1 genes in the Pakistani population. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to identify SNPs in the genes. The present study shows that COMT Val and COMT Met allelic frequencies for the controls were p=0.52, q=0.48 and for the schizophrenic cases they were p=0.34, q=0.66 respectively. The distribution of polymorphism in COMT Val158Met genotype by Hardy-Weinberg equilibrium (HWE) was P=0.61 for controls and P=0.005 for cases. The data reveal that SNP rs1130214 T allele mutation was found neither in patients nor in controls in the 5' untranslated region (UTR). This proves that no association of AKT1 and positive association of COMT with schizophrenia exist in the population of Pakistan. Moreover, a study based on a single family showed COMT Met allele inheritance in schizophrenic offspring. This suggested that COMT allele alteration influences susceptibility to at least some forms of psychosis in the Pakistani population. Interestingly, according to our socio-economical survey, COMT genotype has no association with cannabis but it is strongly associated with tobacco. The Pakistani population with Val158Met SNP showed more susceptibility towards developing schizophrenia. This study highlights the genetic differences between Pakistani and other Caucasian populations.

  16. Diabetic nephropathy is resistant to oral l-arginine or l-citrulline supplementation

    PubMed Central

    You, Hanning; Gao, Ting; Cooper, Timothy K.; Morris, Sidney M.

    2014-01-01

    Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide (NO) synthase (NOS)3-dependent mechanism. Arginase competes with endothelial NOS (eNOS) for the common substrate l-arginine. Lack of l-arginine results in reduced NO production and eNOS uncoupling, which lead to endothelial dysfunction. Therefore, we hypothesized that l-arginine or l-citrulline supplementation would ameliorate diabetic nephropathy. DBA mice injected with multiple low doses of vehicle or streptozotocin (50 mg/kg ip for 5 days) were provided drinking water with or without l-arginine (1.5%, 6.05 g·kg−1·day−1) or l-citrulline (1.66%, 5.73 g·kg−1·day−1) for 9 wk. Nonsupplemented diabetic mice showed significant increases in albuminuria, blood urea nitrogen, glomerular histopathological changes, kidney macrophage recruitment, kidney TNF-α and fibronectin mRNA expression, kidney arginase activity, kidney arginase-2 protein expression, and urinary oxidative stress along with a significant reduction of nephrin and eNOS protein expression and kidney nitrite + nitrate compared with normal mice after 9 wk of diabetes. Surprisingly, l-arginine or l-citrulline supplementation in diabetic mice did not affect any of these parameters despite greatly increasing kidney and plasma arginine levels. These findings demonstrate that chronic l-arginine or l-citrulline supplementation does not prevent or reduce renal injury in a model of type 1 diabetes. PMID:25320354

  17. Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation.

    PubMed

    Thanh Mai Pham, Le; Kim, Yong Hwan

    2016-01-01

    Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5'-adenosyl)-L-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.

  18. Comparative Distribution and Retention of Arsenic in Arsenic (+3 Oxidation State) Methyltransferase Knockout and Wild Type Mice

    EPA Science Inventory

    The mouse arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a ~ 43 kDa protein that catalyzes conversion of inorganic arsenic into methylated products. Heterologous expression of AS3MT or its silencing by RNA interference controls arsenic methylation phenotypes...

  19. A liquid chromatography/mass spectrometry-based generic detection method for biochemical assay and hit discovery of histone methyltransferases.

    PubMed

    Li, Shu; Gu, X Justin; Hao, Qin; Fan, Hong; Li, Ling; Zhou, Shaolian; Zhao, Kehao; Chan, Ho Man; Wang, Y Karen

    2013-12-15

    Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.

  20. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    SciTech Connect

    Song, Yuan; Wu, Keqiang; Dhaubhadel, Sangeeta; An, Lizhe; Tian, Lining

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  1. Detection of a novel arginine vasopression defect by dideoxy fingerprinting

    SciTech Connect

    Krishnamani, M.R.S.; Phillips, J.A. III; Copeland, K.C. Univ. of Vermont College of Medicine, Burlington, VT )

    1993-09-01

    Autosomal dominant neurohypophyseal diabetes insipidus is a familial form of diabetes insipidus. This disorder is associated with variable levels of arginine vasopressin (AVP) and diabetes insipidus of varying severity, which responds to exogenous AVP. To determine the molecular basis of autosomal dominant neurohypophyseal diabetes insipidus, the AVP genes of members of a large kindred were analyzed. A new method, called dideoxy fingerprinting, was used to detect an AVP mutation that was characterized by DNA sequencing. The novel defect found changes the last codon of the AVP signal peptide from alanine to threonine, which should perturb cleavage of mature AVP from its precursor protein and inhibit its secretion or action. 18 refs., 3 figs.

