Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content.

PubMed

Xu, Jingyu; Francis, Tammy; Mietkiewska, Elzbieta; Giblin, E Michael; Barton, Dennis L; Zhang, Yan; Zhang, Meng; Taylor, David C

2008-10-01

A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

PPARγ regulates exocrine pancreas lipase.

PubMed

Danino, Hila; Naor, Ronny Peri-; Fogel, Chen; Ben-Harosh, Yael; Kadir, Rotem; Salem, Hagit; Birk, Ruth

2016-12-01

Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP. Copyright © 2016 Elsevier B.V. All rights reserved.

The DNA Maturation Domain of gpA, the DNA Packaging Motor Protein of Bacteriophage Lambda, Contains an ATPase Site Associated with Endonuclease Activity

PubMed Central

Ortega, Marcos E.; Gaussier, Helene; Catalano, Carlos E.

2007-01-01

Summary Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N-terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in E. coli. Biochemical characterization of gpA-ΔN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N-terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A “P-loop” sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme – DNA maturation and DNA packaging – are discussed. PMID:17870092

Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.

PubMed

Kim, Kye-Won; Smith, Clyde A; Daily, Michael D; Cort, John R; Davin, Laurence B; Lewis, Norman G

2015-01-16

Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (-)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis.

PubMed

Mathupala, S P; Lowe, S E; Podkovyrov, S M; Zeikus, J G

1993-08-05

The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch*

PubMed Central

Harvey, Beth M.; Rana, Nadia A.; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S.

2016-01-01

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

PubMed Central

Gunawardana, Dilantha; Cheng, Heung-Chin; Gayler, Kenwyn R.

2008-01-01

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. PMID:18025047

GATA-1 directly regulates Nanog in mouse embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wen-Zhong; Ai, Zhi-Ying; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100

2015-09-25

Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation.more » Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unterberger, Claudia; Hanson, Steven; Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 tomore » be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.« less

The Serum Response Factor and a Putative Novel Transcription Factor Regulate Expression of the Immediate-Early Gene Arc/Arg3.1 in Cultured Cortical Neurons

PubMed Central

Pintchovski, Sean A.; Peebles, Carol L.; Kim, Hong Joo; Verdin, Eric; Finkbeiner, Steven

2010-01-01

The immediate-early effector gene Arc/Arg3.1 is robustly upregulated by synaptic activity associated with learning and memory. Here we show in primary cortical neuron culture that diverse stimuli induce Arc expression through new transcription. Searching for regulatory regions important for Arc transcription, we found nine DNaseI-sensitive nucleosome-depleted sites at this genomic locus. A reporter gene encompassing these sites responded to synaptic activity in an NMDA receptor–dependent manner, consistent with endogenous Arc mRNA. Responsiveness mapped to two enhancer regions ∼6.5 kb and ∼1.4 kb upstream of Arc. We dissected these regions further and found that the proximal enhancer contains a functional and conserved “Zeste-like” response element that binds a putative novel nuclear protein in neurons. Therefore, activity regulates Arc transcription partly by a novel signaling pathway. We also found that the distal enhancer has a functional and highly conserved serum response element. This element binds serum response factor, which is recruited by synaptic activity to regulate Arc. Thus, Arc is the first target of serum response factor that functions at synapses to mediate plasticity. PMID:19193899

Differences in Transcriptional Activity of Human Papillomavirus Type 6 Molecular Variants in Recurrent Respiratory Papillomatosis

PubMed Central

Measso do Bonfim, Caroline; Simão Sobrinho, João; Lacerda Nogueira, Rodrigo; Salgado Kupper, Daniel; Cardoso Pereira Valera, Fabiana; Lacerda Nogueira, Maurício; Villa, Luisa Lina; Rahal, Paula; Sichero, Laura

2015-01-01

A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied. PMID:26151558

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

PubMed Central

Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

1980-01-01

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

Identification of residues that control Li+ versus Na+ dependent Ca2+ exchange at the transport site of the mitochondrial NCLX.

PubMed

Roy, Soumitra; Dey, Kuntal; Hershfinkel, Michal; Ohana, Ehud; Sekler, Israel

2017-06-01

The Na + /Ca 2+ /Li + exchanger (NCLX) is a member of the Na + /Ca 2+ exchanger family. NCLX is unique in its capacity to transport both Na + and Li + , unlike other members, which are Na + selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li + or Na + selective Ca 2+ transport and map their putative location on NCLX cation transport site. We combined molecular modeling to map transport site of NCLX with euryarchaeal H + /Ca 2+ exchanger, CAX_Af, and fluorescence analysis to monitor Li + versus Na + dependent mitochondrial Ca 2+ efflux of transport site mutants of NCLX in permeabilized cells. Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca 2+ exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li + /Ca 2+ exchange activity but a reduced Na + /Ca 2+ exchange activity. On the other hand, D471A showed dramatically reduced Li + /Ca 2+ exchange, but Na + /Ca 2+ exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca 2+ binding residues was required to completely abolish both Na + /Ca 2+ and Li + /Ca 2+ exchange activities. We identified distinct Na + and Li + selective residues in the NCLX transport site. We propose that functional segregation in Li + and Na + sites reflects the functional properties of NCLX required for Ca 2+ exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. The results of this study provide functional insights into the unique Li + and Na + selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

USDA-ARS?s Scientific Manuscript database

Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp...

Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details.

PubMed

Rosenhouse-Dantsker, Avia

2018-01-01

In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancer.

PubMed

Ceelie, H; Spaargaren-Van Riel, C C; De Jong, M; Bertina, R M; Vos, H L

2003-08-01

Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Analysis of the Mutations in the Active Site of the RNA-Dependent RNA Polymerase of Human Parainfluenza Virus Type 3 (HPIV3)

PubMed Central

Malur, Achut G.; Gupta, Neera K.; De, Bishnu P.; Banerjee, Amiya K.

2002-01-01

The large protein (L) of the human parainfluenza virus type 3 (HPIV3) is the functional RNA-dependent RNA polymerase, which possesses highly conserved residues QGDNQ located within motif C of domain III comprising the putative polymerase active site. We have characterized the role of the QGDNQ residues as well as the residues flanking this region in the polymerase activity of the L protein by site-directed mutagenesis and examining the polymerase activity of the wild-type and mutant L proteins by an in vivo minigenome replication assay and an in vitro mRNA transcription assay. All mutations in the QGDNQ residues abolished transcription while mutations in the flanking residues gave rise to variable polymerase activities. These observations support the contention that the QGDNQ sequence is absolutely required for the polymerase activity of the HPIV3 RNA-dependent RNA polymerase. PMID:12064576

Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product

PubMed Central

Jiménez, Lucía; Rodríguez-Lima, Oscar; Ochoa-Sánchez, Alicia; Landa, Abraham

2015-01-01

Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts. PMID:26090410

Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

PubMed

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-04-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Slack Sodium-activated Potassium Channel Membrane Expression Requires p38 Mitogen-Activated Protein Kinase Phosphorylation

PubMed Central

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-01-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Zhongliang; Liu, Yanfeng; Luo, Mengjun

Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt −85 to −11 in eBHBV1 Cp was critical formore » the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt −64 to −50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt −58 to −54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt −21 to −17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses. - Highlights: • Endogenous budgerigar hepadnavirus (eBHBV) core promoters (Cps) are active in cells. • NF-Y binding site exists in the Cps of eBHBVs and all the extant avihepadnaviruses. • NF-Y binding and mediated upregulation is critical for eBHBV Cp activity.« less

XET Activity is Found Near Sites of Growth and Cell Elongation in Bryophytes and Some Green Algae: New Insights into the Evolution of Primary Cell Wall Elongation

PubMed Central

Van Sandt, Vicky S. T.; Stieperaere, Herman; Guisez, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

2007-01-01

Background and Aims In angiosperms xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is involved in reorganization of the cell wall during growth and development. The location of oligo-xyloglucan transglucosylation activity and the presence of XTH expressed sequence tags (ESTs) in the earliest diverging extant plants, i.e. in bryophytes and algae, down to the Phaeophyta was examined. The results provide information on the presence of an XET growth mechanism in bryophytes and algae and contribute to the understanding of the evolution of cell wall elongation in general. Methods Representatives of the different plant lineages were pressed onto an XET test paper and assayed. XET or XET-related activity was visualized as the incorporation of fluorescent signal. The Physcomitrella genome database was screened for the presence of XTHs. In addition, using the 3′ RACE technique searches were made for the presence of possible XTH ESTs in the Charophyta. Key Results XET activity was found in the three major divisions of bryophytes at sites corresponding to growing regions. In the Physcomitrella genome two putative XTH-encoding cDNA sequences were identified that contain all domains crucial for XET activity. Furthermore, XET activity was located at the sites of growth in Chara (Charophyta) and Ulva (Chlorophyta) and a putative XTH ancestral enzyme in Chara was identified. No XET activity was identified in the Rhodophyta or Phaeophyta. Conclusions XET activity was shown to be present in all major groups of green plants. These data suggest that an XET-related growth mechanism originated before the evolutionary divergence of the Chlorobionta and open new insights in the evolution of the mechanisms of primary cell wall expansion. PMID:17098750

Characterization of a native hammerhead ribozyme derived from schistosomes

PubMed Central

OSBORNE, EDITH M.; SCHAAK, JANELL E.; DEROSE, VICTORIA J.

2005-01-01

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence. PMID:15659358

Putative Prostate Cancer Risk SNP in an Androgen Receptor‐Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

PubMed Central

Bu, Huajie; Narisu, Narisu; Schlick, Bettina; Rainer, Johannes; Manke, Thomas; Schäfer, Georg; Pasqualini, Lorenza; Chines, Peter; Schweiger, Michal R.; Fuchsberger, Christian

2015-01-01

ABSTRACT Genome‐wide association studies have identified genomic loci, whose single‐nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin‐immunoprecipitation‐coupled sequencing and microarray expression profiling in TMPRSS2‐ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor‐binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR‐binding motif, which is enriched in the neighborhood of canonical androgen‐responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor‐suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH. PMID:26411452

Human tRNA genes function as chromatin insulators

PubMed Central

Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

2012-01-01

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

Substrate channel in nitrogenase revealed by a molecular dynamics approach.

PubMed

Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C

2014-04-15

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle; Danyal, Karamatullah; Raugei, Simone

Mo-dependent nitrogenase catalyzes the biological reduction of N 2 to 2NH 3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N 2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel notmore » previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N 2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.« less

Evidence of the Presence of Thyroid Hormones in Achatina fulica Snails.

PubMed

Lustrino, Danilo; Silva, Alba C M; Araujo, Iracema G; Tunholi, Victor M; Tunholi-Alves, Vinícius M; Castro, Rosane N; Carvalho, Denise P; Pinheiro, Jairo; Marassi, Michelle P

2017-01-01

The objective of this study was to identify thyroid hormones and to examine their putative site of synthesis in Achatina fulica snails. For this purpose, radioimmunoassays were performed for T3 and T4 before and after long starvation with or without hemolymph deproteinization. Sodium/iodide symporter activity in vivo was analyzed through 125I administration with and without KClO4 pretreatment. Only T4 was detected, and its concentration decreased due to starvation or deproteinization. However, high-performance liquid chromatography analysis also showed the presence of T2 and T3 apart from T4, but rT3 was not detected in the A. fulica hemolymph. The sodium/iodide symporter activity was greater in cerebral ganglia than digestive gland, but KClO4 treatment did not inhibit iodide uptake in any of the tissues analyzed. Altogether, our data confirm for the first time the presence of thyroid hormones in A. fulica snails and suggest their participation in the metabolism control in this species, although the putative site of hormone biosynthesis remains to be elucidated.

Complete genome sequence of “Enterobacter lignolyticus” SCF1

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeAngelis, Kristen M.; D'Haeseleer, Patrik; Chivian, Dylan

2011-09-23

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated 'Ente-robacter lignolyticus' SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anae-robically, we sequenced the genome. The genome of 'E. lignolyticus' SCF1 is 4.81 Mbpmore » with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohy-drate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degra-dation pathway encoded in a single gene cluster.« less

The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor.

PubMed

Godlewska, Renata; Bujnicki, Janusz M; Ostrowski, Jerzy; Jagusztyn-Krynicka, Elzbieta K

2002-08-14

Screening of the Helicobacter pylori genomic library with sera from infected humans and from immunized rabbits resulted in identification of the 25 kDa protein cell envelope (HppA) which exhibits acid phosphatase activity. Enzyme activity was demonstrated by specific enzymatic assays with whole-cell protein preparations of H. pylori strain N6 and from Escherichia coli carrying the hppA gene (pUWM192). HppA showed optimum activity at pH 5.6 and was resistant to inhibition by EDTA. Bioinformatics analysis and site-directed mutagenesis of two putative active site residues (D73 and D192) provide further insight into the sequence-structure-function relationships of HppA as a member of the DDDD phosphohydrolase superfamily.

Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.

PubMed

Blackler, Ryan J; Gagnon, Susannah M L; Polakowski, Robert; Rose, Natisha L; Zheng, Ruixiang B; Letts, James A; Johal, Asha R; Schuman, Brock; Borisova, Svetlana N; Palcic, Monica M; Evans, Stephen V

2017-04-01

The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

PubMed

Varmanen, P; Rantanen, T; Palva, A

1996-12-01

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides.

PubMed

Bowen, Anthony; Wear, Maggie P; Cordero, Radames J B; Oscarson, Stefan; Casadevall, Arturo

2017-01-13

Studies in the 1980s first showed that some natural antibodies were "catalytic" and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides*

PubMed Central

Wear, Maggie P.; Cordero, Radames J. B.; Oscarson, Stefan

2017-01-01

Studies in the 1980s first showed that some natural antibodies were “catalytic” and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. PMID:27872188

Identification of Anaerobic Aniline-Degrading Bacteria at a Contaminated Industrial Site.

PubMed

Sun, Weimin; Li, Yun; McGuinness, Lora R; Luo, Shuai; Huang, Weilin; Kerkhof, Lee J; Mack, E Erin; Häggblom, Max M; Fennell, Donna E

2015-09-15

Anaerobic aniline biodegradation was investigated under different electron-accepting conditions using contaminated canal and groundwater aquifer sediments from an industrial site. Aniline loss was observed in nitrate- and sulfate-amended microcosms and in microcosms established to promote methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100 days (methanogenic) were observed prior to activity. Time-series DNA-stable isotope probing (SIP) was used to identify bacteria that incorporated (13)C-labeled aniline in the microcosms established to promote methanogenic conditions. In microcosms from heavily contaminated aquifer sediments, a phylotype with 92.7% sequence similarity to Ignavibacterium album was identified as a dominant aniline degrader as indicated by incorporation of (13)C-aniline into its DNA. In microcosms from contaminated canal sediments, a bacterial phylotype within the family Anaerolineaceae, but without a match to any known genus, demonstrated the assimilation of (13)C-aniline. Acidovorax spp. were also identified as putative aniline degraders in both of these two treatments, indicating that these species were present and active in both the canal and aquifer sediments. There were multiple bacterial phylotypes associated with anaerobic degradation of aniline at this complex industrial site, which suggests that anaerobic transformation of aniline is an important process at the site. Furthermore, the aniline degrading phylotypes identified in the current study are not related to any known aniline-degrading bacteria. The identification of novel putative aniline degraders expands current knowledge regarding the potential fate of aniline under anaerobic conditions.

A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan

2012-05-24

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS aremore » tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.« less

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

PubMed

Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

1995-07-03

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein

PubMed Central

Dilley, David R.; Wang, Zhenyong; Kadirjan-Kalbach, Deena K.; Ververidis, Fillipos; Beaudry, Randolph; Padmanabhan, Kallaithe

2013-01-01

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A ‘nest’ comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein–protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner. PMID:24244837

An extension of hypotheses regarding rapid-acting, treatment-refractory, and conventional antidepressant activity of dextromethorphan and dextrorphan.

PubMed

Lauterbach, Edward C

2012-06-01

It was previously hypothesized that dextromethorphan (DM) and dextrorphan (DX) may possess antidepressant properties, including rapid and conventional onsets of action and utility in treatment-refractory depression, based on pharmacodynamic similarities to ketamine. These similarities included sigma-1 (σ(1)) agonist and NMDA antagonist properties, calcium channel blockade, muscarinic binding, serotonin transporter (5HTT) inhibition, and μ receptor potentiation. Here, six specific hypotheses are developed in light of additional mechanisms and evidence. Comparable potencies to ketamine for DM and DX are detailed for σ(1) (DX>DM>ketamine), NMDA PCP site (DX>ketamine>DM), and muscarinic (DX>ketamine>DM) receptors, 5HTT (DM>DX≫ketamine), and NMDA antagonist potentiation of μ receptor stimulation (DM>ketamine). Rapid acting antidepressant properties of DM include NMDA high-affinity site, NMDR-2A, and functional NMDR-2B receptor antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation, μ potentiation, and 5HTT inhibition), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, μ potentiation, and 5HTT inhibition), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Those for dextrorphan include NMDA high-affinity site and NMDR-2A antagonism, σ(1) stimulation, putative mTOR activation (by σ(1) stimulation and ß adrenoreceptor stimulation), putative AMPA receptor trafficking (by mTOR activation, PCP antagonism, σ(1) stimulation, ß stimulation, and μ antagonism), and dendritogenesis, spinogenesis, synaptogenesis, and neuronal survival by NMDA antagonism and σ(1) and mTOR signaling. Conventional antidepressant properties for dextromethorphan and dextrorphan include 5HTT and norepinephrine transporter inhibition, σ(1) stimulation, NMDA and PCP antagonism, and possible serotonin 5HT1b/d receptor stimulation. Additional properties for dextromethorphan include possible presynaptic α(2) adrenoreceptor antagonism or postsynaptic α(2) stimulation and, for dextrorphan, ß stimulation and possible muscarinic and μ antagonism. Treatment-refractory depression properties include increased serotonin and norepinephrine availability, PCP, NMDR-2B, presynaptic alpha-2 antagonism, and the multiplicity of other antidepressant receptor mechanisms. Suggestions for clinical trials are provided for oral high-dose dextromethorphan and Nuedexta (dextromethorphan combined with quinidine to block metabolism to dextrorphan, thereby increasing dextromethorphan plasma concentrations). Suggestions include exclusionary criteria, oral dosing, observation periods, dose-response approaches, and safety and tolerability are considered. Although oral dextromethorphan may be somewhat more likely to show efficacy through complementary antidepressant mechanisms of dextrorphan, a clinical trial will be more logistically complex than one of Nuedexta due to high doses and plasma level variability. Clinical trials may increase our therapeutic armamentarium and our pharmacological understanding of treatment-refractory depression and antidepressant onset of action. Copyright © 2012 Elsevier Ltd. All rights reserved.

Functional overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site.

PubMed

Giger, Julia M; Haddad, Fadia; Qin, Anqi X; Baldwin, Kenneth M

2002-03-01

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, beta-MHC isoform. Different length rat beta-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the betae1, distal muscle CAT (MCAT; betae2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (betae3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the betae3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the betae3 site functions as a putative OL-responsive element in the rat beta-MHC gene promoter.

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

PubMed Central

Ellestad, Laura E.

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland. PMID:23161868

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

PubMed

Ellestad, Laura E; Porter, Tom E

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

PubMed

Hamady, Zaed Z R; Farrar, Mark D; Whitehead, Terence R; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

2008-10-01

The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

The crystal structure of Rv1347c, a putative antibiotic resistance protein from Mycobacterium tuberculosis, reveals a GCN5-related fold and suggests an alternative function in siderophore biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Card, G L; Peterson, N A; Smith, C A

2005-02-15

Mycobacterium tuberculosis, the cause of TB, is a devastating human pathogen. The emergence of multi-drug resistance in recent years has prompted a search for new drug targets and for a better understanding of mechanisms of resistance. Here we focus on the gene product of an open reading frame from M. tuberculosis, Rv1347c, which is annotated as a putative aminoglycoside N-acetyltransferase. The Rv1347c protein does not show this activity, however, and we show from its crystal structure, coupled with functional and bioinformatic data, that its most likely role is in the biosynthesis of mycobactin, the M. tuberculosis siderophore. The crystal structuremore » of Rv1347c was determined by MAD phasing from selenomethionine-substituted protein and refined at 2.2 {angstrom} resolution (R = 0.227, R{sub free} = 0.257). The protein is monomeric, with a fold that places it in the GCN5-related N-acetyltransferase (GNAT) family of acyltransferases. Features of the structure are an acylCoA binding site that is shared with other GNAT family members, and an adjacent hydrophobic channel leading to the surface that could accommodate long-chain acyl groups. Modeling the postulated substrate, the N{sup {var_epsilon}}-hydroxylysine side chain of mycobactin, into the acceptor substrate binding groove identifies two residues at the active site, His130 and Asp168, that have putative roles in substrate binding and catalysis.« less

Structural analysis of the 5{prime} region of mouse and human Huntington disease genes reveals conservation of putative promoter region and Di- and trinucleotide polymorphisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Biaoyang; Nasir, J.; Kalchman, M.A.

1995-02-10

We have previously cloned and characterized the murine homologue of the Huntington disease (HD) gene and shown that it maps to mouse chromosome 5 within a region of conserved synteny with human chromosome 4p16.3. Here we present a detailed comparison of the sequence of the putative promoter and the organization of the 5{prime} genomic region of the murine (Hdh) and human HD genes encompassing the first five exons. We show that in this region these two genes share identical exon boundaries, but have different-size introns. Two dinucleotide (CT) and one trinucleotide intronic polymorphism in Hdh and an intronic CA polymorphismmore » in the HD gene were identified. Comparison of 940-bp sequence 5{prime} to the putative translation start site reveals a highly conserved region (78.8% nucleotide identity) between Hdh and the HD gene from nucleotide -56 to -206 (of Hdh). Neither Hdh nor the HD gene have typical TATA or CCAAT elements, but both show one putative AP2 binding site and numerous potential Sp1 binding sites. The high sequence identity between Hdh and the HD gene for approximately 200 bp 5{prime} to the putative translation start site indicates that these sequences may play a role in regulating expression of the Huntington disease gene. 30 refs., 4 figs., 2 tabs.« less

The sigma factor SigD of Mycobacterium tuberculosis putatively enhances gene expression of the septum site determining protein under stressful environments.

PubMed

Ares, Miguel A; Rios-Sarabia, Nora; De la Cruz, Miguel A; Rivera-Gutiérrez, Sandra; García-Morales, Lázaro; León-Solís, Lizbel; Espitia, Clara; Pacheco, Sabino; Cerna-Cortés, Jorge F; Helguera-Repetto, Cecilia A; García, María Jesús; González-Y-Merchand, Jorge A

2017-07-01

This work examined the expression of the septum site determining gene (ssd) of Mycobacterium tuberculosis CDC1551 and its ∆sigD mutant under different growing conditions. The results showed an up-regulation of ssd during stationary phase and starvation conditions, but not during in vitro dormancy, suggesting a putative role for SigD in the control of ssd expression mainly under lack-of-nutrients environments. Furthermore, we elucidated a putative link between ssd expression and cell elongation of bacilli at stationary phase. In addition, a -35 sigD consensus sequence was found for the ssd promoter region, reinforcing the putative regulation of ssd by SigD, and in turn, supporting this protein role during the adaptation of M. tuberculosis to some stressful environments.

Gene encoding γ-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7

PubMed Central

2010-01-01

Background Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one β-CA and two γ-CAs. Results One of the putative γ-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-γ-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. Conclusions This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized γ-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration. PMID:20598158

Gene encoding gamma-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7.

PubMed

Kaur, Simarjot; Mishra, Mukti N; Tripathi, Anil K

2010-07-04

Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one beta-CA and two gamma-CAs. One of the putative gamma-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

Solution NMR Structures of Pyrenophora tritici-repentis ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity*

PubMed Central

Nyarko, Afua; Singarapu, Kiran K.; Figueroa, Melania; Manning, Viola A.; Pandelova, Iovanna; Wolpert, Thomas J.; Ciuffetti, Lynda M.; Barbar, Elisar

2014-01-01

Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a β-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent β-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin. PMID:25063993

The Evolving Field of Biodefence: Therapeutic Developments and Diagnostics

DTIC Science & Technology

2005-04-01

several ways. One method would be to interfere with the furin -medi- ated cleavage of PA to its active form (PA 63 ) following host-cell receptor binding4...b | The inactive form of protective antigen (PA83) binds to a host-cell receptor, where it is cleaved by a furin -related protease, to give active PA63...explore whether a putative target, such as furin cleavage site of Ebola virus, is essential for viral infection88. Compared with filoviruses, poxvirus

Effectiveness of scat detection dogs for detecting forest carnivores

USGS Publications Warehouse

Long, Robert A.; Donovan, T.M.; MacKay, Paula; Zielinski, William J.; Buzas, Jeffrey S.

2007-01-01

We assessed the detection and accuracy rates of detection dogs trained to locate scats from free-ranging black bears (Ursus americanus), fishers (Martes pennanti), and bobcats (Lynx rufus). During the summers of 2003-2004, 5 detection teams located 1,565 scats (747 putative black bear, 665 putative fisher, and 153 putative bobcat) at 168 survey sites throughout Vermont, USA. Of 347 scats genetically analyzed for species identification, 179 (51.6%) yielded a positive identification, 131 (37.8%) failed to yield DNA information, and 37 (10.7%) yielded DNA but provided no species confirmation. For 70 survey sites where confirmation of a putative target species' scat was not possible, we assessed the probability that ???1 of the scats collected at the site was deposited by the target species (probability of correct identification; P ID). Based on species confirmations or PID values, we detected bears at 57.1% (96) of sites, fishers at 61.3% (103) of sites, and bobcats at 12.5%o (21) of sites. We estimated that the mean probability of detecting the target species (when present) during a single visit to a site was 0.86 for black bears, 0.95 for fishers, and 0.40 for bobcats. The probability of detecting black bears was largely unaffected by site- or visit-specific covariates, but the probability of detecting fishers varied by detection team. We found little or no effect of topographic ruggedness, vegetation density, or local weather (e.g., temp, humidity) on detection probability for fishers or black bears (data were insufficient for bobcat analyses). Detection dogs were highly effective at locating scats from forest carnivores and provided an efficient and accurate method for collecting detection-nondetection data on multiple species.

Searching for putative binding sites of the bispyridinium compound MB327 in the nicotinic acetylcholine receptor.

PubMed

Wein, Thomas; Höfner, Georg; Rappenglück, Sebastian; Sichler, Sonja; Niessen, Karin V; Seeger, Thomas; Worek, Franz; Thiermann, Horst; Wanner, Klaus T

2018-09-01

Irreversible inhibition of the acetylcholine esterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. The bispyridinium compound MB327 has been found to restore functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. In order to optimize the resensitizing effect of MB327 on nAChR, it would be very helpful to know the MB327 specific binding site to apply structure based molecular modeling. The binding site for MB327 at the nAChR is not known and so far goal of speculations, but it has been shown that MB327 does not bind to the orthosteric acetylcholine binding site. We have used docking calculations to screen the surface of nAChR for possible binding sites of MB327. The results indicate that at least two potential binding sites for MB327 at nAChR are present inside the channel pore. In these binding sites, MB327 intercalates between the γ-α and β-δ subunits of nAChR, respectively. Both putative MB327 binding sites show an unsymmetrical distribution of surrounding hydrophilic and lipophilic amino acids. This suggests that substitution of MB327-related bispyridinium compounds on one of the two pyridinium rings with polar substituents should have a favorable effect on the pharmacological function. Copyright © 2017 Elsevier B.V. All rights reserved.

Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

PubMed

Miner, Kyle D; Kurtz, Donald M

2016-02-16

HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases.

PubMed

Volbeda, Anne; Martin, Lydie; Cavazza, Christine; Matho, Michaël; Faber, Bart W; Roseboom, Winfried; Albracht, Simon P J; Garcin, Elsa; Rousset, Marc; Fontecilla-Camps, Juan C

2005-05-01

[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we present the crystal structure of the ready Ni-B state of Desulfovibrio fructosovorans [NiFe] hydrogenase and show it to have a putative mu-hydroxo Ni-Fe bridging ligand at the active site. On the other hand, a new, improved refinement procedure of the X-ray diffraction data obtained for putative unready Ni-A/Ni-SU states resulted in a more elongated electron density for the bridging ligand, suggesting that it is a diatomic species. The slow activation of the Ni-A state, compared with the rapid activation of the Ni-B state, is therefore proposed to result from the different chemical nature of the ligands in the two oxidized species. Our results along with very recent electrochemical studies suggest that the diatomic ligand could be hydro-peroxide.

Effects of drugs of abuse on putative rostromedial tegmental neurons, inhibitory afferents to midbrain dopamine cells.

