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Sample records for protein arginine methyltransferase

  1. Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity.

    PubMed

    Morales, Yalemi; Nitzel, Damon V; Price, Owen M; Gui, Shanying; Li, Jun; Qu, Jun; Hevel, Joan M

    2015-06-12

    Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner. PMID:25911106

  2. 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

  3. The Role of Protein Arginine Methyltransferases in Inflammatory Responses

    PubMed Central

    Kim, Ji Hye; Yoo, Byong Chul; Yang, Woo Seok; Kim, Eunji; Hong, Sungyoul

    2016-01-01

    Protein arginine methyltransferases (PRMTs) mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I–IV). Although most PRMTs do not require posttranslational modification (PTM) to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6) in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses. PMID:27041824

  4. The Role of Protein Arginine Methyltransferases in Inflammatory Responses.

    PubMed

    Kim, Ji Hye; Yoo, Byong Chul; Yang, Woo Seok; Kim, Eunji; Hong, Sungyoul; Cho, Jae Youl

    2016-01-01

    Protein arginine methyltransferases (PRMTs) mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I-IV). Although most PRMTs do not require posttranslational modification (PTM) to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6) in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses. PMID:27041824

  5. Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

    PubMed Central

    Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

    2013-01-01

    The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

  6. 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. PMID:17711414

  7. Aflatoxin B1 induced upregulation of protein arginine methyltransferase 5 in human cell lines.

    PubMed

    Ghufran, Md Sajid; Ghosh, Krishna; Kanade, Santosh R

    2016-09-01

    The exposure of naturally occurring mycotoxins affects human health and play a vital role in cancer initiation and progression. Aflatoxin B1 is a difuranocoumarin mycotoxin, classified as a group I carcinogen. The present study was conducted to assess the effect of aflatoxin B1 on epigenetic regulatory proteins. The protein arginine methyltransferase 5 expression was induced upon aflatoxin B1 treatment in a dose and time dependent manner. Further global arginine methylation was also increased in the same manner. This is the first report showing the induction of epigenetic regulatory protein, protein arginine methyltransferase 5 upon aflatoxin B1 treatment. Further study is required to establish the detailed pathway of PRMT5 induction. PMID:27242039

  8. 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.

  9. 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.

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

    PubMed

    Eram, Mohammad S; Shen, Yudao; Szewczyk, Magdalena M; 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

    2016-03-18

    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 the 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

  11. 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 ...

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

    PubMed

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

    2016-02-23

    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

  13. 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

  14. Cloning, expression, purification and preliminary X-ray crystallographic analysis of mouse protein arginine methyltransferase 7.

    PubMed

    Cura, Vincent; Troffer-Charlier, Nathalie; Lambert, Marie-Annick; Bonnefond, Luc; Cavarelli, Jean

    2014-01-01

    Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 97.4, c = 168.1 Å and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. PMID:24419624

  15. 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

  16. Arginine methyltransferases in normal and malignant hematopoiesis.

    PubMed

    Greenblatt, Sarah M; Liu, Fan; Nimer, Stephen D

    2016-06-01

    Arginine methylation is an abundant covalent modification that regulates diverse cellular processes, including transcription, translation, DNA repair, and RNA processing. The enzymes that catalyze these marks are known as the protein arginine methyltransferases (PRMTs), and they can generate asymmetric dimethyl arginine (type I arginine methyltransferases), symmetric dimethylarginine (type II arginine methyltransferases), or monomethyarginine (type III arginine methyltransferases). The PRMTs are capable of modifying diverse substrates, from histone components to specific nuclear and cytoplasmic proteins. Additionally, the PRMTs can orchestrate chromatin remodeling by blocking the docking of other epigenetic modifying enzymes or by recruiting them to specific gene loci. In the hematopoietic system, PRMTs can regulate cell behavior, including the critical balance between stem cell self-renewal and differentiation, in at least two critical ways, via (i) the covalent modification of transcription factors and (ii) the regulation of histone modifications at promoters critical to cell fate determination. Given these important functions, it is not surprising that these processes are altered in hematopoietic malignancies, such as acute myeloid leukemia, where they promote increased self-renewal and impair hematopoietic stem and progenitor cell differentiation. PMID:27026282

  17. Identification of Methylated Proteins in the Yeast Small Ribosomal Subunit: A Role for SPOUT Methyltransferases in Protein Arginine Methylation†

    PubMed Central

    Young, Brian D.; Weiss, David I.; Zurita-Lopez, Cecilia I.; Webb, Kristofor J.; Clarke, Steven G.; McBride, Anne E.

    2012-01-01

    We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation on Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes. PMID:22650761

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

    PubMed

    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. PMID:25721668

  19. The Arginine Methyltransferase PRMT6 Cooperates with Polycomb Proteins in Regulating HOXA Gene Expression

    PubMed Central

    Bouchard, Caroline; Bauer, Uta-Maria

    2016-01-01

    Protein arginine methyltransferase 6 (PRMT6) catalyses asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a), which has been shown to impede the deposition of histone H3 lysine 4 trimethylation (H3K4me3) by blocking the binding and activity of the MLL1 complex. Importantly, the genomic occurrence of H3R2me2a has been found to coincide with histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone mark generated by the Polycomb repressive complex 2 (PRC2). Therefore, we investigate here a putative crosstalk between PRMT6- and PRC-mediated repression in a cellular model of neuronal differentiation. We show that PRMT6 and subunits of PRC2 as well as PRC1 are bound to the same regulatory regions of rostral HOXA genes and that they control the differentiation-associated activation of these genes. Furthermore, we find that PRMT6 interacts with subunits of PRC1 and PRC2 and that depletion of PRMT6 results in diminished PRC1/PRC2 and H3K27me3 occupancy and in increased H3K4me3 levels at these target genes. Taken together, our data uncover a novel, additional mechanism of how PRMT6 contributes to gene repression by cooperating with Polycomb proteins. PMID:26848759

  20. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma

    PubMed Central

    Yan, Fengting; Alinari, Lapo; Lustberg, Mark E.; Martin, Ludmila Katherine; Cordero-Nieves, Hector M.; Banasavadi-Siddegowda, Yeshavanth; Virk, Selene; Barnholtz-Sloan, Jill; Bell, Erica Hlavin; Wojton, Jeffrey; Jacob, Naduparambil K.; Chakravarti, Arnab; Nowicki, Michal O.; Wu, Xin; Lapalombella, Rosa; Datta, Jharna; Yu, Bo; Gordon, Kate; Haseley, Amy; Patton, John T.; Smith, Porsha L.; Ryu, John; Zhang, Xiaoli; Mo, Xiaokui; Marcucci, Guido; Nuovo, Gerard; Kwon, Chang-Hyuk; Byrd, John C.; Chiocca, E. Antonio; Li, Chenglong; Sif, Said; Jacob, Samson; Lawler, Sean; Kaur, Balveen; Baiocchi, Robert A.

    2014-01-01

    Glioblastoma (GBM) is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric di-methylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell cycle arrest, apoptosis and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Lastly, PRMT5 attenuation enhanced GBM cell survival in a mouse xenograft model of aggressive GBM. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in GBM, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease. PMID:24453002

  1. Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

    PubMed

    Park, Ji Hyun; Szemes, Marianna; Vieira, Gabriella Cunha; Melegh, Zsombor; Malik, Sally; Heesom, Kate J; Von Wallwitz-Freitas, Laura; Greenhough, Alexander; Brown, Keith W; Zheng, Y George; Catchpoole, Daniel; Deery, Michael J; Malik, Karim

    2015-03-01

    Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene. PMID:25475372

  2. Myc and Omomyc functionally associate with the Protein Arginine Methyltransferase 5 (PRMT5) in glioblastoma cells

    PubMed Central

    Mongiardi, Maria Patrizia; Savino, Mauro; Bartoli, Laura; Beji, Sara; Nanni, Simona; Scagnoli, Fiorella; Falchetti, Maria Laura; Favia, Annarita; Farsetti, Antonella; Levi, Andrea; Nasi, Sergio; Illi, Barbara

    2015-01-01

    The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)—the catalyst of the reaction—and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy. PMID:26563484

  3. 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.

  4. 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.

  5. Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing

    PubMed Central

    Hang, Runlai; Liu, Chunyan; Ahmad, Ayaz; Zhang, Yong; Lu, Falong; Cao, Xiaofeng

    2014-01-01

    Ribosome biogenesis is a fundamental and tightly regulated cellular process, including synthesis, processing, and assembly of rRNAs with ribosomal proteins. Protein arginine methyltransferases (PRMTs) have been implicated in many important biological processes, such as ribosome biogenesis. Two alternative precursor rRNA (pre-rRNA) processing pathways coexist in yeast and mammals; however, how PRMT affects ribosome biogenesis remains largely unknown. Here we show that Arabidopsis PRMT3 (AtPRMT3) is required for ribosome biogenesis by affecting pre-rRNA processing. Disruption of AtPRMT3 results in pleiotropic developmental defects, imbalanced polyribosome profiles, and aberrant pre-rRNA processing. We further identify an alternative pre-rRNA processing pathway in Arabidopsis and demonstrate that AtPRMT3 is required for the balance of these two pathways to promote normal growth and development. Our work uncovers a previously unidentified function of PRMT in posttranscriptional regulation of rRNA, revealing an extra layer of complexity in the regulation of ribosome biogenesis. PMID:25352672

  6. Protein arginine methyltransferase 5 is associated with malignant phenotype and peritoneal metastasis in gastric cancer.

    PubMed

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

    2016-09-01

    Identification of novel gastric cancer (GC)-related molecules is necessary to improve management of patients with GC in both diagnostic and therapeutic aspects. The aim of the present study was to determine whether protein arginine methyltransferase 5 (PRMT5) acts as an oncogene in the progression of GC and whether it serves as a novel diagnostic marker and therapeutic target. We conducted global expression profiling of GC cell lines and RNA interference experiments to evaluate the effect of PRMT5 expression on the phenotype of GC cells. We analysed tissues of 179 patients with GC to assess the association of PRMT5 mRNA levels with clinicopathological factors. Differential expression of PRMT5 mRNA by GC cell lines correlated positively with the levels of GEMIN2, STAT3 and TGFB3. PRMT5 knockdown reduced the proliferation, invasion and migration of a GC cell line. PRMT5 mRNA levels were significantly higher in GC tissues than the corresponding adjacent normal tissues and were independent of tumour depth, differentiation and lymph node metastasis. High PRMT5 expression was an independent risk factor of positive peritoneal lavage cytology (odds ratio 3.90, P=0.003) and decreased survival. PRMT5 enhances the malignant phenotype of GC cell lines and its expression in gastric tissues may serve as a biomarker for patient stratification and a potential target of therapy. PMID:27315569

  7. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25605249

  9. Novel Protein Arginine Methyltransferase 8 Isoform Is Essential for Cell Proliferation.

    PubMed

    Hernandez, Sarah; Dominko, Tanja

    2016-09-01

    Identification of molecular mechanisms that regulate cellular replicative lifespan is needed to better understand the transition between a normal and a neoplastic cell phenotype. We have previously reported that low oxygen-mediated activity of FGF2 leads to an increase in cellular lifespan and acquisition of regeneration competence in human dermal fibroblasts (iRC cells). Though cells display a more plastic developmental phenotype, they remain non-tumorigenic when injected into SCID mice (Page et al. [2009] Cloning Stem Cells 11:417-426; Page et al. [2011] Eng Part A 17:2629-2640) allowing for investigation of mechanisms that regulate increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by qRT-PCR revealed a 13.3-fold upregulation of the arginine methyltransferase PRMT8 in iRC cells. Increased protein expression was confirmed in both iRC and human embryonic stem cells-the first demonstration of endogenous human PRMT8 expression outside the brain. Furthermore, iRC cells express a novel PRMT8 mRNA variant. Using siRNA-mediated knockdown we demonstrated that this novel variant was required for proliferation of human dermal fibroblasts (hDFs) and grade IV glioblastomas. PRMT8 upregulation in a non-tumorigenic system may offer a potential diagnostic biomarker and a therapeutic target for cells in pre-cancerous and cancerous states. J. Cell. Biochem. 117: 2056-2066, 2016. © 2016 Wiley Periodicals, Inc. PMID:26851891

  10. 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

  11. Protein N-arginine methyltransferase 5 promotes the tumor progression and radioresistance of nasopharyngeal carcinoma.

    PubMed

    Yang, Daoke; Liang, Tiansong; Gu, Yue; Zhao, Yulin; Shi, Yonggang; Zuo, Xiaoxiao; Cao, Qinchen; Yang, Ya; Kan, Quancheng

    2016-03-01

    Radiotherapy resistance is the main cause of the the poor prognosis of some nasopharyngeal carcinoma (NPC) patients. Yet, the exact mechanism is still elusive. In the present study, we explored the clinical and biological role of protein arginine methyltransferase 5 (PRMT5) in NPC. Our results revealed that PRMT5 was overexpressed in NPC tissues when compared with that in adjacent non-tumor tissues by quantitative RT-PCR and immunoblotting. High expression of PRMT5 was correlated with adverse outcomes of NPC patients as determined by the scoring of a tissue microarray. Silencing of PRMT5 promoted the radiosensitivity of 5-8F and CNE2 cells as determined by cell proliferation and colony formation assays. Furthermore, fibroblast growth factor receptor 3 (FGFR3) was identified as one of the downstream targets of PRMT5, and the silencing of PRMT5 decreased the mRNA and protein levels of FGFR3 in the 5-8F and CNE2 cells. Silencing of FGFR3 induced similar phenotypes as the inhibition of PRMT5, and re-expression of FGFR3 in 5-8F/shPRMT5 and CNE2/shPRMT5 cells restored the proliferation and colony formation ability induced by irradiation exposure. Our results indicate that PRMT5 is a marker of poor prognosis in NPC patients. PRMT5 promoted the radioresistance of NPC cells via targeting FGFR3, at least partly if not totally. PRMT5 and its downstream effector FGFR3 may be potential targets for anticancer strategy. PMID:26708443

  12. Genomic insights of protein arginine methyltransferase Hmt1 binding reveals novel regulatory functions

    PubMed Central

    2012-01-01

    Background Protein arginine methylation is a post-translational modification involved in important biological processes such as transcription and RNA processing. This modification is catalyzed by both type I and II protein arginine methyltransferases (PRMTs). One of the most conserved type I PRMTs is PRMT1, the homolog of which is Hmt1 in Saccharomyces cerevisiae. Hmt1 has been shown to play a role in various gene expression steps, such as promoting the dynamics of messenger ribonucleoprotein particle (mRNP) biogenesis, pre-mRNA splicing, and silencing of chromatin. To determine the full extent of Hmt1’s involvement during gene expression, we carried out a genome-wide location analysis for Hmt1. Results A comprehensive genome-wide binding profile for Hmt1 was obtained by ChIP-chip using NimbleGen high-resolution tiling microarrays. Of the approximately 1000 Hmt1-binding sites found, the majority fall within or proximal to an ORF. Different occupancy patterns of Hmt1 across genes with different transcriptional rates were found. Interestingly, Hmt1 occupancy is found at a number of other genomic features such as tRNA and snoRNA genes, thereby implicating a regulatory role in the biogenesis of these non-coding RNAs. RNA hybridization analysis shows that Hmt1 loss-of-function mutants display higher steady-state tRNA abundance relative to the wild-type. Co-immunoprecipitation studies demonstrate that Hmt1 interacts with the TFIIIB component Bdp1, suggesting a mechanism for Hmt1 in modulating RNA Pol III transcription to regulate tRNA production. Conclusions The genome-wide binding profile of Hmt1 reveals multiple potential new roles for Hmt1 in the control of eukaryotic gene expression, especially in the realm of non-coding RNAs. The data obtained here will provide an important blueprint for future mechanistic studies on the described occupancy relationship for genomic features bound by Hmt1. PMID:23268696

  13. Protein arginine methyltransferase CARM1 attenuates the paraspeckle-mediated nuclear retention of mRNAs containing IRAlus.