  2. Targeting Arginine-Dependent Cancers with Arginine-Degrading Enzymes: Opportunities and Challenges

    PubMed Central

    Phillips, Melissa M.; Sheaff, Michael T.

    2013-01-01

    Arginine deprivation is a novel antimetabolite strategy for the treatment of arginine-dependent cancers that exploits differential expression and regulation of key urea cycle enzymes. Several studies have focused on inactivation of argininosuccinate synthetase 1 (ASS1) in a range of malignancies, including melanoma, hepatocellular carcinoma (HCC), mesothelial and urological cancers, sarcomas, and lymphomas. Epigenetic silencing has been identified as a key mechanism for loss of the tumor suppressor role of ASS1 leading to tumoral dependence on exogenous arginine. More recently, dysregulation of argininosuccinate lyase has been documented in a subset of arginine auxotrophic glioblastoma multiforme, HCC and in fumarate hydratase-mutant renal cancers. Clinical trials of several arginine depletors are ongoing, including pegylated arginine deiminase (ADI-PEG20, Polaris Group) and bioengineered forms of human arginase. ADI-PEG20 is furthest along the path of clinical development from combinatorial phase 1 to phase 3 trials and is described in more detail. The challenge will be to identify tumors sensitive to drugs such as ADI-PEG20 and integrate these agents into multimodality drug regimens using imaging and tissue/fluid-based biomarkers as predictors of response. Lastly, resistance pathways to arginine deprivation require further study to optimize arginine-targeted therapies in the oncology clinic. PMID:24453997

  3. Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo.

    PubMed

    Miraki-Moud, Farideh; Ghazaly, Essam; Ariza-McNaughton, Linda; Hodby, Katharine A; Clear, Andrew; Anjos-Afonso, Fernando; Liapis, Konstantinos; Grantham, Marianne; Sohrabi, Fareeda; Cavenagh, Jamie; Bomalaski, John S; Gribben, John G; Szlosarek, Peter W; Bonnet, Dominique; Taussig, David C

    2015-06-25

    The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

  4. Camphorquinone-10-sulfonic acid and derivatives: convenient reagents for reversible modification of arginine residues

    SciTech Connect

    Pande, C.S.; Pelzig, M.; Glass, J.D.

    1980-02-01

    Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonsulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by o-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents.The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.

  5. Reconstitution of an active arginine deiminase pathway in Mycoplasma pneumoniae M129.

    PubMed

    Rechnitzer, Hagai; Rottem, Shlomo; Herrmann, Richard

    2013-10-01

    Some species of the genus Mycoplasma code for the arginine deiminase pathway (ADI), which enables these bacteria to produce ATP from arginine by the successive reaction of three enzymes: arginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB), and carbamate kinase (ArcC). It so far appears that independently isolated strains of Mycoplasma pneumoniae encode an almost identical truncated version of the ADI pathway in which the proteins ArcA and ArcB have lost their original enzymatic activities due to the deletion of significant regions of these proteins. To study the consequences of a functional ADI pathway, M. pneumoniae M129 was successfully transformed with the cloned functional arcA, arcB, and arcC genes from Mycoplasma fermentans. Enzymatic tests showed that while the M. pneumoniae ArcAB and ArcABC transformants possess functional arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase, they were unable to grow on arginine as the sole energy source. Nevertheless, infection of a lung epithelial cell line, A549, with the M. pneumoniae transformants showed that almost 100% of the infected host cells were nonviable, while most of the lung cells infected with nontransformed M. pneumoniae were viable under the same experimental conditions.

  6. Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA

    PubMed Central

    Bikkavilli, Rama Kamesh; Malbon, Craig C.

    2011-01-01

    Wnt/β-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/β-catenin pathway through stabilization of β-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA. PMID:21652632

  7. Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

    PubMed

    Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

    2017-04-01

    Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein.

  8. Arginine, scurvy and Cartier's "tree of life"

    PubMed Central

    Durzan, Don J

    2009-01-01

    Several conifers have been considered as candidates for "Annedda", which was the source for a miraculous cure for scurvy in Jacques Cartier's critically ill crew in 1536. Vitamin C was responsible for the cure of scurvy and was obtained as an Iroquois decoction from the bark and leaves from this "tree of life", now commonly referred to as arborvitae. Based on seasonal and diurnal amino acid analyses of candidate "trees of life", high levels of arginine, proline, and guanidino compounds were also probably present in decoctions prepared in the severe winter. The semi-essential arginine, proline and all the essential amino acids, would have provided additional nutritional benefits for the rapid recovery from scurvy by vitamin C when food supply was limited. The value of arginine, especially in the recovery of the critically ill sailors, is postulated as a source of nitric oxide, and the arginine-derived guanidino compounds as controlling factors for the activities of different nitric oxide synthases. This review provides further insights into the use of the candidate "trees of life" by indigenous peoples in eastern Canada. It raises hypotheses on the nutritional and synergistic roles of arginine, its metabolites, and other biofactors complementing the role of vitamin C especially in treating Cartier's critically ill sailors. PMID:19187550