PubMed

Lecca, Salvatore; Melis, Miriam; Luchicchi, Antonio; Ennas, Maria Grazia; Castelli, Maria Paola; Muntoni, Anna Lisa; Pistis, Marco

2011-02-01

Recent findings have underlined the rostromedial tegmental nucleus (RMTg), a structure located caudally to the ventral tegmental area, as an important site involved in the mechanisms of aversion. RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding midbrain dopamine (DA) neurons. One of the key features of drug addiction is the perseverance of drug seeking in spite of negative and unpleasant consequences, likely mediated by response suppression within neural pathways mediating aversion. To investigate whether the RMTg has a function in the mechanisms of addicting drugs, we studied acute effects of morphine, cocaine, the cannabinoid agonist WIN55212-2 (WIN), and nicotine on putative RMTg neurons. We utilized single unit extracellular recordings in anesthetized rats and whole-cell patch-clamp recordings in brain slices to identify and characterize putative RMTg neurons and their responses to drugs of abuse. Morphine and WIN inhibited both firing rate in vivo and excitatory postsynaptic currents (EPSCs) evoked by stimulation of rostral afferents in vitro, whereas cocaine inhibited discharge activity without affecting EPSC amplitude. Conversely, nicotine robustly excited putative RMTg neurons and enhanced EPSCs, an effect mediated by α7-containing nicotinic acetylcholine receptors. Our results suggest that activity of RMTg neurons is profoundly influenced by drugs of abuse and, as important inhibitory afferents to midbrain DA neurons, they might take place in the complex interplay between the neural circuits mediating aversion and reward.

/sup 3/H)pirenzepine and (-)-(/sup 3/H)quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. I. Characterization and regulation of agonist binding to putative muscarinic subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, M.; Yamamura, H.I.; Roeske, W.R.

The binding and regulation of selected muscarinic agonists to putative subtypes in rat cerebral cortex and heart were studied. Parallel inhibition studies of (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and (-)-(/sup 3/H)quinuclidinylbenzilate ((-)-(/sup 3/H)QNB)-labeled membranes were done with and without 30 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) at 25 degrees C in 10 mM Na-K-phosphate buffer which enhances PZ binding affinity and in modified Krebs-phosphate buffer, which mimics physiological conditions. Classical agonists such as carbachol, oxotremorine and acetylcholine inhibited (-)-(/sup 3/H)QNB binding to membranes with shallow Hill values (nH less than 1), were better fit to a 2-state model, were Gpp(NH)p-regulated and showed lowermore » affinity in modified Krebs-phosphate buffer than in 10 mM Na-K-phosphate buffer. Some agonists were not significantly better fit to a 2-state model in (/sup 3/H)PZ-labeled cortical membranes, especially in 10 mM Na-K-phosphate buffer. Whereas putative M1 and M2 binding sites distinguished by PZ possessed multiple agonist affinity states, as judged by carbachol, and agonist binding to (/sup 3/H)PZ-labeled sites were Gpp(NH)p modulated, the partial agonist pilocarpine and nonclassical agonist McN-A-343 (3-(m-chlorophenylcarbamoyloxy)-2-butynyl trimethylammonium chloride) showed little Gpp(NH)p-induced shift in (/sup 3/H)PZ-labeled cortical membranes in physiological conditions. Agonist binding to (-)-(/sup 3/H)QNB-labeled putative M2 cardiac sites was more sensitive to Gpp(NH)p than (-)-(/sup 3/H)QNB-labeled cortical sites. Carbachol and acetylcholine showed significant selectivity for putative M2 sites.« less

The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum

DOE PAGES

Choi, Jinlyung; Klingeman, Dawn M.; Brown, Steven D.; ...

2017-06-24

In this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivomore » transcription of the celC–glyR3–licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription.« less

Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarilymore » at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.« less

Proteomic identification of an embryo-specific 1Cys-Prx promoter and analysis of its activity in transgenic rice.

PubMed

Kim, Je Hein; Jung, In Jung; Kim, Dool Yi; Fanata, Wahyu Indra; Son, Bo Hwa; Yoo, Jae Yong; Harmoko, Rikno; Ko, Ki Seong; Moon, Jeong Chan; Jang, Ho Hee; Kim, Woe Yeon; Kim, Jae-Yean; Lim, Chae Oh; Lee, Sang Yeol; Lee, Kyun Oh

2011-04-29

Proteomic analysis of a rice callus led to the identification of 10 abscisic acid (ABA)-induced proteins as putative products of the embryo-specific promoter candidates. 5'-flanking sequence of 1 Cys-Prx, a highly-induced protein gene, was cloned and analyzed. The transcription initiation site of 1 Cys-Prx maps 96 nucleotides upstream of the translation initiation codon and a TATA-box and putative seed-specific cis-acting elements, RYE and ABRE, are located 26, 115 and 124 bp upstream of the transcription site, respectively. β-glucuronidase (GUS) expression driven by the 1 Cys-Prx promoters was strong in the embryo and aleurone layer and the activity reached up to 24.9 ± 3.3 and 40.5 ± 2.1 pmol (4 MU/min/μg protein) in transgenic rice seeds and calluses, respectively. The activity of the 1 Cys-Prx promoters is much higher than that of the previously-identified embryo-specific promoters, and comparable to that of strong endosperm-specific promoters in rice. GUS expression driven by the 1 Cys-Prx promoters has been increased by ABA treatment and rapidly induced by wounding in callus and at the leaf of the transgenic plants, respectively. Furthermore, ectopic expression of the GUS construct in Arabidopsis suggested that the 1 Cys-Prx promoter also has strong activity in seeds of dicot plants. Copyright © 2011 Elsevier Inc. All rights reserved.

Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) of Ureaplasma parvum serovar 3 with specific DNA

PubMed Central

Zimmerman, Carl-Ulrich R; Rosengarten, Renate; Spergser, Joachim

2013-01-01

Phase variation of two loci (‘mba locus’ and ‘UU172 phase-variable element’) in Ureaplasma parvum serovar 3 has been suggested as result of site-specific DNA inversion occurring at short inverted repeats. Three potential tyrosine recombinases (RipX, XerC, and CodV encoded by the genes UU145, UU222, and UU529) have been annotated in the genome of U. parvum serovar 3, which could be mediators in the proposed recombination event. We document that only orthologs of the gene xerC are present in all strains that show phase variation in the two loci. We demonstrate in vitro binding of recombinant maltose-binding protein fusions of XerC to the inverted repeats of the phase-variable loci, of RipX to a direct repeat that flanks a 20-kbp region, which has been proposed as putative pathogenicity island, and of CodV to a putative dif site. Co-transformation of the model organism Mycoplasma pneumoniae M129 with both the ‘mba locus’ and the recombinase gene xerC behind an active promoter region resulted in DNA inversion in the ‘mba locus’. Results suggest that XerC of U. parvum serovar 3 is a mediator in the proposed DNA inversion event of the two phase-variable loci. PMID:23305333

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

PubMed

Lobanova, Juliya S; Gak, Evgueni R; Andreeva, Irina G; Rybak, Konstantin V; Krylov, Alexander A; Mashko, Sergey V

2017-08-01

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1» programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

SITEHOUND-web: a server for ligand binding site identification in protein structures.

PubMed

Hernandez, Marylens; Ghersi, Dario; Sanchez, Roberto

2009-07-01

SITEHOUND-web (http://sitehound.sanchezlab.org) is a binding-site identification server powered by the SITEHOUND program. Given a protein structure in PDB format SITEHOUND-web will identify regions of the protein characterized by favorable interactions with a probe molecule. These regions correspond to putative ligand binding sites. Depending on the probe used in the calculation, sites with preference for different ligands will be identified. Currently, a carbon probe for identification of binding sites for drug-like molecules, and a phosphate probe for phosphorylated ligands (ATP, phoshopeptides, etc.) have been implemented. SITEHOUND-web will display the results in HTML pages including an interactive 3D representation of the protein structure and the putative sites using the Jmol java applet. Various downloadable data files are also provided for offline data analysis.

Bioinformatic prediction of gene functions regulated by quorum sensing in the bioleaching bacterium Acidithiobacillus ferrooxidans.

PubMed

Banderas, Alvaro; Guiliani, Nicolas

2013-08-16

The biomining bacterium Acidithiobacillus ferrooxidans oxidizes sulfide ores and promotes metal solubilization. The efficiency of this process depends on the attachment of cells to surfaces, a process regulated by quorum sensing (QS) cell-to-cell signalling in many Gram-negative bacteria. At. ferrooxidans has a functional QS system and the presence of AHLs enhances its attachment to pyrite. However, direct targets of the QS transcription factor AfeR remain unknown. In this study, a bioinformatic approach was used to infer possible AfeR direct targets based on the particular palindromic features of the AfeR binding site. A set of Hidden Markov Models designed to maintain palindromic regions and vary non-palindromic regions was used to screen for putative binding sites. By annotating the context of each predicted binding site (PBS), we classified them according to their positional coherence relative to other putative genomic structures such as start codons, RNA polymerase promoter elements and intergenic regions. We further used the Multiple EM for Motif Elicitation algorithm (MEME) to further filter out low homology PBSs. In summary, 75 target-genes were identified, 34 of which have a higher confidence level. Among the identified genes, we found afeR itself, zwf, genes encoding glycosyltransferase activities, metallo-beta lactamases, and active transport-related proteins. Glycosyltransferases and Zwf (Glucose 6-phosphate-1-dehydrogenase) might be directly involved in polysaccharide biosynthesis and attachment to minerals by At. ferrooxidans cells during the bioleaching process.

Bioinformatic Prediction of Gene Functions Regulated by Quorum Sensing in the Bioleaching Bacterium Acidithiobacillus ferrooxidans

PubMed Central

Banderas, Alvaro; Guiliani, Nicolas

2013-01-01

The biomining bacterium Acidithiobacillus ferrooxidans oxidizes sulfide ores and promotes metal solubilization. The efficiency of this process depends on the attachment of cells to surfaces, a process regulated by quorum sensing (QS) cell-to-cell signalling in many Gram-negative bacteria. At. ferrooxidans has a functional QS system and the presence of AHLs enhances its attachment to pyrite. However, direct targets of the QS transcription factor AfeR remain unknown. In this study, a bioinformatic approach was used to infer possible AfeR direct targets based on the particular palindromic features of the AfeR binding site. A set of Hidden Markov Models designed to maintain palindromic regions and vary non-palindromic regions was used to screen for putative binding sites. By annotating the context of each predicted binding site (PBS), we classified them according to their positional coherence relative to other putative genomic structures such as start codons, RNA polymerase promoter elements and intergenic regions. We further used the Multiple EM for Motif Elicitation algorithm (MEME) to further filter out low homology PBSs. In summary, 75 target-genes were identified, 34 of which have a higher confidence level. Among the identified genes, we found afeR itself, zwf, genes encoding glycosyltransferase activities, metallo-beta lactamases, and active transport-related proteins. Glycosyltransferases and Zwf (Glucose 6-phosphate-1-dehydrogenase) might be directly involved in polysaccharide biosynthesis and attachment to minerals by At. ferrooxidans cells during the bioleaching process. PMID:23959118

Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de

1994-04-01

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-pointmore » sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.« less

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments[OPEN

PubMed Central

Hafemeister, Christoph; Nicotra, Adrienne B.; Jagadish, S.V. Krishna; Bonneau, Richard; Purugganan, Michael

2016-01-01

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference. PMID:27655842

Cloning and characterization of microbial activated Aedes aegypti MEK4 (AaMEK4): influences of noncatalytic domains on enzymatic activity.

PubMed

Wu, R C-C; Cho, W-L

2014-10-01

Protein kinases are known to be involved in a number of signal transduction cascades. Both the stress-activated Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) p38 pathways have been shown to correlate with the insect immune response to microbial infection. MAP kinase kinase 4 (MEK4) is an upstream kinase of JNK and p38 kinase. The cDNA of AaMEK4 was cloned and characterized. AaMEK4 was activated by microbial lysates of Gram-positive, Gram-negative bacteria and yeast. The conserved lysine (K112 ) and the putative phosphorylation sites (S238 and T242 ) were shown to be important for kinase activity by site-directed mutagenesis. A common MAPK docking site (MAPK_dsA) was found and in addition, a new nearby docking site, MAPK_dsB, was identified in the N-terminal noncatalytic domain of AaMEK4. MAPK_dsB was shown to be a unique element in the MEK4 family. In this study, both MAPK_dsA and _dsB were demonstrated to be important to AaMEK4 enzymatic activity for the downstream protein kinase, Aap38. © 2014 The Royal Entomological Society.

Transcriptional analysis of the bglP gene from Streptococcus mutans.

PubMed

Cote, Christopher K; Honeyman, Allen L

2006-04-21

An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript.

Transcriptional analysis of the bglP gene from Streptococcus mutans

PubMed Central

Cote, Christopher K; Honeyman, Allen L

2006-01-01

Background An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. Results To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. Conclusion The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript. PMID:16630357

Identification and Characterization of Sites Where Persistent Atrial Fibrillation Is Terminated by Localized Ablation.

PubMed

Zaman, Junaid A B; Sauer, William H; Alhusseini, Mahmood I; Baykaner, Tina; Borne, Ryan T; Kowalewski, Christopher A B; Busch, Sonia; Zei, Paul C; Park, Shirley; Viswanathan, Mohan N; Wang, Paul J; Brachmann, Johannes; Krummen, David E; Miller, John M; Rappel, Wouter Jan; Narayan, Sanjiv M; Peters, Nicholas S

2018-01-01

The mechanisms by which persistent atrial fibrillation (AF) terminates via localized ablation are not well understood. To address the hypothesis that sites where localized ablation terminates persistent AF have characteristics identifiable with activation mapping during AF, we systematically examined activation patterns acquired only in cases of unequivocal termination by ablation. We recruited 57 patients with persistent AF undergoing ablation, in whom localized ablation terminated AF to sinus rhythm or organized tachycardia. For each site, we performed an offline analysis of unprocessed unipolar electrograms collected during AF from multipolar basket catheters using the maximum -dV/dt assignment to construct isochronal activation maps for multiple cycles. Additional computational modeling and phase analysis were used to study mechanisms of map variability. At all sites of AF termination, localized repetitive activation patterns were observed. Partial rotational circuits were observed in 26 of 57 (46%) cases, focal patterns in 19 of 57 (33%), and complete rotational activity in 12 of 57 (21%) cases. In computer simulations, incomplete segments of partial rotations coincided with areas of slow conduction characterized by complex, multicomponent electrograms, and variations in assigning activation times at such sites substantially altered mapped mechanisms. Local activation mapping at sites of termination of persistent AF showed repetitive patterns of rotational or focal activity. In computer simulations, complete rotational activation sequence was observed but was sensitive to assignment of activation timing particularly in segments of slow conduction. The observed phenomena of repetitive localized activation and the mechanism by which local ablation terminates putative AF drivers require further investigation. © 2018 American Heart Association, Inc.

Driving Protein Conformational Changes with Light: Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase.

PubMed

Maugeri, Pearson T; Griese, Julia J; Branca, Rui M; Miller, Effie K; Smith, Zachary R; Eirich, Jürgen; Högbom, Martin; Shafaat, Hannah S

2018-01-31

The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine cross-link near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Φ = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligand-to-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting nomore » conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.« less

Identification of regulatory targets for the bacterial Nus factor complex.

PubMed

Baniulyte, Gabriele; Singh, Navjot; Benoit, Courtney; Johnson, Richard; Ferguson, Robert; Paramo, Mauricio; Stringer, Anne M; Scott, Ashley; Lapierre, Pascal; Wade, Joseph T

2017-12-11

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Excavation and aggregation as organizing factors in de novo construction by mound-building termites.

PubMed

Green, Ben; Bardunias, Paul; Turner, J Scott; Nagpal, Radhika; Werfel, Justin

2017-06-14

Termites construct complex mounds that are orders of magnitude larger than any individual and fulfil a variety of functional roles. Yet the processes through which these mounds are built, and by which the insects organize their efforts, remain poorly understood. The traditional understanding focuses on stigmergy, a form of indirect communication in which actions that change the environment provide cues that influence future work. Termite construction has long been thought to be organized via a putative 'cement pheromone': a chemical added to deposited soil that stimulates further deposition in the same area, thus creating a positive feedback loop whereby coherent structures are built up. To investigate the detailed mechanisms and behaviours through which termites self-organize the early stages of mound construction, we tracked the motion and behaviour of major workers from two Macrotermes species in experimental arenas. Rather than a construction process focused on accumulation of depositions, as models based on cement pheromone would suggest, our results indicated that the primary organizing mechanisms were based on excavation. Digging activity was focused on a small number of excavation sites, which in turn provided templates for soil deposition. This behaviour was mediated by a mechanism of aggregation, with termites being more likely to join in the work at an excavation site as the number of termites presently working at that site increased. Statistical analyses showed that this aggregation mechanism was a response to active digging, distinct from and unrelated to putative chemical cues that stimulate deposition. Agent-based simulations quantitatively supported the interpretation that the early stage of de novo construction is primarily organized by excavation and aggregation activity rather than by stigmergic deposition. © 2017 The Author(s).

Excavation and aggregation as organizing factors in de novo construction by mound-building termites

PubMed Central

Bardunias, Paul; Turner, J. Scott; Nagpal, Radhika; Werfel, Justin

2017-01-01

Termites construct complex mounds that are orders of magnitude larger than any individual and fulfil a variety of functional roles. Yet the processes through which these mounds are built, and by which the insects organize their efforts, remain poorly understood. The traditional understanding focuses on stigmergy, a form of indirect communication in which actions that change the environment provide cues that influence future work. Termite construction has long been thought to be organized via a putative ‘cement pheromone’: a chemical added to deposited soil that stimulates further deposition in the same area, thus creating a positive feedback loop whereby coherent structures are built up. To investigate the detailed mechanisms and behaviours through which termites self-organize the early stages of mound construction, we tracked the motion and behaviour of major workers from two Macrotermes species in experimental arenas. Rather than a construction process focused on accumulation of depositions, as models based on cement pheromone would suggest, our results indicated that the primary organizing mechanisms were based on excavation. Digging activity was focused on a small number of excavation sites, which in turn provided templates for soil deposition. This behaviour was mediated by a mechanism of aggregation, with termites being more likely to join in the work at an excavation site as the number of termites presently working at that site increased. Statistical analyses showed that this aggregation mechanism was a response to active digging, distinct from and unrelated to putative chemical cues that stimulate deposition. Agent-based simulations quantitatively supported the interpretation that the early stage of de novo construction is primarily organized by excavation and aggregation activity rather than by stigmergic deposition. PMID:28615497

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

DOE PAGES

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.; ...

2016-06-01

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Effects of Detergents on Catalytic Activity of Human Endometase/Matrilysin-2, a Putative Cancer Biomarker†

PubMed Central

Park, Hyun I.; Lee, Seakwoo; Ullah, Asad; Cao, Qiang; Sang, Qing-Xiang Amy

2009-01-01

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration-dependent exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matirilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (~90 μM). Their IC50 values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was mixed-type as determined by Dixon’s plot, however, that of endometase was non-competitive with a Ki value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block substrate binding site to impede catalysis. Under physiological conditions lipid or membrane microenvironment may regulate enzymatic activity. PMID:19818727

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stojanoski, Vlatko; Sankaran, Banumathi; Prasad, B. V. Venkataram

Due to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity. The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of relatedmore » phosphoethanolamine transferases. The putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.« less

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse.

PubMed

Jacobsen, Jacob P R; Plenge, Per; Sachs, Benjamin D; Pehrson, Alan L; Cajina, Manuel; Du, Yunzhi; Roberts, Wendy; Rudder, Meghan L; Dalvi, Prachiti; Robinson, Taylor J; O'Neill, Sharon P; Khoo, King S; Morillo, Connie Sanchez; Zhang, Xiaodong; Caron, Marc G

2014-12-01

Escitalopram appears to be a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, there by curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and hence anti-depressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram's inhibition here of. Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Recombinant generation of hSERT transgenic mice; in vivo microdialysis; SERT binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). We generated mice expressing either the wild-type human SERT (hSERT(WT)) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERT(ALI/VFL+SI/TT)). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. The hSERT mice showed normal basal 5-HTExt levels. Escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment and was unaffected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small and tended to be enhanced by R-citalopram co-administration. We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram.

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

PubMed Central

Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M.; Bartlam, Mark

2016-01-01

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate. PMID:26919520

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

PubMed

Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M; Bartlam, Mark

2016-03-01

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

Crystal Structure of Phosphatidylglycerophosphatase (PGPase), a Putative Membrane-Bound Lipid Phosphatase, Reveals a Novel Binuclear Metal Binding Site and Two Proton Wires

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumaran,D.; Bonnano, J.; Burley, S.

2006-01-01

Phosphatidylglycerophosphatase (PGPase), an enzyme involved in lipid metabolism, catalyzes formation of phosphatidylglycerol from phosphatidylglycerophosphate. Phosphatidylglycerol is a multifunctional phospholipid, found in the biological membranes of many organisms. Here, we report the crystal structure of Listeria monocytogenes PGPase at 1.8 Angstroms resolution. PGPase, an all-helical molecule, forms a homotetramer. Each protomer contains an independent active site with two metal ions, Ca{sup 2+} and Mg{sup 2+}, forming a hetero-binuclear center located in a hydrophilic cavity near the surface of the molecule. The binuclear center, conserved ligands, metal-bound water molecules, and an Asp-His dyad form the active site. The catalytic mechanism of thismore » enzyme is likely to proceed via binuclear metal activated nucleophilic water. The binuclear metal-binding active-site environment of this structure should provide insights into substrate binding and metal-dependent catalysis. A long channel with inter-linked linear water chains, termed 'proton wires', is observed at the tetramer interface. Comparison of similar water chain structures in photosynthetic reaction centers (RCs), Cytochrome f, gramicidin, and bacteriorhodopsin, suggests that PGPase may conduct protons via proton wires.« less

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

A short region of the promoter of the breast cancer associated PLU-1 gene can regulate transcription in vitro and in vivo.

PubMed

Catteau, Aurélie; Rosewell, Ian; Solomon, Ellen; Taylor-Papadimitriou, Joyce

2004-07-01

The recently cloned gene PLU-1 shows restricted expression in adult tissues, with high expression being found in testis, and transiently in the pregnant mammary gland. However, both the gene and the protein product are specifically up-regulated in breast cancer. To investigate the control of expression of the PLU-1 gene, we have cloned and functionally characterised the 5' flanking region of the gene, which was found to contain another putative gene. Two transcription start sites of the PLU-1 gene were mapped by 5' RACE. A short proximal 249 bp region was defined using reporter gene assays, which encompasses the major transcription start site and exhibits a strong constitutive promoter activity in all cell lines tested. However, regions upstream of this sequence repress transcription more effectively in a non-malignant breast cell line as compared to breast cancer cell lines. The 249 bp region is GC-rich and includes consensus Sp1 sites, GC boxes, cAMP-responsive element (CRE) and other putative cis-elements. Mutational analysis showed that two intact conserved Sp1 binding sites (shown here to bind Sp1 and/or Sp3) are critical for constitutive promoter activity, while a negative role for a neighbouring GC box is indicated. The sequence of the core promoter is highly conserved in the mouse and Plu-1 expression in the mouse embryo has been documented. Using transgenesis, we therefore examined the ability of the 249 bp fragment to control expression of a reporter gene during embryogenesis. We found that not only is the core promoter sufficient to activate transcription in vivo, but that the expression of the reporter gene coincides both temporally and spatially with regions where endogenous Plu-1 is highly expressed. This suggests that tissue specific controlling elements are found within the short fragment and are functional in the embryonic environment.

Spectroscopic and Computational Investigation of Iron(III) Cysteine Dioxygenase: Implications for the Nature of the Putative Superoxo-Fe(III) Intermediate

PubMed Central

2015-01-01

Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron-dependent enzyme that converts exogenous cysteine (Cys) to cysteine sulfinic acid using molecular oxygen. Although the complete catalytic mechanism is not yet known, several recent reports presented evidence for an Fe(III)-superoxo reaction intermediate. In this work, we have utilized spectroscopic and computational methods to investigate the as-isolated forms of CDO, as well as Cys-bound Fe(III)CDO, both in the absence and presence of azide (a mimic of superoxide). An analysis of our electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance data of the azide-treated as-isolated forms of CDO within the framework of density functional theory (DFT) computations reveals that azide coordinates directly to the Fe(III), but not the Fe(II) center. An analogous analysis carried out for Cys-Fe(III)CDO provides compelling evidence that at physiological pH, the iron center is six coordinate, with hydroxide occupying the sixth coordination site. Upon incubation of this species with azide, the majority of the active sites retain hydroxide at the iron center. Nonetheless, a modest perturbation of the electronic structure of the Fe(III) center is observed, indicating that azide ions bind near the active site. Additionally, for a small fraction of active sites, azide displaces hydroxide and coordinates directly to the Cys-bound Fe(III) center to generate a low-spin (S = 1/2) Fe(III) complex. In the DFT-optimized structure of this complex, the central nitrogen atom of the azide moiety lies within 3.12 Å of the cysteine sulfur. A similar orientation of the superoxide ligand in the putative Fe(III)-superoxo reaction intermediate would promote the attack of the distal oxygen atom on the sulfur of substrate Cys. PMID:25093959

High Prevalence of Quorum-Sensing and Quorum-Quenching Activity among Cultivable Bacteria and Metagenomic Sequences in the Mediterranean Sea

PubMed Central

López-Pérez, Mario; Mayer, Celia; Parga, Ana; Amaro-Blanco, Jaime

2018-01-01

There is increasing evidence being accumulated regarding the importance of N-acyl homoserine lactones (AHL)-mediated quorum-sensing (QS) and quorum-quenching (QQ) processes in the marine environment, but in most cases, data has been obtained from specific microhabitats, and subsequently little is known regarding these activities in free-living marine bacteria. The QS and QQ activities among 605 bacterial isolates obtained at 90 and 2000 m depths in the Mediterranean Sea were analyzed. Additionally, putative QS and QQ sequences were searched in metagenomic data obtained at different depths (15–2000 m) at the same sampling site. The number of AHL producers was higher in the 90 m sample (37.66%) than in the 2000 m sample (4.01%). However, the presence of QQ enzymatic activity was 1.63-fold higher in the 2000 m sample. The analysis of putative QQ enzymes in the metagenomes supports the relevance of QQ processes in the deepest samples, found in cultivable bacteria. Despite the unavoidable biases in the cultivation methods and biosensor assays and the possible promiscuous activity of the QQ enzymes retrieved in the metagenomic analysis, the results indicate that AHL-related QS and QQ processes could be common activity in the marine environment. PMID:29462892

Quantitative Analysis of Focused A-To-I RNA Editing Sites by Ultra-High-Throughput Sequencing in Psychiatric Disorders

PubMed Central

Zhu, Hu; Urban, Daniel J.; Blashka, Jared; McPheeters, Matthew T.; Kroeze, Wesley K.; Mieczkowski, Piotr; Overholser, James C.; Jurjus, George J.; Dieter, Lesa; Mahajan, Gouri J.; Rajkowska, Grazyna; Wang, Zefeng; Sullivan, Patrick F.; Stockmeier, Craig A.; Roth, Bryan L.

2012-01-01

A-to-I RNA editing is a post-transcriptional modification of single nucleotides in RNA by adenosine deamination, which thereby diversifies the gene products encoded in the genome. Thousands of potential RNA editing sites have been identified by recent studies (e.g. see Li et al, Science 2009); however, only a handful of these sites have been independently confirmed. Here, we systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites in three sets of normal human brain samples by ultra-high-throughput sequencing (uHTS). Forty of 109 putative sites, including 25 previously confirmed sites, were validated as truly edited in our brain samples, suggesting an overestimation of A-to-I RNA editing in these putative sites by Li et al (2009). To evaluate RNA editing in human disease, we analyzed 29 of the confirmed sites in subjects with major depressive disorder and schizophrenia using uHTS. In striking contrast to many prior studies, we did not find significant alterations in the frequency of RNA editing at any of the editing sites in samples from these patients, including within the 5HT2C serotonin receptor (HTR2C). Our results indicate that uHTS is a fast, quantitative and high-throughput method to assess RNA editing in human physiology and disease and that many prior studies of RNA editing may overestimate both the extent and disease-related variability of RNA editing at the sites we examined in the human brain. PMID:22912834

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

PubMed

García Guerreiro, M P; Fontdevila, A

2007-01-01

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

PubMed Central

Fournier, Jean-Baptiste; Rebuffet, Etienne; Delage, Ludovic; Grijol, Romain; Meslet-Cladière, Laurence; Rzonca, Justyna; Potin, Philippe; Michel, Gurvan; Czjzek, Mirjam

2014-01-01

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family. PMID:25261522

Molecular cloning and identification of the transcriptional regulatory domain of the goat neurokinin B gene TAC3.