    PubMed

    Hu, Shi-Bin; Xiang, Jian-Feng; Li, Xiang; Xu, Yefen; Xue, Wei; Huang, Min; Wong, Catharine C; Sagum, Cari A; Bedford, Mark T; Yang, Li; Cheng, Donghang; Chen, Ling-Ling

    2015-03-15

    In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1. PMID:25792598

  14. Protein arginine methyltransferase CARM1 attenuates the paraspeckle-mediated nuclear retention of mRNAs containing IRAlus

    PubMed Central

    Hu, Shi-Bin; Xiang, Jian-Feng; Li, Xiang; Xu, Yefen; Xue, Wei; Huang, Min; Wong, Catharine C.; Sagum, Cari A.; Bedford, Mark T.; Yang, Li

    2015-01-01

    In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3′ untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54nrb. However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54nrb, resulting in reduced binding of p54nrb to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein–RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1. PMID:25792598

  15. The protein arginine methyltransferase PRMT6 inhibits HIV-1 Tat nucleolar retention.

    PubMed

    Fulcher, Alex J; Sivakumaran, Haran; Jin, Hongping; Rawle, Daniel J; Harrich, David; Jans, David A

    2016-02-01

    The human immunodeficiency virus (HIV)-1 transactivator protein Tat is known to play a key role in HIV infection, integrally related to its role in the host cell nucleus/nucleolus. Here we show for the first time that Tat localisation can be modulated by specific methylation, whereby overexpression of active but not catalytically inactive PRMT6 methyltransferase specifically leads to exclusion of Tat from the nucleolus. An R52/53A mutated Tat derivative does not show this redistribution, implying that R52/53, within Tat's nuclear/nucleolar localisation signal, are the targets of PRMT6 activity. Analysis using fluorescence recovery after photobleaching indicate that Tat nucleolar accumulation is largely through binding to nucleolar components, with methylation of Tat by PRMT6 preventing this. To our knowledge, this is the first report of specific protein methylation inhibiting nucleolar retention. PMID:26611710

  16. 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

  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

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

    2015-05-01

    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 Arg(71), Arg(75), and Arg(92) residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys(88) and Lys(91) 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 is an essential component of the hypoxia-inducible factor 1 signaling pathway

    SciTech Connect

    Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

  19. Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution

    SciTech Connect

    Chang, Yuan-I; Hsu, Sheng-Chieh; Chau, Gar-Yang; Huang, Chi-Ying F.; Sung, Jung-Sung; Hua, Wei-Kai; Lin, Wey-Jinq

    2011-01-21

    Research highlights: {yields} Verifying by direct methylation assay the substrate sites of PRMT1 in the hnRNP K protein. {yields} Identifying the preferred PMRT1 methylation regions in hnRNP K by kinetic analysis. {yields} Linking methylation in regulating nuclear localization of hnRNP K. -- Abstract: Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.

  20. 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.

  1. 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

  2. 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. PMID:25843627

  3. Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats.

    PubMed

    Sun, Qingzhu; Yang, Xudong; Zhong, Bo; Jiao, Fangfang; Li, Chenyan; Li, Dongmin; Lan, Xi; Sun, Jian; Lu, Shemin

    2012-04-01

    Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis. PMID:22387551

  4. 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. PMID:27012206

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

    PubMed

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

    2016-01-29

    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

  6. Exchange Factor TBL1 and Arginine Methyltransferase PRMT6 Cooperate in Protecting G Protein Pathway Suppressor 2 (GPS2) from Proteasomal Degradation*

    PubMed Central

    Huang, Jiawen; Cardamone, M. Dafne; Johnson, Holly E.; Neault, Mathieu; Chan, Michelle; Floyd, Z. Elizabeth; Mallette, Frédérick A.; Perissi, Valentina

    2015-01-01

    G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNFα pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment. PMID:26070566

  7. Protein Arginine Methyltransferase 6 (Prmt6) Is Essential for Early Zebrafish Development through the Direct Suppression of gadd45αa Stress Sensor Gene.

    PubMed

    Zhao, Xin-Xi; Zhang, Yun-Bin; Ni, Pei-Li; Wu, Zhi-Li; Yan, Yuan-Chang; Li, Yi-Ping

    2016-01-01

    Histone lysine methylation is important in early zebrafish development; however, the role of histone arginine methylation in this process remains unclear. H3R2me2a, generated by protein arginine methyltransferase 6 (Prmt6), is a repressive mark. To explore the role of Prmt6 and H3R2me2a during zebrafish embryogenesis, we identified the maternal characteristic of prmt6 and designed two prmt6-specific morpholino-oligos (MOs) to study its importance in early development, application of which led to early epiboly defects and significantly reduced the level of H3R2me2a marks. prmt6 mRNA could rescue the epiboly defects and the H3R2me2a reduction in the prmt6 morphants. Functionally, microarray data demonstrated that growth arrest and DNA damage-inducible, α, a (gadd45αa) was a significantly up-regulated gene in MO-treated embryos, the activity of which was linked to the activation of the p38/JNK pathway and apoptosis. Importantly, gadd45αa MO and p38/JNK inhibitors could partially rescue the defect of prmt6 morphants, the downstream targets of Prmt6, and the apoptosis ratios of the prmt6 morphants. Moreover, the results of ChIP quantitative real time PCR and luciferase reporter assay indicated that gadd45αa is a repressive target of Prmt6. Taken together, these results suggest that maternal Prmt6 is essential to early zebrafish development by directly repressing gadd45αa. PMID:26487724

  8. 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

  9. Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

    PubMed Central

    Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

    2007-01-01

    Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

  10. Type I Arginine Methyltransferases PRMT1 and PRMT-3 Act Distributively*S⃞

    PubMed Central

    Kölbel, Knut; Ihling, Christian; Bellmann-Sickert, Kathrin; Neundorf, Ines; Beck-Sickinger, Annette G.; Sinz, Andrea; Kühn, Uwe; Wahle, Elmar

    2009-01-01

    Asymmetric dimethylation of arginine residues is a common posttranslational modification of proteins carried out by type I protein arginine methyltransferases, including PRMT1 and -3. We report that the consecutive transfer of two methyl groups to a single arginine side chain by PRMT1 and -3 occurs in a distributive manner, i.e. with intermittent release of the monomethylated intermediate. The oligomeric state of PRMTs together with the clustering of methylated arginine residues in most proteins carrying this type of modification suggests that multiple methyl transfers to a single polypeptide chain might proceed in a processive manner by cooperation of multiple active sites. However, three different types of experiments provide evidence that the reaction is distributive even with substrates containing multiple methyl-accepting arginines, including one with 13 such residues. PRMT1 also does not prefer substrates already containing one or more singly or doubly methylated arginine residues. Even though the reaction is distributive, the efficiency of methylation of one particular protein strongly depends on the number of methyl-accepting arginine residues it contains. PMID:19158082

  11. pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication

    PubMed Central

    Thomas, Marco; Sonntag, Eric; Müller, Regina; Schmidt, Stefanie; Zielke, Barbara; Fossen, Torgils

    2015-01-01

    ABSTRACT The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus. IMPORTANCE The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a

  12. 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.

  13. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond

    PubMed Central

    Stopa, Nicole

    2015-01-01

    Post-translational arginine methylation is responsible for regulation of many biological processes. The protein arginine methyltransferase 5 (PRMT5, also known as Hsl7, Jbp1, Skb1, Capsuleen, or Dart5) is the major enzyme responsible for mono- and symmetric dimethylation of arginine. An expanding literature demonstrates its critical biological function in a wide range of cellular processes. Histone and other protein methylation by PRMT5 regulate genome organization, transcription, stem cells, primordial germ cells, differentiation, the cell cycle, and spliceosome assembly. Metazoan PRMT5 is found in complex with the WD-repeat protein MEP50 (also known as Wdr77, androgen receptor coactivator p44, or Valois). PRMT5 also directly associates with a range of other protein factors, including pICln, Menin, CoPR5 and RioK1 that may alter its subcellular localization and protein substrate selection. Protein substrate and PRMT5–MEP50 post-translation modifications induce crosstalk to regulate PRMT5 activity. Crystal structures of C. elegans PRMT5 and human and frog PRMT5–MEP50 complexes provide substantial insight into the mechanisms of substrate recognition and procession to dimethylation. Enzymo-logical studies of PRMT5 have uncovered compelling insights essential for future development of specific PRMT5 inhibitors. In addition, newly accumulating evidence implicates PRMT5 and MEP50 expression levels and their methyltransferase activity in cancer tumorigenesis, and, significantly, as markers of poor clinical outcome, marking them as potential oncogenes. Here, we review the substantial new literature on PRMT5 and its partners to highlight the significance of understanding this essential enzyme in health and disease. PMID:25662273

  14. GABAAergic stimulation modulates intracellular protein arginine methylation.

    PubMed

    Denman, Robert B; Xie, Wen; Merz, George; Sung, Ying-Ju

    2014-06-20

    Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome. PMID:24793772

  15. 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.

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

    PubMed

    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

  17. 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

  18. The arginine methyltransferase PRMT5 regulates CIITA-dependent MHC II transcription.

    PubMed

    Fan, Zhiwen; Kong, Xiaocen; Xia, Jun; Wu, Xiaoyan; Li, He; Xu, Huihui; Fang, Mingming; Xu, Yong

    2016-05-01

    Class II major histocompatibility complex (MHC II) dependent antigen presentation serves as a key step in mammalian adaptive immunity and host defense. In antigen presenting cells (e.g., macrophages), MHC II transcription can be activated by interferon gamma (IFN-γ) and mediated by class II transactivator (CIITA). The underlying epigenetic mechanism, however, is not completely understood. Here we report that following IFN-γ stimulation, symmetrically dimethylated histone H3 arginine 2 (H3R2Me2s) accumulated on the MHC II promoter along with CIITA. IFN-γ augmented expression, nuclear translocation, and promoter binding of the protein arginine methyltransferase PRMT5 in macrophages. Over-expression of PRMT5 potentiated IFN-γ induced activation of MHC II transcription in an enzyme activity-dependent manner. In contrast, PRMT5 silencing or inhibition of PRMT5 activity by methylthioadenosine (MTA) suppressed MHC II transactivation by IFN-γ. CIITA interacted with and recruited PRMT5 to the MHC II promoter and mediated the synergy between PRMT5 and ASH2/WDR5 to activate MHC II transcription. PRMT5 expression was down-regulated in senescent and H2O2-treated macrophages rendering ineffectual induction of MHC II transcription by IFN-γ. Taken together, our data reveal a pathophysiologically relevant role for PRMT5 in MHC II transactivation in macrophages. PMID:26972221

  19. 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}.

  20. Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-gamma-inducible MHC-II gene expression.

    PubMed

    Zika, Eleni; Fauquier, Lucas; Vandel, Laurence; Ting, Jenny P-Y

    2005-11-01

    Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-gamma. IFN-gamma activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-gamma-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, CARM1, and the acetyltransferase cyclic-AMP response element binding (CREB)-binding protein (CBP) during this process. CARM1 synergizes with CIITA in activating MHC-II transcription and synergy is abrogated when an arginine methyltransferase-defective CARM1 mutant is used. Protein-arginine methyltransferase 1 has much less effect on MHC-II transcription. Specific RNA interference reduced CARM1 expression as well as MHC-II expression. The recruitment of CARM1 to the promoter requires endogenous CIITA and results in methylation of histone H3-R17; hence, CIITA is an upstream regulator of histone methylation. Previous work has shown that CARM1 can methylate CBP at three arginine residues. Using wild-type CBP and a mutant of CBP lacking the CARM1-targeted arginine residues (R3A), we show that arginine methylation of CBP is required for IFN-gamma induction of MHC-II. A kinetic analysis shows that CIITA, CARM1, and H3-R17 methylation all precede CBP loading on the MHC-II promoter during IFN-gamma treatment. These results suggest functional and temporal relationships among CIITA, CARM1, and CBP for IFN-gamma induction of MHC-II. PMID:16254053

  1. Improving treatment of guanidinoacetate methyltransferase deficiency: reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation.

    PubMed

    Schulze, A; Ebinger, F; Rating, D; Mayatepek, E

    2001-12-01

    Guanidinoacetate methyltransferase (GAMT) deficiency (McKusick 601240), an inborn error of creatine biosynthesis, is characterized by creatine depletion and accumulation of guanidinoacetate (GAA) in the brain. Treatment by oral creatine supplementation had no effect on the intractable seizures. Based on the possible role of GAA as an epileptogenic agent, we evaluated a dietary treatment with arginine restriction and ornithine supplementation in order to achieve reduction of GAA. In an 8-year-old Kurdish girl with GAMT deficiency arginine intake was restricted to 15 mg/kg/day (0.4 g natural protein/kg/day) and ornithine was supplemented with 100 mg/kg/day over a period of 14 months. The diet was enriched with 0.4 g/kg/day of arginine-free essential amino acid mixture and creatine treatment remained unchanged (1.1 g/kg/day). Guanidino compounds in blood, urine, and CSF were measured by means of cation-exchange chromatography. The combination of arginine restriction and ornithine supplementation led to a substantial and permanent decrease of arginine without disturbance of nitrogen detoxification. Formation of GAA was effectively reduced after 4 weeks of treatment and sustained thereafter. Biochemical effects were accompanied by a marked clinical improvement. Distinctly reduced epileptogenic activities in electroencephalography accompanied by almost complete disappearance of seizures demonstrates the positive effect of GAA reduction. This indicates for the first time that GAA may exert an important epileptogenic potential in man. Arginine restriction in combination with ornithine supplementation represents a new and rationale therapeutic approach in GAMT deficiency. PMID:11749046

  2. Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

    PubMed Central

    Wigle, Tim J.; Provencher, Laurel M.; Norris, Jacqueline L.; Jin, Jian; Brown, Peter J.; Frye, Stephen V.; Janzen, William P.

    2010-01-01

    Summary The discovery of small molecules targeting the > 80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a novel and highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules. PMID:20659682

  3. Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila.

    PubMed

    Jäckel, Sandra; Summerer, Anna K; Thömmes, Catherine M; Pan, Xia; Voigt, Aaron; Schulz, Jörg B; Rasse, Tobias M; Dormann, Dorothee; Haass, Christian; Kahle, Philipp J

    2015-02-01

    Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS

  4. 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.

  5. 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.

  6. Beyond PAINs: Chemotype Sensitivity of Protein Methyltransferases in Screens.

    PubMed

    Gao, Cen; Margolis, Brandon J; Strelow, John M; Vidler, Lewis R; Mader, Mary M

    2016-02-11

    Screening of the relatively new target class, the lysine and arginine methyltransferases (MTases), presents unique challenges in the identification and confirmation of active chemical matter. Examination of high throughput screening data generated using Scintillation Proximity Assay (SPA) format for a number of protein MTase targets reveals sensitivity to both the known pan assay interference compounds (PAINS) and also other scaffolds not currently precedented as assay interferers. We find that, in general, true actives show significant selectivity within the MTase family. With the exception of slight modifications of SAM-like compounds, scaffolds that are observed frequently in multiple MTase assays should be viewed with caution and should be carefully validated before following up. PMID:26985291

  7. 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...