  9. Arginine, scurvy and Cartier's "tree of life".

    PubMed

    Durzan, Don J

    2009-02-02

    Several conifers have been considered as candidates for "Annedda", which was the source for a miraculous cure for scurvy in Jacques Cartier's critically ill crew in 1536. Vitamin C was responsible for the cure of scurvy and was obtained as an Iroquois decoction from the bark and leaves from this "tree of life", now commonly referred to as arborvitae. Based on seasonal and diurnal amino acid analyses of candidate "trees of life", high levels of arginine, proline, and guanidino compounds were also probably present in decoctions prepared in the severe winter. The semi-essential arginine, proline and all the essential amino acids, would have provided additional nutritional benefits for the rapid recovery from scurvy by vitamin C when food supply was limited. The value of arginine, especially in the recovery of the critically ill sailors, is postulated as a source of nitric oxide, and the arginine-derived guanidino compounds as controlling factors for the activities of different nitric oxide synthases. This review provides further insights into the use of the candidate "trees of life" by indigenous peoples in eastern Canada. It raises hypotheses on the nutritional and synergistic roles of arginine, its metabolites, and other biofactors complementing the role of vitamin C especially in treating Cartier's critically ill sailors.

  10. Arginine Deiminase Enzyme Evolving As A Potential Antitumor Agent.

    PubMed

    Somani, Rakesh; Chaskar, Pratip K

    2016-08-17

    Some melanomas and hepatocellular carcinomas have been shown to be auxotrophic for arginine. Arginine deiminase (ADI), an arginine degrading enzyme isolated from Mycoplasma, can inhibit the growth of these tumors. It is a catabolizing enzyme which catabolizes arginine to citrulline. Tumor cells do not express an enzyme called arginosuccinate synthetase (ASS) and hence, these cells becomes auxotrophic for arginine. It is found that ADI is specific for arginine and did not degrade other amino acid. This review covers various aspects of ADIs like origin, properties and chemical modifications for better antitumor activity.

  11. Structural Analysis and Insights into the Oligomeric State of an Arginine-Dependent Transcriptional Regulator from Bacillus halodurans

    PubMed Central

    Park, Young Woo; Kang, Jina; Yeo, Hyun Ku; Lee, Jae Young

    2016-01-01

    The arginine repressor (ArgR) is an arginine-dependent transcription factor that regulates the expression of genes encoding proteins involved in the arginine biosynthesis and catabolic pathways. ArgR is a functional homolog of the arginine-dependent repressor/activator AhrC from Bacillus subtilis, and belongs to the ArgR/AhrC family of transcriptional regulators. In this research, we determined the structure of the ArgR (Bh2777) from Bacillus halodurans at 2.41 Å resolution by X-ray crystallography. The ArgR from B. halodurans appeared to be a trimer in a size exclusion column and in the crystal structure. However, it formed a hexamer in the presence of L-arginine in multi-angle light scattering (MALS) studies, indicating the oligomerization state was dependent on the presence of L-arginine. The trimeric structure showed that the C-terminal domains form the core, which was made by inter-subunit interactions mainly through hydrophobic contacts, while the N-terminal domains containing a winged helix-turn-helix DNA binding motif were arranged around the periphery. The arrangement of trimeric structure in the B. halodurans ArgR was different from those of other ArgR homologs previously reported. We finally showed that the B. halodurans ArgR has an arginine-dependent DNA binding property by an electrophoretic mobility shift assay. PMID:27171430

  12. Improving cancer immunotherapy with DNA methyltransferase inhibitors.

    PubMed

    Saleh, Mohammad H; Wang, Lei; Goldberg, Michael S

    2016-07-01

    Immunotherapy confers durable clinical benefit to melanoma, lung, and kidney cancer patients. Challengingly, most other solid tumors, including ovarian carcinoma, are not particularly responsive to immunotherapy, so combination with a complementary therapy may be beneficial. Recent findings suggest that epigenetic modifying drugs can prime antitumor immunity by increasing expression of tumor-associated antigens, chemokines, and activating ligands by cancer cells as well as cytokines by immune cells. This review, drawing from both preclinical and clinical data, describes some of the mechanisms of action that enable DNA methyltransferase inhibitors to facilitate the establishment of antitumor immunity.