PubMed

Suetomi, Yuta; Matsuda, Fuko; Uenoyama, Yoshihisa; Maeda, Kei-ichiro; Tsukamura, Hiroko; Ohkura, Satoshi

2013-10-01

Neurokinin B (NKB), encoded by TAC3, is thought to be an important accelerator of pulsatile gonadotropin-releasing hormone release. This study aimed to clarify the transcriptional regulatory mechanism of goat TAC3. First, we determined the full-length mRNA sequence of goat TAC3 from the hypothalamus to be 820 b, including a 381 b coding region, with the putative transcription start site located 143-b upstream of the start codon. The deduced amino acid sequence of NKB, which is produced from preproNKB, was completely conserved among goat, cattle, and human. Next, we cloned 5'-upstream region of goat TAC3 up to 3400 b from the translation initiation site, and this region was highly homologous with cattle TAC3 (89%). We used this goat TAC3 5'-upstream region to perform luciferase assays. We created a luciferase reporter vector containing DNA constructs from -2706, -1837, -834, -335, or -197 to +166 bp (the putative transcription start site was designated as +1) of goat TAC3 and these were transiently transfected into mouse hypothalamus-derived N7 cells and human neuroblastoma-derived SK-N-AS cells. The luciferase activity gradually increased with the deletion of the 5'-upstream region, suggesting that the transcriptional suppressive region is located between -2706 and -336 bp and that the core promoter exists downstream of -197 bp. Estradiol treatment did not lead to significant suppression of luciferase activity of any constructs, suggesting the existence of other factor(s) that regulate goat TAC3 transcription.

Direct activation of the olfactory cyclic nucleotide-gated channel through modification of sulfhydryl groups by NO compounds.

PubMed

Broillet, M C; Firestein, S

1996-02-01

The activation of a cyclic nucleotide-gated channel is the final step in sensory transduction in olfaction. Normally, this channel is opened by the intracellular cyclic nucleotide second messenger cAMP or cGMP. However, in single channel recordings we found that donors of nitric oxide, a putative intercellular messenger, could directly activate the native olfactory neuron channel. Its action was independent of the presence of the normal ligand and did not involve the cyclic nucleotide binding site, suggesting an alternate site on the molecule that is critical in channel gating. The biochemical pathway appears to utilize nitric oxide in one of its alternate redox states, the nitrosonium ion, transnitrosylating a free sulfhydryl group belonging to a cysteine residue tentatively identified as being in the region linking the S6 transmembrane domain to the ligand binding domain.

The orphan nuclear receptor NR4A1 attenuates oxidative stress-induced β cells apoptosis via up-regulation of glutathione peroxidase 1.

PubMed

Yang, Yingfeng; Xie, Fangyu; Qin, Dandan; Zong, Chen; Han, Feng; Pu, Zeqing; Liu, Dong; Li, Xia; Zhang, Yuchao; Liu, Yuantao; Wang, Xiangdong

2018-06-15

Our previous study showed that NR4A1 protects against oxidative stress-induced cell apoptosis. However, the targets downstream of NR4A1 are incompletely known. Glutathione peroxidase 1 (GPX1) is the most common antioxidant enzyme in the glutathione peroxidase class. In this study, we aimed to investigate whether GPX1 is a mediator of the protective effects of NR4A1 in pancreatic β cells. A pancreatic β cell line, MIN6, was used to generate NR4A1 over-expression cell line. GPX1 expression and GPX1 promoter trans-activation in these cells was determined. These cells were then treated with H 2 O 2 , and the active caspase3 level was determined. NR4A1 over-expression in MIN6 cells resulted in increased GPX1 expression at both mRNA and protein levels. Dual luciferase assay showed that NR4A1 over-expression was able to enhance the trans-activation of GPX1 promoter, and the critical regulatory elements were narrowed down between 0 to -2000 bp in GPX1 promoter with a putative NR4A1 binding site (-273 to -268). ChIP assays demonstrated that NR4A1 physically associates with the GPX1 promoter. Over-expression of GPX1 reduced the active level of Caspase3 after H 2 O 2 treatment. NR4A1 increases the expression of GPX1 by enhancing the trans-activation of GPX1 promoter through binding to the putative binding site on GPX1 promoter. NR4A1 potentially protects pancreatic β cells against oxidative stress-induced apoptosis by increasing GPX1 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

PubMed

Dayan, Avraham; Babin, Gilad; Ganoth, Assaf; Kayouf, Nivin Samir; Nitoker Eliaz, Neta; Mukkala, Srijana; Tsfadia, Yossi; Fleminger, Gideon

2017-08-01

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO 2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO 2 -binding capabilities with TiBP. Intrigued by the unique TiO 2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO 2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO 2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO 2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO 2 through similar regions located far from the active site and the dimerization sites. The putative TiO 2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins. Copyright © 2017 John Wiley & Sons, Ltd.

Penetration Peg Formation and Invasive Hyphae Development Require Stage-Specific Activation of MoGTI1 in Magnaporthe oryzae.

PubMed

Li, Yang; Wang, Guanghui; Xu, Jin-Rong; Jiang, Cong

2016-01-01

The hemibiotrophic pathogen Magnaporthe oryzae causes one of the most destructive diseases in cultivated rice. Complex infection-related morphogenesis and production of various effectors are known to be important for successful colonization and disease development. In this study, we characterized the activation of the MoGTI1 transcription factor and its role in infection-related morphogenesis and effector gene expression. The Mogti1 mutant was nonpathogenic, although it was normal in appressorium formation and turgor generation. Close examination showed that Mogti1 was defective in penetration and growth of normal invasive hyphae. Deletion of MoGTI1 affected the expression of the majority of effector genes. The expression of MoGti1 appeared to be controlled by the Mps1 but not Pmk1 mitogen-activated protein kinase (MAPK), and the mps1 and Mogti1 mutants had similar phenotypes in plant infection and cell wall integrity defects. However, lack of MAPK phosphorylation sites and dispensability of the putative MAPK docking site suggested that MoGti1 is not a direct target of Mps1. Site-specific mutagenesis analyses showed that the putative protein kinase A phosphorylation site was not essential for localization of MoGti1 to the nucleus but important for its normal function. Although the cyclin-dependent kinase (CDK) phosphorylation site of MoGti1 is dispensable during vegetative growth and appressorium formation, the S77A mutation affected penetration and invasive growth. Localization of MoGti1(S77A)-green fluorescent protein to the nucleus in late stages of appressorium formation and during invasive growth was not observed, suggesting a stage-specific CDK phosphorylation of MoGti1. Overall, our data indicate that Mps1 may indirectly regulate the expression of MoGti1 in maintaining cell wall integrity, conidiation, and plant infection. MoGti1 is likely a stage-specific target of CDK and plays a crucial role in effector gene expression and morphogenesis related to the development of penetration pegs and invasive hyphae.

Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis.

PubMed

Hidalgo, A R; Akond, M A; Kita, K; Kataoka, M; Shimizu, S

2001-12-01

Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

PubMed Central

2010-01-01

In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications. PMID:21054894

Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

PubMed Central

Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

2011-01-01

Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - C ata L ytic A ctive S ite P rediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID:22174814

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

PubMed Central

Ananvoranich, S; Lafontaine, D A; Perreault, J P

1999-01-01

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway. PMID:10037808

C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells.

PubMed

Fujiwara, Miho; Kato, Shintaro; Niwa, Yuki; Suzuki, Takehiro; Tsuchiya, Miyu; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

2016-08-01

R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp(153) and Trp(156) ; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp(153) and Trp(156) by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling. © 2016 Federation of European Biochemical Societies.

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

PubMed

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

Structure of the novel monomeric glyoxalase I from Zea mays

PubMed Central

Turra, Gino L.; Agostini, Romina B.; Fauguel, Carolina M.; Presello, Daniel A.; Andreo, Carlos S.; González, Javier M.; Campos-Bermudez, Valeria A.

2015-01-01

The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined. PMID:26457425

Structure of the novel monomeric glyoxalase I from Zea mays.

PubMed

Turra, Gino L; Agostini, Romina B; Fauguel, Carolina M; Presello, Daniel A; Andreo, Carlos S; González, Javier M; Campos-Bermudez, Valeria A

2015-10-01

The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined.

Discrimination of putative M1 and M2 muscarinic receptor subtypes in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Norman, A.B.; Creese, I.

1986-03-01

The EC/sub 50/ of EEDQ for the inhibition of (/sup 3/H)(-)QNB binding in vitro was approximately 3 fold lower for homogenates of hippocampus than brainstem (containing predominantly putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes respectively). Furthermore, the time-dependent loss of (/sup 3/H)(-)QNB binding produced by 100 ..mu..M EEDQ was faster in homogenates of hippocampus than brainstem. Administration of EEDQ (20 mg/kg i.p.) irreversibly reduced the Bmax of (/sup 3/H)(-)QNB binding by 56% and 34% in hippocampus and brainstem respectively. Pirenzepine competition for the remaining (/sup 3/H)(-)QNB binding sites following in vitro and in vivo treatment with EEDQ revealedmore » a significant increase in the proportion of (/sup 3/H)(-)QNB binding sites having low affinity for pirenzepine (M/sub 2/ receptors), indicating that the high affinity pirenzepine binding sites (M/sub 1/ receptors) were selectively and irreversibly lost. Thus, EEDQ discriminates the same putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes that are discriminated by pirenzepine. The reduction of (/sup 3/H)(-)QNB binding could be prevented both in vitro and in vivo by atropine or scopolamine. These data may indicate differences in the accessibility of these putative receptor subtypes to EEDQ or, alternatively, differences in the availability of carboxyl groups able to interact with EEDQ at the ligand recognition site of M/sub 1/ and M/sub 2/ muscarinic receptors.« less

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays.

PubMed

Fortes-Dias, Consuelo Latorre; Santos, Roberta Márcia Marques dos; Magro, Angelo José; Fontes, Marcos Roberto de Mattos; Chávez-Olórtegui, Carlos; Granier, Claude

2009-01-01

Crotoxin (CA.CB) is a beta-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA(2) counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein-protein interactions in these PLA(2)-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and beta-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF.

Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

NASA Astrophysics Data System (ADS)

Li, Shengjie; Bai, Junjie; Wang, Lin

2008-08-01

Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse

PubMed Central

Jacobsen, Jacob P.R.; Plenge, Per; Sachs, Benjamin D.; Pehrson, Alan L.; Cajina, Manuel; Du, Yunzhi; Roberts, Wendy; Rudder, Meghan L.; Dalvi, Prachiti; Robinson, Taylor J.; O’Neill, Sharon P.; Khoo, King S.; Morillo, Connie Sanchez; Zhang, Xiaodong; Caron, Marc G.

2015-01-01

Rationale Escitalopram is a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and antidepressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram’s inhibition hereof. Objectives Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Methods Recombinant technology; in vivo microdialysis; receptor binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). Results We generated mice expressing either the wild-type human SERT (hSERTWT) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERTALI/VFL+SI/TT). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. Importantly, escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment. Further, escitalopram-induced 5-HTExt elevation was not affected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small, tending to be enhanced by R-citalopram co-administration. Conclusions We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram. Our findings points to mechanisms for R-citalopram antagonism of escitalopram’s antidepressant action other than direct antagonistic binding interactions at the hSERT. PMID:24810106

Structure of the catalytic domain of the colistin resistance enzyme MCR-1

DOE PAGES

Stojanoski, Vlatko; Sankaran, Banumathi; Prasad, B. V. Venkataram; ...

2016-09-21

Due to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity. The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of relatedmore » phosphoethanolamine transferases. The putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.« less

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Heterogeneity of signal transduction by Na-K-ATPase α-isoforms: role of Src interaction.

PubMed

Yu, Hui; Cui, Xiaoyu; Zhang, Jue; Xie, Joe X; Banerjee, Moumita; Pierre, Sandrine V; Xie, Zijian

2018-02-01

Of the four Na-K-ATPase α-isoforms, the ubiquitous α1 Na-K-ATPase possesses both ion transport and Src-dependent signaling functions. Mechanistically, we have identified two putative pairs of domain interactions between α1 Na-K-ATPase and Src that are critical for α1 signaling function. Our subsequent report that α2 Na-K-ATPase lacks these putative Src-binding sites and fails to carry on Src-dependent signaling further supported our proposed model of direct interaction between α1 Na-K-ATPase and Src but fell short of providing evidence for a causative role. This hypothesis was specifically tested here by introducing key residues of the two putative Src-interacting domains present on α1 but not α2 sequence into the α2 polypeptide, generating stable cell lines expressing this mutant, and comparing its signaling properties to those of α2-expressing cells. The mutant α2 was fully functional as a Na-K-ATPase. In contrast to wild-type α2, the mutant gained α1-like signaling function, capable of Src interaction and regulation. Consistently, the expression of mutant α2 redistributed Src into caveolin-1-enriched fractions and allowed ouabain to activate Src-mediated signaling cascades, unlike wild-type α2 cells. Finally, mutant α2 cells exhibited a growth phenotype similar to that of the α1 cells and proliferated much faster than wild-type α2 cells. These findings reveal the structural requirements for the Na-K-ATPase to function as a Src-dependent receptor and provide strong evidence of isoform-specific Src interaction involving the identified key amino acids. The sequences surrounding the putative Src-binding sites in α2 are highly conserved across species, suggesting that the lack of Src binding may play a physiologically important and isoform-specific role.

Molecular analysis of the human SLC13A4 sulfate transporter gene promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jefferis, J.; Rakoczy, J.; School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland

2013-03-29

Highlights: ► Basal promoter activity of SLC13A4 −57 to −192 nt upstream of transcription initiation site. ► Human SLC13A4 5′-flanking region has conserved motifs with other placental species. ► Putative NFY, SP1 and KLF7 motifs in SLC13A4 5′-flanking region enhance transcription. -- Abstract: The human solute linked carrier (SLC) 13A4 gene is primarily expressed in the placenta where it is proposed to mediate the transport of nutrient sulfate from mother to fetus. The molecular mechanisms involved in the regulation of SLC13A4 expression remain unknown. To investigate the regulation of SLC13A4 gene expression, we analysed the transcriptional activity of the humanmore » SLC13A4 5′-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Basal transcriptional activity was identified in the region −57 to −192 nucleotides upstream of the SLC13A4 transcription initiation site. Mutational analysis of the minimal promoter region identified Nuclear factor Y (NFY), Specificity protein 1 (SP1) and Krüppel like factor 7 (KLF7) motifs which conferred positive transcriptional activity, as well as Zinc finger protein of the cerebellum 2 (ZIC2) and helix–loop–helix protein 1 (HEN1) motifs that repressed transcription. The conserved NFY, SP1, KLF7, ZIC2 and HEN1 motifs in the SLC13A4 promoter of placental species but not in non-placental species, suggests a potential role for these putative transcriptional factor binding motifs in the physiological control of SLC13A4 mRNA expression.« less

Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Brien, D.A.

1992-11-01

A putative transcription factor in Rhodobactor capsulatus which binds upstream of the crt and bch pigment biosynthesis operons and appears to play a role in the adaptation of the organism from the aerobic to the anaerobic-photosynthetic growth mode was characterized. Chapter 2 describes the identification of this factor through an in vitro mobility shift assay, as well as the determination of its binding properties and sequence specificity. Chapter 3 focuses on the isolation of this factor. Biochemistry of later carotenoid biosynthesis enzymes derived from the non-photosynthetic bacterium, Erwinia herbicola. Chapter 4 describes the separate overexpression and in vitro analysis ofmore » two enzymes involved in the main sequence of the carotenoid biosynthesis pathway, lycopene cyclase and 5-carotene hydroxylase. Chapter 5 examines the overexpression and enzymology of functionally active zeaxanthin glucosyltransferase, an enzyme which carries out a more unusual transformation, converting a carotenoid into its more hydrophilic mono- and diglucoside derivatives. In addition, amino acid homology with other glucosyltransferases suggests a putative binding site for the UDP-activated glucose substrate.« less

Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent.

PubMed

Cochrane, Rachel V K; Sanichar, Randy; Lambkin, Gareth R; Reiz, Béla; Xu, Wei; Tang, Yi; Vederas, John C

2016-01-11

The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl-tRNA synthetase, and exhibits activity against both blood- and liver-stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non-reducing (NR) iterative type I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced-intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl-tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baumgold, J.

1986-05-01

Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and themore » remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.« less

Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

PubMed Central

2017-01-01

TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B. PMID:28046119

The pure anti-oestrogen ICI 182,780 (Faslodex™) activates large conductance Ca2+-activated K+ channels in smooth muscle

PubMed Central

Dick, Gregory M

2002-01-01

Oestrogen and tamoxifen activate large conductance Ca2+-activated K+ (BKCa) channels in smooth muscle through a non-genomic mechanism that depends on the regulatory β1 subunit and an extracellular binding site. It is unknown whether a ‘pure' anti-oestrogen such as ICI 182,780 (Faslodex™), that has no known oestrogenic properties, would have any effect on BKCa channels. Using single channel patch clamp techniques on canine colonic myocytes, the hypothesis that ICI 182,780 would activate BKCa channels was tested. ICI 182,780 increased the open probability of BKCa channels in inside-out patches with an EC50 of 1 μM. These data suggest that molecules with the ability to bind nuclear oestrogen receptors, regardless of oestrogenic or anti-oestrogenic nature, activate BKCa channels through this nongenomic, membrane-delimited mechanism. The identity and characteristics of this putative binding site remain unclear; however, it has pharmacological similarity to oestrogen receptors α and β, as ICI 182,780 interacts with it. PMID:12145095

Proton transfer and protein quake in photoreceptor activation

NASA Astrophysics Data System (ADS)

Xie, Aihua

2002-03-01

Proteins are able to perform an enormous variety of functions, while using only a limited number of underlying processes. One of these is proton transfer, found in a range of receptors and enzymes. It is conceivable that proton transfer is essential in biological energy transduction, but it is less evident how proton transfer is employed in receptor activation during biological signal transduction. An important question regarding receptor activation is how a localized event of detecting a stimulus at the active site drives global conformational changes involving protein surface for signal relay. We will present structural, kinetic and energetic studies on the activation mechanism of a prototype PAS domain photoreceptor, photoactive yellow protein (PYP). Our data reveal that the putative signaling state of PYP upon absorption of a blue photon is formed during a large-amplitude protein quake triggered by the formation of a new buried charge in a hydrophobic pocket at the active site of PYP via intramolecular proton transfer. This mechanism for protein quakes driven by proton transfer and electrostatic interactions may play roles during the functioning of other receptor proteins and non-receptor proteins that require large conformational changes.

Assessing impacts of unconventional natural gas extraction on microbial communities in headwater stream ecosystems in Northwestern Pennsylvania

PubMed Central

Trexler, Ryan; Solomon, Caroline; Brislawn, Colin J.; Wright, Justin R.; Rosenberger, Abigail; McClure, Erin E.; Grube, Alyssa M.; Peterson, Mark P.; Keddache, Mehdi; Mason, Olivia U.; Hazen, Terry C.; Grant, Christopher J.; Lamendella, Regina

2014-01-01

Hydraulic fracturing and horizontal drilling have increased dramatically in Pennsylvania Marcellus shale formations, however the potential for major environmental impacts are still incompletely understood. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community structure of water, sediment, bryophyte, and biofilm samples from 26 headwater stream sites in northwestern Pennsylvania with different histories of fracking activity within Marcellus shale formations. Further, we describe the relationship between microbial community structure and environmental parameters measured. Approximately 3.2 million 16S rRNA gene sequences were retrieved from a total of 58 samples. Microbial community analyses showed significant reductions in species richness as well as evenness in sites with Marcellus shale activity. Beta diversity analyses revealed distinct microbial community structure between sites with and without Marcellus shale activity. For example, operational taxonomic units (OTUs) within the Acetobacteracea, Methylocystaceae, Acidobacteriaceae, and Phenylobacterium were greater than three log-fold more abundant in MSA+ sites as compared to MSA− sites. Further, several of these OTUs were strongly negatively correlated with pH and positively correlated with the number of wellpads in a watershed. It should be noted that many of the OTUs enriched in MSA+ sites are putative acidophilic and/or methanotrophic populations. This study revealed apparent shifts in the autochthonous microbial communities and highlighted potential members that could be responding to changing stream conditions as a result of nascent industrial activity in these aquatic ecosystems. PMID:25408683

hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

PubMed Central

Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

2015-01-01

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5′-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site. PMID:26237581

Absence of specific binding of several putative neuro-transmitters to human fibroblasts.

PubMed

Berrettini, W H; Nadi, N S; Gershon, E S

1983-01-01

Fibroblasts were examined for specific binding sites of ten putative neurotransmitters to determine whether this tissue could be used in receptor studies of neurologic and psychiatric disorders. Stereospecific saturable binding was not found for any of the ligands: arginine vasopressin, neurotensin, somatostatin, angiotensin II, thyrotropin-releasing hormone (TRH), alpha-bungarotoxin, LSD, dihydromorphine, muscimol and spiperone.

Can PET-CT imaging and radiokinetic analyses provide useful clinical information on atypical femoral shaft fracture in osteoporotic patients?

PubMed

Chesnut, C Haile; Chesnut, Charles H

2012-03-01

Atypical femoral shaft fractures are associated with the extended usage of nitrogen-containing bisphosphonates as therapy for osteoporosis. For such fractures, the positron emission tomography (PET) procedure, coupled with computerized tomography (CT), provides a potential imaging modality for defining aspects of the pathogenesis, site specificity, and possible prodromal abnormalities prior to fracture. PET-CT may assess the radiokinetic variables K1 (a putative marker for skeletal blood flow) and Ki (a putative marker for skeletal bone formation), and when combined with PET imaging modalities and CT skeletal site localization, may define the site of such radiokinetic findings. Further studies into the clinical usage of PET-CT in patients with atypical femoral shaft fractures are warranted.

Putative melatonin receptors in a human biological clock

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.

In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completelymore » inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.« less

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin.

PubMed

Magro, Massimiliano; Fasolato, Luca; Bonaiuto, Emanuela; Andreani, Nadia Andrea; Baratella, Davide; Corraducci, Vittorino; Miotto, Giovanni; Cardazzo, Barbara; Vianello, Fabio

2016-10-01

Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe(3+) sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens. SAMNs were applied for mimicking Fe(3+) ions in solution, acting as magnetically drivable probes to evaluate putative Fe(3+) recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV-Vis spectroscopy and mass spectrometry. The whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores. In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation. The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

PubMed

Gou, Xiangbo; Wang, Wenying; Zou, Sihao; Qi, Yajuan; Xu, Yanfang

2018-03-01

The slowly activating delayed rectifier K + current (I Ks ) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates I Ks through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of I Ks by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record I Ks in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited I Ks in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human I Ks in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on I Ks and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism underlying the modulation of the KCNQ1/KCNE1 channel. Copyright © 2018 Elsevier Ltd. All rights reserved.

Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.

PubMed

Kawabata, Yutaka; Murata, Kousaku; Kawai, Shigeyuki

2015-12-25

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization and localization of arginine vasotocin receptors in the brain and kidney of an amphibian

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyd, S.K.

1987-01-01

Because arginine vasotocin (AVT) activates male sexual behaviors in the rough-skinned newt (Taricha granulosa), quantitative autoradiography with radiolabeled arginine vasopressin (/sup 3/H-AVP) was used to localize and characterize putative AVT receptors in the brain of this amphibian. Binding of /sup 3/H-AVP to sites within the medial pallium was saturable, specific, reversible, of high affinity and low capacity. These binding sites appear to represent authentic central nervous system receptors for AVT. Furthermore, ligand specificity for the binding sites in this amphibian differs from that reported for AVP binding sites in rat brains. Dense concentrations of specific binding sites were located inmore » the olfactory nerve as it entered the olfactory bulb within the medial pallium, dorsal pallium, and amygdala pars lateralis of the telencephalon, and in the tegmental region of the medulla. Concentrations of binding sites differed significantly among various brain regions. A comparison of male and female newts collected during the breeding season revealed no sexual dimorphism. These areas may represent site(s) of action where AVT elicits sexual behaviors in male T. granulosa.« less

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter.

PubMed

Li, Peizhen; Wang, Bo; Cao, Dandan; Liu, Yuezhong; Zhang, Quanqi; Wang, Xubo

2017-10-01

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

PubMed Central

Liu, Tingting; Yao, Ruolan; Zhao, Yucheng; Xu, Sheng; Huang, Chuanlong; Luo, Jun; Kong, Lingyi

2017-01-01

Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure–function relationships of this important family of proteins. PMID:28144249

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

PubMed Central

Faehnle, Christopher R.; Liu, Xuying; Pavlovsky, Alexander; Viola, Ronald E.

2006-01-01

The activation of the β-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate l-­aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups. PMID:17012784

Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa)

PubMed Central

Perdomo-Sabogal, Alvaro; Nowick, Katja; Piccini, Ilaria; Sudbrak, Ralf; Lehrach, Hans; Yaspo, Marie-Laure; Warnatz, Hans-Jörg; Querfurth, Robert

2016-01-01

A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes. PMID:26814189

Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

PubMed Central

Assadi-Porter, Fariba M.; Maillet, Emeline L.; Radek, James T.; Quijada, Jeniffer; Markley, John L.; Max, Marianna

2010-01-01

The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N- and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9–19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9–19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models. PMID:20302879

Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin

2010-10-28

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences aremore » located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Zhang; G Buchko; L Qin

2011-12-31

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are locatedmore » at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

PubMed Central

Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

2014-01-01

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

Integrated analysis of bacterial and microeukaryotic communities from differentially active mud volcanoes in the Gulf of Cadiz

NASA Astrophysics Data System (ADS)

Coelho, Francisco J. R. C.; Louvado, António; Domingues, Patrícia M.; Cleary, Daniel F. R.; Ferreira, Marina; Almeida, Adelaide; Cunha, Marina R.; Cunha, Ângela; Gomes, Newton C. M.

2016-10-01

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults.

Integrated analysis of bacterial and microeukaryotic communities from differentially active mud volcanoes in the Gulf of Cadiz

PubMed Central

Coelho, Francisco J. R. C.; Louvado, António; Domingues, Patrícia M.; Cleary, Daniel F. R.; Ferreira, Marina; Almeida, Adelaide; Cunha, Marina R.; Cunha, Ângela; Gomes, Newton C. M.

2016-01-01

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults. PMID:27762306

Integrated analysis of bacterial and microeukaryotic communities from differentially active mud volcanoes in the Gulf of Cadiz.

PubMed

Coelho, Francisco J R C; Louvado, António; Domingues, Patrícia M; Cleary, Daniel F R; Ferreira, Marina; Almeida, Adelaide; Cunha, Marina R; Cunha, Ângela; Gomes, Newton C M

2016-10-20

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults.

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

PubMed

Doblas, Verónica G; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M; Botella, Miguel A

2013-02-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

PubMed Central

Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

2013-01-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

Copper-zinc-superoxide dismutase (CuZnSOD), an antioxidant gene from seahorse (Hippocampus abdominalis); molecular cloning, sequence characterization, antioxidant activity and potential peroxidation function of its recombinant protein.

PubMed

Perera, N C N; Godahewa, G I; Lee, Jehee

2016-10-01

Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5' untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3' UTR of 313 bp. The putative peptide consists of 154 amino acids. HaCuZnSOD had a predicted molecular mass of 15.94 kDa and a theoretical pI value of 5.73, which is favorable for copper binding activity. In silico analysis revealed that HaCuZnSOD had a prominent Cu-Zn_superoxide_dismutase domain, two Cu/Zn signature sequences, a putative N-glycosylation site, and several active sites including Cu(2+) and Zn(2+) binding sites. The three dimensional structure indicated a β-sheet barrel with 8 β-sheets and two short α-helical regions. Multiple alignment analyses revealed many conserved regions and active sites among its orthologs. The highest amino acid identity to HaCuZnSOD was found in Siniperca chuatsi (87.4%), while Maylandia zebra shared a close relationship in the phylogenetic analysis. Functional assays were performed to assess the antioxidant, biophysical and biochemical properties of overexpressed recombinant (r) HaCuZnSOD. A xanthine/XOD assay gave optimum results at pH 9 and 25 °C indicating these may be the best conditions for its antioxidant action in the seahorse. An MTT assay and flow cytometry confirmed that rHaCuZnSOD showed peroxidase activity in the presence of HCO3(-). In all the functional assays, the level of antioxidant activity of rHaCuZnSOD was concentration dependent; metal ion supplementation also increased its activity. The highest mRNA expressional level of HaCuZnSOD was found in blood. Temporal assessment under pathological stress showed a delay response by HaCuZnSOD. Our findings demonstrated that HaCuZnSOD is an important antioxidant, which might be involved in the host antioxidant defense mechanism against oxidative stress. Copyright © 2016 Elsevier Ltd. All rights reserved.

CREB, NF-Y and MEIS1 conserved binding sites are essential to balance Myostatin promoter/enhancer activity during early myogenesis.