  8. 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. PMID:20666457

  9. Toxoplasma gondii Arginine Methyltransferase 1 (PRMT1) Is Necessary for Centrosome Dynamics during Tachyzoite Cell Division

    PubMed Central

    El Bissati, Kamal; Suvorova, Elena S.; Xiao, Hui; Lucas, Olivier; Upadhya, Rajendra; Ma, Yanfen; Hogue Angeletti, Ruth; White, Michael W.

    2016-01-01

    ABSTRACT The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T. gondii mutants lacking PRMT1 (Δprmt1) by deletion of the PRMT1 gene. Δprmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the Δprmt::PRMT1mRFP complemented strain. Tagged PRMT1 localizes primarily in the cytoplasm with enrichment at the pericentriolar material, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, Δprmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole-genome expression profiling demonstrated differences in expression of cell-cycle-regulated genes in the Δprmt1 strain relative to the complemented Δprmt1::PRMT1mRFP and parental wild-type strains, but these changes do not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our studies suggest that PRMT1 plays an important role within the centrosome to ensure the proper replication of the parasite. PMID:26838719

  10. Preferential interactions between protein and arginine: effects of arginine on tertiary conformational and colloidal stability of protein solution.

    PubMed

    Wen, Lili; Chen, Yan; Liao, Jie; Zheng, Xianxian; Yin, Zongning

    2015-01-30

    The purpose of this study was to better understand the preferential binding behavior of arginine to protein as well as the impact of arginine on the conformational and colloidal stability of protein solution. Physical stabilities of model proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were investigated by fluorescence-based and dynamic light scattering techniques in the absence and presence of arginine. We investigated the interactions between arginine and tryptophan or tyrosine residues by conducting solubility and fluorescence studies of two amino acid derivatives, N-acetyl-l-tryptophanamide (NATA) and N-acetyl-l-tyrosinamide (NAYA), in arginine solutions. The result showed that arginine preferentially bond to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals' forces, while the binding constant K of arginine with BSA and OVA at 298K was 41.92 and 5.77L/mol, respectively. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS peak position revealed that arginine perturbed the local environment of tryptophan and tyrosine residues. We also found the attenuated electrostatic repulsion among BSA and OVA molecules after adding arginine. These findings provided strong evidence that arginine possessed negative effects on tertiary conformational and colloidal stability of BSA and OVA during the preferential binding process. PMID:25529432

  11. Arginine Depletion by Arginine Deiminase Does Not Affect Whole Protein Metabolism or Muscle Fractional Protein Synthesis Rate in Mice

    PubMed Central

    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. PMID:25775142

  12. 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

  13. Protein Arginine Methylation and Citrullination in Epigenetic Regulation.

    PubMed

    Fuhrmann, Jakob; Thompson, Paul R

    2016-03-18

    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

  14. The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice.

    PubMed

    Li, Ziwei; Yu, Juehua; Hosohama, Linzi; Nee, Kevin; Gkountela, Sofia; Chaudhari, Sonal; Cass, Ashley A; Xiao, Xinshu; Clark, Amander T

    2015-03-12

    PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1-Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells. PMID:25519955

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

    PubMed

    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, Ca(2+)/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. PMID:27466704

  16. 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

  17. The NSD family of protein methyltransferases in human cancer.

    PubMed

    Vougiouklakis, Theodore; Hamamoto, Ryuji; Nakamura, Yusuke; Saloura, Vassiliki

    2015-08-01

    The NSD family of protein lysine methyltransferases consists of NSD1, NSD2/WHSC1/MMSET and NSD3/WHSC1L1. NSD2 haploinsufficiency causes Wolf-Hirschhorn syndrome, while NSD1 mutations lead to the Sotos syndrome. Recently, a number of studies showed that the NSD methyltransferases were overexpressed, amplified or somatically mutated in multiple types of cancer, suggesting their critical role in cancer. These enzymes methylate specific lysine residues on histone tails and their dysfunction results in epigenomic aberrations which play a fundamental role in oncogenesis. Furthermore, NSD1 was also reported to methylate a nonhistone protein substrate, RELA/p65 subunit of NF-κB, implying its regulatory function through nonhistone methylation pathways. In this review, we summarize the current research regarding the role of the NSD family proteins in cancer and underline their potential as targets for novel cancer therapeutics. PMID:25942451

  18. 1 Protein Methyltransferases: Their Distribution Among the Five Structural Classes of AdoMet-Dependent Methyltransferases.

    PubMed

    Schubert, Heidi L; Blumenthal, Robert M; Cheng, Xiaodong

    2006-01-01

    S-adenosyl-l-methionine (AdoMet) dependent methyltransferases (MTases) are involved in biosynthesis, signal transduction, protein repair, chromatin regulation, and gene silencing. Five different structural folds (designated I through V) have been described that bind AdoMet and catalyze methyltransfer to diverse substrates, although the great majority of known MTases have the Class I fold. Even within a particular MTase class the amino-acid sequence similarity can be as low as 10%. Thus, the structural and catalytic requirements for methyltransfer from AdoMet appear to be remarkably flexible. MTases that act on protein substrates have been found to date among three of the five structural classes (I, the classical fold; III, the corrin MTase fold; and V, the SET fold). "There are many paths to the top of the mountain, but the view is always the same."-Chinese proverb The Columbia World of Quotations, New York, Columbia University Press, 1996. PMID:26718035

  19. Protein arginine deiminase 4: a target for an epigenetic cancer therapy.

    PubMed

    Slack, Jessica L; Causey, Corey P; Thompson, Paul R

    2011-02-01

    The recent approvals of anticancer therapeutic agents targeting the histone deacetylases and DNA methyltransferases have highlighted the important role that epigenetics plays in human diseases, and suggested that the factors controlling gene expression are novel drug targets. Protein arginine deiminase 4 (PAD4) is one such target because its effects on gene expression parallel those observed for the histone deacetylases. We demonstrated that F- and Cl-amidine, two potent PAD4 inhibitors, display micromolar cytotoxic effects towards several cancerous cell lines (HL-60, MCF7 and HT-29); no effect was observed in noncancerous lines (NIH 3T3 and HL-60 granulocytes). These compounds also induced the differentiation of HL-60 and HT29 cells. Finally, these compounds synergistically potentiated the cell killing effects of doxorubicin. Taken together, these findings suggest PAD4 inhibition as a novel epigenetic approach for the treatment of cancer, and suggest that F- and Cl-amidine are candidate therapeutic agents for this disease. PMID:20706768

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

    PubMed

    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

  1. 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

  2. West Nile virus methyltransferase domain interacts with protein kinase G

    PubMed Central

    2013-01-01

    Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication. PMID:23876037

  3. Arginine selective reagents for ligation to peptides and proteins.

    PubMed

    Thompson, Darren A; Ng, Raymond; Dawson, Philip E

    2016-05-01

    A new class of arginine-specific bioconjugation reagents for protein labeling has been developed. This method utilizes a triazolyl-phenylglyoxal group on the probe molecule that reacts selectively with the guandinyl group of Arg residues in a protein or peptide. The reaction proceeds in neutral to basic bicarbonate buffers and is selective for arginine residues in peptides and folded proteins. Importantly, the triazolyl-phenylglyoxal group can be introduced into complex molecules containing alkyne groups using CuAAC chemistry, providing a robust approach for the generation of phenylglyoxal reactive groups into molecules to be covalently attached onto the surface of proteins. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27005702

  4. The regulatory PII protein controls arginine biosynthesis in Arabidopsis.

    PubMed

    Ferrario-Méry, Sylvie; Besin, Evelyne; Pichon, Olivier; Meyer, Christian; Hodges, Michael

    2006-04-01

    In higher plants, PII is a nuclear-encoded plastid protein which is homologous to bacterial PII signalling proteins known to be involved in the regulation of nitrogen metabolism. A reduced ornithine, citrulline and arginine accumulation was observed in two Arabidopsis PII knock-out mutants in response to NH4+ resupply after N starvation. This difference could be explained by the regulation of a key enzyme of the arginine biosynthesis pathway, N-acetyl glutamate kinase (NAGK) by PII. In vitro assays using purified recombinant proteins showed the catalytic activation of Arabidopsis NAGK by PII giving the first evidence of a physiological role of the PII protein in higher plants. Using Arabidopsis transcriptome microarray (CATMA) and RT-PCR analyses, it was found that none of the genes involved in the arginine biosynthetic or catabolic pathways were differentially expressed in a PII knock-out mutant background. In conclusion, the observed changes in metabolite levels can be explained by the reduced activation of NAGK by PII. PMID:16545809

  5. The protein arginine deiminases (PADs): Structure, Function, Inhibition, and Disease

    PubMed Central

    Bicker, Kevin L.

    2012-01-01

    The post translational modification of histones has significant effects on overall chromatin function. One such modification is citrullination, which is catalyzed by the protein arginine deiminases (PADs), a unique family of enzymes that catalyzes the hydrolysis of peptidyl-arginine to form peptidyl-citrulline on histones, fibrinogen, and other biologically relevant proteins. Overexpression and/or increased PAD activity is observed in several diseases, including rheumatoid arthritis, Alzheimer’s disease, multiple sclerosis, lupus, Parkinson’s disease, and cancer. This review discusses the important structural and mechanistic characteristics of the PADs, as well as recent investigations into the role of the PADs in increasing disease severity in RA and colitis and the importance of PAD activity in mediating neutrophil extracellular trap (NET) formation through chromatin decondensation. Lastly, efforts to develop PAD inhibitors with excellent potency, selectivity and in vivo efficacy are discussed, highlighting the most promising inhibitors. PMID:23175390

  6. 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.

  7. Protein lysine methylation by seven-β-strand methyltransferases.

    PubMed

    Falnes, Pål Ø; Jakobsson, Magnus E; Davydova, Erna; Ho, Angela; Małecki, Jędrzej

    2016-07-15

    Methylation of biomolecules is a frequent biochemical reaction within the cell, and a plethora of highly specific methyltransferases (MTases) catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to various substrates. The posttranslational methylation of lysine residues, catalysed by numerous lysine (K)-specific protein MTases (KMTs), is a very common and important protein modification, which recently has been subject to intense studies, particularly in the case of histone proteins. The majority of KMTs belong to a class of MTases that share a defining 'SET domain', and these enzymes mostly target lysines in the flexible tails of histones. However, the so-called seven-β-strand (7BS) MTases, characterized by a twisted beta-sheet structure and certain conserved sequence motifs, represent the largest MTase class, and these enzymes methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dot1/DOT1L was the only identified eukaryotic 7BS KMT. However, a number of novel 7BS KMTs have now been discovered, and, in particular, several recently characterized human and yeast members of MTase family 16 (MTF16) have been found to methylate lysines in non-histone proteins. Here, we review the status and recent progress on the 7BS KMTs, and discuss these enzymes at the levels of sequence/structure, catalytic mechanism, substrate recognition and biological significance. PMID:27407169

  8. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    PubMed

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  9. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

    PubMed Central

    Elsholz, Alexander K. W.; Turgay, Kürşad; Michalik, Stephan; Hessling, Bernd; Gronau, Katrin; Oertel, Dan; Mäder, Ulrike; Bernhardt, Jörg; Becher, Dörte; Hecker, Michael; Gerth, Ulf

    2012-01-01

    Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria. PMID:22517742

  10. 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

  11. 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. PMID:25771539

  12. 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.

  13. A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex.

    PubMed

    Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

    2008-11-21

    The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro. PMID:18829457

  14. Regulating the regulators: serine/arginine-rich proteins under scrutiny.

    PubMed

    Risso, Guillermo; Pelisch, Federico; Quaglino, Ana; Pozzi, Berta; Srebrow, Anabella

    2012-10-01

    Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation. PMID:22941908

  15. Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

    PubMed Central

    Villalba, M; Pajares, M A; Renart, M F; Mato, J M

    1987-01-01

    When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed. Images Fig. 1. Fig. 4. PMID:3593229

  16. Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

    PubMed

    Wanigasekara, Maheshika S K; Chowdhury, Saiful M

    2016-09-01

    Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. PMID:27543028

  17. 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. PMID:26801560

  18. Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis

    SciTech Connect

    Cherney, Leonid T.; Cherney, Maia M.; Garen, Craig R.; Lu, George J.; James, Michael N. G.

    2008-09-01

    The structure of the core domain of the arginine repressor protein from M. tuberculosis has been determined with (1.85 Å resolution) and without (2.15 Å resolution) the arginine corepressor bound. Three additional arginine molecules have been found to bind to the core domain hexamer at high (0.2 M) arginine concentration. 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 Å resolution with bound arginine and at 2.15 Å 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° 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. 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. PMID:26512127

  20. The CASTOR proteins are arginine sensors for the mTORC1 pathway

    PubMed Central

    Chantranupong, Lynne; Scaria, Sonia M.; Saxton, Robert A.; Gygi, Melanie P.; Shen, Kuang; Wyant, Gregory A.; Wang, Tim; Harper, J. Wade; Gygi, Steven P.; Sabatini, David M.

    2016-01-01

    Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. PMID:26972053

  1. The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway.

    PubMed

    Chantranupong, Lynne; Scaria, Sonia M; Saxton, Robert A; Gygi, Melanie P; Shen, Kuang; Wyant, Gregory A; Wang, Tim; Harper, J Wade; Gygi, Steven P; Sabatini, David M

    2016-03-24

    Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ∼30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. PMID:26972053

  2. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

    PubMed Central

    Ridler, Charlotte E.; Clayton, Emma L.; Devoy, Anny; Moens, Thomas; Norona, Frances E.; Woollacott, Ione O.C.; Pietrzyk, Julian; Cleverley, Karen; Nicoll, Andrew J.; Pickering-Brown, Stuart; Dols, Jacqueline; Cabecinha, Melissa; Hendrich, Oliver; Fratta, Pietro; Fisher, Elizabeth M.C.; Partridge, Linda; Isaacs, Adrian M.

    2016-01-01

    An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration. PMID:25103406

  3. PRMT9 is a Type II methyltransferase that methylates the splicing factor SAP145

    PubMed Central

    Yang, Yanzhong; Hadjikyriacou, Andrea; Xia, Zheng; Gayatri, Sitaram; Kim, Daehoon; Zurita-Lopez, Cecilia; Kelly, Ryan; Guo, Ailan; Li, Wei; Clarke, Steven G.; Bedford, Mark T.

    2015-01-01

    The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9), which members can catalyze three distinct types of methylation on arginine residues. Here, we identify two spliceosome-associated proteins – SAP145 and SAP49 – as PRMT9 binding partners, linking PRMT9 to U2snRNP maturation. We show that SAP145 is methylated by PRMT9 at arginine 508, which takes the form of monomethylated arginine (MMA) and symmetrically dimethylated arginine (SDMA). PRMT9 thus joins PRMT5 as the only mammalian enzymes capable of depositing the SDMA mark. Methylation of SAP145 on Arg508 generates a binding site for the Tudor domain of the Survival of Motor Neuron (SMN) protein, and RNA-seq analysis reveals gross splicing changes when PRMT9 levels are attenuated. These results identify PRMT9 as a non-histone methyltransferase that primes the U2snRNP for interaction with SMN. PMID:25737013

  4. Facile synthesis of SAM–peptide conjugates through alkyl linkers targeting protein N-terminal methyltransferase 1†

    PubMed Central

    Zhang, Gang

    2016-01-01

    We report the first chemical synthesis of SAM–peptide conjugates through alkyl linkers to prepare bisubstrate analogs for protein methyltransferases. We demonstrate its application by developing a series of bisubstrate inhibitors for protein N-terminal methyltransferase 1 and the most potent one exhibits a Ki value of 310 ± 55 nM.