  13. In vivo specificity of EcoRI DNA methyltransferase.

    PubMed Central

    Smith, D W; Crowder, S W; Reich, N O

    1992-01-01

    The EcoRI adenine DNA methyltransferase forms part of a bacterial restriction/modification system; the methyltransferase modifies the second adenine within the canonical site GAATTC, thereby preventing the EcoRI endonuclease from cleaving this site. We show that five noncanonical EcoRI sites (TAATTC, CAATTC, GTATTC, GGATTC and GAGTTC) are not methylated in vivo under conditions when the canonical site is methylated. Only when the methyltransferase is overexpressed is partial in vivo methylation of the five sites detected. Our results suggest that the methyltransferase does not protect host DNA against potential endonuclease-mediated cleavage at noncanonical sites. Our related in vitro analysis of the methyltransferase reveals a low level of sequence-discrimination. We propose that the high in vivo specificity may be due to the active removal of methylated sequences by DNA repair enzymes (J. Bacteriology (1987), 169 3243-3250). Images PMID:1461739

  14. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    SciTech Connect

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  15. Methylated nucleosides in tRNA and tRNA methyltransferases

    PubMed Central

    Hori, Hiroyuki

    2014-01-01

    To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

  16. MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode

    PubMed Central

    Wagner, Tristan; Ermler, Ulrich; Shima, Seigo

    2016-01-01

    In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA–H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change. PMID:27324530

  17. Methyltransferase and demethylase profiling studies during brown adipocyte differentiation.

    PubMed

    Son, Min Jeong; Kim, Won Kon; Oh, Kyoung-Jin; Park, Anna; Lee, Da Som; Han, Baek Soo; Lee, Sang Chul; Bae, Kwang-Hee

    2016-07-01

    Although brown adipose tissue is important with regard to energy balance, the molecular mechanism of brown adipocyte differentiation has not been extensively studied. Specifically, regulation factors at the level of protein modification are largely unknown. In this study, we examine the changes in the expression level of enzymes which are involved in protein lysine methylation during brown adipocyte differentiation. Several enzymes, in this case SUV420H2, PRDM9, MLL3 and JHDM1D, were found to be up-regulated. On the other hand, Set7/9 was significantly down-regulated. In the case of SUV420H2, the expression level increased sharply during brown adipocyte differentiation, whereas the expression of SUV420H2 was marginally enhanced during the white adipocyte differentiation. The knock-down of SUV420H2 caused the suppression of brown adipocyte differentiation, as compared to a scrambled control. These results suggest that SUV420H2, a methyltransferase, is involved in brown adipocyte differentiation, and that the methylation of protein lysine is important in brown adipocyte differentiation. [BMB Reports 2016; 49(7): 388-393].

  18. Methyltransferase and demethylase profiling studies during brown adipocyte differentiation

    PubMed Central

    Son, Min Jeong; Kim, Won Kon; Oh, Kyoung-Jin; Park, Anna; Lee, Da Som; Han, Baek Soo; Lee, Sang Chul; Bae, Kwang-Hee

    2016-01-01

    Although brown adipose tissue is important with regard to energy balance, the molecular mechanism of brown adipocyte differentiation has not been extensively studied. Specifically, regulation factors at the level of protein modification are largely unknown. In this study, we examine the changes in the expression level of enzymes which are involved in protein lysine methylation during brown adipocyte differentiation. Several enzymes, in this case SUV420H2, PRDM9, MLL3 and JHDM1D, were found to be up-regulated. On the other hand, Set7/9 was significantly down-regulated. In the case of SUV420H2, the expression level increased sharply during brown adipocyte differentiation, whereas the expression of SUV420H2 was marginally enhanced during the white adipocyte differentiation. The knock-down of SUV420H2 caused the suppression of brown adipocyte differentiation, as compared to a scrambled control. These results suggest that SUV420H2, a methyltransferase, is involved in brown adipocyte differentiation, and that the methylation of protein lysine is important in brown adipocyte differentiation. [BMB Reports 2016; 49(7): 388-393] PMID:27157542

  19. Methionine cycle kinetics and arginine supplementation in endothelial dysfunction of ESRD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate the effect of arginine supplementation on metabolic pathways involved in endothelial dysfunction of end stage renal disease (ESRD), we conducted a study on 11 ESRD patients age 49+/-16; wt 93+/-26 kg receiving an adequate protein and energy intake for 1 week, followed by a primed, con...

  20. Role of Environmental Conditions on the Interaction of L-Arginine with Oxide Mineral Surfaces

    NASA Astrophysics Data System (ADS)

    Klochko, K.; Jonsson, C. M.; Jonsson, C. L.; Lee, N.; Cleaves, H. J., II; Sverjensky, D. A.; Hazen, R. M.

    2010-04-01

    The current study is focused on surface interactions between L-arginine, the most basic protein amino acid, and rutile in NaCl media over a wide range of solution pH conditions, amino acid concentrations, and solution ionic strengths.