PubMed

Grade, Carla Vermeulen Carvalho; Mantovani, Carolina Stefano; Fontoura, Marina Alves; Yusuf, Faisal; Brand-Saberi, Beate; Alvares, Lúcia Elvira

2017-10-01

Myostatin (MSTN) is a strong inhibitor of skeletal muscle growth in human and other vertebrates. Its transcription is controlled by a proximal promoter/enhancer (Mstn P/E) containing a TATA box besides CREB, NF-Y, MEIS1 and FXR transcription factor binding sites (TFBSs), which are conserved throughout evolution. The aim of this work was to investigate the role of these TFBSs on Mstn P/E activity and evaluate the potential of their putative ligands as Mstn trans regulators. Mstn P/E mutant constructs were used to establish the role of conserved TFBSs using dual-luciferase assays. Expression analyses were performed by RT-PCR and in situ hybridization in C2C12 myoblasts and E10.5 mouse embryos, respectively. Our results revealed that CREB, NF-Y and MEIS1 sites are required to balance Mstn P/E activity, keeping Mstn transcription within basal levels during myoblast proliferation. Furthermore, our data showed that NF-Y site is essential, although not sufficient, to mediate Mstn P/E transcriptional activity. In turn, CREB and MEIS1 binding sites seem to depend on the presence of NF-Y site to induce Mstn P/E. FXR appears not to confer any effect on Mstn P/E activity, except in the absence of all other conserved TFBS. Accordingly, expression studies pointed to CREB, NF-Y and MEIS1 but not to FXR factors as possible regulators of Mstn transcription in the myogenic context. Altogether, our findings indicated that CREB, NF-Y and MEIS1 conserved sites are essential to control basal Mstn transcription during early myogenesis, possibly by interacting with these or other related factors.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 thatmore » forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less

Dynamic HypA zinc site is essential for acid viability and proper urease maturation in Helicobacter pylori.

PubMed

Johnson, Ryan C; Hu, Heidi Q; Merrell, D Scott; Maroney, Michael J

2015-04-01

Helicobacter pylori requires urease activity in order to survive in the acid environment of the human stomach. Urease is regulated in part by nickelation, a process that requires the HypA protein, which is a putative nickel metallochaperone that is generally associated with hydrogenase maturation. However, in H. pylori, HypA plays a dual role. In addition to an N-terminal nickel binding site, HypA proteins also contain a structural zinc site that is coordinated by two rigorously conserved CXXC sequences, which in H. pylori are flanked by His residues. These structural Zn sites are known to be dynamic, converting from Zn(Cys)4 centers at pH 7.2 to Zn(Cys)2(His)2 centers at pH 6.3 in the presence of Ni(ii) ions. In this study, mutant strains of H. pylori that express zinc site variants of the HypA protein are used to show that the structural changes in the zinc site are important for the acid viability of the bacterium, and that a reduction in acid viability in these variants can be traced in large measure to deficient urease activity. This in turn leads to a model that connects the Zn(Cys)4 coordination to urease maturation.

Evolving artificial metalloenzymes via random mutagenesis

NASA Astrophysics Data System (ADS)

Yang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; Moellering, Raymond E.; Lewis, Jared C.

2018-03-01

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

3 Benzyl-6-chloropyrone: a suicide inhibitor of cholesterol esterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saint, C.; Gallo, I.; Kantorow, M.

Cholesterol, absorbed from the intestine, appears in lymph as the ester. Cholesterol esterase is essential for this process, since depletion of the enzyme blocks and repletion restores, absorption. Selective inhibitors of cholesterol esterase may thus prove useful in reducing cholesterol uptake. A series of potential suicide substrates were synthesized which, following cleavage by the enzyme, would attack the putative nucleophile in the active site. One of these, 3-benzyl-6-chloropyrone (3BCP), inhibited both synthesis and hydrolysis of /sup 14/C-cholesteryl oleate with an I/sub 50/ of approximately 150 ..mu..M. The inactivation was time-dependent and characteristic of a suicide mechanism. The ..cap alpha.. pyronemore » structure (lactone analog) is cleaved by a serine-hydroxyl in the active site. This generates an enoyl chloride which inactivates the imidazole believed to play a part in the catalytic function of the enzyme. Inhibition by 3BCP is selective for cholesterol esterase. The activity of pancreatic lipase as not affected by concentrations up to 1 mM.« less

Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

PubMed Central

Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

2016-01-01

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

Molecular modeling and identification of novel glucokinase activators through stepwise virtual screening.

PubMed

Behera, Pabitra Mohan; Behera, Deepak Kumar; Satpati, Suresh; Agnihotri, Geetanjali; Nayak, Sanghamitra; Padhi, Payodhar; Dixit, Anshuman

2015-04-01

The glucose phosphorylating enzyme glucokinase (GK) is a 50kD monomeric protein having 465 amino acids. It maintains glucose homeostasis inside cells, acts as a glucose sensor in pancreatic β-cells and as a rate controlling enzyme for hepatic glucose clearance and glycogen synthesis. It has two binding sites, one for binding d-glucose and the other for a putative allosteric activator named glucokinase activator (GKA). The GKAs interact with the same region of the GK enzyme that is commonly affected by naturally occurring mutations in humans. However, many GKAs do not bind to GK in the absence of glucose. Recently, it has been reported that GKAs are highly effective in patients with type 2 diabetes mellitus. In this milieu a molecular modeling study has been carried out on three natural variants of GK that lie in the GKA binding site and are known to cause maturity onset diabetes of young (MODY). Additionally, a 10ns molecular dynamics simulation was done on each of the modeled variant in order to explore the flexibility of this site. Subsequently, a systematic virtual screening study was done to identify compounds which can bind with high affinity at GKA binding site of mutant GK. Copyright © 2015 Elsevier Inc. All rights reserved.

Virus-like attachment sites as structural landmarks of plants retrotransposons.

PubMed

Ochoa Cruz, Edgar Andres; Cruz, Guilherme Marcello Queiroga; Vieira, Andréia Prata; Van Sluys, Marie-Anne

2016-01-01

The genomic data available nowadays has enabled the study of repetitive sequences and their relationship to viruses. Among them, long terminal repeat retrotransposons (LTR-RTs) are the largest component of most plant genomes, the Gypsy and Copia superfamilies being the most common. Recently it has been found that Del lineage, an LTR-RT of Gypsy superfamily, has putative virus-like attachment (vl-att) sites. This signature, originally described for retroviruses, is recognized by retroviral integrase conferring specificity to the integration process. Here we retrieved 26,092 putative complete LTR-RTs from 10 lineages found in 10 fully sequenced angiosperm genomes and found putative vl-att sites that are a conserved structural landmark across these genomes. Furthermore, we reveal that each plant genome has a distinguishable LTR-RT lineage amplification pattern that could be related to the vl-att sites diversity. We used these patterns to generate a specific quick-response (QR) code for each genome that could be used as a barcode of identification of plants in the future. The universal distribution of vl-att sites represents a new structural feature common to plant LTR-RTs and retroviruses. This is an important finding that expands the information about the structural similarity between LTR-RT and retroviruses. We speculate that the sequence diversity of vl-att sites could be important for the life cycle of retrotransposons, as it was shown for retroviruses. All the structural vl-att site signatures are strong candidates for further functional studies. Moreover, this is the first identification of specific LTR-RT content and their amplification patterns in a large dataset of LTR-RT lineages and angiosperm genomes. These distribution patterns could be used in the future with biotechnological identification purposes.

Structural studies of viperin, an antiviral radical SAM enzyme.

PubMed

Fenwick, Michael K; Li, Yue; Cresswell, Peter; Modis, Yorgo; Ealick, Steven E

2017-06-27

Viperin is an IFN-inducible radical S -adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S -adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα) 6 -barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.

Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase

PubMed Central

Liu, Binbin; Banavali, Nilesh K.; Jones, Susan A.; Zhang, Jing; Li, Zhong; Kramer, Laura D.; Li, Hongmin

2015-01-01

The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2’-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition. PMID:26098995

Systemic and Mucosal Differences in HIV Burden, Immune and Therapeutic Responses

PubMed Central

Wahl, Sharon M.; Redford, Maryann; Christensen, Shawna; Mack, Wendy; Cohn, Jon; Janoff, Edward N.; Mestecky, Jiri; Jenson, Hal B.; Navazesh, Mahvash; Cohen, Mardge; Reichelderfer, Patricia; Kovacs, Andrea

2011-01-01

Background Mucosal tissues represent major targets for HIV transmission, but differ in susceptibility and reservoir function by unknown mechanisms. Methods In a cross-sectional study, HIV RNA and infectious virus were compared between oral and genital compartments and blood in HIV-infected women, in association with clinical parameters, co-pathogens and putative innate and adaptive HIV inhibitors. Results HIV RNA was detectable in 24.5% of women from all 3 compartments, whereas 45% had RNA in only one or two sites. By comparison, infectious HIV, present in blood of the majority, was rare in mucosal sites. Innate mediators, SLPI and TSP, were highest in mucosae. Highly active antiretroviral therapy (HAART) was associated with an 80% decreased probability of shedding. Multivariate logistic regression models revealed that mucosal HIV RNA was associated with higher plasma RNA, infectious virus, and total mucosal IgA, but not IgG. There was a 37-fold increased probability of detecting RNA in both genital and oral specimens (P=0.008;P=0.02, respectively) among women in highest vs lowest IgA tertiles. Conclusions Mucosal sites exhibit distinct characteristics of infectious HIV, viral shedding and responses to therapy, dependent upon both systemic and local factors. Of the putative innate and adaptive mucosal defense factors examined, only IgA was associated with HIV RNA shedding. However, rather than being protective, there was a striking increase in probability of detectable HIV RNA shedding in women with highest total IgA. PMID:21239996

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

PubMed

Prigozhin, Daniil M; Papavinasasundaram, Kadamba G; Baer, Christina E; Murphy, Kenan C; Moskaleva, Alisa; Chen, Tony Y; Alber, Tom; Sassetti, Christopher M

2016-10-28

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer

PubMed Central

2014-01-01

Background Protein coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs including long non-coding RNAs. A growing volume of literature has proposed that lncRNAs are important players in cancer. HOTAIR was previously shown to be an oncogene and negative prognostic factor in a variety of cancers. However, the factors that contribute to its upregulation and the interaction between HOTAIR and miRNAs are largely unknown. Methods A computational screen of HOTAIR promoter was conducted to search for transcription-factor-binding sites. HOTAIR promoter activities were examined by luciferase reporter assay. The function of the c-Myc binding site in the HOTAIR promoter region was tested by a promoter assay with nucleotide substitutions in the putative E-box. The association of c-Myc with the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay and Electrophoretic mobility shift assay. A search for miRNAs with complementary base paring with HOTAIR was performed utilizing online software program. Gain and loss of function approaches were employed to investigate the expression changes of HOTAIR or miRNA-130a. The expression levels of HOTAIR, c-Myc and miRNA-130a were examined in 65 matched pairs of gallbladder cancer tissues. The effects of HOTAIR and miRNA-130a on gallbladder cancer cell invasion and proliferation was tested using in vitro cell invasion and flow cytometric assays. Results We demonstrate that HOTAIR is a direct target of c-Myc through interaction with putative c-Myc target response element (RE) in the upstream region of HOTAIR in gallbladder cancer cells. A positive correlation between c-Myc and HOTAIR mRNA levels was observed in gallbladder cancer tissues. We predicted that HOTAIR harbors a miRNA-130a binding site. Our data showed that this binding site is vital for the regulation of miRNA-130a by HOTAIR. Moreover, a negative correlation between HOTAIR and miRNA-130a was observed in gallbladder cancer tissues. Finally, we demonstrate that the oncogenic activity of HOTAIR is in part through its negative regulation of miRNA-130a. Conclusion Together, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a. PMID:24953832

NF-{kappa}B p65 represses {beta}-catenin-activated transcription of cyclin D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Injoo; Choi, Yong Seok; Jeon, Mi-Ya

2010-12-03

Research highlights: {yields} Cyclin D1 transcription is directly activated by {beta}-catenin; however, {beta}-catenin-induced cyclin D1 transcription is reduced by NF-{kappa}B p65. {yields} Protein-protein interaction between NF-{kappa}B p65 and {beta}-catenin might be responsible for p65-mediated repression of cyclin D1. {yields} One of five putative binding sites, located further upstream of other sites, is the major {beta}-catenin binding site in the cyclin D1 promoter. {yields} NF-{kappa}B binding site in cyclin D1 is occupied not only by p65 but also by {beta}-catenin, which is dynamically regulated by the signal. -- Abstract: Signaling crosstalk between the {beta}-catenin and NF-{kappa}B pathways represents a functional network.more » To test whether the crosstalk also occurs on their common target genes, the cyclin D1 promoter was used as a model because it contains binding sites for both proteins. {beta}-catenin activated transcription from the cyclin D1 promoter, while co-expression of NF-{kappa}B p65 reduced {beta}-catenin-induced transcription. Chromatin immunoprecipitation revealed lithium chloride-induced binding of {beta}-catenin on one of the T-cell activating factor binding sites. More interestingly, {beta}-catenin binding was greatly reduced by NF-{kappa}B p65, possibly by the protein-protein interaction between the two proteins. Such a dynamic and complex binding of {beta}-catenin and NF-{kappa}B on promoters might contribute to the regulated expression of their target genes.« less

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia.

PubMed

Reis, Marta I R; do Vale, Ana; Pinto, Cristina; Nascimento, Diana S; Costa-Ramos, Carolina; Silva, Daniela S P; Silva, Manuel T; Dos Santos, Nuno M S

2007-03-01

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rascle, Anne; Neumann, Tanja; Raschta, Anne-Sarah

2009-01-01

LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-{kappa}B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-{kappa}B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-{kappa}B activity by TNF-{alpha} synergizes induction of NF-{kappa}B target genes by LMX1B. Chromatin immunoprecipitation demonstratedmore » that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized {kappa}B site, and that LMX1B recruitment correlates with increased NF-{kappa}B DNA association. IL-6 promoter-reporter assays showed that the {kappa}B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-{kappa}B target genes is affected in the kidney of Lmx1b{sup -/-} knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-{kappa}B target genes in cooperation with nuclear p50/p65 NF-{kappa}B.« less

Differential Activation of Fast-Spiking and Regular-Firing Neuron Populations During Movement and Reward in the Dorsal Medial Frontal Cortex

PubMed Central

Insel, Nathan; Barnes, Carol A.

2015-01-01

The medial prefrontal cortex is thought to be important for guiding behavior according to an animal's expectations. Efforts to decode the region have focused not only on the question of what information it computes, but also how distinct circuit components become engaged during behavior. We find that the activity of regular-firing, putative projection neurons contains rich information about behavioral context and firing fields cluster around reward sites, while activity among putative inhibitory and fast-spiking neurons is most associated with movement and accompanying sensory stimulation. These dissociations were observed even between adjacent neurons with apparently reciprocal, inhibitory–excitatory connections. A smaller population of projection neurons with burst-firing patterns did not show clustered firing fields around rewards; these neurons, although heterogeneous, were generally less selective for behavioral context than regular-firing cells. The data suggest a network that tracks an animal's behavioral situation while, at the same time, regulating excitation levels to emphasize high valued positions. In this scenario, the function of fast-spiking inhibitory neurons is to constrain network output relative to incoming sensory flow. This scheme could serve as a bridge between abstract sensorimotor information and single-dimensional codes for value, providing a neural framework to generate expectations from behavioral state. PMID:24700585

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

PubMed Central

Mayer, Kimberly M; Shanklin, John

2007-01-01

Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented. PMID:17201914

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

PubMed

Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

2016-05-21

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

Pharmacophore screening of the protein data bank for specific binding site chemistry.

PubMed

Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

2010-03-22

A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

Use of steric encumbrance to develop conjugated nanoporous polymers for metal-free catalytic hydrogenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Chengcheng; Zhu, Xiang; Abney, Carter W.

The design and synthesis of metal-free heterogeneous catalysts for efficient hydrogenation remains a great challenge. Here we report a novel approach to create conjugated nanoporous polymers with efficient hydrogenation activities toward unsaturated ketones by leveraging the innate steric encumbrance. The steric bulk of the framework as well as the local sterics of the Lewis basic sites within the polymeric skeleton result in the generation of the putative catalyst. This approach opens up new possibilities for the development of innovative metal-free heterogeneous catalysts.

Use of steric encumbrance to develop conjugated nanoporous polymers for metal-free catalytic hydrogenation

DOE PAGES

Tian, Chengcheng; Zhu, Xiang; Abney, Carter W.; ...

2016-09-08

The design and synthesis of metal-free heterogeneous catalysts for efficient hydrogenation remains a great challenge. Here we report a novel approach to create conjugated nanoporous polymers with efficient hydrogenation activities toward unsaturated ketones by leveraging the innate steric encumbrance. The steric bulk of the framework as well as the local sterics of the Lewis basic sites within the polymeric skeleton result in the generation of the putative catalyst. Lastly, this approach opens up new possibilities for the development of innovative metal-free heterogeneous catalysts.

Use of steric encumbrance to develop conjugated nanoporous polymers for metal-free catalytic hydrogenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Chengcheng; Zhu, Xiang; Abney, Carter W.

The design and synthesis of metal-free heterogeneous catalysts for efficient hydrogenation remains a great challenge. Here we report a novel approach to create conjugated nanoporous polymers with efficient hydrogenation activities toward unsaturated ketones by leveraging the innate steric encumbrance. The steric bulk of the framework as well as the local sterics of the Lewis basic sites within the polymeric skeleton result in the generation of the putative catalyst. Lastly, this approach opens up new possibilities for the development of innovative metal-free heterogeneous catalysts.

Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells.

PubMed

Bae, Eunjin; Sato, Misako; Kim, Ran-Ju; Kwak, Mi-Kyung; Naka, Kazuhito; Gim, Jungsoo; Kadota, Mitsutaka; Tang, Binwu; Flanders, Kathleen C; Kim, Tae-Aug; Leem, Sun-Hee; Park, Taesung; Liu, Fang; Wakefield, Lalage M; Kim, Seong-Jin; Ooshima, Akira

2014-11-01

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer. ©2014 American Association for Cancer Research.

Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains.

PubMed

Mullen, Lauren; Boerrigter, Kim; Ferriero, Nicholas; Rosalsky, Jeff; Barrett, Abigail van Buren; Murray, Patrick J; Steen, Andrew D

2018-01-01

Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N -terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases.

Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains

PubMed Central

Mullen, Lauren; Boerrigter, Kim; Ferriero, Nicholas; Rosalsky, Jeff; Barrett, Abigail van Buren; Murray, Patrick J.; Steen, Andrew D.

2018-01-01

Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N-terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases. PMID:29559961

Structure of C3b reveals conformational changes that underlie complement activity.

PubMed

Janssen, Bert J C; Christodoulidou, Agni; McCarthy, Andrew; Lambris, John D; Gros, Piet

2006-11-09

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rice, E.A.; Bannon, G.A.; Glenn, K.C.

2008-11-21

The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysinemore » revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.« less

Engineering synthetic TAL effectors with orthogonal target sites

PubMed Central

Garg, Abhishek; Lohmueller, Jason J.; Silver, Pamela A.; Armel, Thomas Z.

2012-01-01

The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression. PMID:22581776

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

PubMed

Kamalakaran, Sitharthan; Radhakrishnan, Senthil K; Beck, William T

2005-06-03

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

Characterization of member of DUF1888 protein family, self-cleaving and self-assembling endopeptidase.

PubMed

Osipiuk, Jerzy; Mulligan, Rory; Bargassa, Monireh; Hamilton, John E; Cunningham, Mark A; Joachimiak, Andrzej

2012-06-01

The crystal structure of SO1698 protein from Shewanella oneidensis was determined by a SAD method and refined to 1.57 Å. The structure is a β sandwich that unexpectedly consists of two polypeptides; the N-terminal fragment includes residues 1-116, and the C-terminal one includes residues 117-125. Electron density also displayed the Lys-98 side chain covalently linked to Asp-116. The putative active site residues involved in self-cleavage were identified; point mutants were produced and characterized structurally and in a biochemical assay. Numerical simulations utilizing molecular dynamics and hybrid quantum/classical calculations suggest a mechanism involving activation of a water molecule coordinated by a catalytic aspartic acid.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Tingting; Chang, Chin -Yuan; Lohman, Jeremy R.

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us tomore » propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. In conclusion, these findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.« less

Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane

PubMed Central

Merlino, Giuseppe; Marzorati, Massimo; Rizzi, Aurora; Lavazza, Davide; de Ferra, Francesca; Carpani, Giovanna

2015-01-01

The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales. Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community. PMID:26273600

Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

PubMed Central

Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

2011-01-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

Affinity cleavage at the putative metal-binding site of pigeon liver malic enzyme by the Fe(2+)-ascorbate system.

PubMed

Wei, C H; Chou, W Y; Huang, S M; Lin, C C; Chang, G G

1994-06-28

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells

PubMed Central

Camp, J. Gray; Weiser, Matthew; Cocchiaro, Jordan L.; Kingsley, David M.; Furey, Terrence S.; Sheikh, Shehzad Z.; Rawls, John F.

2017-01-01

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology. PMID:28850571

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses.

PubMed

Komatsu, Ken; Hirata, Hisae; Fukagawa, Takako; Yamaji, Yasuyuki; Okano, Yukari; Ishikawa, Kazuya; Adachi, Tatsushi; Maejima, Kensaku; Hashimoto, Masayoshi; Namba, Shigetou

2012-07-01

The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution. Copyright © 2012 Elsevier B.V. All rights reserved.

Genome-Wide SNP Discovery, Genotyping and Their Preliminary Applications for Population Genetic Inference in Spotted Sea Bass (Lateolabrax maculatus)

PubMed Central

Wang, Juan; Xue, Dong-Xiu; Zhang, Bai-Dong; Li, Yu-Long; Liu, Bing-Jian; Liu, Jin-Xian

2016-01-01

Next-generation sequencing and the collection of genome-wide single-nucleotide polymorphisms (SNPs) allow identifying fine-scale population genetic structure and genomic regions under selection. The spotted sea bass (Lateolabrax maculatus) is a non-model species of ecological and commercial importance and widely distributed in northwestern Pacific. A total of 22 648 SNPs was discovered across the genome of L. maculatus by paired-end sequencing of restriction-site associated DNA (RAD-PE) for 30 individuals from two populations. The nucleotide diversity (π) for each population was 0.0028±0.0001 in Dandong and 0.0018±0.0001 in Beihai, respectively. Shallow but significant genetic differentiation was detected between the two populations analyzed by using both the whole data set (FST = 0.0550, P < 0.001) and the putatively neutral SNPs (FST = 0.0347, P < 0.001). However, the two populations were highly differentiated based on the putatively adaptive SNPs (FST = 0.6929, P < 0.001). Moreover, a total of 356 SNPs representing 298 unique loci were detected as outliers putatively under divergent selection by FST-based outlier tests as implemented in BAYESCAN and LOSITAN. Functional annotation of the contigs containing putatively adaptive SNPs yielded hits for 22 of 55 (40%) significant BLASTX matches. Candidate genes for local selection constituted a wide array of functions, including binding, catalytic and metabolic activities, etc. The analyses with the SNPs developed in the present study highlighted the importance of genome-wide genetic variation for inference of population structure and local adaptation in L. maculatus. PMID:27336696

Genome-Wide SNP Discovery, Genotyping and Their Preliminary Applications for Population Genetic Inference in Spotted Sea Bass (Lateolabrax maculatus).

PubMed

Wang, Juan; Xue, Dong-Xiu; Zhang, Bai-Dong; Li, Yu-Long; Liu, Bing-Jian; Liu, Jin-Xian

2016-01-01

Next-generation sequencing and the collection of genome-wide single-nucleotide polymorphisms (SNPs) allow identifying fine-scale population genetic structure and genomic regions under selection. The spotted sea bass (Lateolabrax maculatus) is a non-model species of ecological and commercial importance and widely distributed in northwestern Pacific. A total of 22 648 SNPs was discovered across the genome of L. maculatus by paired-end sequencing of restriction-site associated DNA (RAD-PE) for 30 individuals from two populations. The nucleotide diversity (π) for each population was 0.0028±0.0001 in Dandong and 0.0018±0.0001 in Beihai, respectively. Shallow but significant genetic differentiation was detected between the two populations analyzed by using both the whole data set (FST = 0.0550, P < 0.001) and the putatively neutral SNPs (FST = 0.0347, P < 0.001). However, the two populations were highly differentiated based on the putatively adaptive SNPs (FST = 0.6929, P < 0.001). Moreover, a total of 356 SNPs representing 298 unique loci were detected as outliers putatively under divergent selection by FST-based outlier tests as implemented in BAYESCAN and LOSITAN. Functional annotation of the contigs containing putatively adaptive SNPs yielded hits for 22 of 55 (40%) significant BLASTX matches. Candidate genes for local selection constituted a wide array of functions, including binding, catalytic and metabolic activities, etc. The analyses with the SNPs developed in the present study highlighted the importance of genome-wide genetic variation for inference of population structure and local adaptation in L. maculatus.

Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

PubMed

Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

2018-06-01

To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

Microscopic characterization of orchid mycorrhizal fungi: Scleroderma as a putative novel orchid mycorrhizal fungus of Vanilla in different crop systems.

PubMed

González-Chávez, Ma Del Carmen A; Torres-Cruz, Terry J; Sánchez, Samantha Albarrán; Carrillo-González, Rogelio; Carrillo-López, Luis Manuel; Porras-Alfaro, Andrea

2018-02-01

Vanilla is an orchid of economic importance widely cultivated in tropical regions and native to Mexico. We sampled three species of Vanilla (V. planifolia, V. pompona, and V. insignis) in different crop systems. We studied the effect of crop system on the abundance, type of fungi, and quality of pelotons found in the roots using light and electron microscopy and direct sequencing of mycorrhizal structures. Fungi were identified directly from pelotons obtained from terrestrial roots of vanilla plants in the flowering stage. Root samples were collected from plants in crop systems located in the Totonacapan area in Mexico (states of Puebla and Veracruz). DNA was extracted directly from 40 pelotons and amplified using ITS rRNA sequencing. Peloton-like structures were observed, presenting a combination of active pelotons characterized by abundant hyphal coils and pelotons in various stages of degradation. The most active pelotons were observed in crop systems throughout living tutors (host tree) in comparison with roots collected from dead or artificial tutors. Fungi identified directly from pelotons included Scleroderma areolatum, a common ectomycorrhizal fungus that has not been reported as a mycorrhizal symbiont in orchids. Direct amplification of pelotons also yielded common plant pathogens, including Fusarium and Pyrenophora seminiperda, especially in those sites with low colonization rates, and where large numbers of degraded pelotons were observed. This research reports for the first time the potential colonization of Vanilla by Scleroderma, as a putative orchid mycorrhizal symbiont in four sites in Mexico and the influence of crop system on mycorrhizal colonization on this orchid.

A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions.

PubMed

Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Hisano, Tamao; Abe, Hideki; Tsuge, Takeharu

2015-06-01

Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

PubMed Central

Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

2009-01-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

PubMed

Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

2009-09-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

PubMed Central

Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

2013-01-01

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

PubMed

Azad, Abul Kalam; Katsuhara, Maki; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2008-08-01

We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

PubMed

Thom, R; Dixon, D P; Edwards, R; Cole, D J; Lapthorn, A J

2001-05-18

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed. Copyright 2001 Academic Press.

Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity.

PubMed Central

Yamamoto-Katayama, S; Sato, A; Ariyoshi, M; Suyama, M; Ishihara, K; Hirano, T; Nakamura, H; Morikawa, K; Jingami, H

2001-01-01

Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate. In mammals ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus-insect-cell system. In this study, we have purified the sBST-1, and it migrated as two major bands by SDS/PAGE, suggesting that it is post-translationally modified. BST-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1-N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies. PMID:11439087

Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium.

PubMed Central

Langin, D; Laurell, H; Holst, L S; Belfrage, P; Holm, C

1993-01-01

The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis. Images Fig. 5 PMID:8506334

MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.

2004-10-28

We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33756a

Stress-Responsive Mitogen-Activated Protein Kinases Interact with the EAR Motif of a Poplar Zinc Finger Protein and Mediate Its Degradation through the 26S Proteasome1[W][OA

PubMed Central

Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T.; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand

2011-01-01

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors. PMID:21873571

Investigating the Differences in the Total and Active Microbial Community of Mid-Atlantic Ridge Sediments

NASA Astrophysics Data System (ADS)

Sobol, M. S.; Zinke, L. A.; Orcutt, B.; Mills, H. J.; Edwards, K. J.; Girguis, P. R.; Reese, B. K.

2016-02-01

Microbes in the marine deep subsurface are key mediators of many geochemical cycles. It is important to understand how microbial communities and the diversity of those communities impacts geochemical cycling. Sediment cores were collected from IODP (Integrated Ocean Drilling Program) Expedition 336 to the western flank of the mid-Atlantic ridge also referred to as North Pond. The dissolved oxygen concentration decreased with depth for 60-70 mbsf, followed by a sharp increase in oxygen until it terminated at the basement. The 16S rRNA genes (DNA) and transcripts (RNA) were extracted simultaneously using a method designed by Reese et al. (2013) to differentiate between the total and active microbial community structures, respectively, as well as correlate the putative metabolism with the geochemistry. We observed many differences between the active and total communities. Sequences most closely related to Cyanobacteria were found to dominate the total community at both sites, but were found in small numbers in the active community. The most abundant phyla in the active community were Alphaproteobacteria, which suggests that they may have high activity even though the abundance was not as great in the total community. This suggests that, even in small numbers, bacteria are capable of contributing greatly to their environment. Principal Component Analysis (PCA) and Singular Value Decomposition (SVD) showed that iron-reducing bacteria in the active (RNA) community correlated strongly with solid phase iron oxides. SVD also showed that the putative nitrate reducers in the active community were found in greater abundance where porewater NO3- and NO2- total concentrations were elevated. Overall, the active (RNA) community correlated significantly with the geochemistry whereas the total (DNA) community did not. Therefore, RNA analysis yields a more accurate representation of how microbial communities impact geochemical cycling.