  5. Dysregulation of protein methyltransferases in human cancer: An emerging target class for anticancer therapy.

    PubMed

    Hamamoto, Ryuji; Nakamura, Yusuke

    2016-04-01

    Protein methylation is one of the important post-translational modifications. Although its biological and physiological functions were unknown for a long time, we and others have characterized a number of protein methyltransferases, which have unveiled the critical functions of protein methylation in various cellular processes, in particular, in epigenetic regulation. In addition, it had been believed that protein methylation is an irreversible phenomenon, but through identification of a variety of protein demethylases, protein methylation is now considered to be dynamically regulated similar to protein phosphorylation. A large amount of evidence indicated that protein methylation has a pivotal role in post-translational modification of histone proteins as well as non-histone proteins and is involved in various processes of cancer development and progression. As dysregulation of this modification has been observed frequently in various types of cancer, small-molecule inhibitors targeting protein methyltransferases and demethylases have been actively developed as anticancer drugs; clinical trials for some of these drugs have already begun. In this review, we discuss the biological and physiological importance of protein methylation in human cancer, especially focusing on the significance of protein methyltransferases as emerging targets for anticancer therapy. PMID:26751963

  6. 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

  7. 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

  8. Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

    PubMed Central

    Mininno, Morgane; Brugière, Sabine; Gilgen, Annabelle; Ma, Sheng; Mazzoleni, Meryl; Gigarel, Océane; Martin-Laffon, Jacqueline; Ferro, Myriam; Ravanel, Stéphane

    2014-01-01

    Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology. PMID:24748391

  9. Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts.

    PubMed

    Alban, Claude; Tardif, Marianne; Mininno, Morgane; Brugière, Sabine; Gilgen, Annabelle; Ma, Sheng; Mazzoleni, Meryl; Gigarel, Océane; Martin-Laffon, Jacqueline; Ferro, Myriam; Ravanel, Stéphane

    2014-01-01

    Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology. PMID:24748391

  10. Yeast Hmt1 catalyses asymmetric dimethylation of histone H3 arginine 2 in vitro.

    PubMed

    Li, Hong-Tao; Gong, Ting; Zhou, Zhen; Liu, Yu-Ting; Cao, Xiongwen; He, Yongning; Chen, Charlie Degui; Zhou, Jin-Qiu

    2015-05-01

    Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate protein arginine residues. PRMTs' substrates include histones and a variety of non-histone proteins. Previous studies have shown that yeast Hmt1 is a type I PRMT and methylates histone H4 arginine 3 and several mRNA-binding proteins. Hmt1 forms dimers or oligomers, but how dimerization or oligomerization affects its activity remains largely unknown. We now report that Hmt1 can methylate histone H3 arginine 2 (H3R2) in vitro. The dimerization but not hexamerization is essential for Hmt1's activity. Interestingly, the methyltransferase activity of Hmt1 on histone H3R2 requires reciprocal contributions from two Hmt1 molecules. Our results suggest an intermolecular trans-complementary mechanism by which Hmt1 dimer methylates its substrates. PMID:25715670

  11. Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction.

    PubMed

    Whitfield, Jason H; Zhang, William H; Herde, Michel K; Clifton, Ben E; Radziejewski, Johanna; Janovjak, Harald; Henneberger, Christian; Jackson, Colin J

    2015-09-01

    Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue. PMID:26061224

  12. Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction

    PubMed Central

    Whitfield, Jason H; Zhang, William H; Herde, Michel K; Clifton, Ben E; Radziejewski, Johanna; Janovjak, Harald; Henneberger, Christian; Jackson, Colin J

    2015-01-01

    Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding “core” that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue. PMID:26061224

  13. Comprehensive identification of arginine methylation in primary T cells reveals regulatory roles in cell signalling

    PubMed Central

    Geoghegan, Vincent; Guo, Ailan; Trudgian, David; Thomas, Benjamin; Acuto, Oreste

    2015-01-01

    The impact of protein arginine methylation on the regulation of immune functions is virtually unknown. Here, we apply a novel method—isomethionine methyl-SILAC—coupled with antibody-mediated arginine-methylated peptide enrichment to identify methylated peptides in human T cells by mass spectrometry. This approach allowed the identification of 2,502 arginine methylation sites from 1,257 tissue-specific and housekeeping proteins. We find that components of T cell antigen receptor signal machinery and several key transcription factors that regulate T cell fate determination are methylated on arginine. Moreover, we demonstrate changes in arginine methylation stoichiometry during cellular stimulation in a subset of proteins critical to T cell differentiation. Our data suggest that protein arginine methyltransferases exert key regulatory roles in T cell activation and differentiation, opening a new field of investigation in T cell biology. PMID:25849564

  14. [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. PMID:26552158

  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. The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

    PubMed Central

    Sardana, Richa; Johnson, Arlen W.

    2012-01-01

    We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates. PMID:22956767

  17. A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens.

    PubMed

    Borders, C L; Broadwater, J A; Bekeny, P A; Salmon, J E; Lee, A S; Eldridge, A M; Pett, V B

    1994-04-01

    We propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a "structural" arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase II, Arg 31 in Streptomyces rubiginosus xylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the "mantle" (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV-1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8003972

  18. Glucose Autoxidation Induces Functional Damage to Proteins via Modification of Critical Arginine Residues†

    PubMed Central

    Chetyrkin, Sergei; Mathis, Missy; Pedchenko, Vadim; Sanchez, Otto A.; McDonald, W. Hayes; Hachey, David L.; Madu, Hartman; Stec, Donald; Hudson, Billy; Voziyan, Paul

    2011-01-01

    Non-enzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to αVβ3 integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while non-oxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation. PMID:21661747

  19. Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes

    SciTech Connect

    Gilbert, J.M.; Fowler, A.; Bleibaum, J.; Clarke, S.

    1988-07-12

    The authors have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. They found sequence identity or probable identity in 111 out of 112 residues when they compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.

  20. 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

    2016-03-01

    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.

  1. 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

  2. A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase.

    PubMed

    Hatzixanthis, Kostas; Palmer, Tracy; Sargent, Frank

    2003-09-01

    A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins. PMID:12940994

  3. NRMT is an α-N-methyltransferase that methylates RCC1 and Retinoblastoma Protein

    PubMed Central

    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.

    2010-01-01

    The post-translational methylation of α-amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins L16 and L331–2, 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 α-N-methylated3–10. However, the Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of α-N-methylation has been identified3, 11. Methylation-defective mutants of RCC1 have reduced affinity for DNA and cause mitotic defects3, 11, 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 they may be targets of the same, unknown enzyme3,12. The initiating Met is cleaved, and the exposed α-amino group is mono-, di-, or trimethylated. Here we report the discovery of the first α-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/TAF-I/PHAPII and the retinoblastoma protein, RB. Knockdown of NRMT recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants3, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation. PMID:20668449

  4. Twin-arginine-dependent translocation of SufI in the absence of cytosolic helper proteins.

    PubMed

    Holzapfel, Eva; Moser, Michael; Schiltz, Emile; Ueda, Takuya; Betton, Jean-Michel; Müller, Matthias

    2009-06-16

    The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Folding of some Tat precursor proteins requires dedicated chaperones that also sequester the signal sequence during the maturation process. Whether or not signal sequence-binding chaperones are a general prerequisite for all Tat substrate proteins is not known. Here, we have studied the propensity of Tat signal sequences of Escherichia coli to interact with general chaperones and peptidyl-prolyl-cis,trans-isomerases. Site-specific photocross-linking revealed a clear specificity for FK506-binding proteins. Nevertheless transport of the Tat substrate SufI into inverted inner membrane vesicles of E. coli was found to occur in the bona fide absence of any cytosolic chaperone. Our results suggest that in E. coli, cytosolic chaperones are not essential for the twin-arginine-dependent export of cofactor-less substrates. PMID:19432418

  5. Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

    PubMed Central

    Qui, H F; Dubois, E; Messenguy, F

    1991-01-01

    ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain. Images PMID:2005903

  6. Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations.

    PubMed Central

    Morimoto, B H; Brady, J F; Atkinson, D E

    1990-01-01

    Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate. PMID:2268294

  7. Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

    PubMed

    Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-03-18

    Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1. PMID:26797129

  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. PMID:7715456

  9. Design, synthesis, and protein methyltransferase activity of a unique set of constrained amine containing compounds.

    PubMed

    Zhou, Hao; Che, Xin; Bao, Guochen; Wang, Na; Peng, Li; Barnash, Kimberly D; Frye, Stephen V; James, Lindsey I; Bai, Xu

    2016-09-15

    Epigenetic alterations relate to various human diseases, and developing inhibitors of Kme regulatory proteins is considered to be a new frontier for drug discovery. We were inspired by the known multicyclic ligands, UNC669 and UNC926, which are the first reported small molecule ligands for a methyl-lysine binding domain. We hypothesized that reducing the conformational flexibility of the key amine moiety of UNC669 would result in a unique set of ligands. Twenty-five novel compounds containing a fused bi- or tricyclic amine or a spirocyclic amine were designed and synthesized. To gauge the potential of these amine-containing compounds to interact with Kme regulatory proteins, the compounds were screened against a panel of 24 protein methyltransferases. Compound 13 was discovered as a novel scaffold that interacts with SETD8 and could serve as a starting point for the future development of PKMT inhibitors. PMID:27528434

  10. Low molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native flaxseed protein.

    PubMed

    Udenigwe, Chibuike C; Adebiyi, Abayomi P; Doyen, Alain; Li, Huan; Bazinet, Laurent; Aluko, Rotimi E

    2012-05-01

    Flaxseed protein isolate (FPI) contains high amount of arginine, which plays important physiological roles especially as nitric oxide precursor in the vascular endothelium. Arginine-rich peptides can be generated from FPI and used as a source of nitric oxide, which can produce in vivo vasodilatory effects during hypertension. Enzymatic hydrolysis of FPI with trypsin and pronase resulted in a hydrolysate that was fractionated using electrodialysis-ultrafiltration (EDUF). EDUF experiment resulted in migration of peptides to the anionic and cationic recovery compartments. Compared to FPI with 11% arginine, about one-third of the cationic fraction was composed of arginine. Thirteen potential peptide sequences were identified to be present in the cationic compartment of which 12 contained at least one arginine residue. None of the peptides identified from the anionic compartment contained arginine. Oral administration of the cationic peptides (200mg/kgbodywt.) to spontaneously hypertensive rats resulted in a more rapid decrease in systolic blood pressure when compared to similar amounts of FPI or the amino acid form of arginine. It was concluded that the rapid effect of the arginine-rich peptide product suggests faster rate of peptide absorption than amino acids and this may be exploited to provide fast relief from hypertension. PMID:26434317

  11. Arginine, a key residue for the enhancing ability of an antifreeze protein of the beetle Dendroides canadensis†

    PubMed Central

    Wang, Sen; Amornwittawat, Natapol; Juwita, Vonny; Kao, Yu; Duman, John G.; Pascal, Tod A.; Goddard, William A.; Wen, Xin

    2009-01-01

    Antifreeze proteins (AFPs) can produce a difference between the nonequilibrium freezing point and the melting point is termed thermal hysteresis (TH). The TH activity of an antifreeze protein (AFP) depends on the specific AFP, its concentration as well as the presence of co-solutes including low-molecular-mass solutes and/or proteins. We recently identified series of carboxylates and polyols as efficient enhancers for an AFP from the beetle Dendroides canadensis. In this study, we chemically modified DAFP-1 using the arginine-specific reagent 1,2-cyclohexanedione. We demonstrated that 1,2-cyclohexanedione specifically modifies one arginine residue and the modified DAFP-1 loses its enhancing ability completely or partially in the presence of previously identified enhancers. The stronger the enhancement ability of the enhancer on the native DAFP-1, the stronger the enhancement effect of the enhancer has on the modified DAFP-1. The weaker enhancers (e.g., glycerol) completely lose their enhancement effect on the modified DAFP-1 due to their inability to compete with 1,2-cyclohexanedione for the arginine residue. Regeneration of the arginine residue using hydroxylamine fully restored the enhancing ability of DAFP-1. These studies indicated that an arginine residue is critical for the enhancing ability of DAFP-1 and the guanidinium group of the arginine residue is important for its interaction with the enhancers, where the general mechanism of arginine-ligand interaction is borne. This work may initiate a complete mechanistic study of the enhancement effect in AFPs. PMID:19746966

  12. 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.

  13. Charge state of arginine as an additive on heat-induced protein aggregation.

    PubMed

    Miyatake, Takumi; Yoshizawa, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2016-06-01

    Arginine (Arg) is one of the most versatile solvent additives, such as suppressing protein aggregation, increasing solubility of small aromatic compounds and peptides, and preventing protein binding on solid surfaces. In this study, we investigated the role of the charged state of α-amino group of Arg for the prevention of protein aggregation. As expected, Arg effectively suppressed thermal aggregation of hen egg-white lysozyme at neutral pH, whereas the suppression effect diminished at and above pH 9.0, which corresponds to the pK of Arg's α-amino group. The pH dependence of Arg as an aggregation suppressor was confirmed by additional experiments with neutral proteins, bovine hemoglobin and bovine γ-globulin. Interestingly, N-acetylated arginine, which lacks the α-amino group, showed a weaker suppressive effect on protein aggregation than Arg, even at neutral pH. These results indicate that both positively charged α-amino group and guanidinium group play important roles in suppressing heat-induced protein aggregation by Arg. The elucidated limitation of Arg at alkaline pH provides new insight in the application as well as the mechanism of Arg as a solvent additive. PMID:26987431

  14. Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story.

    PubMed

    Ausili, Alessio; Staiano, Maria; Dattelbaum, Jonathan; Varriale, Antonio; Capo, Alessandro; D'Auria, Sabato

    2013-01-01

    Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications. PMID:25371336

  15. Mannitol/l-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals: Effect of the l-Arginine Counter Ion and Formulation Composition on the Formulation Properties and the Physical State of Mannitol.

    PubMed

    Stärtzel, Peter; Gieseler, Henning; Gieseler, Margit; Abdul-Fattah, Ahmad M; Adler, Michael; Mahler, Hanns-Christian; Goldbach, Pierre

    2016-10-01

    Previous studies have shown that protein storage stability in freeze-dried l-arginine-based systems improved in the presence of chloride ions. However, chloride ions reduced the glass transition temperature of the freeze concentrate (Tg') and made freeze drying more challenging. In this study, l-arginine was freeze dried with mannitol to obtain partially crystalline solids that can be freeze dried in a fast process and result in elegant cakes. We characterized the effect of different l-arginine counter ions on physicochemical properties of mannitol compared with mannitol/sucrose systems. Thermal properties of formulations with different compositions were correlated to thermal history during freeze drying and to physicochemical properties (cake appearance, residual moisture, reconstitution time, crystallinity). Partially crystalline solids were obtained even at the highest l-arginine level (mannitol:l-arginine of 2:1) used in this study. All l-arginine-containing formulations yielded elegant cakes. Only cakes containing l-arginine chloride and succinate showed a surface "crust" formed by phase separation. X-ray powder diffraction showed that inhibition of mannitol crystallization was stronger for l-arginine compared with sucrose and varied with the type of l-arginine counter ion. The counter ion affected mannitol polymorphism and higher levels of mannitol hemi-hydrate were obtained at high levels of l-arginine chloride. PMID:27506270

  16. Structure of the TatC core of the twin-arginine protein transport system.

    PubMed

    Rollauer, Sarah E; Tarry, Michael J; Graham, James E; Jääskeläinen, Mari; Jäger, Franziska; Johnson, Steven; Krehenbrink, Martin; Liu, Sai-Man; Lukey, Michael J; Marcoux, Julien; McDowell, Melanie A; Rodriguez, Fernanda; Roversi, Pietro; Stansfeld, Phillip J; Robinson, Carol V; Sansom, Mark S P; Palmer, Tracy; Högbom, Martin; Berks, Ben C; Lea, Susan M

    2012-12-13

    The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism. PMID:23201679

  17. Freeze-Drying of L-Arginine/Sucrose-Based Protein Formulations, Part 2: Optimization of Formulation Design and Freeze-Drying Process Conditions for an L-Arginine Chloride-Based Protein Formulation System.

    PubMed

    Stärtzel, Peter; Gieseler, Henning; Gieseler, Margit; Abdul-Fattah, Ahmad M; Adler, Michael; Mahler, Hanns-Christian; Goldbach, Pierre

    2015-12-01

    We recently reported that the presence of chloride counter ions in freeze-dried l-arginine/sucrose formulations provided the greatest protein stability, but led to low collapse temperatures and glass transition temperatures of the freeze concentrates. The objectives of this study were to identify l-arginine chloride-based formulations and optimize freeze-drying process conditions to deliver a freeze-dried product with good physical quality attributes (including cake appearance, residual moisture, and reconstitution time). Additional properties were tested such as thermal properties, cake microstructure, and protein physical stability. Excipient concentrations were varied with and without a model protein (bovine serum albumin, BSA). Formulations were frozen with and without annealing or with and without controlled nucleation. Primary drying was conducted at high and low shelf temperature. Cakes with least defects and optimum physical attributes were achieved when protein to excipient ratios were high. Controlled nucleation led to elegant cakes for most systems at a low shelf temperature. Replacing BSA by a monoclonal antibody showed that protein (physical) stability was slightly improved under stress storage temperature (i.e., 40°C) in the presence of a low concentration of l-arginine in a sucrose-based formulation. At higher l-arginine concentrations, cake defects increased. Using optimized formulation design, addition of l-arginine chloride to a sucrose-based formulation provided elegant cakes and benefits for protein stability. PMID:26422647

  18. Endothelial transcriptome in response to pharmacological methyltransferase inhibition.

    PubMed

    Okabe, Jun; Fernandez, Ana Z; Ziemann, Mark; Keating, Samuel T; Balcerczyk, Aneta; El-Osta, Assam

    2014-08-01

    The enzymatic activities of protein methyltransferases serve to write covalent modifications on histone and non-histone proteins in the control of gene transcription. Here, we describe gene expression changes in human endothelial cells caused by treatment with methyltransferase inhibitors 7,7'-carbonylbis (azanediyl) bis(4-hydroxynaphthalene-2 -sulfonic acid (AMI-1) and disodium-2-(2,4,5,7- tetrabromo-3-oxido-6-oxoxanthen-9-yl) benzoate trihydrate (AMI-5). Deep sequencing of mRNA indicated robust change on transcription following AMI-5 treatment compared with AMI-1. Functional annotation analysis revealed that both compounds suppress the expression of genes associated with translational regulation, suggesting arginine methylation by protein arginine methyltransferases (PRMTs) could be associated with regulation of this pathway. Interestingly, AMI-5 but not AMI-1 was found to decrease methylation of H3 histones at lysine 4 and down-regulate gene expression associated with interleukin-6 (IL-6) and activator protein-1 (AP-1) signaling pathways. These results imply that inhibition of protein methylation by AMI-1 and AMI-5 can differentially regulate specific pathways with potential to interrupt pathological signaling in the vascular endothelium. PMID:24850797

  19. The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7.

    PubMed

    Ali, Ibraheem; Ramage, Holly; Boehm, Daniela; Dirk, Lynnette M A; Sakane, Naoki; Hanada, Kazuki; Pagans, Sara; Kaehlcke, Katrin; Aull, Katherine; Weinberger, Leor; Trievel, Raymond; Schnoelzer, Martina; Kamada, Masafumi; Houtz, Robert; Ott, Melanie

    2016-07-29

    The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat. PMID:27235396

  20. Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

    PubMed Central

    Haynes, Chad; Iakoucheva, Lilia M.

    2006-01-01

    Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence. PMID:16407336

  1. Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation

    PubMed Central

    Gao, Wei-wei; Xiao, Rong-quan; Peng, Bing-ling; Xu, Huan-teng; Shen, Hai-feng; Huang, Ming-feng; Shi, Tao-tao; Yi, Jia; Zhang, Wen-juan; Wu, Xiao-nan; Gao, Xiang; Lin, Xiang-zhi; Dorrestein, Pieter C.; Rosenfeld, Michael G.; Liu, Wen

    2015-01-01

    Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control. PMID:26080448

  2. An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.

    PubMed

    Dorgan, Kathleen M; Wooderchak, Whitney L; Wynn, Donraphael P; Karschner, Erin L; Alfaro, Joshua F; Cui, Yinqiu; Zhou, Zhaohui Sunny; Hevel, Joan M

    2006-03-15

    Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase. PMID:16460659

  3. 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.

  4. Arabidopsis Protein Repair L-Isoaspartyl Methyltransferases: Predominant Activities at Lethal Temperatures.

    PubMed

    Villa, Sarah T; Xu, Qilong; Downie, A Bruce; Clarke, Steven G

    2006-12-01

    Protein L-isoaspartyl (D-aspartyl) O-methyltransferases (EC 2.1.1.77; PIMT or PCMT) are enzymes that initiate the full or partial repair of damaged L-aspartyl and L-asparaginyl residues, respectively. These enzymes are found in most organisms and maintain a high degree of sequence conservation. Arabidopsis thaliana (Arabidopsis L. Heynh.) is unique among eukaryotes in that it contains two genes, rather than one, that encode PIMT isozymes. We describe a novel Arabidopsis PIMT isozyme, designated AtPIMT2αω, encoded by the PIMT2 gene (At5g50240). We characterized the enzymatic activity of the recombinant AtPIMT2αω in comparison to the other AtPIMT2 isozymes, AtPIMT1, and to the human PCMT ortholog, to better understand its role in Arabidopsis. All Arabidopsis PIMT isozymes are active over a relatively wide pH range. For AtPIMT2αω maximal activity is observed at 50 °C (a lethal temperature for Arabidopsis); this activity is almost ten times greater than the activity at the growth temperature of 25 °C. Interestingly, enzyme activity decreases after pre-incubation at temperatures above 30°C. A similar situation is found for the recombinant AtPIMT2ψ and the AtPIMT2ω isozymes, as well as for the AtPIMT1 and human PCMT1 enzymes. These results suggest that the short-term ability of these methyltransferases to initiate repair under extreme temperature conditions may be a common feature of both the plant and animal species. PMID:21076691

  5. An In Silico Approach for Characterization of an Aminoglycoside Antibiotic-Resistant Methyltransferase Protein from Pyrococcus furiosus (DSM 3638)

    PubMed Central

    Oany, Arafat Rahman; Jyoti, Tahmina Pervin; Ahmad, Shah Adil Ishtiyaq

    2014-01-01

    Pyrococcus furiosus is a hyperthermophilic archaea. A hypothetical protein of this archaea, PF0847, was selected for computational analysis. Basic local alignment search tool and multiple sequence alignment (MSA) tool were employed to search for related proteins. Both the secondary and tertiary structure prediction were obtained for further analysis. Three-dimensional model was assessed by PROCHECK and QMEAN6 programs. To get insights about the physical and functional associations of the protein, STRING network analysis was performed. Binding of the SAM (S-adenosyl-l-methionine) ligand with our protein, fetched from an antibiotic-related methyltransferase (PDB code: 3P2K: D), showed high docking energy and suggested the function of the protein as methyltransferase. Finally, we tried to look for a specific function of the proposed methyltransferase, and binding of the geneticin bound to the eubacterial 16S rRNA A-site (PDB code: 1MWL) in the active site of the PF0847 gave us the indication to predict the protein responsible for aminoglycoside antibiotic resistance. PMID:24683305

  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. Overexpression of DNA methyltransferase 1 (DNMT1) protein in astrocytic tumour and its correlation with O6-methylguanine-DNA methyltransferase (MGMT) expression

    PubMed Central

    Rahman, Wan Faiziah Wan Abdul; Rahman, Khairul Shakir Ab; Nafi, Siti Norasikin Mohd; Fauzi, Mohd Hashairi; Jaafar, Hasnan

    2015-01-01

    Background: The relationship between DNA methyltransferase (DNMT) and O6-methylguanine-DNA methyltransferase (MGMT) in mediating tumorigenesis is still poorly understood. This study was carried out to investigate a correlation between DNMT1 and MGMT immunoexpression in astrocytic tumour samples. Methods: Formalin-fixed paraffin embedded tissues of astrocytic tumour patients was obtained from an observational study conducted in Hospital Universiti Sains Malaysia (USM), which was performed from January 1997 until May 2012. Patient’s histological information was retrieved from the accessible Pathology Registry. Immunohistochemistry (IHC) staining was performed to assess DNMT1 and MGMT expressions in patients’ tumours. Results: Our data showed that DNMT1 was highly expressed in high grade astrocytic tumours. A multiple regression analysis demonstrated a significant association of DNMT1 overexpression with tumour grade III and IV (GIII: OR=5.802; 95% CI: 1.059, 31.785; p value=0.043; GIV: OR=40.663; 95% CI=4.069, 406.347; p value=0.002). The MGMT protein was downregulated in tumours with higher grade as evident by a reduction mean H-score for MGMT expression from GI to GIV [28.36 ± 43.88, 28.08 ± 33.67, 26.00 ± 48.70 and 16.20 ± 35.61]. However, a good negative correlation was observed between DNMT1 and MGMT in high grade tumour [Spearman correlation test: r=-0.561, p value≤0.001 in percentage expression and r=-0.576, p value≤0.001 in H score]. Conclusion: DNMT1 overexpression was seen correlated with a reduction of MGMT protein expression in high grade astrocytic tumour. Understanding the role of these markers could be important to overcome astrocytic tumour aggresiveness. PMID:26261487

  8. Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation.

    PubMed Central

    Tang, Zhaohua; Käufer, Norbert F; Lin, Ren-Jang

    2002-01-01

    The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1-Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast. PMID:12186627

  9. Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain.

    PubMed

    Mouille, Grégory; Ralet, Marie-Christine; Cavelier, Céline; Eland, Cathlene; Effroy, Delphine; Hématy, Kian; McCartney, Lesley; Truong, Hoai Nam; Gaudon, Virginie; Thibault, Jean-François; Marchant, Alan; Höfte, Herman

    2007-05-01

    Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions. PMID:17425712

  10. Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein Lysine Methyltransferase SMYD2

    PubMed Central

    Spellmon, Nicholas; Sun, Xiaonan; Sirinupong, Nualpun; Edwards, Brian; Li, Chunying; Yang, Zhe

    2015-01-01

    SMYD proteins are an exciting field of study as they are linked to many types of cancer-related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative correlated inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allosteric paths for the correlated dynamics. There are nine communities in the dynamical network with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging β hairpin. This study provides insight into the dynamical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity. PMID:26717235

  11. Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (βAPP).

    PubMed

    Jęśko, Henryk; Wilkaniec, Anna; Cieślik, Magdalena; Hilgier, Wojciech; Gąssowska, Magdalena; Lukiw, Walter J; Adamczyk, Agata

    2016-01-01

    Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer's disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing human Aβ precursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD. PMID:26971935

  12. 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

  13. Autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell activation

    PubMed Central

    Andrade, Felipe; Darrah, Erika; Gucek, Marjan; Cole, Robert N.; Rosen, Antony; Zhu, Xiaoming

    2010-01-01

    Objective To address mechanisms that control the activity of human peptidyl arginine deiminase type 4 (PAD-4). Methods PAD-4 autocitrullination was determined by anti–modified citrulline immunoblotting, using purified recombinant and endogenous PAD-4 from activated human primary neutrophils and cell lines expressing PAD-4. The citrullination sites in PAD-4 were determined by mass spectrometry. Mechanisms of autocitrullination-induced inactivation and the functional consequences of autocitrullination in PAD-4 polymorphic variants were addressed using purified components and cell lines expressing PAD-4 wild-type, PAD-4 mutant, and PAD-4 polymorphic variants relevant to rheumatoid arthritis (RA). Results PAD-4 is autocitrullinated in vitro and during activation of primary cells and cell lines expressing PAD-4. Interestingly, this modification inactivated the function of the enzyme. The efficiency of inactivation differed among genetically defined PAD-4 variants relevant to RA. PAD-4 was citrullinated at 10 sites, which are clustered into 3 distinct regions, including a cluster of arginines around the active site cleft where Arg-372 and -374 were identified as the potential autocitrullination targets that inactivate the enzyme. Autocitrullination also modified the structure of PAD-4, abrogating its recognition by multiple rabbit antibodies, but augmenting its recognition by human anti–PAD-4 autoantibodies. Conclusion Our findings suggest that autocitrullination regulates the production of citrullinated proteins during cell activation, and that this is affected by structural polymorphisms in PAD-4. Autocitrullination also influences PAD-4 structure and immune response. PMID:20201080

  14. 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.

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

    DOE PAGESBeta

    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; et al

    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

  16. 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

  17. 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. PMID:27087524

  18. 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

  19. Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics.

    PubMed

    Olsen, Jonathan B; Cao, Xing-Jun; Han, Bomie; Chen, Lisa Hong; Horvath, Alexander; Richardson, Timothy I; Campbell, Robert M; Garcia, Benjamin A; Nguyen, Hannah

    2016-03-01

    The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. PMID:26750096

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Four Human Thiopurine S-Methyltransferase Alleles Severely Affect Protein Structure and Dynamics

    PubMed Central

    Rutherford, Karen; Daggett, Valerie

    2008-01-01

    Summary Thiopurine S-methyltransferase (TPMT) metabolizes cytotoxic thiopurine drugs used in the treatment of leukemia and inflammatory bowel disease. TPMT is a major pharmacogenomic target with 23 alleles identified to date. Several of these alleles cause rapid protein degradation and/or aggregation, making it extremely difficult to study the structural impact of the TPMT polymorphisms experimentally. We, therefore, have performed multiple molecular dynamics simulations of the four most common alleles (TPMT 2 (A80P), 3A (A154T/Y240C), 3B (A154T) and 3C (Y240C)) to investigate the molecular mechanism of TPMT inactivation at an atomic level. The A80P polymorphism in TPMT *2 disrupts helix α3 bordering the active site, which breaks several salt-bridge interactions and opens up a large cleft in the protein. The A154T polymorphism is located within the co-substrate binding-site. The larger threonine alters the packing of substrate binding residues (P68, L69, Y166), increasing the solvent exposure of the polymorphic site. This packing rearrangement may account for the complete lack of activity in the A154T mutant. The Y240C polymorphism is located in β-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix α8 are lost in TPMT *3C. Residues 154 and 240 in TPMT *3A are connected through a hydrogen-bonding network. The dual polymorphisms result in a flattened, slightly distorted protein structure and an increase in the thiopurine-binding site solvent accessibility. The two variants that undergo the most rapid degradation in vivo, TPMT*2 and *3A, are also the most deformed in the simulations. PMID:18482735

  6. Techniques in protein methylation.

    PubMed

    Lee, Jaeho; Cheng, Donghang; Bedford, Mark T

    2004-01-01

    Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated. PMID:15173617

  7. Structural model for the protein-translocating element of the twin-arginine transport system

    PubMed Central

    Rodriguez, Fernanda; Rouse, Sarah L.; Tait, Claudia E.; Harmer, Jeffrey; De Riso, Antonio; Timmel, Christiane R.; Sansom, Mark S. P.; Berks, Ben C.; Schnell, Jason R.