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

PubMed Central

Sammond, Deanne W.; Payne, Christina M.; Brunecky, Roman; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions. PMID:23139804

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bodea, Smaranda; Funk, Michael A.; Balskus, Emily P.

We report that deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structuralmore » data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. Lastly, this work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production.« less

Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

PubMed

Hwang, D C; Kim, D H; Lee, J S; Kang, B H; Han, J; Kim, W; Song, B D; Choi, K Y

2000-10-31

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus.

PubMed

Ferro, Ana Margarida; Ramos, Patrícia; Guerra, Ângela; Parreira, Paula; Brás, Teresa; Guerreiro, Olinda; Jerónimo, Eliana; Capel, Carmen; Capel, Juan; Yuste-Lisbona, Fernando J; Duarte, Maria F; Lozano, Rafael; Oliveira, M Margarida; Gonçalves, Sónia

2018-04-01

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

EARLY BUD-BREAK 1 (EBB1) is a regulator of release from seasonal dormancy in poplar trees

PubMed Central

Yordanov, Yordan S.; Ma, Cathleen; Strauss, Steven H.; Busov, Victor B.

2014-01-01

Trees from temperate latitudes transition between growth and dormancy to survive dehydration and freezing stress during winter months. We used activation tagging to isolate a dominant mutation affecting release from dormancy and identified the corresponding gene EARLY BUD-BREAK 1 (EBB1). We demonstrate through positioning of the tag, expression analysis, and retransformation experiments that EBB1 encodes a putative APETALA2/Ethylene responsive factor transcription factor. Transgenic up-regulation of the gene caused early bud-flush, whereas down-regulation delayed bud-break. Native EBB1 expression was highest in actively growing apices, undetectable during the dormancy period, but rapidly increased before bud-break. The EBB1 transcript was localized in the L1/L2 layers of the shoot meristem and leaf primordia. EBB1-overexpressing transgenic plants displayed enlarged shoot meristems, open and poorly differentiated buds, and a higher rate of cell division in the apex. Transcriptome analyses of the EBB1 transgenics identified 971 differentially expressed genes whose expression correlated with the EBB1 expression changes in the transgenic plants. Promoter analysis among the differentially expressed genes for the presence of a canonical EBB1-binding site identified 65 putative target genes, indicative of a broad regulatory context of EBB1 function. Our results suggest that EBB1 has a major and integrative role in reactivation of meristem activity after winter dormancy. PMID:24951507

A site-directed mutagenesis analysis of tNOX functional domains

NASA Technical Reports Server (NTRS)

Chueh, Pin-Ju; Morre, Dorothy M.; Morre, D. James

2002-01-01

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

Quantitative proteome-based systematic identification of SIRT7 substrates.

PubMed

Zhang, Chaohua; Zhai, Zichao; Tang, Ming; Cheng, Zhongyi; Li, Tingting; Wang, Haiying; Zhu, Wei-Guo

2017-07-01

SIRT7 is a class III histone deacetylase that is involved in numerous cellular processes. Only six substrates of SIRT7 have been reported thus far, so we aimed to systematically identify SIRT7 substrates using stable-isotope labeling with amino acids in cell culture (SILAC) coupled with quantitative mass spectrometry (MS). Using SIRT7 +/+ and SIRT7 -/- mouse embryonic fibroblasts as our model system, we identified and quantified 1493 acetylation sites in 789 proteins, of which 261 acetylation sites in 176 proteins showed ≥2-fold change in acetylation state between SIRT7 -/- and SIRT7 +/+ cells. These proteins were considered putative SIRT7 substrates and were carried forward for further analysis. We then validated the predictive efficiency of the SILAC-MS experiment by assessing substrate acetylation status in vitro in six predicted proteins. We also performed a bioinformatic analysis of the MS data, which indicated that many of the putative protein substrates were involved in metabolic processes. Finally, we expanded our list of candidate substrates by performing a bioinformatics-based prediction analysis of putative SIRT7 substrates, using our list of putative substrates as a positive training set, and again validated a subset of the proteins in vitro. In summary, we have generated a comprehensive list of SIRT7 candidate substrates. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Putative lung adenocarcinoma with epidermal growth factor receptor mutation presenting as carcinoma of unknown primary site

PubMed Central

Yamasaki, Masahiro; Funaishi, Kunihiko; Saito, Naomi; Sakano, Ayaka; Fujihara, Megumu; Daido, Wakako; Ishiyama, Sayaka; Deguchi, Naoko; Taniwaki, Masaya; Ohashi, Nobuyuki; Hattori, Noboru

2018-01-01

Abstract Rationale: Only a few cases of putative lung adenocarcinoma presenting as carcinoma of unknown primary site (CUP) with epidermal growth factor receptor (EGFR) mutation have been reported, and the efficacy of EGFR-tyrosine kinase inhibitors (TKIs) for these cases is unclear. Patient concerns and diagnoses: A 67-year-old man complained of paresis of the right lower extremity, dysarthria, and memory disturbance. Computed tomography and magnetic resonance imaging showed multiple brain tumors with brain edema and swelling of the left supraclavicular, mediastinal, and upper abdominal lymph nodes. Moreover, a metastatic duodenal tumor was detected via upper gastrointestinal endoscopy examination. The biopsy specimen of the lesion was examined and was diagnosed as adenocarcinoma with CK7 and TTF-1 positivity. Finally, the case was diagnosed as EGFR mutation-positive putative lung adenocarcinoma presenting as CUP. Interventions and outcomes: Oral erlotinib, an EGFR-TKI, was administered at 150 mg daily. Five weeks later, the brain lesions and several swollen lymph nodes showed marked improvement, and the symptoms of the patient also improved. Three months later, the duodenal lesion was undetected on upper gastrointestinal endoscopy. After an 8-month follow-up, the patient was well with no disease progression. Lessons: Putative lung adenocarcinoma presenting as CUP may have EGFR mutation, and EGFR-TKI therapy may be effective for such malignancy. PMID:29443782

Regulation of TNF-Related Apoptosis-Inducing Ligand Signaling by Glycosylation

PubMed Central

2018-01-01

Tumor necrosis-factor related apoptosis-inducing ligand, also known as TRAIL or APO2L (Apo-2 ligand), is a cytokine of the TNF superfamily acknowledged for its ability to trigger selective apoptosis in tumor cells while being relatively safe towards normal cells. Its binding to its cognate agonist receptors, namely death receptor 4 (DR4) and/or DR5, can induce the formation of a membrane-bound macromolecular complex, coined DISC (death-signaling inducing complex), necessary and sufficient to engage the apoptotic machinery. At the very proximal level, TRAIL DISC formation and activation of apoptosis is regulated both by antagonist receptors and by glycosylation. Remarkably, though, despite the fact that all membrane-bound TRAIL receptors harbor putative glycosylation sites, only pro-apoptotic signaling through DR4 and DR5 has, so far, been found to be regulated by N- and O-glycosylation, respectively. Because putative N-glycosylation sequons and O-glycosylation sites are also found and conserved in all these receptors throughout all animal species (in which these receptors have been identified), glycosylation is likely to play a more prominent role than anticipated in regulating receptor/receptor interactions or trafficking, ultimately defining cell fate through TRAIL stimulation. This review aims to present and discuss these emerging concepts, the comprehension of which is likely to lead to innovative anticancer therapies. PMID:29498673

Occurrence of putative virulence genes on Arcobacter butzleri isolated from three different environmental sites throughout the dairy chain.

PubMed

Piva, S; Gariano, G R; Bonilauri, P; Giacometti, F; Decastelli, L; Florio, D; Massella, E; Serraino, A

2017-04-01

This comparative study investigated the occurrence of cadF, cj1349, ciaB, pldA, tlyA, hecA, hecB, mviN, irgA and IroE genes in 212 Arcobacter butzleri isolated from three different environmental sites linked to the dairy chain (farms, industrial and artisanal dairy plants) located in three Italian regions (Lombardy, Emilia-Romagna and Calabria). According to the presence of these genes, different pathotypes (P-types) were determined. The main genes detected were ciaB, mviN, tlyA, cj1349, pldA and cadF, while the least common genes were iroE, hecA, hecB and irgA. TlyA, irgA, hecA, hecB and iroE, which were significantly more frequent in isolates recovered in industrial dairy plants. Twelve P-types were detected. The occurrence of the most frequently detected P-types (P-types 1, 2, 3 and 5) differed significantly (P < 0·001) in relation to both the environmental site and geographical area of isolation. The highest diversity in P-types was observed in industrial dairy plants and in the Calabria region. The results of this study show a correlation between the occurrence of putative virulence genes and virulence genotype variability depending on the environmental site and geographical origin of the isolates. The present study provides insights into the similar distribution of putative virulence genes in a dairy chain and other sources' isolates and also into a geographical distribution of some P-types. We have shown that industrial dairy plants may represent an environmental site favouring a selection of the isolates with a higher pathogenetic pattern. © 2017 The Society for Applied Microbiology.

Characterization of Member of DUF1888 Protein Family, Self-cleaving and Self-assembling Endopeptidase*

PubMed Central

Osipiuk, Jerzy; Mulligan, Rory; Bargassa, Monireh; Hamilton, John E.; Cunningham, Mark A.; Joachimiak, Andrzej

2012-01-01

The crystal structure of SO1698 protein from Shewanella oneidensis was determined by a SAD method and refined to 1.57 Å. The structure is a β sandwich that unexpectedly consists of two polypeptides; the N-terminal fragment includes residues 1–116, and the C-terminal one includes residues 117–125. Electron density also displayed the Lys-98 side chain covalently linked to Asp-116. The putative active site residues involved in self-cleavage were identified; point mutants were produced and characterized structurally and in a biochemical assay. Numerical simulations utilizing molecular dynamics and hybrid quantum/classical calculations suggest a mechanism involving activation of a water molecule coordinated by a catalytic aspartic acid. PMID:22493430

Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

PubMed

Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

2016-01-01

Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

PubMed

Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

2016-10-01

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.

PubMed

Chae, J P; Valeriano, V D; Kim, G-B; Kang, D-K

2013-01-01

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l(-1) isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni(2+) -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes. © 2012 The Society for Applied Microbiology.

Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1.

PubMed

Stanislauskienė, Rūta; Kutanovas, Simonas; Kalinienė, Laura; Bratchikov, Maksim; Meškys, Rolandas

2018-06-25

An inducible promoter region, P TTMP (tetramethylpyrazine [TTMP]), has been identified upstream of the tpdABC operon, which contains the genes required for the initial degradation of 2,3,5,6-tetramethylpyrazine in Rhodococcus jostii TMP1 bacteria. In this work, the promoter region was fused with the gene for the enhanced green fluorescent protein (EGFP) to investigate the activity of P TTMP by measuring the fluorescence of bacteria. The highest promoter activity was observed when bacteria were grown in a nutrient broth (NB) medium supplemented with 5 mM 2,3,5,6-tetramethylpyrazine for 48 h. Using a primer extension reaction, two transcriptional start sites for tpdA were identified, and the putative −35 and −10 promoter motifs were determined. The minimal promoter along with two 15 bp long direct repeats and two 7 bp inverted sequences were identified. Also, the influence of the promoter elements on the activity of P TTMP were determined using site-directed mutagenesis. Furthermore, P TTMP was shown to be induced by pyrazine derivatives containing methyl groups in the 2- and 5-positions of the heterocyclic ring, in the presence of the LuxR family transcriptional activator TpdR.

Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum.

PubMed

Chen, Zhuo; Luo, Ling; Chen, Runfa; Hu, Hanhua; Pan, Yufang; Jiang, Haibo; Wan, Xia; Jin, Hu; Gong, Yangmin

2018-03-01

N ε -lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD + -dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of activation and processing of the cystic fibrosis transmembrane conductance regulator (CFTR) by a complex electrostatic interaction between the regulatory domain and cytoplasmic loop 3.

PubMed

Wang, Guangyu; Duan, Dayue Darrel

2012-11-23

NEG2 regulates CFTR gating but the mechanism is unknown. A putative NEG2-CL3 electrostatic attraction, possibly weakened by Arg-764/Arg-766 of the R domain, prohibited CFTR activation. A charge exchange between NEG2 and CL3 caused misprocessing. Electrostatic regulation of CFTR activation and processing may be asymmetric at the CL3-R interface. The CL3-R interface is optimally designed for multiple regulations of CFTR functions. NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

Mutational Analysis of Escherichia coli MoeA: Two Functional Activities Map to the Active Site Cleft

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nichols,J.; Xiang, S.; Schindelin, H.

2007-01-01

The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe amore » detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA{sup -} crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.« less

Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

PubMed Central

Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kumar, Konidala Kranthi; Bhaskar, Matcha

2015-01-01

Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis. PMID:25834405

VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

PubMed

Argemi, X; Prévost, G; Riegel, P; Keller, D; Meyer, N; Baldeyrou, M; Douiri, N; Lefebvre, N; Meghit, K; Ronde Oustau, C; Christmann, D; Cianférani, S; Strub, J M; Hansmann, Y

2017-05-01

Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.).

PubMed

Takakura, Y; Ito, T; Saito, H; Inoue, T; Komari, T; Kuwata, S

2000-04-01

A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils. Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs. OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants. The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice. The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding. The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate. Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family. The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated. The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis. The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation. The flower-predominant gene expression was identified also in the transgenic rice plants. The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants. The possible functions of OsChia 1;175 are discussed.

Structure and Activity of the Flagellar Rotor Protein FliY

PubMed Central

Sircar, Ria; Greenswag, Anna R.; Bilwes, Alexandrine M.; Gonzalez-Bonet, Gabriela; Crane, Brian R.

2013-01-01

Rotating flagella propel bacteria toward favorable environments. Sense of rotation is determined by the intracellular response regulator CheY, which when phosphorylated (CheY-P) interacts directly with the flagellar motor. In many different types of bacteria, the CheC/CheX/FliY (CXY) family of phosphatases terminates the CheY-P signal. Unlike CheC and CheX, FliY is localized in the flagellar switch complex, which also contains the stator-coupling protein FliG and the target of CheY-P, FliM. The 2.5 Å resolution crystal structure of the FliY catalytic domain from Thermotoga maritima bears strong resemblance to the middle domain of FliM. Regions of FliM that mediate contacts within the rotor compose the phosphatase active sites in FliY. Despite the similarity between FliY and FliM, FliY does not bind FliG and thus is unlikely to be a substitute for FliM in the center of the switch complex. Solution studies indicate that FliY dimerizes through its C-terminal domains, which resemble the Escherichia coli switch complex component FliN. FliY differs topologically from the E. coli chemotaxis phosphatase CheZ but appears to utilize similar structural motifs for CheY dephosphorylation in close analogy to CheX. Recognition properties and phosphatase activities of site-directed mutants identify two pseudosymmetric active sites in FliY (Glu35/Asn38 and Glu132/Asn135), with the second site (Glu132/Asn135) being more active. A putative N-terminal CheY binding domain conserved with FliM is not required for binding CheY-P or phosphatase activity. PMID:23532838

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

PubMed

Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

2015-10-01

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.

Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway

PubMed Central

Yamamoto, Katsuyoshi

2015-01-01

To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor. PMID:26598606

Identification of Candidate Transcription Factor Binding Sites in the Cattle Genome

PubMed Central

Bickhart, Derek M.; Liu, George E.

2013-01-01

A resource that provides candidate transcription factor binding sites (TFBSs) does not currently exist for cattle. Such data is necessary, as predicted sites may serve as excellent starting locations for future omics studies to develop transcriptional regulation hypotheses. In order to generate this resource, we employed a phylogenetic footprinting approach—using sequence conservation across cattle, human and dog—and position-specific scoring matrices to identify 379,333 putative TFBSs upstream of nearly 8000 Mammalian Gene Collection (MGC) annotated genes within the cattle genome. Comparisons of our predictions to known binding site loci within the PCK1, ACTA1 and G6PC promoter regions revealed 75% sensitivity for our method of discovery. Additionally, we intersected our predictions with known cattle SNP variants in dbSNP and on the Illumina BovineHD 770k and Bos 1 SNP chips, finding 7534, 444 and 346 overlaps, respectively. Due to our stringent filtering criteria, these results represent high quality predictions of putative TFBSs within the cattle genome. All binding site predictions are freely available at http://bfgl.anri.barc.usda.gov/BovineTFBS/ or http://199.133.54.77/BovineTFBS. PMID:23433959

Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology*

PubMed Central

Ramírez-Escudero, Mercedes; del Pozo, Mercedes V.; Marín-Navarro, Julia; González, Beatriz; Golyshin, Peter N.; Polaina, Julio; Ferrer, Manuel; Sanz-Aparicio, Julia

2016-01-01

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials. PMID:27679487

Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

PubMed

Shinkai, Akeo; Kira, Satoshi; Nakagawa, Noriko; Kashihara, Aiko; Kuramitsu, Seiki; Yokoyama, Shigeyuki

2007-05-01

The extremely thermophilic bacterium Thermus thermophilus HB8, which belongs to the phylum Deinococcus-Thermus, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named T. thermophilus CRP is highly homologous to the CRP family proteins from the phyla Firmicutes, Actinobacteria, and Cyanobacteria, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this strain, which did not drastically fluctuate during cultivation in a rich medium. The expression of several genes was altered upon disruption of the T. thermophilus CRP gene. We found six CRP-cAMP-dependent promoters in in vitro transcription assays involving DNA fragments containing the upstream regions of the genes exhibiting decreased expression in the CRP disruptant, indicating that the CRP is a transcriptional activator. The consensus T. thermophilus CRP-binding site predicted upon nucleotide sequence alignment is 5'-(C/T)NNG(G/T)(G/T)C(A/C)N(A/T)NNTCACAN(G/C)(G/C)-3'. This sequence is unique compared with the known consensus binding sequences of CRP family proteins. A putative -10 hexamer sequence resides at 18 to 19 bp downstream of the predicted T. thermophilus CRP-binding site. The CRP-regulated genes found in this study comprise clustered regularly interspaced short palindromic repeat (CRISPR)-associated (cas) ones, and the genes of a putative transcriptional regulator, a protein containing the exonuclease III-like domain of DNA polymerase, a GCN5-related acetyltransferase homolog, and T. thermophilus-specific proteins of unknown function. These results suggest a role for cAMP signal transduction in T. thermophilus and imply the T. thermophilus CRP is a cAMP-responsive regulator.

Neurotoxic properties of the anabolic androgenic steroids nandrolone and methandrostenolone in primary neuronal cultures.

PubMed

Caraci, Filippo; Pistarà, V; Corsaro, A; Tomasello, Flora; Giuffrida, Maria Laura; Sortino, Maria Angela; Nicoletti, Ferdinando; Copani, Agata

2011-04-01

Anabolic-androgenic steroid (AAS) abuse is associated with multiple neurobehavioral disturbances. The sites of action and the neurobiological sequels of AAS abuse are unclear at present. We investigated whether two different AASs, nandrolone and methandrostenolone, could affect neuronal survival in culture. The endogenous androgenic steroid testosterone was used for comparison. Both testosterone and nandrolone were neurotoxic at micromolar concentrations, and their effects were prevented by blockade of androgen receptors (ARs) with flutamide. Neuronal toxicity developed only over a 48-hr exposure to the steroids. The cell-impermeable analogues testosterone-BSA and nandrolone-BSA, which preferentially target membrane-associated ARs, were also neurotoxic in a time-dependent and flutamide-sensitive manner. Testosterone-BSA and nandrolone-BSA were more potent than their parent compounds, suggesting that membrane-associated ARs were the relevant sites for the neurotoxic actions of the steroids. Unlike testosterone and nandrolone, toxicity by methandrostenolone and methandrostenolone-BSA was insensitive to flutamide, but it was prevented by the glucocorticoid receptor (GR) antagonist RU-486. Methandrostenolone-BSA was more potent than the parent compound, suggesting that its toxicity relied on the preferential activation of putative membrane-associated GRs. Consistently with the evidence that membrane-associated GRs can mediate rapid effects, a brief challenge with methandrostenolone-BSA was able to promote neuronal toxicity. Activation of putative membrane steroid receptors by nontoxic (nanomolar) concentrations of either nandrolone-BSA or methandrostenolone-BSA became sufficient to increase neuronal susceptibility to the apoptotic stimulus provided by β-amyloid (the main culprit of AD). We speculate that AAS abuse might facilitate the onset or progression of neurodegenerative diseases not usually linked to drug abuse. Copyright © 2011 Wiley-Liss, Inc.

PUTATIVE GENE PROMOTER SEQUENCES IN THE CHLORELLA VIRUSES

PubMed Central

Fitzgerald, Lisa A.; Boucher, Philip T.; Yanai-Balser, Giane; Suhre, Karsten; Graves, Michael V.; Van Etten, James L.

2008-01-01

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication. PMID:18768195

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions

PubMed Central

Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae

2017-01-01

ABSTRACT There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima. We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn2+. In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the characterization and functional annotation of a putative nonphosphorylated sugar 3-epimerase from a hyperthermophilic bacterium. Furthermore, we determined its crystal structures in complex with divalent metal ions and l-erythrulose, highlighting its metal-dependent, bifunctional, sugar-isomerizing activity. This hyperthermophilic R3E exhibited d-erythrose/d-threose isomerase activity, with structural features near the substrate-binding site distinct from those of its mesophilic counterparts. Moreover, this metalloenzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose at 70°C. Therefore, TM0416 can be functionally classified as a novel type of promiscuous R3E with a potential for the production of rare sugars for the food and pharmaceutical industries. PMID:28258150

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

PubMed

Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sung Haeng; Lee, Dong-Woo

2017-05-15

There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn 2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the characterization and functional annotation of a putative nonphosphorylated sugar 3-epimerase from a hyperthermophilic bacterium. Furthermore, we determined its crystal structures in complex with divalent metal ions and l-erythrulose, highlighting its metal-dependent, bifunctional, sugar-isomerizing activity. This hyperthermophilic R3E exhibited d-erythrose/d-threose isomerase activity, with structural features near the substrate-binding site distinct from those of its mesophilic counterparts. Moreover, this metalloenzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose at 70°C. Therefore, TM0416 can be functionally classified as a novel type of promiscuous R3E with a potential for the production of rare sugars for the food and pharmaceutical industries. Copyright © 2017 American Society for Microbiology.

Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access.

PubMed

Fritz, Timothy A; Liu, Lu; Finer-Moore, Janet S; Stroud, Robert M

2002-06-04

Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.

Structural and Functional Analysis of the Pyocyanin Biosynthetic Protein PhzM from Pseudomonas aeruginosa†‡

PubMed Central

Parsons, James F.; Greenhagen, Bryan T.; Shi, Katherine; Calabrese, Kelly; Robinson, Howard; Ladner, Jane E.

2008-01-01

Pyocyanin is a biologically active phenazine produced by the human pathogen Pseudomonas aeruginosa. It is thought to endow P. aeruginosa with a competitive growth advantage in colonized tissue and is also thought to be a virulence factor in diseases such as cystic fibrosis and AIDS where patients are commonly infected by pathogenic Pseudomonads due to their immunocompromised state. Pyocyanin is also a chemically interesting compound due to its unusual oxidation-reduction activity. Phenazine-1-carboxylic acid, the precursor to the bioactive phenazines, is synthesized from chorismic acid by enzymes encoded in a seven-gene cistron in Pseudomonas aeruginosa and in other Pseudomonads. Phenzine-1-carboxylic acid is believed to be converted to pyocyanin by the sequential actions of the putative S-adenosylmethionine dependent N-methyltransferase PhzM and the putative flavin-dependent hydroxylase PhzS. Here we report the 1.8 Å crystal structure of PhzM solved by single anomalous dispersion. Unlike many methyltransferases, PhzM is a dimer in solution. The 36 kDa PhzM polypeptide folds into three domains. The C-terminal domain exhibits the α/β-hydrolase fold typical of small molecule methyltransferases. Two smaller N-terminal domains form much of the dimer interface. Structural alignments with known methyltransferases show that PhzM is most similar to the plant O-methyltransferases that are characterized by an unusual intertwined dimer interface. The structure of PhzM contains no ligands and the active site is open and solvent exposed when compared to structures of similar enzymes. In vitro experiments using purified PhzM alone demonstrate that it has little or no ability to methylate phenzine-1-carboxylic acid. However, when the putative hydroxylase PhzS is included, pyocyanin is readily produced. This observation suggests that a mechanism has evolved in P. aeruginosa that ensures efficient production of pyocyanin by preventing the formation and release of an unstable and potentially deleterious intermediate. PMID:17253782

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

PubMed

Toporkova, Yana Y; Gorina, Svetlana S; Bessolitsyna, Elena K; Smirnova, Elena O; Fatykhova, Valeria S; Brühlmann, Fredi; Ilyina, Tatiana M; Mukhtarova, Lucia S; Grechkin, Alexander N

2018-04-01

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs. Copyright © 2018 Elsevier B.V. All rights reserved.

Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karakas, E.; Simorowski, N; Furukawa, H

2009-01-01

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-freemore » and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.« less

Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

PubMed

Middleton, Adam J; Wright, Joshua D; Day, Catherine L

2017-11-10

Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1.

PubMed

Hamid, Azzmer Azzar Abdul; Hamid, Tengku Haziyamin Tengku Abdul; Wahab, Roswanira Abdul; Huyop, Fahrul

2015-03-01

The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

DOE PAGES

Huang, Tingting; Chang, Chin -Yuan; Lohman, Jeremy R.; ...

2016-10-01

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us tomore » propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. In conclusion, these findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.« less

A novel site-specific recombination system derived from bacteriophage phiMR11.

PubMed

Rashel, Mohammad; Uchiyama, Jumpei; Ujihara, Takako; Takemura, Iyo; Hoshiba, Hiroshi; Matsuzaki, Shigenobu

2008-04-04

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.

Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

PubMed

Kitchen, Sheila A; Bourdelais, Andrea J; Taylor, Alison R

2018-01-01

The potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na + ) and calcium (Ca 2+ ) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na + VGCs. We examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na + /Ca 2+ VGC of the non-toxic diatom Odontella sinensi s using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin's effect on voltage gated Na + /Ca 2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na + /Ca 2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na + VGCs, previously identified as the PbTx binding site in animals. Up to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na + /Ca 2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na + /Ca 2+ VGCs than the 4-domain eukaryote channels. Although membrane excitability and the kinetics of action potential currents were unaffected, the permeation of the channels underlying the diatom action potential was significantly altered in the presence of PbTx-3. However, at environmentally relevant concentrations the effects of PbTx- on diatom voltage activated currents and interference of cell signaling through this pathway may be limited. The relative insensitivity of phytoplankton VGCs may be due to divergence of site-5 (the putative PbTx binding site), and in some cases, such as O. sinensis , resistance to toxin effects may be because of evolutionary loss of the 4-domain eukaryote channel, while retaining a single domain bacterial-like VGC that can substitute in the generation of fast action potentials.

The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

PubMed Central

2010-01-01

Background The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function. Results A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. Conclusions We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression. PMID:20487545

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenberg, C.R.; Taylor, C.D.; Haworth, J.C.

The authors have discovered a single homoallelic nucleotide substitution as the putative cause of the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Previous linkage and haplotype analysis in this population suggested that a single mutational event was responsible for this autosomal recessive form of hypophosphatasia. The mutation is a guanosine-to-adenosine substitution at nucleotide position 1177 in exon 10 of the tissue nonspecific (liver/bone/kidney) alkaline phosphatase gene. This Gly[sup 317] [yields] Asp mutation segregates exclusively with the heterozygote phenotype previously assigned by biochemical testing (maximum combined lod score of 18.24 at [theta] = 0.00). This putative disease-causing mutation has notmore » been described in controls nor in other non-Mennonite probands with both lethal and nonlethal forms of hypophosphatasia studied to date. This Gly[sup 317] [yields] Asp mutation changes a polar glycine to an acidic aspartate at amino acid position 317 within the highly conserved active site region of the 507-amino-acid polypeptide. Carrier screening for this lethal mutation in a high-risk population is now feasible. 15 refs., 2 figs.« less

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis.

PubMed

Jing, Lan; Guo, Dandan; Hu, Wenjie; Niu, Xiaofan

2017-03-11

Many plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome. 908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen. This investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.

Structure-function studies on the active site of the coelenterazine-dependent luciferase from Renilla.

PubMed

Woo, Jongchan; Howell, Matthew H; von Arnim, Albrecht G

2008-04-01

Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the alpha/beta hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220--three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB--also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate.

Differential Sox10 Genomic Occupancy in Myelinating Glia

PubMed Central

Lopez-Anido, Camila; Sun, Guannan; Koenning, Matthias; Srinivasan, Rajini; Hung, Holly A.; Emery, Ben; Keles, Sunduz; Svaren, John

2015-01-01

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells, and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. PMID:25974668

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.

PubMed

Montgomery, H J; Romanov, V; Guillemette, J G

2000-02-18

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

PubMed

Waszak, Sebastian M; Kilpinen, Helena; Gschwind, Andreas R; Orioli, Andrea; Raghav, Sunil K; Witwicki, Robert M; Migliavacca, Eugenia; Yurovsky, Alisa; Lappalainen, Tuuli; Hernandez, Nouria; Reymond, Alexandre; Dermitzakis, Emmanouil T; Deplancke, Bart

2014-01-15

High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

Analysis of the site-specific integration system of the Streptomyces aureofaciens phage μ1/6.

PubMed

Farkašovská, Jarmila; Godány, Andrej

2012-03-01

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

PubMed

Bansal, Ankita; Kar, Debasish; Murugan, Rajagopal A; Mallick, Sathi; Dutta, Mouparna; Pandey, Satya Deo; Chowdhury, Chiranjit; Ghosh, Anindya S

2015-05-01

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities. © 2015 The Authors.

[Characterization of a recombinant aminopeptidase Lmo1711 from Listeria monocytogenes].

PubMed

He, Zhan; Wang, Hang; Han, Xiao; Ma, Tiantian; Hang, Yi; Yu, Huifei; Wei, Fangfang; Sun, Jing; Yang, Yongchun; Cheng, Changyong; Song, Houhui

2018-05-25

We aimed to obtain the recombinant aminopeptidase encoded by Listeria monocytogenes (L. monocytogenes) gene lmo1711, and characterized the enzyme. First, the amino acid sequences of Lmo1711 from L. monocytogenes EGD-e and its homologues in other microbial species were aligned and the putative active sites were analyzed. The putative model of Lmo1711 was constructed through the SWISS-MODEL Workspace. Then, the plasmid pET30a-Lmo1711 was constructed and transformed into E. coli for expression of the recombinant Lmo1711. The his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography. With the amino acid-p-nitroaniline as the substrate, Lmo1711 hydrolyzed the substrate to free p-nitroaniline monomers, whose absorbance measured at 405 nm reflected the aminopeptidase activity. The specificity of Lmo1711 to substrates was then examined by changing various substrates, and the effect of metal ions on the catalytic efficiency of this enzyme was further determined. Based on the bioinformatics data, Lmo1711 is a member of the M29 family aminopeptidases, containing a highly conserved catalytic motif (Glu-Glu-His-Tyr-His-Asp) with typical structure arrangements of the peptidase family. The recombinant Lmo1711 with a size of about 49.3 kDa exhibited aminopeptidase activity and had a selectivity to the substrates, with the highest degree of affinity for leucine-p-nitroaniline. Interestingly, the enzymatic activity of Lmo1711 can be activated by Cd²⁺, Zn²⁺, and is strongly stimulated by Co²⁺. We here, for the first time demonstrate that L. monocytogenes lmo1711 encodes a cobalt-activated aminopeptidase of M29 family.

Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance.

PubMed

Sporer, Abigail J; Beierschmitt, Christopher; Bendebury, Anastasia; Zink, Katherine E; Price-Whelan, Alexa; Buzzeo, Marisa C; Sanchez, Laura M; Dietrich, Lars E P

2018-05-01

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [L-(+)-lactate dehydrogenase

PubMed Central

Genet, R; Lederer, F

1990-01-01

Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM. No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase. Propionate is a competitive inhibitor, with a Ki of 28 mM. The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed. PMID:2178603

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

PubMed

Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

2006-05-19

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

Parallel synthesis of a series of potentially brain penetrant aminoalkyl benzoimidazoles.

PubMed

Micco, Iolanda; Nencini, Arianna; Quinn, Joanna; Bothmann, Hendrick; Ghiron, Chiara; Padova, Alessandro; Papini, Silvia

2008-03-01

Alpha7 agonists were identified via GOLD (CCDC) docking in the putative agonist binding site of an alpha7 homology model and a series of aminoalkyl benzoimidazoles was synthesised to obtain potentially brain penetrant drugs. The array was prepared starting from the reaction of ortho-fluoronitrobenzenes with a selection of diamines, followed by reduction of the nitro group to obtain a series of monoalkylated phenylene diamines. N,N'-Carbonyldiimidazole (CDI) mediated acylation, followed by a parallel automated work-up procedure, afforded the monoacylated phenylenediamines which were cyclised under acidic conditions. Parallel work-up and purification afforded the array products in good yields and purities with a robust parallel methodology which will be useful for other libraries. Screening for alpha7 activity revealed compounds with agonist activity for the receptor.

Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome.

PubMed

Nikolaev, Sergey I; Santoni, Federico; Vannier, Anne; Falconnet, Emilie; Giarin, Emanuela; Basso, Giuseppe; Hoischen, Alexander; Veltman, Joris A; Groet, Jurgen; Nizetic, Dean; Antonarakis, Stylianos E

2013-07-25

Some neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires "third-hit driver" mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC.

Identification of three protein disulfide isomerase members from Haemaphysalis longicornis tick.

PubMed

Liao, Min; Hatta, Takeshi; Umemiya, Rika; Huang, Penglong; Jia, Honglin; Gong, Haiyan; Zhou, Jinlin; Nishikawa, Yoshifumi; Xuan, Xuenan; Fujisaki, Kozo

2007-07-01

Three genes encoding putative protein disulfide isomerase (PDI) were isolated from the Haemaphysalis longicornis EST database and designed as HlPDI-1, HlPDI-2, and HlPDI-3. All three PDI genes contain two typical PDI active sites CXXC and encode putative 435, 499, and 488 amino acids, respectively. The recombinant proteins expressed in Escherichia coli all show PDI activities, and the activities were inhibited by a PDI-specific inhibitor, zinc bacitracin. Western blot analysis and real-time PCR revealed that three HlPDIs were present in all the developmental stages of the tick as well as in the midgut, salivary glands, ovary, hemolymph, and fatbody of adult female ticks, but the three genes were expressed at the highest level in the egg stage. HlPDI-1 is expressed primarily in the ovary and secondarily in the salivary glands. HlPDI-2 and HlPDI-3 are expressed primarily in the salivary gland, suggesting that the PDI genes are important for tick biology, especially for egg development, and that they play distinct roles in different tissues. Blood feeding induced significantly increased expression of HlPDI-1 and HlPDI-3 in both partially fed nymphs and adults. Babesia gibsoni-infected larval ticks expressed HlPDI-1 and HlPDI-3 2.0 and 4.0 times higher than uninfected normal larval ticks, respectively. The results indicate that HlPDI-1 and HlPDI-3 might be involved in tick blood feeding and Babesia parasite infection in ticks.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed

Sakoda, H; Imanaka, T

1992-02-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed Central

Sakoda, H; Imanaka, T

1992-01-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH. Images PMID:1735726

EROD activity measured in flatfish from the area of the Sea Empress oil spill.

PubMed

Kirby, M F; Neall, P; Tylor, T

1999-05-01

Dab (Limanda limanda) and plaice (Pleuronectes platessa) were collected at five stations near to the site of the Sea Empress oil spill within two weeks of the incident and a further fourteen stations three months after the spillage. Ethoxyresorufin-O-deethylase (EROD) activity was determined in the livers of the specimens to determine whether induction could be detected. Statistically significant inter-site differences in EROD levels in both species were demonstrated. Elevated levels of EROD activity in dab were found at the two stations nearest to the incident up to three months after the spill but no clear relationship to putative contaminant levels was determined. EROD levels in plaice showed a generally similar pattern of induction as in dab. Correlation of EROD levels with other variables showed that sexual maturity had the greatest influence on dab during the study period. The plaice specimens were sexually immature and, therefore, did not demonstrate a corresponding relationship. It was concluded that, for EROD monitoring purposes, fish should be sampled during their sexually inactive phase and that close attention needs to be paid to other variables (depth, temperature, GSI, length, influential contaminants etc.) when interpreting the results.

Structure, Regulation, and Putative Function of the Arginine Deiminase System of Streptococcus suis

PubMed Central

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative β-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions. PMID:16385025

Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

PubMed

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus.

PubMed

Sivakamavalli, Jeyachandran; Tripathi, Sunil Kumar; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

2015-01-01

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

PubMed

Halmillawewa, Anupama P; Restrepo-Córdoba, Marcela; Perry, Benjamin J; Yost, Christopher K; Hynes, Michael F

2016-02-01

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

Horizontal transfer of a large and highly toxic secondary metabolic gene cluster between fungi.

PubMed

Slot, Jason C; Rokas, Antonis

2011-01-25

Genes involved in intermediary and secondary metabolism in fungi are frequently physically linked or clustered. For example, in Aspergillus nidulans the entire pathway for the production of sterigmatocystin (ST), a highly toxic secondary metabolite and a precursor to the aflatoxins (AF), is located in a ∼54 kb, 23 gene cluster. We discovered that a complete ST gene cluster in Podospora anserina was horizontally transferred from Aspergillus. Phylogenetic analysis shows that most Podospora cluster genes are adjacent to or nested within Aspergillus cluster genes, although the two genera belong to different taxonomic classes. Furthermore, the Podospora cluster is highly conserved in content, sequence, and microsynteny with the Aspergillus ST/AF clusters and its intergenic regions contain 14 putative binding sites for AflR, the transcription factor required for activation of the ST/AF biosynthetic genes. Examination of ∼52,000 Podospora expressed sequence tags identified transcripts for 14 genes in the cluster, with several expressed at multiple life cycle stages. The presence of putative AflR-binding sites and the expression evidence for several cluster genes, coupled with the recent independent discovery of ST production in Podospora [1], suggest that this HGT event probably resulted in a functional cluster. Given the abundance of metabolic gene clusters in fungi, our finding that one of the largest known metabolic gene clusters moved intact between species suggests that such transfers might have significantly contributed to fungal metabolic diversity. PAPERFLICK: Copyright © 2011 Elsevier Ltd. All rights reserved.

Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass.

PubMed

Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

2016-03-15

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.

Design and synthesis of inositolphosphoglycan putative insulin mediators.

PubMed

López-Prados, Javier; Cuevas, Félix; Reichardt, Niels-Christian; de Paz, José-Luis; Morales, Ezequiel Q; Martín-Lomas, Manuel

2005-03-07

The binding modes of a series of molecules, containing the glucosamine (1-->6) myo-inositol structural motif, into the ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase using the same intermediates has been explored. The results from the biological evaluation of these molecules provide additional support to an insulin-mediated signalling system which involves the intermediacy of inositolphosphoglycans as putative insulin mediators.

Mutagenesis of the C2 domain of protein kinase C-alpha. Differential roles of Ca2+ ligands and membrane binding residues.

PubMed

Medkova, M; Cho, W

1998-07-10

The C2 domains of conventional protein kinase C (PKC) have been implicated in their Ca2+-dependent membrane binding. The C2 domain of PKC-alpha contains several Ca2+ ligands that bind multiple Ca2+ ions and other putative membrane binding residues. To understand the roles of individual Ca2+ ligands and protein-bound Ca2+ ions in the membrane binding and activation of PKC-alpha, we mutated five putative Ca2+ ligands (D187N, D193N, D246N, D248N, and D254N) and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration of PKC-alpha. Altered properties of these mutants indicate that individual Ca2+ ions and their ligands have different roles in the membrane binding and activation of PKC-alpha. The binding of Ca2+ to Asp187, Asp193, and Asp246 of PKC-alpha is important for the initial binding of protein to membrane surfaces. On the other hand, the binding of another Ca2+ to Asp187, Asp246, Asp248, and Asp254 induces the conformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp246 was shown to be most essential for both membrane binding and activation of PKC-alpha, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane binding residues (Trp245, Trp247, Arg249, and Arg252). Membrane binding and enzymatic properties of two double-site mutants (W245A/W247A and R249A/R252A) indicate that Arg249 and Arg252 are involved in electrostatic interactions of PKC-alpha with anionic membranes, whereas Trp245 and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of conventional PKC as a membrane docking unit as well as a module that triggers conformational changes to activate the protein.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, R.N.; Robinson, H.; Klauer, A. A.

The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonicalmore » helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases.« less

A Novel Collection of snRNA-Like Promoters with Tissue-Specific Transcription Properties

PubMed Central

Garritano, Sonia; Gigoni, Arianna; Costa, Delfina; Malatesta, Paolo; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2012-01-01

We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III. PMID:23109855

A novel collection of snRNA-like promoters with tissue-specific transcription properties.

PubMed

Garritano, Sonia; Gigoni, Arianna; Costa, Delfina; Malatesta, Paolo; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2012-01-01

We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III.

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

PubMed

Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

2015-03-02

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The 5' non-translated region of Varroa destructor virus 1 (genus Iflavirus): structure prediction and IRES activity in Lymantria dispar cells.

PubMed

Ongus, Juliette R; Roode, Els C; Pleij, Cornelis W A; Vlak, Just M; van Oers, Monique M

2006-11-01

Structure prediction of the 5' non-translated region (NTR) of four iflavirus RNAs revealed two types of potential internal ribosome entry site (IRES), which are discriminated by size and level of complexity, in this group of viruses. In contrast to the intergenic IRES of dicistroviruses, the potential 5' IRES structures of iflaviruses do not have pseudoknots. To test the activity of one of these, a bicistronic construct was made in which the 5' NTR of Varroa destructor virus 1 (VDV-1) containing a putative IRES was cloned in between two reporter genes, enhanced green fluorescent protein and firefly luciferase (Fluc). The presence of the 5' NTR of VDV-1 greatly enhanced the expression levels of the second reporter gene (Fluc) in Lymantria dispar Ld652Y cells. The 5' NTR was active in a host-specific manner, as it showed lower activity in Spodoptera frugiperda Sf21 cells and no activity in Drosophila melanogaster S2 cells.

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

IspE Inhibitors Identified by a Combination of In Silico and In Vitro High-Throughput Screening

PubMed Central

Tidten-Luksch, Naomi; Grimaldi, Raffaella; Torrie, Leah S.; Frearson, Julie A.; Hunter, William N.; Brenk, Ruth

2012-01-01

CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens. PMID:22563402

Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

NASA Astrophysics Data System (ADS)

Ibrahim, Ahmed M. A.; Kim, Yonggyun

2008-01-01

Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth.

PubMed

Goschzik, Tobias; Cremer, Harold; Gnanapragassam, Vinayaga S; Horstkorte, Rüdiger; Bork, Kaya; Diestel, Simone

2017-07-01

The cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

PubMed

Masukagami, Yumiko; Tivendale, Kelly Anne; Browning, Glenn Francis; Sansom, Fiona Margaret

2018-02-01

The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

Active site characterization and molecular cloning of Tenebrio molitor midgut trehalase and comments on their insect homologs.

PubMed

Gomez, Ana; Cardoso, Christiane; Genta, Fernando A; Terra, Walter R; Ferreira, Clélia

2013-08-01

The soluble midgut trehalase from Tenebrio molitor (TmTre1) was purified after several chromatographic steps, resulting in an enzyme with 58 kDa and pH optimum 5.3 (ionizing active groups in the free enzyme: pK(e1) = 3.8 ± 0.2 pK(e2) = 7.4 ± 0.2). The purified enzyme corresponds to the deduced amino acid sequence of a cloned cDNA (TmTre1-cDNA), because a single cDNA coding a soluble trehalase was found in the T. molitor midgut transcriptome. Furthermore, the mass of the protein predicted to be coded by TmTre1-cDNA agrees with that of the purified enzyme. TmTre1 has the essential catalytic groups Asp 315 and Glu 513 and the essential Arg residues R164, R217, R282. Carbodiimide inactivation of the purified enzyme at different pH values reveals an essential carboxyl group with pKa = 3.5 ± 0.3. Phenylglyoxal modified a single Arg residue with pKa = 7.5 ± 0.2, as observed in the soluble trehalase from Spodoptera frugiperda (SfTre1). Diethylpyrocarbonate modified a His residue that resulted in a less active enzyme with pK(e1) changed to 4.8 ± 0.2. In TmTre1 the modified His residue (putatively His 336) is more exposed than the His modified in SfTre1 (putatively His 210) and that affects the ionization of an Arg residue. The architecture of the active site of TmTre1 and SfTre1 is different, as shown by multiple inhibition analysis, the meaning of which demands further research. Trehalase sequences obtained from midgut transcriptomes (pyrosequencing and Illumina data) from 8 insects pertaining to 5 different orders were used in a cladogram, together with other representative sequences. The data suggest that the trehalase gene went duplication and divergence prior to the separation of the paraneopteran and holometabolan orders and that the soluble trehalase derived from the membrane-bound one by losing the C-terminal transmembrane loop. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles

PubMed Central

1994-01-01

Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein- DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus. PMID:8113685

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

PubMed Central

Yang, C-S; Sinenko, S A; Thomenius, M J; Robeson, A C; Freel, C D; Horn, S R; Kornbluth, S

2014-01-01

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin–proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation. PMID:24362437

Binding site feature description of 2-substituted benzothiazoles as potential AcrAB-TolC efflux pump inhibitors in E. coli.

PubMed

Yilmaz, S; Altinkanat-Gelmez, G; Bolelli, K; Guneser-Merdan, D; Ufuk Over-Hasdemir, M; Aki-Yalcin, E; Yalcin, I

2015-01-01

The resistance-nodulation-division (RND) family efflux pumps are important in the antibiotic resistance of Gram-negative bacteria. However, although a number of bacterial RND efflux pump inhibitors have been developed, there has been no clinically available RND efflux pump inhibitor to date. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combinations with ciprofloxacin (CIP) against the AcrAB-TolC overexpressor Escherichia coli AG102 clinical strain. The results indicated that the BSN compounds did not show intrinsic antimicrobial activity when tested alone. However, when used in combinations with CIP, a reversal in the antibacterial activity of CIP with up to 10-fold better MIC values was observed. In order to describe the binding site features of these BSN compounds with AcrB, docking studies were performed using the CDocker method. The performed docking poses and the calculated binding energy scores revealed that the tested compounds BSN-006, BSN-023, and BSN-004 showed significant binding interactions with the phenylalanine-rich region in the distal binding site of the AcrB binding monomer. Moreover, the tested compounds BSN-006 and BSN-023 possessed stronger binding energies than CIP, verifying that BSN compounds are acting as the putative substrates of AcrB.

Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

NASA Astrophysics Data System (ADS)

Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

2014-07-01

Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.

Endocrine active contaminants in aquatic systems and intersex in common sport fishes

USGS Publications Warehouse

Lee Pow, Crystal S. D.; Law, J. Mac; Kwak, Thomas J.; Cope, W. Gregory; Rice, James A.; Kullman, Seth W.; Aday, D. Derek

2017-01-01

Male fish are susceptible to developing intersex, a condition characterized by the presence of testicular oocytes. In the present study, the relationship between intersex and exposure to estrogenic endocrine active contaminants (EACs) was assessed for 2 genera of sport fish, Micropterus and Lepomis, at 20 riverine sites. Seasonal trends and relationships between EACs and intersex (prevalence and severity) were examined at varying putative sources of EACs throughout North Carolina, identified as point sources, nonpoint sources, and reference sites. Intersex was identified in both genera, which was documented for the first time in wild-caught Lepomis. Intersex was more prevalent (59.8%) and more severe (1.6 mean rank) in Micropterus, which was highly correlation to EACs in sediment. In contrast, intersex was less common (9.9%) and less severe (0.2 mean rank) in Lepomis and was highly correlated to EACs in the water column. The authors found that concentrations of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, industrial EACs, and estrogens were highest at point source sites; however, no source type variation was identified in the prevalence or severity of intersex, nor were there seasonal trends in intersex or EAC concentrations. The authors’ results associate genus-specific prevalence of intersex with specific EAC classes in common sport fishes having biological, ecological, and conservation implications.

Influence of codon usage bias on FGLamide-allatostatin mRNA secondary structure.

PubMed

Martínez-Pérez, Francisco; Bendena, William G; Chang, Belinda S W; Tobe, Stephen S

2011-03-01

The FGLamide allatostatins (ASTs) are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders. They also show myomodulatory activity. FGLamide AST nucleotide frequencies and codon bias were investigated with respect to possible effects on mRNA secondary structure. 367 putative FGLamide ASTs and their potential endoproteolytic cleavage sites were identified from 40 species of crustaceans, chelicerates and insects. Among these, 55% comprised only 11 amino acids. An FGLamide AST consensus was identified to be (X)(1→16)Y(S/A/N/G)FGLGKR, with a strong bias for the codons UUU encoding for Phe and AAA for Lys, which can form strong Watson-Crick pairing in all peptides analyzed. The physical distance between these codons favor a loop structure from Ser/Ala-Phe to Lys-Arg. Other loop and hairpin loops were also inferred from the codon frequencies in the N-terminal motif, and the first amino acids from the C-terminal motif, or the dibasic potential endoproteolytic cleavage site. Our results indicate that nucleotide frequencies and codon usage bias in FGLamide ASTs tend to favor mRNA folds in the codon sequence in the C-terminal active peptide core and at the dibasic potential endoproteolytic cleavage site. Copyright © 2010 Elsevier Inc. All rights reserved.

Effect of substance P injection into the nucleus tractus solitarius of rats on cricothyroid and thyroarytenoid motor activity and cardiovascular and respiratory systems.

PubMed

Bauman, Nancy M; Wang, DeQiang; Luschei, Erich S; Talman, William T

2002-10-01

Identification of central neurotransmitters that mediate laryngeal adductor and/or tensor activity may prove useful in managing pathological laryngeal adduction as occurs in laryngospasm or apparent life-threatening events. The putative transmitter substance P (SP) is found in the nucleus tractus solitarius (NTS), in which laryngeal afferents terminate. Therefore, we studied the laryngeal, cardiovascular, and respiratory effects of SP injected into the NTS of rats. We completed bilateral stereotactic injections of 20 nL of SP (15 micromol) or control solution into the region of the NTS, the dorsal motor nucleus (DMN), or the nucleus gracilis (GR) in 30 anesthetized rats. Changes in diaphragm, cricothyroid (CT), and thyroarytenoid (TA) electromyography (EMG), as well as blood pressure (BP), were compared. The injection sites were verified histologically. Injection of SP into the NTS altered CT and/or TA EMG activity in all animals. The change ranged from complete inhibition, to a phasic increase, to a tonic increase. No change in laryngeal adductor EMG activity was seen in 8 of 9 animals after SP injections into the DMN (4/5) or GR (4/4), but 1 animal demonstrated brief inhibition of CT and TA EMG activity after SP injection into the DMN. Injection of SP into the NTS induced central apnea and a significant decrease in BP in all animals. The duration of apnea tended to be longer after NTS injections than after DMN or GR injections (p < .10 and p < .05, respectively). We conclude that stereotactic injections of putative neurotransmitters in rats may be accomplished to identify effects on laryngeal motor activity. Direct application of SP into the NTS consistently elicits a change in CT and/or TA EMG activity, ranging from inhibition to excitation. This model may prove useful in evaluating pharmacological targets of central reflex activity to manage life-threatening laryngeal reflex activity.

Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel

PubMed Central

Kubo, Yoshihiro; Murata, Yoshimichi

2001-01-01

The rectification property of the inward rectifier K+ channel is chiefly due to the block of outward current by cytoplasmic Mg2+ and polyamines. In the cloned inward rectifier K+ channel Kir2.1 (IRK1), Asp172 in the second transmembrane region (M2) and Glu224 in the putative cytoplasmic region after M2 are reported to be critical for the sensitivity to these blockers. However, the difference in the inward rectification properties between Kir2.1 and a very weak inward rectifier sWIRK could not be explained by differences at these two sites. Following sequence comparison of Kir2.1 and sWIRK, we focused this study on Glu299 located in the centre of the putative cytoplasmic region after M2. Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224Gly-Glu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells. Their electrophysiological properties were compared with those of wild-type (WT) Kir2.1 and the following observations were made. (a) Glu299Ser showed a weaker inward rectification, a slower activation upon hyperpolarization, a slower decay of the outward current upon depolarization, a lower sensitivity to block by cytoplasmic spermine and a smaller single-channel conductance than WT. (b) The features of Glu224Gly were similar to those of Glu299Ser. (c) In the double mutant (Glu224Gly-Glu299Ser), the differences from WT described above were more prominent. These results demonstrate that Glu299 as well as Glu224 control rectification and permeation, and suggest the possibility that the two sites contribute to the inner vestibule of the channel pore. The slowing down of the on- and off-blocking processes by mutation of these sites implies that Glu224 and Glu299 function to facilitate the entry (and exit) of spermine to (and from) the blocking site. PMID:11251047

Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.

PubMed

Xu, Li; Liu, Xiaohong; Yin, Zhenhao; Liu, Qian; Lu, Lili; Xiao, Min

2016-12-01

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp 252 , Asp 257 , Asp 264 , Glu 530 , Arg 548 , His 553 , and Trp 555 ) and may form the hydrophobic pocket in stabilizing donor (Trp 261 , Tyr 302 , Tyr 316 , and Trp 369 ) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp 257 . Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

γ-secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aβ.

PubMed

Liu, Fei; Xue, Zhi-Qin; Deng, Si-Hao; Kun, Xiong; Luo, Xue-Gang; Patrylo, Peter R; Rose, Gregory M; Cai, Huaibin; Struble, Robert G; Cai, Yan; Yan, Xiao-Xin

2013-05-01

Deposition of β -amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aβ peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored the possibility of Aβ production and secretion by the choroid plexus (CP). The specific binding density of [(3) H]-L-685,458, a radiolabeled high-affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post-mortem delays. The CP in post-mortem samples exhibited exceptionally high [(3) H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aβ40 and Aβ42 into the medium. Overall, our results suggest that γ-secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non-neuronal contributor to CSF Aβ, probably at reduced levels in AD. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

γ-Secretase binding sites in aged and Alzheimer’s disease human cerebrum: The choroid plexus as a putative origin of CSF Aβ

PubMed Central

Liu, Fei; Xue, Zhi-Qin; Deng, Si-Hao; Kun, Xiong; Luo, Xue-Gang; Patrylo, Peter R.; Rose, Gregory M.; Cai, Huaibin; Struble, Robert G.; Cai, Yan; Yan, Xiao-Xin

2013-01-01

Deposition of β-amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer’s disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although less is clear about the change of the enzyme’s activity in AD brain. Cerebrospinal fluid (CSF) Aβ peptides are considered to derive from brain parenchyma, thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored a possibility of Aβ production and secretion by the choroid plexus (CP). Specific binding density of [3H]-L-685,458, a radiolabeled high affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with comparable ages and postmortem delays. The CP in postmortem samples exhibited exceptionally high [3H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins but released Aβ40 and Aβ42 into the medium. These results suggest that γ-secretase activity appears not altered in the cerebrum in AD related to aged control, nor correlated with regional amyloid plaque pathology. The choroid plexus appears to represent a novel non-neuronal source in the brain that may contribute Aβ into cerebrospinal fluid, probably at reduced levels in AD. PMID:23432732

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

A catalog of putative adverse outcome pathways (AOPs) that ...

EPA Pesticide Factsheets

A number of putative AOPs for several distinct MIEs of thyroid disruption have been formulated for amphibian metamorphosis and fish swim bladder inflation. These have been entered into the AOP knowledgebase on the OECD WIKI. The EDSP has been actively advancing high-throughput screening for chemical activity toward estrogen, androgen and thyroid targets. However, it has been recently identified that coverage for thyroid-related targets is lagging behind estrogen and androgen assay coverage. As thyroid-related medium-high throughput assays are actively being developed for inclusion in the ToxCast chemical screening program, a parallel effort is underway to characterize putative adverse outcome pathways (AOPs) specific to these thyroid-related targets. This effort is intended to provide biological and ecological context that will enhance the utility of ToxCast high throughput screening data for hazard identification.

Functional elements in the minimal promoter of the human proton-coupled folate transporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stark, Michal; Gonen, Nitzan; Assaraf, Yehuda G., E-mail: assaraf@tx.technion.ac.il

2009-10-09

The proton-coupled folate transporter (PCFT) is the dominant intestinal folate transporter, however, its promoter has yet to be revealed. Hence, we here cloned a 3.1 kb fragment upstream to the first ATG of the human PCFT gene and generated sequential deletion constructs evaluated in luciferase reporter assay. This analysis mapped the minimal promoter to 157 bp upstream to the first ATG. Crucial GC-box sites were identified within the minimal promoter and in its close vicinity which substantially contribute to promoter activity, as their disruption resulted in 94% loss of luciferase activity. We also identified upstream enhancer elements including YY1 andmore » AP1 which, although distantly located, prominently transactivated the minimal promoter, as their inactivation resulted in 50% decrease in reporter activity. This is the first functional identification of the minimal PCFT promoter harboring crucial GC-box elements that markedly contribute to its transcriptional activation via putative interaction with distal YY1 and AP1 enhancer elements.« less

Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.

PubMed

Ohba, M

1997-06-09

In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

DNase I nick translation in situ on meiotic chromosomes of the mouse, Mus musculus.