    2013-01-01

    The twin-arginine translocase (Tat) carries out the remarkable process of translocating fully folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Tat is required for bacterial pathogenesis and for photosynthesis in plants. TatA, the protein-translocating element of the Tat system, is a small transmembrane protein that assembles into ring-like oligomers of variable size. We have determined a structural model of the Escherichia coli TatA complex in detergent solution by NMR. TatA assembly is mediated entirely by the transmembrane helix. The amphipathic helix extends outwards from the ring of transmembrane helices, permitting assembly of complexes with variable subunit numbers. Transmembrane residue Gln8 points inward, resulting in a short hydrophobic pore in the center of the complex. Simulations of the TatA complex in lipid bilayers indicate that the short transmembrane domain distorts the membrane. This finding suggests that TatA facilitates protein transport by sensitizing the membrane to transient rupture. PMID:23471988

  8. 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

  9. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  10. The T. brucei TRM5 methyltransferase plays an essential role in mitochondrial protein synthesis and function

    PubMed Central

    Paris, Zdeněk; Horáková, Eva; Rubio, Mary Anne T.; Sample, Paul; Fleming, Ian M.C.; Armocida, Stephanie; Lukeš, Julius; Alfonzo, Juan D.

    2013-01-01

    All tRNAs undergo post-transcriptional chemical modifications as part of their natural maturation pathway. Some modifications, especially those in the anticodon loop, play important functions in translational efficiency and fidelity. Among these, 1-methylguanosine, at position 37 (m1G37) of the anticodon loop in several tRNAs, is evolutionarily conserved and participates in translational reading frame maintenance. In eukaryotes, the tRNA methyltransferase TRM5 is responsible for m1G formation in nucleus-encoded as well as mitochondria-encoded tRNAs, reflecting the universal importance of this modification for protein synthesis. However, it is not clear what role, if any, mitochondrial TRM5 serves in organisms that do not encode tRNAs in their mitochondrial genomes. These organisms may easily satisfy the m1G37 requirement through their robust mitochondrial tRNA import mechanisms. We have explored this possibility in the parasitic protist Trypanosoma brucei and show that down-regulation of TRM5 by RNAi leads to the expected disappearance of m1G37, but with surprisingly little effect on cytoplasmic translation. On the contrary, lack of TRM5 causes a marked growth phenotype and a significant decrease in mitochondrial functions, including protein synthesis. These results suggest mitochondrial TRM5 may be needed to mature unmethylated tRNAs that reach the mitochondria and that could pose a problem for translational fidelity. This study also reveals an unexpected lack of import specificity between some fully matured and potentially defective tRNA species. PMID:23520175

  11. MEP50/PRMT5 reduces gene expression by histone arginine methylation and this is reversed by PKCδ/p38δ signaling

    PubMed Central

    Saha, Kamalika; Adhikary, Gautam; Eckert, Richard L.

    2016-01-01

    PKCδ and p38δ are key proteins in a cascade that stimulates keratinocyte differentiation. This cascade activates transcription of involucrin (hINV) and other genes associated with differentiation. Protein arginine methyltransferase 5 (PRMT5) is an arginine methyltransferase that symmetrically dimethylates arginine residues. This protein interacts with a cofactor, MEP50, and symmetrically dimethylates arginine eight of histone 3 (H3R8me2s) and arginine three of histone 4 (H4R3me2s) to silence gene expression. We use the involucrin gene as a tool to understand the relationship between PKCδ/p38δ and PRMT5/MEP50 signaling. MEP50 suppresses hINV mRNA level and promoter activity. This is associated with increased arginine dimethylation of hINV gene-associated H3/H4. We further show that the PKCδ/p38δ keratinocyte differentiation cascade reduces PRMT5 and MEP50 expression, association with the hINV gene promoter, and H3R8me2s and H4R2me2s formation. We propose that PRMT5/MEP50-dependent methylation is an epigenetic mechanism that assists in silencing of hINV expression, and that PKCδ signaling activates gene expression by directly activating transcription and by suppressing PRMT5/MEP50 dependent arginine dimethylation of promoter associated histones. This is an example of crosstalk between PKCδ/p38δ signaling and PRMT5/MEP50 epigenetic silencing. PMID:26763441

  12. CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

    PubMed

    Nishii, Y; Morishima, M; Kakehi, Y; Umehara, K; Kioka, N; Terano, Y; Amachi, T; Ueda, K

    2000-01-14

    A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth. PMID:10631324

  13. 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

  14. Weaver Syndrome-Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro.

    PubMed

    Cohen, Ana S A; 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

    2016-03-01

    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

  15. Response of maize serine/arginine-rich protein gene family in seedlings to drought stress.

    PubMed

    Li, Jiao; Guo, Yuqi; Cui, Weiling; Xu, Aihua; Tian, Zengyuan

    2014-07-01

    Alternative splicing (AS) in eukaryotic organisms is closely related to the gene regulation in plant abiotic stress responses, in which serine/arginine-rich proteins (SR proteins) act as key regulators. The genome sequence of maize inbred line B73 was analyzed, showing that the promoter regions of SR genes possess about three to eight kinds of cis-acting regulatory elements. Twenty-seven SR genes encode alkaline proteins, and 23 of which are divided into five subgroups in terms of the first RNA recognition motif (RRM) at the amino terminal. The expression of SR genes showed tissue-specific and genotype-dependent features under drought stress in the hybrid Zhengdan-958 and its parents, Zheng-58 and Chang-7-2 via bidirectional hierarchical clustering. SR genes were down-regulated in roots while they were up-regulated in shoots under drought stress. However, SR genes were down-regulated in both roots and shoots in three different rehydration stages after severe drought stress. Additionally, a widespread alternative splicing exists in all SR genes although SR genes showed differential expression tendency under drought stress and/or during rehydration stages. Results above will deepen our understanding of the molecular mechanisms of plant response to abiotic stress from the perspective of AS-network. PMID:25076035

  16. Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

    PubMed Central

    Cimmino, Amelia; Capasso, Rosanna; Muller, Fabbri; Sambri, Irene; Masella, Lucia; Raimo, Marianna; De Bonis, Maria Luigia; D'Angelo, Stefania; Zappia, Vincenzo; Galletti, Patrizia; Ingrosso, Diego

    2008-01-01

    Background Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). Methodology/Principal Findings Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H2O2; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H2O2 concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H2O2 treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative

  17. Conserved Arginines of Bovine Adenovirus-3 33K Protein Are Important for Transportin-3 Mediated Transport and Virus Replication

    PubMed Central

    Islam, Azharul; Tikoo, Suresh K.

    2014-01-01

    The L6 region of bovine adenovirus (BAdV)-3 encodes a spliced protein designated 33K. The 33K specific sera detected five major proteins and three minor proteins in transfected or virus infected cells, which could arise by internal initiation of translation and alternative splicing. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells. The 33K nuclear transport utilizes both classical importin-α/-β and importin-β dependent nuclear import pathways and preferentially binds to importin-α5 and transportin-3 receptors, respectively. Analysis of mutant 33K proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing RS repeat are required for nuclear localization and, binding to both importin-α5 and transportin-3 receptors. Interestingly, the arginine residues of conserved RS repeat are required for binding to transportin-3 receptor but not to importin-α5 receptor. Moreover, mutation of arginines residues of RS repeat proved lethal for production of progeny virus. Our results suggest that arginines of RS repeat are required for efficient nuclear transport of 33K mediated by transportin-3, which appears to be essential for replication and production of infectious virion. PMID:25019945

  18. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone 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. PMID:26258118

  19. 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

    2016-01-01

    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 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. PMID:27113483

  20. 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

  1. 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...

  2. Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome

    PubMed Central

    Lott, Kaylen; Mukhopadhyay, Shreya; Li, Jun; Wang, Jie; Yao, Jin; Sun, Yijun; Qu, Jun; Read, Laurie K.

    2015-01-01

    Arginine methylation is a posttranslational modification that impacts wide-ranging cellular functions, including transcription, mRNA splicing and translation. RNA binding proteins (RBPs) represent one of the largest classes of arginine methylated proteins in both mammals and the early diverging parasitic protozoan, Trypanosoma brucei. Here, we report the effects of arginine methylation on the functions of the essential and previously uncharacterized T. brucei RBP, DRBD18. RNAseq analysis shows that DRBD18 depletion causes extensive rearrangement of the T. brucei transcriptome, with increases and decreases in hundreds of mRNAs. DRBD18 contains three methylated arginines, and we used complementation of DRBD18 knockdown cells with methylmimic or hypomethylated DRBD18 to assess the functions of these methylmarks. Methylmimic and hypomethylated DRBD18 associate with different ribonucleoprotein complexes. These altered macromolecular interactions translate into differential impacts on the T. brucei transcriptome. Methylmimic DRBD18 preferentially stabilizes target RNAs, while hypomethylated DRBD18 is more efficient at destabilizing RNA. The protein arginine methyltransferase, TbPRMT1, interacts with DRBD18 and knockdown of TbPRMT1 recapitulates the effects of hypomethylated DRBD18 on mRNA levels. Together, these data support a model in which arginine methylation acts as a switch that regulates T. brucei gene expression. PMID:25940618

  3. Distinct patterns of expression but similar biochemical properties of protein L-isoaspartyl methyltransferase in higher plants.

    PubMed

    Thapar, N; Kim, A K; Clarke, S

    2001-02-01

    Protein L-isoaspartyl methyltransferase is a widely distributed repair enzyme that initiates the conversion of abnormal L-isoaspartyl residues to their normal L-aspartyl forms. Here we show that this activity is expressed in developing corn (Zea mays) and carrot (Daucus carota var. Danvers Half Long) plants in patterns distinct from those previously seen in winter wheat (Triticum aestivum cv Augusta) and thale cress (Arabidopsis thaliana), whereas the pattern of expression observed in rice (Oryza sativa) is similar to that of winter wheat. Although high levels of activity are found in the seeds of all of these plants, relatively high levels of activity in vegetative tissues are only found in corn and carrot. The activity in leaves was found to decrease with aging, an unexpected finding given the postulated role of this enzyme in repairing age-damaged proteins. In contrast with the situation in wheat and Arabidopsis, we found that osmotic or salt stress could increase the methyltransferase activity in newly germinated seeds (but not in seeds or seedlings), whereas abscisic acid had no effect. We found that the corn, rice, and carrot enzymes have comparable affinity for methyl-accepting substrates and similar optimal temperatures for activity of 45 degrees C to 55 degrees C as the wheat and Arabidopsis enzymes. These experiments suggest that this enzyme may have specific roles in different plant tissues despite a common catalytic function. PMID:11161058

  4. 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

  5. LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2.

    PubMed

    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

    2015-05-29

    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. 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. PMID:25825497

  6. 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

  7. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    PubMed Central

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  8. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins.

    PubMed

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-04-19

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  9. Involvement of the "A" isozyme of methyltransferase II and the 29-kilodalton corrinoid protein in methanogenesis from monomethylamine.

    PubMed Central

    Burke, S A; Krzycki, J A

    1995-01-01

    An assay which allowed detection of proteins involved in the trimethylamine- or monomethylamine (MMA)-dependent methylation of coenzyme M (CoM) was developed. The two activities could be separated by anion-exchange chromatography. The unresolved activity responsible for MMA:CoM methyl transfer eluted from a gel permeation column in the molecular mass range of 32 kDa. The activity was purified to two monomeric proteins of 40 and 29 kDa. The preparation contained protein-bound corrinoid in a mixture of Co(II) and Co(III) states, as well as methyl-B12:CoM methyltransferase (MT2) activity. N-terminal sequence analysis demonstrated that the polypeptides were two previously identified proteins of undefined physiological function. The smaller polypeptide was the monomeric 29-kDa corrinoid protein. The larger polypeptide was the "A" isozyme of MT2. Individually purified preparations of both proteins increased the rate of MMA-dependent CoM methylation by approximately 1.7 mumol/min/mg of purified protein above background activity in the extract of methanol-grown cells. These results indicate that the 29-kDa corrinoid protein and the "A" isozyme of MT2 function in methanogenesis from MMA. A likely mechanism is that the 29-kDa corrinoid is methylated by MMA and the methyl group is then transferred by the "A" isozyme of MT2 to CoM. PMID:7635826

  10. 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. PMID:23381944

  11. The l-Isoaspartyl Protein Repair Methyltransferase Enhances Survival of Aging Escherichia coli Subjected to Secondary Environmental Stresses

    PubMed Central

    Visick, Jonathan E.; Cai, Hui; Clarke, Steven

    1998-01-01

    Like its homologs throughout the biological world, the l-isoaspartyl protein repair methyltransferase of Escherichia coli, encoded by the pcm gene, can convert abnormal l-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885–9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation in rpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158–4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcm mutant lacks these phenotypes. Interestingly, the newly constructed pcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42°C. The pcm mutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functional pcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo. PMID:9573145

  12. Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase.

    PubMed

    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

    2016-02-12

    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

  13. 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

  14. Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.

    PubMed

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

    2015-01-16

    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

  15. Mapping precursor-binding site on TatC subunit of twin arginine-specific protein translocase by site-specific photo cross-linking.

    PubMed

    Zoufaly, Stefan; Fröbel, Julia; Rose, Patrick; Flecken, Tobias; Maurer, Carlo; Moser, Michael; Müller, Matthias

    2012-04-13

    A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase. PMID:22362773

  16. 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. PMID:26438231

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

    PubMed

    Eggert, Erik; Hillig, Roman C; Koehr, Silke; Stöckigt, Detlef; Weiske, Jörg; Barak, Naomi; Mowat, Jeffrey; Brumby, Thomas; Christ, Clara D; Ter Laak, Antonius; Lang, Tina; Fernandez-Montalvan, Amaury E; Badock, Volker; Weinmann, Hilmar; Hartung, Ingo V; Barsyte-Lovejoy, Dalia; Szewczyk, Magdalena; Kennedy, Steven; Li, Fengling; Vedadi, Masoud; Brown, Peter J; Santhakumar, Vijayaratnam; Arrowsmith, Cheryl H; Stellfeld, Timo; Stresemann, Carlo

    2016-05-26

    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

  18. 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

  19. Arginine methylation modulates autophagic degradation of PGL granules in C. elegans.

    PubMed

    Li, Sihui; Yang, Peiguo; Tian, E; Zhang, Hong

    2013-11-01

    The selective degradation of intracellular components by autophagy involves sequential interactions of the cargo with a receptor, which also binds the autophagosomal protein Atg8 and a scaffold protein. Here, we demonstrated that mutations in C. elegans epg-11, which encodes an arginine methyltransferase homologous to PRMT1, cause the defective removal of PGL-1 and PGL-3 (cargo)-SEPA-1 (receptor) complexes, known as PGL granules, from somatic cells during embryogenesis. Autophagic degradation of the PGL granule scaffold protein EPG-2 and other protein aggregates was unaffected in epg-11/prmt-1 mutants. Loss of epg-11/prmt-1 activity impairs the association of PGL granules with EPG-2 and LGG-1 puncta. EPG-11/PRMT-1 directly methylates arginines in the RGG domains of PGL-1 and PGL-3. Autophagic removal of PGL proteins is impaired when the methylated arginines are mutated. Our study reveals that posttranslational arginine methylation regulates the association of the cargo-receptor complex with the scaffold protein, providing a mechanism for modulating degradation efficiency in selective autophagy. PMID:24140420

  20. 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

  1. 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

  2. Quantification of Arginine and Its Methylated Derivatives in Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

    PubMed

    Vicente, Faye B; Vespa, Gina; Miller, Alan; Haymond, Shannon

    2016-01-01

    Arginine is the substrate for nitric oxide synthases (NOS), thus the production of nitric oxide (NO) is based on arginine availability. Arginine is methylated through the activity of protein arginine methyltransferases (PRMT1 and PRMT2), to form asymmetrical dimethylarginine (ADMA) and symmetrical dimethylarginine (SDMA). These compounds have gained interest in recent years due to their influence on NO production rates and association with cardiovascular and renal diseases. The accurate and precise measurement of arginine and its methylated derivatives is needed for research studies investigating their role(s) in NO bioavailability and development of disease. We describe a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for quantifying arginine, ADMA, and SDMA requiring only 50 μL of plasma. The sample preparation involves addition of internal standards (ADMA-d7 for ADMA and SDMA, and (13)C6 -arginine for arginine) prior to protein precipitation with LCMS grade acetonitrile. Samples are centrifuged and supernatant is dried under nitrogen gas at 50 °C. Samples are reconstituted with mobile phase (ammonium acetate-formic acid-water). Arginine, ADMA, and SDMA are separated using an isocratic HPLC method on a 3 μM silica analytical column. MS/MS detection is performed in the multiple-reaction monitoring (MRM) mode and the transitions monitored are m/z 203 to m/z 70 for ADMA and SDMA, m/z 210 to m/z 77 for ADMA-d7, m/z 175 to m/z 70 for arginine, and m/z 181 to m/z 74 for (13)C6-arginine. PMID:26602113

  3. Inhibition of Cellular Methyltransferases Promotes Endothelial Cell Activation by Suppressing Glutathione Peroxidase 1 Protein Expression*

    PubMed Central

    Barroso, Madalena; Florindo, Cristina; Kalwa, Hermann; Silva, Zélia; Turanov, Anton A.; Carlson, Bradley A.; de Almeida, Isabel Tavares; Blom, Henk J.; Gladyshev, Vadim N.; Hatfield, Dolph L.; Michel, Thomas; Castro, Rita; Loscalzo, Joseph; Handy, Diane E.