PubMed

Raman, R; Singh, A P; Nanda, I

1988-08-01

DNase-I-sensitive sites have been located on the meiotic chromosomes of the mouse, Mus musculus, by the in situ DNase I nick-translation method. We find that: (1) of all the cell types studied, pachytene nuclei are the most sensitive to DNase I; (2) in diplotene the nicks occur preferentially in the vicinity of chiasmata; (3) the sex chromosomes are also sensitive to the enzyme despite their transcriptional quiescence; and (4) in the sex bivalent the nicks are primarily observed in the putative region of recombination. We conclude that, in addition to discriminating between the transcriptionally active and inactive states of chromatin, DNase I identifies recombination-specific chromatin changes in meiotic prophase.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

PubMed

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase.

PubMed

Paradisi, Francesca; Dean, Jonathan L E; Geoghegan, Kieran F; Engel, Paul C

2005-03-08

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.

Prime-Boost Immunization of Rabbits with HIV-1 gp120 Elicits Potent Neutralization Activity against a Primary Viral Isolate

PubMed Central

Narayan, Kristin M.; Agrawal, Nitish; Du, Sean X.; Muranaka, Janelle E.; Bauer, Katherine; Leaman, Daniel P.; Phung, Pham; Limoli, Kay; Chen, Helen; Boenig, Rebecca I.; Wrin, Terri; Zwick, Michael B.; Whalen, Robert G.

2013-01-01

Development of a vaccine for HIV-1 requires a detailed understanding of the neutralizing antibody responses that can be experimentally elicited to difficult-to-neutralize primary isolates. Rabbits were immunized with the gp120 subunit of HIV-1 JR-CSF envelope (Env) using a DNA-prime protein-boost regimen. We analyzed five sera that showed potent autologous neutralizing activity (IC50s at ∼103 to 104 serum dilution) against pseudoviruses containing Env from the primary isolate JR-CSF but not from the related isolate JR-FL. Pseudoviruses were created by exchanging each variable and constant domain of JR-CSF gp120 with that of JR-FL or with mutations in putative N-glycosylation sites. The sera contained different neutralizing activities dependent on C3 and V5, C3 and V4, or V4 regions located on the glycan-rich outer domain of gp120. All sera showed enhanced neutralizing activity toward an Env variant that lacked a glycosylation site in V4. The JR-CSF gp120 epitopes recognized by the sera are generally distinct from those of several well characterized mAbs (targeting conserved sites on Env) or other type-specific responses (targeting V1, V2, or V3 variable regions). The activity of one serum requires specific glycans that are also important for 2G12 neutralization and this serum blocked the binding of 2G12 to gp120. Our findings show that different fine specificities can achieve potent neutralization of HIV-1, yet this strong activity does not result in improved breadth. PMID:23326351

Dihydroartemisinin as a Putative STAT3 Inhibitor, Suppresses the Growth of Head and Neck Squamous Cell Carcinoma by Targeting Jak2/STAT3 Signaling

PubMed Central

Jia, Lifeng; Song, Qi; Zhou, Chenyang; Li, Xiaoming; Pi, Lihong; Ma, Xiuru; Li, Hui; Lu, Xiuying; Shen, Yupeng

2016-01-01

Developing drugs that can effectively block STAT3 activation may serve as one of the most promising strategy for cancer treatment. Currently, there is no putative STAT3 inhibitor that can be safely and effectively used in clinic. In the present study, we investigated the potential of dihydroartemisinin (DHA) as a putative STAT3 inhibitor and its antitumor activities in head and neck squamous cell carcinoma (HNSCC). The inhibitory effects of DHA on STAT3 activation along with its underlying mechanisms were studied in HNSCC cells. The antitumor effects of DHA against HNSCC cells were explored both in vitro and in vivo. An investigation on cooperative effects of DHA with cisplatin in killing HNSCC cells was also implemented. DHA exhibited remarkable and specific inhibitory effects on STAT3 activation via selectively blocking Jak2/STAT3 signaling. Besides, DHA significantly inhibited HNSCC growth both in vitro and in vivo possibly through induction of apoptosis and attenuation of cell migration. DHA also synergized with cisplatin in tumor inhibition in HNSCC cells. Our findings demonstrate that DHA is a putative STAT3 inhibitor that may represent a new and effective drug for cancer treatment and therapeutic sensitization in HNSCC patients. PMID:26784960

Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

PubMed

Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

1996-08-01

The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

Conservation Status of the Indo-Pacific Humpback Dolphin (Sousa chinensis) in the Northern Beibu Gulf, China.

PubMed

Chen, Bingyao; Xu, Xinrong; Jefferson, Thomas A; Olson, Paula A; Qin, Qiurong; Zhang, Hongke; He, Liwen; Yang, Guang

2016-01-01

There has been very little previous research on Indo-Pacific humpback dolphins (Sousa chinensis) in the Beibu Gulf of southern China. Here, we report on the population size, habitat and ecology, threats, and overall conservation status of this putative population. 'Population size' was estimated based on photo-identification mark/recapture analysis. It was estimated to number a total of 398-444 individuals (95% CI: 393-506), with two apparently distinct groups in the Dafengjiang-Nanliujiang Estuary and at Shatian-Caotan. Movements of dolphins in the Beibu Gulf appear to be limited, with high site fidelity. These dolphins were found to occur mainly in shallow coastal waters near estuaries. The main threats are fisheries interactions (including by-catch), vessel traffic, mariculture operations, dolphin-watching tourism, and habitat degradation (including marine construction activities and large-scale land reclamation). Although the conservation status of this putative population has been considered to be better than that of other populations of the species in more northern areas of China, there is still reason for strong concern about its future, and several management recommendations are made. © 2016 Elsevier Ltd. All rights reserved.

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures[OPEN

PubMed Central

Zhu, Bo; Zhang, Wenli; Jiang, Jiming

2015-01-01

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants. PMID:26373455

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

PubMed

Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baig, M.; Brown, A.; Eswaramoorthy, S.

Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) atmore » Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.« less

Putative pyramidal neurons and interneurons in the monkey parietal cortex make different contributions to the performance of a visual grouping task.

PubMed

Yokoi, Isao; Komatsu, Hidehiko

2010-09-01

Visual grouping of discrete elements is an important function for object recognition. We recently conducted an experiment to study neural correlates of visual grouping. We recorded neuronal activities while monkeys performed a grouping detection task in which they discriminated visual patterns composed of discrete dots arranged in a cross and detected targets in which dots with the same contrast were aligned horizontally or vertically. We found that some neurons in the lateral bank of the intraparietal sulcus exhibit activity related to visual grouping. In the present study, we analyzed how different types of neurons contribute to visual grouping. We classified the recorded neurons as putative pyramidal neurons or putative interneurons, depending on the duration of their action potentials. We found that putative pyramidal neurons exhibited selectivity for the orientation of the target, and this selectivity was enhanced by attention to a particular target orientation. By contrast, putative interneurons responded more strongly to the target stimuli than to the nontargets, regardless of the orientation of the target. These results suggest that different classes of parietal neurons contribute differently to the grouping of discrete elements.

Redox-Stratified Bacterial Communities in Sediments Associated with Multiple Lucinid Bivalve Species: Implications for Symbiosis in Changing Coastal Habitats

NASA Astrophysics Data System (ADS)

Paterson, A. T.; Fortier, C. M.; Long, B.; Kokesh, B. S.; Lim, S. J.; Campbell, B. J.; Anderson, L. C.; Engel, A. S.

2017-12-01

Lucinids, chemosymbiotic marine bivalves, occupy strong redox gradient habitats, including the rhizosphere of coastal seagrass beds and mangrove forests in subtropical to tropical ecosystems. Lucinids and their sulfide-oxidizing gammaproteobacterial endosymbionts, which are acquired from the environment, provide a critical ecosystem service by removing toxic reduced sulfur compounds from the surrounding environment, and lucinids may be an important food source to economically valuable fisheries. The habitats of Phacoides pectinatus, Stewartia floridana, Codakia orbicularis, Ctena orbiculata, and Lucina pensylvanica lucinids in Florida and San Salvador in The Bahamas were evaluated in comprehensive malacological, microbiological, and geochemical surveys. Vegetation cover included different seagrass species or calcareous green macroalgae. All sites were variably affected by anthropogenic activities, as evidenced by visible prop scars in seagrass beds, grain size distributions atypical of low energy environments (i.e., artificial fill or dredge material from nearby channels), and high levels of pyrogenic hydrocarbon compounds in sediment indicative of urbanization impact. Where present, lucinid population densities frequently exceeded 2000 individuals per cubic meter, and were typically more abundant underlying seagrass compared to unvegetated, bare sand. Dissolved oxygen and sulfide levels varied from where lucinids were recovered. The sediment bacterial communities from classified 16S rRNA gene sequences indicated that the diversity of putative anaerobic groups increased with sediment depth, but putative aerobes, including of Gammaproteobacteria related to the lucinid endosymbionts, decreased with depth. Where multiple seagrass species co-occurred, retrieved bacterial community compositions correlated to overlying seagrass species, but diversity differed from bare sand patches, including among putative free-living endosymbiont groups. As such, continued sea-level rise and urbanization near shallow marine ecosystems can negatively impact the lucinid chemosymbiotic system and lucinid habitat because of changes to the redox gradients and/or vegetation cover that support putative lucinid endosymbiont diversity in the environment.

The SBP-Box Gene VpSBP11 from Chinese Wild Vitis Is Involved in Floral Transition and Affects Leaf Development.

PubMed

Hou, Hongmin; Yan, Xiaoxiao; Sha, Ting; Yan, Qin; Wang, Xiping

2017-07-13

Flowering occurs in angiosperms during a major developmental transition from vegetative growth to the reproductive phase. Squamosa promoter binding protein (SBP)-box genes have been found to play critical roles in regulating flower and fruit development, but their roles in grapevine have remained unclear. To better understand the functions of the grape SBP-box genes in both vegetative and reproductive growth phases, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP11 , was obtained from Chinese wild grapevine Vitis pseudoreticulata Wen Tsai Wang (W. T. Wang) clone 'Baihe-35-1'. VpSBP11 encoded a putative polypeptide of 170 amino acids with a highly conserved SBP-domain with two zinc-binding sites of the Cx2C-x3-H-x11-C-x6-H (C2HCH) type and a nuclear localization signal. We confirmed that the VpSBP11 protein was targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans -activation assay. Over-expression of VpSBP11 in Arabidopsis thaliana was shown to activate the FUL gene, and subsequently the AP1 and LFY genes, all of which were floral meristem identity genes, and to cause earlier flowering than in wild type (WT) plants. The pattern of vegetative growth was also different between the transgenic and WT plants. For example, in the VpSBP11 over-expressing transgenic plants, the number of rosette leaves was less than that of WT; the petiole was significantly elongated; and the rosette and cauline leaves curled upwards or downwards. These results were consistent with VpSBP11 acting as a transcription factor during the transition from the vegetative stage to the reproductive stage.

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

PubMed

Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

2015-02-01

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

Highly conformationally constrained halogenated 6-spiroepoxypenicillins as probes for the bioactive side-chain conformation of benzylpenicillin

NASA Astrophysics Data System (ADS)

Shute, Richard E.; Jackson, David E.; Bycroft, Barrie W.

1989-06-01

The halogenated 6-spiroepoxypenicillins are a series of novel semisynthetic β-lactam compounds with highly conformationally restricted side chains incorporating an epoxide. Their biological activity profiles depend crucially on the configuration at position C-3 of that epoxide. In derivatives with aromatic-containing side chains, e.g., anilide, the 3 R-compounds possess notable Gram-positive antibacterial activity and potent β-lactamase inhibitory properties. The comparable 3S-compounds are antibacterially inactive, but retain β-lactamase inhibitory activity. Using the molecular simulation programs COSMIC and ASTRAL, we attempted to map a putative, lipophilic accessory binding site on the PBPs that must interact with the side-chain aromatic residue. Comparative computer-assisted modelling of the 3 R, and 3 S-anilides, along with benzylpenicillin, indicated that the available conformational space at room temperature for the side chains of the 3 R and the 3 S-anilides was mutually exclusive. The conformational space for the more flexible benzylpenicillin could accommodate the side chains of both the constrained penicillin derivatives. By a combination of van der Waals surface calculations and a pharmacophoric distance approach, closely coincident conformers of the 3 R-anilide and benzylpenicillin were identified. These conformers must be related to the antibacterial, `bioactive' conformer for the classical β-lactam antibiotics. From these proposed bioactive conformations, a model for the binding of benzylpenicillin to the PBPs relating the three-dimensional arrangement of a putative lipophilic S2-subsite, specific for the side-chain aromatic moiety, and the 3 α-carboxylate functionality is presented.

SMA-MAP: a plasma protein panel for spinal muscular atrophy.

PubMed

Kobayashi, Dione T; Shi, Jing; Stephen, Laurie; Ballard, Karri L; Dewey, Ruth; Mapes, James; Chung, Brett; McCarthy, Kathleen; Swoboda, Kathryn J; Crawford, Thomas O; Li, Rebecca; Plasterer, Thomas; Joyce, Cynthia; Chung, Wendy K; Kaufmann, Petra; Darras, Basil T; Finkel, Richard S; Sproule, Douglas M; Martens, William B; McDermott, Michael P; De Vivo, Darryl C; Walker, Michael G; Chen, Karen S

2013-01-01

Spinal Muscular Atrophy (SMA) presents challenges in (i) monitoring disease activity and predicting progression, (ii) designing trials that allow rapid assessment of candidate therapies, and (iii) understanding molecular causes and consequences of the disease. Validated biomarkers of SMA motor and non-motor function would offer utility in addressing these challenges. Our objectives were (i) to discover additional markers from the Biomarkers for SMA (BforSMA) study using an immunoassay platform, and (ii) to validate the putative biomarkers in an independent cohort of SMA patients collected from a multi-site natural history study (NHS). BforSMA study plasma samples (N = 129) were analyzed by immunoassay to identify new analytes correlating to SMA motor function. These immunoassays included the strongest candidate biomarkers identified previously by chromatography. We selected 35 biomarkers to validate in an independent cohort SMA type 1, 2, and 3 samples (N = 158) from an SMA NHS. The putative biomarkers were tested for association to multiple motor scales and to pulmonary function, neurophysiology, strength, and quality of life measures. We implemented a Tobit model to predict SMA motor function scores. 12 of the 35 putative SMA biomarkers were significantly associated (p<0.05) with motor function, with a 13(th) analyte being nearly significant. Several other analytes associated with non-motor SMA outcome measures. From these 35 biomarkers, 27 analytes were selected for inclusion in a commercial panel (SMA-MAP) for association with motor and other functional measures. Discovery and validation using independent cohorts yielded a set of SMA biomarkers significantly associated with motor function and other measures of SMA disease activity. A commercial SMA-MAP biomarker panel was generated for further testing in other SMA collections and interventional trials. Future work includes evaluating the panel in other neuromuscular diseases, for pharmacodynamic responsiveness to experimental SMA therapies, and for predicting functional changes over time in SMA patients.

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

PubMed Central

2011-01-01

Background Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation. PMID:21689454

Novel compound heterozygous Thyroglobulin mutations c.745+1G>A/c.7036+2T>A associated with congenital goiter and hypothyroidism in a Vietnamese family. Identification of a new cryptic 5' splice site in the exon 6.

PubMed

Citterio, Cintia E; Morales, Cecilia M; Bouhours-Nouet, Natacha; Machiavelli, Gloria A; Bueno, Elena; Gatelais, Frédérique; Coutant, Regis; González-Sarmiento, Rogelio; Rivolta, Carina M; Targovnik, Héctor M

2015-03-15

Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6 + 1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40 + 2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein molecule that alter the biosynthesis of thyroid hormones. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB

PubMed Central

Badrinarayanan, Anjana; Cisse, Ibrahim I.

2017-01-01

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400–2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair–the attenuation of DNA-end processing and the initiation of homology search by RecA–thereby helping to ensure that genomic integrity is maintained during DSB repair. PMID:28489851

Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

PubMed

Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

2011-07-01

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.

2008-06-23

Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 {angstrom}. The structure of the apoenzyme reveals that the protein is composed of five -helicesmore » with connecting loops and is a member of the {alpha}-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between {alpha}-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.« less

Genetic Signatures of HIV-1 Envelope-mediated Bystander Apoptosis

PubMed Central

Joshi, Anjali; Lee, Raphael T. C.; Mohl, Jonathan; Sedano, Melina; Khong, Wei Xin; Ng, Oon Tek; Maurer-Stroh, Sebastian; Garg, Himanshu

2014-01-01

The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4+ T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4+ T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype. PMID:24265318

A multiyear dust devil vortex survey using an automated search of pressure time series

NASA Astrophysics Data System (ADS)

Jackson, Brian; Lorenz, Ralph

2015-03-01

Dust devils occur in arid climates on the Earth and ubiquitously on Mars, where they likely dominate the supply of atmospheric dust and influence climate. Martian dust devils have been studied with a combination of orbiting and landed spacecraft, while most studies of terrestrial dust devils have involved manned monitoring of field sites, which can be costly both in time and personnel. As an alternative approach, we describe a multiyear in situ survey of terrestrial dust devils using pressure loggers deployed at El Dorado Playa in Nevada, USA, a site known for dust devil activity. Analogous to previous surveys for Martian dust devils, we conduct a posthoc analysis of the barometric data to search for putative dust devil pressure dips using a new automated detection algorithm. We investigate the completeness and false positive rates of our new algorithm and conduct several statistically robust analyses of the resulting population of dips. We also investigate possible seasonal, annual, and spatial variability of the putative dust devil dips, possible correlations with precipitation, and the influence of sample size on the derived population statistics. Our results suggest that large numbers of dips (>1000) collected over multiple seasons are probably required for accurate assessment of the underlying dust devil population. Correlating long-term barometric time series with other data streams (e.g., solar flux measurements from photovoltaic cells) can uniquely elucidate the natures and origins of dust devils, and accurately assessing their influence requires consideration of the full distribution of dust devil properties, rather than average values.

Putative kappa opioid heteromers as targets for developing analgesics free of adverse effects.

PubMed

Le Naour, Morgan; Lunzer, Mary M; Powers, Michael D; Kalyuzhny, Alexander E; Benneyworth, Michael A; Thomas, Mark J; Portoghese, Philip S

2014-08-14

It is now generally recognized that upon activation by an agonist, β-arrestin associates with G protein-coupled receptors and acts as a scaffold in creating a diverse signaling network that could lead to adverse effects. As an approach to reducing side effects associated with κ opioid agonists, a series of β-naltrexamides 3-10 was synthesized in an effort to selectively target putative κ opioid heteromers without recruiting β-arrestin upon activation. The most potent derivative 3 (INTA) strongly activated KOR-DOR and KOR-MOR heteromers in HEK293 cells. In vivo studies revealed 3 to produce potent antinociception, which, when taken together with antagonism data, was consistent with the activation of both heteromers. 3 was devoid of tolerance, dependence, and showed no aversive effect in the conditioned place preference assay. As immunofluorescence studies indicated no recruitment of β-arrestin2 to membranes in coexpressed KOR-DOR cells, this study suggests that targeting of specific putative heteromers has the potential to identify leads for analgesics devoid of adverse effects.

Tracing Cytoplasmic Ca2+ Ion and Water Access Points in the Ca2+-ATPase

PubMed Central

Musgaard, Maria; Thøgersen, Lea; Schiøtt, Birgit; Tajkhorshid, Emad

2012-01-01

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) transports two Ca2+ ions across the membrane of the sarco(endo)plasmic reticulum against the concentration gradient, harvesting the required energy by hydrolyzing one ATP molecule during each transport cycle. Although SERCA is one of the best structurally characterized membrane transporters, it is still largely unknown how the transported Ca2+ ions reach their transmembrane binding sites in SERCA from the cytoplasmic side. Here, we performed extended all-atom molecular dynamics simulations of SERCA. The calculated electrostatic potential of the protein reveals a putative mechanism by which cations may be attracted to and bind to the Ca2+-free state of the transporter. Additional molecular dynamics simulations performed on a Ca2+-bound state of SERCA reveal a water-filled pathway that may be used by the Ca2+ ions to reach their buried binding sites from the cytoplasm. Finally, several residues that are involved in attracting and guiding the cations toward the possible entry channel are identified. The results point to a single Ca2+ entry site close to the kinked part of the first transmembrane helix, in a region loaded with negatively charged residues. From this point, a water pathway outlines a putative Ca2+ translocation pathway toward the transmembrane ion-binding sites. PMID:22339863

SOX2 as a New Regulator of HPV16 Transcription.

PubMed

Martínez-Ramírez, Imelda; Del-Castillo-Falconi, Víctor; Mitre-Aguilar, Irma B; Amador-Molina, Alfredo; Carrillo-García, Adela; Langley, Elizabeth; Zentella-Dehesa, Alejandro; Soto-Reyes, Ernesto; García-Carrancá, Alejandro; Herrera, Luis A; Lizano, Marcela

2017-07-05

Persistent infections with high-risk human papillomavirus (HPV) constitute the main risk factor for cervical cancer development. HPV16 is the most frequent type associated to squamous cell carcinomas (SCC), followed by HPV18. The long control region (LCR) in the HPV genome contains the replication origin and sequences recognized by cellular transcription factors (TFs) controlling viral transcription. Altered expression of E6 and E7 viral oncogenes, modulated by the LCR, causes modifications in cellular pathways such as proliferation, leading to malignant transformation. The aim of this study was to identify specific TFs that could contribute to the modulation of high-risk HPV transcriptional activity, related to the cellular histological origin. We identified sex determining region Y (SRY)-box 2 (SOX2) response elements present in HPV16-LCR. SOX2 binding to the LCR was demonstrated by in vivo and in vitro assays. The overexpression of this TF repressed HPV16-LCR transcriptional activity, as shown through reporter plasmid assays and by the down-regulation of endogenous HPV oncogenes. Site-directed mutagenesis revealed that three putative SOX2 binding sites are involved in the repression of the LCR activity. We propose that SOX2 acts as a transcriptional repressor of HPV16-LCR, decreasing the expression of E6 and E7 oncogenes in a SCC context.

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

PubMed Central

Witzgall, R; O'Leary, E; Gessner, R; Ouellette, A J; Bonventre, J V

1993-01-01

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins. Images PMID:8382778

LOW MOLECULAR WEIGHT PROTEIN TYROSINE PHOSPHATASE (LMW-PTP) AND ITS POSSIBLE PHYSIOLOGICAL FUNCTIONS OF REDOX SIGNALING IN THE EYE LENS*

PubMed Central

Xing, Kuiyi; Raza, Ashraf; Löfgren, Stefan.; Fernando, M. Rohan.; Ho, Ye-Shih; Lou, Marjorie F.

2007-01-01

Low molecular weight protein tyrosine phosphatase (LMW-PTP) was cloned from human lens epithelial B3 cells (HLE B3) and the recombinant enzyme was purified to homogeneity. The pure enzyme reacted positively with anti-LMW-PTP antibody, displayed tyrosine-specific phosphatase activity and was extremely sensitive to H2O2. The inactivated LMW-PTP could be regenerated by thioltransferase (TTase)/GSH system as demonstrated by both activity assay and by mass spectrometry (MS). The MS study also showed that an intramolecular disulfide bond was formed between C13 and C18 at the active site, and was reduced by the TTase/GSH system. The putative role of LMW-PTP in regulating platelet derived growth factor (PDGF)-stimulated cell signaling was demonstrated in wild type mouse lens epithelial cells (LEC) in which LMW-PTP was transiently inactivated, corroborated with the transient phosphorylation of Tyr857 at the active site of PDGF receptor and the downstream signaling components of Akt and ERK1/2. In contrast, LMW-PTP activity in PDGF-stimulated LEC from TTase −/− mice was progressively lost, concomitant with the high basal and sustained high phosphorylation levels at Tyr857, Akt and ERK1/2. We conclude that the reversible LMW-PTP activity regulated by ROS-mediated oxidation and TTase/GSH reduction is the likely mechanism of redox signaling in lens epithelial cells. PMID:17428749

Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea.

PubMed Central

Davis, M T; Vakharia, V N; Henry, J; Kempe, T G; Raina, A K

1992-01-01

Pheromone biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone biosynthesis in female Helicoverpa (Heliothis) zea. Two oligonucleotide probes representing two overlapping amino acid regions of PBAN were used to screen 2.5 x 10(5) recombinant plaques, and a positive recombinant clone was isolated. Sequence analysis of the isolated clone showed that the PBAN gene is interrupted after the codon encoding amino acid 14 by a 0.63-kilobase (kb) intron. Preceding the PBAN amino acid sequence is a 10-amino acid sequence containing a pentapeptide Phe-Thr-Pro-Arg-Leu, which is followed by a Gly-Arg-Arg processing site. Immediately after the PBAN amino acid sequence is a Gly-Arg processing site and a short stretch of 10 amino acids. This 10-amino acid sequence contains a repeat of the PBAN C-terminal pentapeptide Phe-Ser-Pro-Arg-Leu and is terminated by another Gly-Arg processing site. It is suggested that the PBAN gene in H. zea might carry, besides PBAN, a 7- and an 8-residue amidated peptide, which share with PBAN the core C-terminal pentapeptide Phe-(Ser or Thr)-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence of PBAN represents the minimum sequence required for pheromonotropic activity in H. zea and also bears a high degree of homology to the pyrokinin family of insect peptides with myotropic activity. It is possible that the putative heptapeptide and octapeptide might be new members of the pyrokinin family, with pheromonotropic and/or myotropic activities. Thus, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea. Images PMID:1729680

Catalytic site identification—a web server to identify catalytic site structural matches throughout PDB

PubMed Central

Kirshner, Daniel A.; Nilmeier, Jerome P.; Lightstone, Felice C.

2013-01-01

The catalytic site identification web server provides the innovative capability to find structural matches to a user-specified catalytic site among all Protein Data Bank proteins rapidly (in less than a minute). The server also can examine a user-specified protein structure or model to identify structural matches to a library of catalytic sites. Finally, the server provides a database of pre-calculated matches between all Protein Data Bank proteins and the library of catalytic sites. The database has been used to derive a set of hypothesized novel enzymatic function annotations. In all cases, matches and putative binding sites (protein structure and surfaces) can be visualized interactively online. The website can be accessed at http://catsid.llnl.gov. PMID:23680785

Catalytic site identification--a web server to identify catalytic site structural matches throughout PDB.

PubMed

Kirshner, Daniel A; Nilmeier, Jerome P; Lightstone, Felice C

2013-07-01

The catalytic site identification web server provides the innovative capability to find structural matches to a user-specified catalytic site among all Protein Data Bank proteins rapidly (in less than a minute). The server also can examine a user-specified protein structure or model to identify structural matches to a library of catalytic sites. Finally, the server provides a database of pre-calculated matches between all Protein Data Bank proteins and the library of catalytic sites. The database has been used to derive a set of hypothesized novel enzymatic function annotations. In all cases, matches and putative binding sites (protein structure and surfaces) can be visualized interactively online. The website can be accessed at http://catsid.llnl.gov.

MUC4 is negatively regulated through the Wnt/β-catenin pathway via the Notch effector Hath1 in colorectal cancer

PubMed Central

Pai, Priya; Rachagani, Satyanarayana; Dhawan, Punita; Sheinin, Yuri M.; Mallya, Kavita; Pothuraju, Ramesh; Batra, Surinder K.

2016-01-01

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC. PMID:27551331

MUC4 is negatively regulated through the Wnt/β-catenin pathway via the Notch effector Hath1 in colorectal cancer.

PubMed

Pai, Priya; Rachagani, Satyanarayana; Dhawan, Punita; Sheinin, Yuri M; Macha, Muzafar A; Qazi, Asif Khurshid; Chugh, Seema; Ponnusamy, Moorthy P; Mallya, Kavita; Pothuraju, Ramesh; Batra, Surinder K

2016-05-01

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/β-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by β-catenin in CRC. We first knocked down (KD) β-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant β-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear β-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of β-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/β-catenin and Notch signaling. The KD of β-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/β-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.

DJ-1 Is a Copper Chaperone Acting on SOD1 Activation*

PubMed Central

Girotto, Stefania; Cendron, Laura; Bisaglia, Marco; Tessari, Isabella; Mammi, Stefano; Zanotti, Giuseppe; Bubacco, Luigi

2014-01-01

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis. PMID:24567322

High cancer-specific expression of mesothelin (MSLN) is attributable to an upstream enhancer containing a transcription enhancer factor dependent MCAT motif.

PubMed

Hucl, Tomas; Brody, Jonathan R; Gallmeier, Eike; Iacobuzio-Donahue, Christine A; Farrance, Iain K; Kern, Scott E

2007-10-01

Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies.