    2014-01-01

    S-Adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNASec to the Um34 form. The formation of methylated tRNASec facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNASec and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNASec hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype. PMID:24719327

  4. 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.

  5. Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins.

    PubMed

    Blobel, Jascha; Brath, Ulrika; Bernadó, Pau; Diehl, Carl; Ballester, Lidia; Sornosa, Alejandra; Akke, Mikael; Pons, Miquel

    2011-12-01

    Addition of a 50 mM mixture of L: -arginine and L: -glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE. PMID:21390527

  6. Protein L-isoaspartyl methyltransferase catalyzes in vivo racemization of Aspartate-25 in mammalian histone H2B.

    PubMed

    Young, Glen W; Hoofring, Sarah A; Mamula, Mark J; Doyle, Hester A; Bunick, Gerard J; Hu, Yonglin; Aswad, Dana W

    2005-07-15

    Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (D/L < or = 0.01). Racemization of Asp25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (D/L = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age. PMID:15908425

  7. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain.

    PubMed

    Potisopon, Supanee; Priet, Stéphane; Collet, Axelle; Decroly, Etienne; Canard, Bruno; Selisko, Barbara

    2014-10-01

    Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis. PMID:25209234

  8. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

    PubMed Central

    Potisopon, Supanee; Priet, Stéphane; Collet, Axelle; Decroly, Etienne; Canard, Bruno; Selisko, Barbara

    2014-01-01

    Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis. PMID:25209234

  9. The juxtamembrane lysine and arginine residues of surfactant protein C precursor influence palmitoylation via effects on trafficking.

    PubMed

    ten Brinke, A; Batenburg, J J; Gadella, B M; Haagsman, H P; Vaandrager, A B; van Golde, L M

    2001-08-01

    Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cysteines 5 and 6 before mature SP-C is formed by several proteolytic steps. To study the structural requirements for the palmitoylation of proSP-C, his-tagged human proSP-C (his-proSP-C) and his-proSP-C mutants were expressed in Chinese hamster ovary cells and analyzed by metabolic labeling with [(3)H]palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by serines showed that these were the only two cysteine residues palmitoylated in his-proSP-C. Substitution of the juxtamembrane basic residues lysine and arginine by uncharged glutamines led to a large decrease in palmitoylation level of proSP-C. The addition of brefeldin A nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutant increased also, but not to wild-type (WT) levels. Fluorescence immunocytochemistry showed that WT proSP-C was localized in punctate vesicles throughout the cell, whereas the mutant lacking the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). This indicates that the two basic juxtamembrane residues influence palmitoylation of proSP-C by preventing the transport of proSP-C out of the ER, implying that proSP-C becomes palmitoylated normally in a compartment distal to the ER. PMID:11509324

  10. Design, Synthesis, and Biological Evaluation of Tetrazole Analogs of Cl-Amidine as Protein Arginine Deiminase Inhibitors

    PubMed Central

    2016-01-01

    Protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also block the formation of neutrophil extracellular traps. These bioisosteres represent an important step in our efforts to develop stable, bioavailable, and selective inhibitors for the PADs. PMID:25559347

  11. 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.

  12. Linking electrostatic effects and protein motions in enzymatic catalysis. A theoretical analysis of catechol o-methyltransferase.

    PubMed

    García-Meseguer, Rafael; Zinovjev, Kirill; Roca, Maite; Ruiz-Pernía, Javier J; Tuñón, Iñaki

    2015-01-22

    The role of protein motions in enzymatic catalysis is the subject of a hot scientific debate. We here propose the use of an explicit solvent coordinate to analyze the impact of environmental motions during the reaction process. The example analyzed here is the reaction catalyzed by catechol O-methyltransferase, a methyl transfer reaction from S-adenosylmethionine (SAM) to the nucleophilic oxygen atom of catecholate. This reaction proceeds from a charged reactant to a neutral product, and then a large electrostatic coupling with the environment could be expected. By means of a two-dimensional free energy surface, we show that a large fraction of the environmental motions needed to attain the transition state happens during the first stages of the reaction because most of the environmental motions are slower than changes in the substrate. The incorporation of the solvent coordinate in the definition of the transition state improves the transmission coefficient and the committor histogram in solution, while the changes are much less significant in the enzyme. The equilibrium solvation approach seems then to work better in the enzyme than in aqueous solution because the enzyme provides a preorganized environment where the reaction takes place. PMID:25159911

  13. 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

  14. The human interferon-regulated ISG95 protein interacts with RNA polymerase II and shows methyltransferase activity

    SciTech Connect

    Haline-Vaz, Thais; Lima Silva, Tereza Cristina; Zanchin, Nilson I.T.

    2008-08-08

    A major mechanism of cellular resistance to viral invasion involves genes from the interferon signaling pathway, called ISGs (interferon stimulated genes). Global transcriptional profiling studies have linked increased expression of ISG95 (KIAA0082) to response to interferon treatment and viral infection, suggesting that it may be part of the cellular defense against viral replication. In this work, we show that the ISG95 promoter can drive interferon-induced transcription of a reporter gene in Vero cells. Recombinant ISG95 shows RNA- and S-adenosyl-methionine binding and protein methyltransferase activity in vitro. ISG95 interacts with the C-terminal domain of RNA polymerase II, which is consistent with its nuclear localization and with the predicted function of the WW domain found in the C-terminal region of ISG95. The results presented in this work indicate that ISG95 is part of the interferon response pathway and functions in the pre-mRNA processing events mediated by the C-terminal domain of the RNA polymerase II.

  15. Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface

    PubMed Central

    Klema, Valerie J.; Ye, Mengyi; Hindupur, Aditya; Teramoto, Tadahisa; Gottipati, Keerthi; Padmanabhan, Radhakrishnan; Choi, Kyung H.

    2016-01-01

    Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication. PMID:26895240

  16. Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine.

    PubMed

    de la Fuente, A; Martín, J F; Rodríguez-García, A; Liras, P

    2004-10-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations. PMID:15375131

  17. Arginine transport in catabolic disease states.

    PubMed

    Pan, Ming; Choudry, Haroon A; Epler, Mark J; Meng, Qinghe; Karinch, Anne; Lin, Chengmao; Souba, Wiley

    2004-10-01

    Arginine appears to be a semiessential amino acid in humans during critical illness. Catabolic disease states such as sepsis, injury, and cancer cause an increase in arginine utilization, which exceeds body production, leading to arginine depletion. This is aggravated by the reduced nutrient intake that is associated with critical illness. Arginine depletion may have negative consequences on tissue function under these circumstances. Nutritional regimens containing arginine have been shown to improve nitrogen balance and lymphocyte function, and stimulate arginine transport in the liver. We have studied the effects of stress mediators on arginine transport in vascular endothelium, liver, and gut epithelium. In vascular endothelium, endotoxin stimulates arginine uptake, an effect that is mediated by the cytokine tumor necrosis factor-alpha (TNF-alpha) and by the cyclo-oxygenase pathway. This TNF-alpha stimulation involves the activation of intracellular protein kinase C (PKC). A significant increase in hepatic arginine transport activity also occurs following burn injury and in rats with progressive malignant disease. Surgical removal of the growing tumor results in a normalization of the accelerated hepatic arginine transport within days. Chronic metabolic acidosis and sepsis individually augment intestinal arginine transport in rats and Caco-2 cell culture. PKC and mitogen-activated protein kinases are involved in mediating the sepsis/acidosis stimulation of arginine transport. Understanding the regulation of plasma membrane arginine transport will enhance our knowledge of nutrition and metabolism in seriously ill patients and may lead to the design of improved nutritional support formulas. PMID:15465794

  18. Arginine and nitrogen storage.

    PubMed

    Llácer, José L; Fita, Ignacio; Rubio, Vicente

    2008-12-01

    When nitrogen is abundant, prokaryotic and eukaryotic oxygen-producing photosynthetic organisms store nitrogen as arginine, by relieving feedback inhibition of the arginine biosynthesis controlling enzyme, N-acetylglutamate kinase (NAGK). The signalling protein PII, an ancient and widely distributed nitrogen/carbon/ADP/ATP sensor, mediates feedback inhibition relief of NAGK by binding to this enzyme. PII phosphorylation or PII binding of ADP or 2-oxoglutarate prevents PII-NAGK complex formation. Crystal structures of NAGK, cyanobacterial and plant PII and corresponding PII-NAGK complexes have been recently determined. In these complexes, two polar PII trimers sandwich one ring-like NAGK hexamer. Each PII subunit contacts one NAGK subunit, triggering a symmetry-restricted narrowing of the NAGK ring, with concomitant adoption by the arginine sites of a low-affinity conformation. PMID:19013524

  19. Effect of counter ions of arginine as an additive for the solubilization of protein and aromatic compounds.

    PubMed

    Yoshizawa, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2016-10-01

    Arginine is widely used in biotechnological application, but mostly with chloride counter ion. Here, we examined the effects of various anions on solubilization of aromatic compounds and reduced lysozyme and on refolding of the lysozyme. All arginine salts tested increased the solubility of propyl gallate with acetate much more effectively than chloride. The effects of arginine salts were compared with those of sodium or guanidine salts, indicating that the ability of anions to modulate the propyl gallate solubility is independent of the cation. Comparison of transfer free energy of propyl gallate between sodium and arginine salts indicates that the interaction of propyl gallate is more favorable with arginine than sodium. On the contrary, the solubility of aromatic amino acids is only slightly modulated by anions, implying that there is specific interaction between acetic acid and propyl gallate. Unlike their effects on the solubility of small aromatic compounds, the solubility of reduced lysozyme was much higher in arginine chloride than in arginine acetate or sulfate. Consistent with high solubility, refolding of reduced lysozyme was most effective in arginine chloride. These results suggest potential broader applications of arginine modulated by different anions. PMID:27234496

  20. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    PubMed

    Henderson, Morgan L; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones. PMID:25993347

  1. The reductive acetyl coenzyme A pathway: sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum.

    PubMed Central

    Roberts, D L; Zhao, S; Doukov, T; Ragsdale, S W

    1994-01-01

    The methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur protein in the acetyl coenzyme A pathway. MeTr was purified to homogeneity and shown to lack metals. The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell protein. Regions in the sequence of MeTr and the E. coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains. PMID:7928975

  2. 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

    Apicella, Alessandra; Heunemann, Peggy; Bolisetty, Sreenath; Marascio, Matteo; Gemperli Graf, Anja; Garamszegi, Laszlo; Mezzenga, Raffaele; Fischer, Peter; Plummer, Christopher J; 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

  3. 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

  4. Recruitment of histone methyltransferase G9a mediates transcriptional repression of Fgf21 gene by E4BP4 protein.

    PubMed

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

    2013-02-22

    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

  5. Arginine Metabolism in Developing Soybean Cotyledons

    PubMed Central

    Micallef, Barry J.; Shelp, Barry J.

    1989-01-01

    Tracerkinetic experiments were performed using l-[guanidino-14C]arginine, l-[U-14C]arginine, l-[ureido-14C]citrulline, and l-[1-14C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free 14C compounds and incubated in sealed vials containing a CO2 trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the 14C metabolized from [guanidino-14C]arginine and [U-14C]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 14CO2 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 14CO2 was evolved during the catabolism of ornithine-derived glutamate; 14C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented. The maximum specific radioactivity ratios between arginine in newly synthesized protein and total free arginine in the 14C-citrulline and 14C-ornithine experiments indicated that only 3% of the free arginine was in the protein precursor pool, and that argininosuccinate and citrulline were present in multiple pools. PMID:16666991

  6. 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

  7. Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.

    PubMed

    Baranauskė, Simona; Mickutė, Milda; Plotnikova, Alexandra; Finke, Andreas; Venclovas, Česlovas; Klimašauskas, Saulius; Vilkaitis, Giedrius

    2015-03-11

    Methylation of 3'-terminal nucleotides of miRNA/miRNA* is part of miRNAs biogenesis in plants but is not found in animals. In Arabidopsis thaliana this reaction is carried out by a multidomain AdoMet-dependent 2'-O-methyltransferase HEN1. Using deletion and structure-guided mutational analysis, we show that the double-stranded RNA-binding domains R(1) and R(2) of HEN1 make significant but uneven contributions to substrate RNA binding, and map residues in each domain responsible for this function. Using GST pull-down assays and yeast two-hybrid analysis we demonstrate direct HEN1 interactions, mediated by its FK506-binding protein-like domain and R(2) domain, with the microRNA biogenesis protein HYL1. Furthermore, we find that HEN1 forms a complex with DICER-LIKE 1 (DCL1) ribonuclease, another key protein involved in miRNA biogenesis machinery. In contrast, no direct interaction is detectable between HEN1 and SERRATE. On the basis of these findings, we propose a mechanism of plant miRNA maturation which involves binding of the HEN1 methyltransferase to the DCL1•HYL1•miRNA complex excluding the SERRATE protein. PMID:25680966

  8. 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...

  9. Interaction of the hepatitis B virus X protein with the lysine methyltransferase SET and MYND domain-containing 3 induces activator protein 1 activation.

    PubMed

    Hayashi, Miwako; Deng, Lin; Chen, Ming; Gan, Xiang; Shinozaki, Kenta; Shoji, Ikuo; Hotta, Hak

    2016-01-01

    Hepatitis B virus (HBV) is a widespread human pathogen that often causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The detailed mechanisms underlying HBV pathogenesis remain poorly understood. The HBV X protein (HBx) is a multifunctional regulator that modulates viral replication and host cell functions, such as cell cycle progression, apoptosis and protein degradation through interaction with a variety of host factors. Recently, the nonstructural protein 5A (NS5A) of hepatitis C virus has been reported to interact with methyltransferase SET and MYND domain-containing 3 (SMYD3), which is implicated in chromatin modification and development of cancer. Because HBx shares fundamental regulatory functions concerning viral replication and pathogenesis with NS5A, it was decided to examine whether HBx interacts with SMYD3. In the present study, it was demonstrated by co-immunoprecipitation analysis that HBx interacts with both ectopically and endogenously expressed SMYD3 in Huh-7.5 cells. Deletion mutation analysis revealed that the C-terminal region of HBx (amino acids [aa] 131-154) and an internal region of SMYD3 (aa 269-288) are responsible for their interaction. Immunofluorescence and proximity ligation assays showed that HBx and SMYD3 co-localize predominantly in the cytoplasm. Luciferase reporter assay demonstrated that the interaction between HBx and SMYD3 activates activator protein 1 (AP-1) signaling, but not that of nuclear factor-kappa B (NF-κB). On the other hand, neither overexpression nor knockdown of SMYD3 altered production of HBV transcripts and HBV surface antigen (HBsAg). In conclusion, a novel HBx-interacting protein, SMYD3, was identified, leading to proposal of a novel mechanism of AP-1 activation in HBV-infected cells. PMID:26616333

  10. Milk protein responses in dairy cows to changes in postruminal supplies of arginine, isoleucine, and valine.

    PubMed

    Haque, M N; Rulquin, H; Lemosquet, S

    2013-01-01

    An ideal profile of essential AA (EAA) can improve the efficiency of metabolizable protein (or PDIE, the equivalent in the INRA feeding system) utilization in dairy cows. Compared with other EAA, existing recommendations for the requirements of Arg, Ile, and Val are few and inconsistent. Four multiparous Holstein dairy cows at 22±6 wk of lactation received 4 treatments (duodenal infusions of 445±22.4 g/d of an EAA mixture complementing a low-protein diet in a 4×4 Latin square design with a period length of 1 wk). The control treatment provided a balanced supply (in % of PDIE) of 5.1% Arg, 5.2% Ile, and 5.9% Val, whereas in the 3 subsequent treatments of -Arg, -Ile, and -Val, the concentrations of these 3 EAA were reduced to 3.5, 4.1, and 4.5%, respectively. All treatments were made isonitrogenous and were balanced to provide 7 other EAA (Lys, Met, His, Leu, Phe, Thr, and Trp), according to the recommendations described in the literature. Combined, the diet and the infusions provided 14.3±0.1% crude protein on a dry matter basis, and 66.0±1.2 g of PDIE/Mcal of net energy for lactation. Neither dry matter intake (19.2 kg/d) nor milk yield (30.4±0.4 kg/d) was affected by treatments. The -Arg and -Ile treatments did not modify milk protein synthesis or the efficiency of N utilization. However, the -Val treatment decreased milk protein content by 4.9% and milk crude protein content by 4.3%, and tended to decrease the efficiency of N use for milk protein yield by 3.7% (compared with the control). These effects of Val were related to a decrease in the plasma concentration of Val as well as a trend toward decreasing plasma concentrations of Met, His, and the sum of all EAA and nonessential AA in the -Val treatment, which indicates a different utilization of all AA in response to the Val deficit. The deletion of Ile, compared with the deletion of Val, tended to decrease the milk protein-to-fat ratio by 3.8%. In conclusion, the supply of Arg at 3.5% of PDIE was not

  11. 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

  12. Proline 235 plays a key role in the regulation of the oligomeric states of Thermotoga maritima Arginine Binding Protein.

    PubMed

    Smaldone, Giovanni; Vigorita, Marilisa; Ruggiero, Alessia; Balasco, Nicole; Dattelbaum, Jonathan D; D'Auria, Sabato; Del Vecchio, Pompea; Graziano, Giuseppe; Vitagliano, Luigi

    2016-07-01

    The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBP(P235GK)) or a Gly residue (TmArgBP(P235G)). Different forms of these mutants were generated and extensively characterized using biophysical techniques. For both TmArgBP(P235GK) and TmArgBP(P235G) mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBP(P235G) monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye. The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties. PMID:27087545

  13. Arginine kinase: differentiation of gene expression and protein activity in the red imported fire ant, Solenopsis invicta.

    PubMed

    Wang, Haichuan; Zhang, Lan; Zhang, Lee; Lin, Qin; Liu, Nannan

    2009-02-01

    Arginine kinase (AK), a primary enzyme in cell metabolism and adenosine 5'-triphosphate (ATP)-consuming processes, plays an important role in cellular energy metabolism and maintaining constant ATP levels in invertebrate cells. In order to identify genes that are differentially expressed between larvae and adults, queens and workers, and female alates (winged) and queens (wingless), AK cDNA was obtained from the red imported fire ant. The cDNA sequence of the gene has open reading frames of 1065 nucleotides, encoding a protein of 355 amino acid residues that includes the substrate recognition region, the signature sequence pattern of ATP:guanidino kinases, and an "actinin-type" actin binding domain. Northern blot analysis and protein activity analysis demonstrated that the expression of the AK gene and its protein activity were developmentally, caste specifically, and tissue specifically regulated in red imported fire ants with a descending order of worker> alate (winged adult) female> alate (winged adult) male> larvae> worker pupae approximately alate pupae. These results suggest a different demand for energy-consumption and production in the different castes of the red imported fire ant, which may be linked to their different missions and physiological activities in the colonies. The highest level of the AK gene expression and activity was identified in head tissue of both female alates and workers and thorax tissue of workers, followed by thorax tissue of female alates and abdomen tissue of male alates, suggesting the main tissues or cells in these body parts, such as brain, neurons and muscles, which have been identified as the major tissues and/or cells that display high and variable rates of energy turnover in other organisms, play a key role in energy production and its utilization in the fire ant. In contrast, in the male alate, the highest AK expression and activity were found in the abdomen, suggesting that here energy demand may relate to sperm formation

  14. 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...

  15. 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

  16. Identification of essential arginine residues of Escherichia coli DedA/Tvp38 family membrane proteins YqjA and YghB.

    PubMed

    Kumar, Sujeet; Bradley, Cersten L; Mukashyaka, Patience; Doerrler, William T

    2016-07-01

    Escherichia coli DedA/Tvp38 family proteins YghB and YqjA are putative membrane transporters with 62% amino acid identity and overlapping functions. An E. coli strain (BC202) with nonpolar ΔyghB and ΔyqjA mutations displays cell-division defects and temperature sensitivity and is sensitive to antibiotics and alkaline pH. In this study, we performed site-directed mutagenesis on conserved, charged amino acids of YqjA and YghB. We discovered two conserved predicted membrane-embedded arginines (R130 and R136) that are critical for function in both proteins as defined by their ability to complement BC202 phenotypes, when expressed from a plasmid. Lysine can substitute for arginine at position R130 indicating a charge dependence at this position, but could not substitute at R136. In light of the established role that arginine plays in the translocation mechanism of numerous membrane transporters, we hypothesize that these amino acids play a role in the transport mechanism of these DedA/Tvp38 family proteins. PMID:27190159

  17. Arginine residues at internal positions in a protein are always charged.

    PubMed

    Harms, Michael J; Schlessman, Jamie L; Sue, Gloria R; García-Moreno, Bertrand

    2011-11-22

    Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK(a) values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK(a) values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK(a); they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments. PMID:22080604

  18. Arginine residues at internal positions in a protein are always charged

    PubMed Central

    Harms, Michael J.; Schlessman, Jamie L.; Sue, Gloria R.; García-Moreno E., Bertrand

    2011-01-01

    Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pKa values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pKa values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pKa; they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments. PMID:22080604

  19. Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis.

    PubMed

    Mazzoleni, Meryl; Figuet, Sylvie; Martin-Laffon, Jacqueline; Mininno, Morgane; Gilgen, Annabelle; Leroux, Mélanie; Brugière, Sabine; Tardif, Marianne; Alban, Claude; Ravanel, Stéphane

    2015-09-01

    Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function. PMID:26116422

  20. 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 Central

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

    2015-01-01

    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. PMID:25592393

  1. The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.

    PubMed

    Tsai, Chi-Neu; Tsai, Chia-Lung; Tse, Ka-Po; Chang, Hwan-You; Chang, Yu-Sun

    2002-07-23

    The latent membrane protein (LMP1) of Epstein-Barr virus (EBV) is expressed in EBV-associated nasopharyngeal carcinoma, which is notoriously metastatic. Although it is established that LMP1 represses E-cadherin expression and enhances the invasive ability of carcinoma cells, the mechanism underlying this repression remains to be elucidated. In this study, we demonstrate that LMP1 induces the expression and activity of the DNA methyltransferases 1, 3a, and 3b, using real-time reverse transcription-PCR and enzyme activity assay. This results in hypermethylation of the E-cadherin promoter and down-regulation of E-cadherin gene expression, as revealed by methylation-specific PCR, real-time reverse transcription-PCR and Western blotting data. The DNA methyltransferase inhibitor, 5'-Aza-2'dC, restores E-cadherin promoter activity and protein expression in LMP1-expressing cells, which in turn blocks cell migration ability, as demonstrated by the Transwell cell migration assay. Our findings suggest that LMP1 down-regulates E-cadherin gene expression and induces cell migration activity by using cellular DNA methylation machinery. PMID:12110730

  2. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.

    PubMed Central

    Clarke, S; Vogel, J P; Deschenes, R J; Stock, J

    1988-01-01

    The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the alpha-carboxyl group is also methyl esterified. To determine if ras proteins are themselves modified by a similar type of methylation reaction, we incubated rat embryo fibroblasts transformed with p53 and activated Ha-ras oncogenes with L-[methyl-3H]methionine under conditions in which the isotope was converted to the methyl donor S-adenosyl-L-[methyl-3H]methionine. By using an assay that detects methyl ester linkages, we found that immunoprecipitated ras proteins are in fact esterified and that the stability of these esters is consistent with a carboxyl-terminal localization. This methylation reaction may be important in regulating the interaction of ras proteins with plasma membrane components. The presence of analogous carboxyl-terminal tetrapeptide sequences in other proteins may provide a general recognition sequence for lipidation and methylation modification reactions. Images PMID:3290900

  3. Serine-arginine protein kinase 1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer.

    PubMed

    Mavrou, A; Brakspear, K; Hamdollah-Zadeh, M; Damodaran, G; Babaei-Jadidi, R; Oxley, J; Gillatt, D A; Ladomery, M R; Harper, S J; Bates, D O; Oltean, S

    2015-08-13

    Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor A (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa. PMID:25381816

  4. 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. PMID:25424656

  5. Jian carp (Cyprinus carpio var. Jian) intestinal immune responses, antioxidant status and tight junction protein mRNA expression are modulated via Nrf2 and PKC in response to dietary arginine deficiency.

    PubMed

    Wang, Biao; Feng, Lin; Chen, Gang-Fu; Jiang, Wei-Dan; Liu, Yang; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhao, Juan; Zhou, Xiao-Qiu

    2016-04-01

    This study investigated the effect of dietary arginine on the immune response, antioxidant status and tight junction mRNA expression in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with an average initial weight of 6.33 ± 0.03 g were fed graded levels of arginine (9.8-24.5 g kg(-1) diet) for nine weeks. The study showed that arginine deficiency up-regulated interleukin 1, interleukin 8 and transforming growth factor-β and down-regulated tumour necrosis factor α gene expression (P < 0.05). Additionally, arginine deficiency increased malondialdehyde (MDA), protein carbonyl (PC) and glutathione contents and decreased the activities of copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) and glutathione-S-transferase (GST) (P < 0.05). Meanwhile, arginine deficiency significantly increased claudin 7, occludin, protein kinase C, NF-E2-related factor 2 and Kelch-like-ECH- associated protein 1 mRNA expression and decreased SOD1, CAT and GR mRNA expression (P < 0.05). All of these results indicated that arginine deficiency impaired intestinal immune function via the regulation of mRNA expression of cytokines, tight junction proteins, antioxidant enzymes, Nrf2/Keap1 and PKC in fish intestine. PMID:26518504

  6. Identification of protein-protein interactions between the TatB and TatC subunits of the twin-arginine translocase system and respiratory enzyme specific chaperones.

    PubMed

    Kuzniatsova, Lalita; Winstone, Tara M L; Turner, Raymond J

    2016-04-01

    The Twin-arginine translocation (Tat) pathway serves for translocation of fully folded proteins across the cytoplasmic membrane in bacterial and chloroplast thylakoid membranes. The Escherichia coli Tat system consists of three core components: TatA, TatB, and TatC. The TatB and TatC subunits form the receptor complex for Tat dependent proteins. The TatB protein is composed of a single transmembrane helix and cytoplasmic domain. The structure of TatC revealed six transmembrane helices. Redox Enzyme Maturation Proteins (REMPs) are system specific chaperones, which play roles in the maturation of Tat dependent respiratory enzymes. Here we applied the in vivo bacterial two-hybrid technique to investigate interaction of REMPs with the TatBC proteins, finding that all but the formate dehydrogenase REMP dock to TatB or TatC. We focused on the NarJ subfamily, where DmsD--the REMP for dimethyl sulfoxide reductase in E. coli--was previously shown to interact with TatB and TatC. We found that these REMPs interact with TatC cytoplasmic loops 1, 2 and 4, with the exception of NarJ, that only interacts with 1 and 4. An in vitro isothermal titration calorimetry study was applied to confirm the evidence of interactions between TatC fragments and DmsD chaperone. Using a peptide overlapping array, it was shown that the different NarJ subfamily REMPs interact with different regions of the TatB cytoplasmic domains. The results demonstrate a role of REMP chaperones in targeting respiratory enzymes to the Tat system. The data suggests that the different REMPs may have different mechanisms for this task. PMID:26826271

  7. A Cyclized Helix-Loop-Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein-Protein Interactions by Epitope and Arginine Grafting.

    PubMed

    Fujiwara, Daisuke; Kitada, Hidekazu; Oguri, Masahiro; Nishihara, Toshio; Michigami, Masataka; Shiraishi, Kazunori; Yuba, Eiji; Nakase, Ikuhiko; Im, Haeri; Cho, Sunhee; Joung, Jong Young; Kodama, Seiji; Kono, Kenji; Ham, Sihyun; Fujii, Ikuo

    2016-08-26

    The design of inhibitors of intracellular protein-protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop-helix (cHLH) peptide as a scaffold for generating cell-permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs. PMID:27467415

  8. Arginine-mediated stimulation of intestinal epithelial cell protein synthesis is mTOR-dependent but NO-independent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arginine (ARG) is an indispensable amino acid in neonates and required for growth. Evidence indicates that 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 di...

  9. Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

    SciTech Connect

    Soriano, Erika V.; McCloskey, Diane E.; Kinsland, Cynthia; Pegg, Anthony E.; Ealick, Steven E.

    2008-04-01

    The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.

  10. Frameshift events associated with the lysyl-tRNA and the rare arginine codon, AGA, in Escherichia coli: a case study involving the human Relaxin 2 protein.

    PubMed

    Kerrigan, John J; McNulty, Dean E; Burns, Matthew; Allen, Kimberly E; Tang, Xiaoyan; Lu, Quinn; Trulli, Janice M; Johanson, Kyung O; Kane, James F

    2008-08-01

    Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a -1 frameshift associated with 'slippery A' sequence XXA-AAA-AAG accounted for 10% of the product with a mass of 17,118 Da. Presumably, the shift to -1 also occurred because there was a paucity of the arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species. PMID:18474430