Cadmium Induces Liver Cell Apoptosis through Caspase-3A Activation in Purse Red Common Carp (Cyprinus carpio)

PubMed Central

Qiao, Panpan; Liu, Shen; Zhang, Li; He, Penghui; Zhang, Xiaoyan; Wang, Yannan; Min, Weiping

2013-01-01

Caspase-3, the essential effector caspase, plays a pivotal role during caspase-dependent apoptosis. In this study, we isolated and characterized caspase-3A gene from common carp. The common carp caspase-3A comprising 273 amino acids showed 71.8% sequence similarity and 59.3% sequence identity to human caspase-3. It exhibited an evolutionarily conserved structure of mammalian caspase-3 genes, including a pro-domain, a large subunit, a small subunit and other motifs such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. Phylogenetic analysis demonstrated that common carp caspase-3A formed a clade with cyprinid fish caspase-3. To assess whether caspase-3A is involved in cadmium (Cd)-induced cell apoptosis in common carp, a Cd exposure experiment was performed. TUNEL analysis showed that Cd triggered liver cell apoptosis; caspase-3A activity was markedly increased; its proenzyme level was significantly decreased, and the levels of its cleaved forms were markedly increased. However, real-time quantitative PCR analysis revealed that the mRNA transcript level of caspase-3A was not significantly elevated. Immunoreactivities were observed in the cytoplasm of hepatocytes by immunohistochemical detection. The findings indicates that Cd can trigger liver cell apoptosis through the activation of caspase-3A. Caspase-3A may play an essential role in Cd-induced apoptosis. PMID:24349509

Modulation of KvAP Unitary Conductance and Gating by 1-Alkanols and Other Surface Active Agents

PubMed Central

Finol-Urdaneta, Rocio K.; McArthur, Jeffrey R.; Juranka, Peter F.; French, Robert J.; Morris, Catherine E.

2010-01-01

Abstract The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains. PMID:20197029

The Zur regulon of Corynebacterium glutamicum ATCC 13032

PubMed Central

2010-01-01

Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum. PMID:20055984

Toddlers' Duration of Attention toward Putative Threat

ERIC Educational Resources Information Center

Kiel, Elizabeth J.; Buss, Kristin A.

2011-01-01

Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

Three sorghum serpin recombinant proteins inhibit midgut trypsin activity and growth of corn earworm

USDA-ARS?s Scientific Manuscript database

The sorghum (Sorghum bicolor) genome contains at least 17 putative serpin (serine protease inhibitor) open reading frames, some of which are induced by pathogens. Recent transcriptome studies found that most of the putative serpins are expressed but their roles are unknown. Four sorghum serpins were...

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome

PubMed Central

Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

2016-01-01

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. PMID:27401230

Alternative Ways to Think about Cellular Internal Ribosome Entry*

PubMed Central

Gilbert, Wendy V.

2010-01-01

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of “cap-dependent” and “IRES-dependent” should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation. PMID:20576611

Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

PubMed

Gómara, María José; Pérez-Pomeda, Ignacio; Gatell, José María; Sánchez-Merino, Victor; Yuste, Eloisa; Haro, Isabel

2017-02-01

The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

Contemplating the plasmalemmal control center model

NASA Technical Reports Server (NTRS)

Pickard, B. G.

1994-01-01

An abundant epidermal mechanosensory calcium-selective ion channel appears able not only to detect mechanical stimuli such as those that initiate gravitropism but also to detect thermal, electrical, and various chemical stimuli. Because it responds to multimodal input with a second messenger output, this channel system seems likely to be an integrator that can engage in feedbacks with many other systems of the cell--and feedback is the hallmark of regulation. In general, the mechanical tension required for channel activation is likely transmitted from the relatively rigid cell wall to the plasma membrane system via linkage or adhesion sites that display antigenicities recognized by antibodies to animal beta-1 integrin, vitronectin, and fibronectin and which have mechanical connections to the cytoskeleton. Thus, functionally, leverage exerted against any given adhesion site will tend to control channels within a surrounding domain. Reactions initiated by passage of calcium ions through the channels could presumably be more effectively regulated if channels within the domains were somewhat clustered and if appropriate receptors, kinases, porters, pumps, and some key cytoskeletal anchoring sites were in turn clustered about them. Accumulating evidence suggests not only that activity of clusters of channels may contribute to control of cytoskeletal architecture and of regulatory protein function within their domain, but also that both a variety of regulatory proteins and components of the cortical cytoskeleton may contribute to control of channel activity. The emerging capabilities of electronic optical microscopy are well suited for resolving the spatial distributions of many of these cytoskeletal and regulatory molecules in living cells, and for following some of their behaviors as channels are stimulated to open and cytosolic calcium builds in their vicinity. Such microscopy, coupled with biochemical and physiological probing, should help to establish the nature of the feedback loops putatively controlled by the linkage sites and their channel domains.

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa

PubMed Central

Morita, Hiroyuki; Wanibuchi, Kiyofumi; Nii, Hirohiko; Kato, Ryohei; Sugio, Shigetoshi; Abe, Ikuro

2010-01-01

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C6-C7-C6 diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C6-C3 coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H2O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C6-C7-C6 scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes. PMID:21041675

Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent transcriptional regulation of endothelial cell adhesion molecules.

PubMed

Wang, Yu-Ling; Kuo, Je-Hung; Lee, Shao-Chen; Liu, Jai-Shin; Hsieh, Yin-Cheng; Shih, Yu-Tsung; Chen, Chun-Jung; Chiu, Jeng-Jiann; Wu, Wen-Guey

2010-11-26

Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn(2+) at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn(2+)-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn(2+)-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn(2+)-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa.

PubMed

Morita, Hiroyuki; Wanibuchi, Kiyofumi; Nii, Hirohiko; Kato, Ryohei; Sugio, Shigetoshi; Abe, Ikuro

2010-11-16

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C(6)-C(7)-C(6) diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C(6)-C(3) coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H(2)O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C(6)-C(7)-C(6) scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.

A giant X-ray flare on Lambda Eridani (B2e)

NASA Technical Reports Server (NTRS)

Smith, Myron A.; Grady, Carol A.; Peters, Geraldine J.; Feigelson, Eric D.

1993-01-01

A 30 ks observation with the ROSAT PSPC distributed over 39 hr shows that the putatively single, mild B2e star Lambda Eri emits at most times a soft X-ray flux at a rate and temperature consistent with other B stars. However, during the middle of our observations, this star's X-ray flux increased by a factor of 6 before returning to the basal level. This brightening, due entirely to photon energies of 0.7 keV or greater, can be fitted well to a Raymond-Smith temperature parameter of 14 MK and luminosity 4 x 10 exp 31 ergs/s; these are characteristics of giant stellar flares. With an estimated duration of about 50,000 s, this event is arguably the strongest X-ray flare yet observed. We consider several possible scenarios for the site of the flare, including several with an active cool secondary or degenerate companion. We find that IUE and optical spectra do not support a binary picture and that it is most probable that the flare site is on or related to Lambda Eri itself. This supports other evidence for violent magnetic activity on some B-type stars.

Crystal structure of Pistol, a class of self-cleaving ribozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Laura A.; Wang, Jimin; Steitz, Thomas A.

2017-01-17

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile bymore » deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.« less

Understanding allosteric interactions in G protein-coupled receptors using Supervised Molecular Dynamics: A prototype study analysing the human A3 adenosine receptor positive allosteric modulator LUF6000.

PubMed

Deganutti, Giuseppe; Cuzzolin, Alberto; Ciancetta, Antonella; Moro, Stefano

2015-07-15

The search for G protein-coupled receptors (GPCRs) allosteric modulators represents an active research field in medicinal chemistry. Allosteric modulators usually exert their activity only in the presence of the orthosteric ligand by binding to protein sites topographically different from the orthosteric cleft. They therefore offer potentially therapeutic advantages by selectively influencing tissue responses only when the endogenous agonist is present. The prediction of putative allosteric site location, however, is a challenging task. In facts, they are usually located in regions showing more structural variation among the family members. In the present work, we applied the recently developed Supervised Molecular Dynamics (SuMD) methodology to interpret at the molecular level the positive allosteric modulation mediated by LUF6000 toward the human adenosine A3 receptor (hA3 AR). Our data suggest at least two possible mechanisms to explain the experimental data available. This study represent, to the best of our knowledge, the first case reported of an allosteric recognition mechanism depicted by means of molecular dynamics simulations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii.

PubMed

Horibata, Y; Okino, N; Ichinose, S; Omori, A; Ito, M

2000-10-06

Endoglycoceramidase (EC ) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here the purification, characterization, and cDNA cloning of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 and was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxycholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to hydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid sequence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Glu-Pro sequence, which is the putative active site of Rhodococcus endoglycoceramidase, was conserved in the deduced amino acid sequences. This is the first report of the cloning of an endoglycoceramidase from a eukaryote.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

PubMed Central

Ng, Chai Ann; Ke, Ying; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Vandenberg, Jamie I.

2013-01-01

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

Phylogenetic factorization of compositional data yields lineage-level associations in microbiome datasets.

PubMed

Washburne, Alex D; Silverman, Justin D; Leff, Jonathan W; Bennett, Dominic J; Darcy, John L; Mukherjee, Sayan; Fierer, Noah; David, Lawrence A

2017-01-01

Marker gene sequencing of microbial communities has generated big datasets of microbial relative abundances varying across environmental conditions, sample sites and treatments. These data often come with putative phylogenies, providing unique opportunities to investigate how shared evolutionary history affects microbial abundance patterns. Here, we present a method to identify the phylogenetic factors driving patterns in microbial community composition. We use the method, "phylofactorization," to re-analyze datasets from the human body and soil microbial communities, demonstrating how phylofactorization is a dimensionality-reducing tool, an ordination-visualization tool, and an inferential tool for identifying edges in the phylogeny along which putative functional ecological traits may have arisen.

Variation in the Intensity of Selection on Codon Bias over Time Causes Contrasting Patterns of Base Composition Evolution in Drosophila

PubMed Central

Jackson, Benjamin C.; Campos, José L.; Haddrill, Penelope R.; Charlesworth, Brian

2017-01-01

Four-fold degenerate coding sites form a major component of the genome, and are often used to make inferences about selection and demography, so that understanding their evolution is important. Despite previous efforts, many questions regarding the causes of base composition changes at these sites in Drosophila remain unanswered. To shed further light on this issue, we obtained a new whole-genome polymorphism data set from D. simulans. We analyzed samples from the putatively ancestral range of D. simulans, as well as an existing polymorphism data set from an African population of D. melanogaster. By using D. yakuba as an outgroup, we found clear evidence for selection on 4-fold sites along both lineages over a substantial period, with the intensity of selection increasing with GC content. Based on an explicit model of base composition evolution, we suggest that the observed AT-biased substitution pattern in both lineages is probably due to an ancestral reduction in selection intensity, and is unlikely to be the result of an increase in mutational bias towards AT alone. By using two polymorphism-based methods for estimating selection coefficients over different timescales, we show that the selection intensity on codon usage has been rather stable in D. simulans in the recent past, but the long-term estimates in D. melanogaster are much higher than the short-term ones, indicating a continuing decline in selection intensity, to such an extent that the short-term estimates suggest that selection is only active in the most GC-rich parts of the genome. Finally, we provide evidence for complex evolutionary patterns in the putatively neutral short introns, which cannot be explained by the standard GC-biased gene conversion model. These results reveal a dynamic picture of base composition evolution. PMID:28082609

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

NASA Astrophysics Data System (ADS)

Kalid, Ori; Mense, Martin; Fischman, Sharon; Shitrit, Alina; Bihler, Hermann; Ben-Zeev, Efrat; Schutz, Nili; Pedemonte, Nicoletta; Thomas, Philip J.; Bridges, Robert J.; Wetmore, Diana R.; Marantz, Yael; Senderowitz, Hanoch

2010-12-01

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

Genetic variation in the MITF promoter affects skin colour and transcriptional activity in black-boned chickens.

PubMed

Wang, G; Liao, J; Tang, M; Yu, S

2018-02-01

1. Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte development by regulating the transcription of major pigmentation enzymes (e.g. TYR, TYRP1 and DCT). A single-nucleotide polymorphism (SNP), c.-638T>C, was identified in the MITF promoter, and genotyping of a population (n = 426) revealed that SNP c.-638T>C was associated with skin colour in black-boned chickens. 2. Individuals with genotypes CC and TC exhibited greater MTIF expression than those with genotype TT. Luciferase assays also revealed that genotype CC and TC promoters had higher activity levels than genotype TT. Expression of melanogenesis-related gene (TYR) was higher in the skin of chickens with the CC and CT genotype compared to TT chickens (P < 0.05). 3. Transcription factor-binding site analyses showed that the c.-638C allele contains a putative binding site for transcription factor sterol regulatory element-binding transcription factor 2, aryl hydrocarbon receptor nuclear translocator, transcription factor binding to IGHM enhancer 3 and upstream transcription factor 2. In contrast, the c.-638T allele contains binding sites for Sp3 transcription factor and Krüppel-like factor 1. 4. It was concluded that MITF promoter polymorphisms affected chicken skin colour. SNP c.-638T>C could be used for the marker-assisted selection of skin colour in black-boned chicken breeding.

Crystal structures of two novel dye-decolorizing peroxidases reveal a beta-barrel fold with a conserved heme-binding motif.

PubMed

Zubieta, Chloe; Krishna, S Sri; Kapoor, Mili; Kozbial, Piotr; McMullan, Daniel; Axelrod, Herbert L; Miller, Mitchell D; Abdubek, Polat; Ambing, Eileen; Astakhova, Tamara; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C; Duan, Lian; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K; Hale, Joanna; Hampton, Eric; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kumar, Abhinav; Marciano, David; Morse, Andrew T; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L; Schimmel, Paul; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2007-11-01

BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein). (c) 2007 Wiley-Liss, Inc.

Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass

PubMed Central

Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

2016-01-01

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca2+ ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca2+ ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite. PMID:26975884

Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase γ: Novel Mechanisms of Function and Pathogenesis.

PubMed

Euro, Liliya; Haapanen, Outi; Róg, Tomasz; Vattulainen, Ilpo; Suomalainen, Anu; Sharma, Vivek

2017-03-07

DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory β-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol γ-associated neurodegenerative disorders.

Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

NASA Astrophysics Data System (ADS)

Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

2016-05-01

A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana, USA

PubMed Central

King, Gary M.; Judd, Craig; Kuske, Cheryl R.; Smith, Conor

2012-01-01

We used high throughput pyrosequencing to characterize stomach and gut content microbiomes of Crassostrea virginica, the Easter oyster, obtained from two sites, one in Barataria Bay (Hackberry Bay) and the other in Terrebonne Bay (Lake Caillou), Louisiana, USA. Stomach microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by Mollicutes most closely related to Mycoplasma; a more rich community dominated by Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities for oysters from both sites differed from stomach communities, and harbored a relatively diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella and a Chloroflexi strain dominated the Lake Caillou and Hackberry Bay gut microbiota, respectively. While many members of the stomach and gut microbiomes appeared to be transients or opportunists, a putative core microbiome was identified based on phylotypes that occurred in all stomach or gut samples only. The putative core stomach microbiome comprised 5 OTUs in 3 phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla. These results collectively revealed novel microbial communities within the oyster digestive system, the functions of the oyster microbiome are largely unknown. A comparison of microbiomes from Louisiana oysters with bacterial communities reported for other marine invertebrates and fish indicated that molluscan microbiomes were more similar to each other than to microbiomes of polychaetes, decapods and fish. PMID:23251548

Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana, USA

DOE PAGES

King, Gary M.; Judd, Craig; Kuske, Cheryl R.; ...

2012-12-12

In this paper, we used high throughput pyrosequencing to characterize stomach and gut content microbiomes of Crassostrea virginica, the Easter oyster, obtained from two sites, one in Barataria Bay (Hackberry Bay) and the other in Terrebonne Bay (Lake Caillou), Louisiana, USA. Stomach microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by Mollicutes most closely related to Mycoplasma; a more rich community dominated by Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities for oysters from both sites differed from stomach communities, and harbored a relatively diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella and a Chloroflexi strainmore » dominated the Lake Caillou and Hackberry Bay gut microbiota, respectively. While many members of the stomach and gut microbiomes appeared to be transients or opportunists, a putative core microbiome was identified based on phylotypes that occurred in all stomach or gut samples only. The putative core stomach microbiome comprised 5 OTUs in 3 phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla. These results collectively revealed novel microbial communities within the oyster digestive system, the functions of the oyster microbiome are largely unknown. Finally, a comparison of microbiomes from Louisiana oysters with bacterial communities reported for other marine invertebrates and fish indicated that molluscan microbiomes were more similar to each other than to microbiomes of polychaetes, decapods and fish.« less

Characterization of putative class II bacteriocins identified from a non-bacteriocin-producing strain Lactobacillus casei ATCC 334.

PubMed

Kuo, Yang-Cheng; Liu, Cheng-Feng; Lin, Jhao-Fen; Li, An-Chieh; Lo, Ta-Chun; Lin, Thy-Hou

2013-01-01

Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334, all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activity. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121°C for 30 min but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced.

gamma. -Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dam, T.V.; Takeda, Y.; Krause, J.E.

1990-01-01

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide (gamma-PPT-(72-92)-NH2), a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeledmore » Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.« less

Structural analysis of a putative SAM-dependent methyltransferase, YtqB, from Bacillus subtilis.

PubMed

Park, Sun Cheol; Song, Wan Seok; Yoon, Sung-il

2014-04-18

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB. Copyright © 2014 Elsevier Inc. All rights reserved.

Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase.

PubMed

Leferink, Nicole G H; Han, Cong; Antonyuk, Svetlana V; Heyes, Derren J; Rigby, Stephen E J; Hough, Michael A; Eady, Robert R; Scrutton, Nigel S; Hasnain, S Samar

2011-05-17

We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.

In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

PubMed Central

Tanaka, Mizuki; Sakai, Yoshifumi; Yamada, Osamu; Shintani, Takahiro; Gomi, Katsuya

2011-01-01

To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them. PMID:21586533

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Ti; Li, De-Feng; Zhou, Ning-Yi, E-mail: n.zhou@pentium.whiov.ac.cn

2011-07-08

Highlights: {yields} Application of site-directed mutagenesis to probe the active site residues of glutathione-dependent maleylpyruvate isomerase. {yields} Two conserved residues, Arg8 and Arg176, in zeta class glutathione S-transferases are critical for maleylpyruvate orientation and enolization. {yields} Arg109, found exclusively in NagL, participates in k{sub cat} regulation. {yields} The T11A mutant exhibited a significantly decreased K{sub m} value for glutathione with little impact on maleylpyruvate kinetics. {yields} The Thr11 residue appears to have significance in the evolution of glutathione S-transferase classes. -- Abstract: The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerizationmore » of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k{sub cat} regulation. Surprisingly, the T11A mutant had a decreased GSH K{sub m} value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.« less

Portal inflammation during NAFLD is frequent and associated with the early phases of putative hepatic progenitor cell activation.

PubMed

Carotti, Simone; Vespasiani-Gentilucci, Umberto; Perrone, Giuseppe; Picardi, Antonio; Morini, Sergio

2015-11-01

We investigated whether portal tract inflammation observed in non-alcoholic fatty liver disease (NAFLD) is associated with hepatic progenitor cell compartment activation, as thoroughly evaluated with different markers of the staminal lineage. Fifty-two patients with NAFLD were studied. NAFLD activity score, fibrosis and portal inflammation were histologically evaluated. Putative hepatic progenitor cells, intermediate hepatobiliary cells and bile ductules/interlobular bile ducts were evaluated by immunohistochemistry for cytokeratin (CK)-7, CK-19 and epithelial cell adhesion molecule (EpCAM), and a hepatic progenitor cell compartment score was derived. Hepatic stellate cell and myofibroblast activity was determined by immunohistochemistry for α-smooth muscle actin. Portal inflammation was absent in a minority of patients, mild in 40% of cases and more than mild in about half of patients, showing a strong correlation with fibrosis (r=0.76, p<0.001). Portal inflammation correlated with CK-7-counted putative hepatic progenitor cells (r=0.48, p<0.001), intermediate hepatobiliary cells (r=0.6, p<0.001) and bile ductules/interlobular bile ducts (r=0.6, p<0.001), and with the activity of myofibroblasts (r=0.5, p<0.001). Correlations were confirmed when elements were counted by immunostaining for CK-19 and EpCAM. Lobular inflammation, ballooning, myofibroblast activity and hepatic progenitor cell compartment activation were associated with portal inflammation by univariate analysis. In the multivariate model, the only variable independently associated with portal inflammation was hepatic progenitor cell compartment activation (OR 3.7, 95% CI 1.1 to 12.6). Portal inflammation is frequent during NAFLD and strongly associated with activation of putative hepatic progenitor cells since the first steps of their differentiation, portal myofibroblast activity and fibrosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

PubMed Central

2013-01-01

Background Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. Results We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). Conclusions Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3. PMID:24001316

Continuous movement behavior of humpback whales during the breeding season in the southwest Indian Ocean: on the road again!

PubMed

Dulau, Violaine; Pinet, Patrick; Geyer, Ygor; Fayan, Jacques; Mongin, Philippe; Cottarel, Guillaume; Zerbini, Alexandre; Cerchio, Salvatore

2017-01-01

Humpback whales are known to undertake long-distance migration between feeding and breeding sites, but their movement behavior within their breeding range is still poorly known. Satellite telemetry was used to investigate movement of humpback whales during the breeding season and provide further understanding of the breeding ecology and sub-population connectivity within the southwest Indian Ocean (SWIO). Implantable Argos satellite tags were deployed on 15 whales (7 males and 6 females) during the peak of the breeding season in Reunion Island. A switching-state-space model was applied to the telemetry data, in order to discriminate between "transiting" and "localized" movements, the latter of which relates to meandering behavior within putative breeding habitats, and a kernel density analysis was used to assess the spatial scale of the main putative breeding sites. Whales were tracked for up to 71 days from 31/07/2013 to 16/10/2013. The mean transmission duration was 25.7 days and the mean distance travelled was 2125.8 km. The tracks showed consistent movement of whales from Reunion to Madagascar, demonstrating a high level of connectivity between the two sub-regions, and the use of yet unknown breeding sites such as underwater seamounts (La Perouse) and banks (Mascarene Plateau). A localized movement pattern occurred in distinct bouts along the tracks, suggesting that whales were involved in breeding activity for 4.3 consecutive days on average, after which they resume transiting for an average of 6.6 days. Males visited several breeding sites within the SWIO, suggesting for the first time a movement strategy at a basin scale to maximize mating. Unexpectedly, females with calf also showed extensive transiting movement, while they engaged in localized behavior mainly off Reunion and Sainte-Marie (East Madagascar). The results indicated that whales from Reunion do not represent a discrete population. Discrete breeding sites were identified, thereby highlighting priority areas for conservation. The study is a first attempt to quantify movement of humpback whales within the southwestern Indian Ocean breeding range. We demonstrate a wandering behavior with stopovers at areas that likely represent key breeding habitat, a strategy which may enhance likelihood of individual reproductive success.

Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

PubMed Central

Kohler, Samay; Buck, Annika; Dambacher, Christine; König, Armin; Bohnert, Jürgen A.; Kern, Winfried V.

2014-01-01

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. PMID:25182653

Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

PubMed

Garrocho-Villegas, Verónica; Aguilar C, Raúl; Sánchez de Jiménez, Estela

2013-12-23

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Identification of Key Residues for pH Dependent Activation of Violaxanthin De-Epoxidase from Arabidopsis thaliana

PubMed Central

Fufezan, Christian; Simionato, Diana; Morosinotto, Tomas

2012-01-01

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pKa values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121–Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role. PMID:22558195

Identification of key residues for pH dependent activation of violaxanthin de-epoxidase from Arabidopsis thaliana.

PubMed

Fufezan, Christian; Simionato, Diana; Morosinotto, Tomas

2012-01-01

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pK(a) values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121-Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role.

NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

PubMed

Cattaruzza, Sabrina; Nicolosi, Pier Andrea; Braghetta, Paola; Pazzaglia, Laura; Benassi, Maria Serena; Picci, Piero; Lacrima, Katia; Zanocco, Daniela; Rizzo, Erika; Stallcup, William B; Colombatti, Alfonso; Perris, Roberto

2013-06-01

In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

PubMed

Pornbanlualap, Somchai; Chalopagorn, Pornchanok

2011-08-01

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. Copyright © 2011 Elsevier Inc. All rights reserved.

TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens.

PubMed

Marchesan, Julie; Jiao, Yizu; Schaff, Riley A; Hao, Jie; Morelli, Thiago; Kinney, Janet S; Gerow, Elizabeth; Sheridan, Rachel; Rodrigues, Vinicius; Paster, Bruce J; Inohara, Naohiro; Giannobile, William V

2016-06-01

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

TLR4, NOD1 and NOD2 Mediate Immune Recognition of Putative Newly-Identified Periodontal Pathogens

PubMed Central

Schaff, Riley A.; Hao, Jie; Morelli, Thiago; Kinney, Janet S.; Gerow, Elizabeth; Sheridan, Rachel; Rodrigues, Vinicius; Paster, Bruce J.; Inohara, Naohiro; Giannobile, William V.

2015-01-01

SUMMARY Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. While the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, 6 being classical pathogens and 4 putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone marrow–derived macrophages (BMDM) from wild-type (WT) and toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. C. concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney (HEK) cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2-stimulatory activity. These studies allowed us to provide important evidence on newly-identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855). PMID:26177212

Mucin (MUC1) Expression and Function in Prostate Cancer Cells

DTIC Science & Technology

2004-03-01

Biophysical Research Communications 293 (2002) 1183-1190 1189 site, and is a feature of MUC4 metabolism [36]. [12] S. Zotter, P.C. Hageman, A. Lossnitzer...W.J. Mooi, J. Hilgers, Although the cleavage site sequence in MUC4 differs Tissue and tumor distribution of human polymorphic epithelial from that of...family of putative secreted (2001) 715-720. and membrane-associated MUC4 mucins, Eur. J. Biochem. 267 [261 M. Boshell, E-N. Lalani, L. Pemberton, J

The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

PubMed Central

Liu, Xin; Ropp, Susan L.; Jackson, Richard J.; Frey, Teryl K.

1998-01-01

The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn2+, Cd2+, and Co2+, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans. PMID:9557742

Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

PubMed Central

Matsumoto, Yuiko; Buemio, Alvin; Chu, Randy; Vafaee, Mozhgon; Crews, David

2013-01-01

In the red-eared slider turtle (Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5′-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5′-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development. PMID:23762231

The wheat homolog of putative nucleotide-binding site-leucine-rich repeat resistance gene TaRGA contributes to resistance against powdery mildew.

PubMed

Wang, Defu; Wang, Xiaobing; Mei, Yu; Dong, Hansong

2016-03-01

Powdery mildew, one of the most destructive wheat diseases worldwide, is caused by Blumeria graminis f. sp. tritici (Bgt), a fungal species with a consistently high mutation rate that makes individual resistance (R) genes ineffective. Therefore, effective resistance-related gene cloning is vital for breeding and studying the resistance mechanisms of the disease. In this study, a putative nucleotide-binding site-leucine-rich repeat (NBS-LRR) R gene (TaRGA) was cloned using a homology-based cloning strategy and analyzed for its effect on powdery mildew disease and wheat defense responses. Real-time reverse transcription-PCR (RT-PCR) analyses revealed that a Bgt isolate 15 and salicylic acid stimulation significantly induced TaRGA in the resistant variety. Furthermore, the silencing of TaRGA in powdery mildew-resistant plants increased susceptibility to Bgt15 and prompted conidia propagation at the infection site. However, the expression of TaRGA in leaf segments after single-cell transient expression assay highly increased the defense responses to Bgt15 by enhancing callose deposition and phenolic autofluorogen accumulation at the pathogen invading sites. Meanwhile, the expression of pathogenesis-related genes decreased in the TaRGA-silenced plants and increased in the TaRGA-transient-overexpressing leaf segments. These results implied that the TaRGA gene positively regulates the defense response to powdery mildew disease in wheat.

Civilising Recalcitrant Boys' Bodies: Pursuing Social Fitness through the Anti-Obesity Offensive

ERIC Educational Resources Information Center

Monaghan, Lee F.

2014-01-01

Obesity discourse provides a commonly recycled rationale for multiple, ostensibly well-intended, interventions. Formal educational settings sometimes operate as sites for these biopedagogies which putatively promote "good health" among young people as they transition to "responsible" adulthood. Yet, regulation and control, or…

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs

The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. colimore » α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.« less

Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: Functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Xiaokuang; Davis, F.C.; Ingram, L.O.

1997-02-01

Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-{beta}-glucosidase, which appear to form an operon (casRAB).more » Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-{beta}-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation. 63 refs., 4 figs., 4 tabs.« less

Structural and functional characterisation of multi-copper oxidase CueO from lignin-degrading bacterium Ochrobactrum sp. reveal its activity towards lignin model compounds and lignosulfonate.

PubMed

Granja-Travez, Rommel Santiago; Wilkinson, Rachael C; Persinoti, Gabriela Felix; Squina, Fabio M; Fülöp, Vilmos; Bugg, Timothy D H

2018-05-01

The identification of enzymes responsible for oxidation of lignin in lignin-degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin-degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B-type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi-copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase-like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of β-aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca-lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four-coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three-coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase-like multicopper oxidases in some lignin-degrading bacteria. Structural data are available in the PDB under the accession number 6EVG. © 2018 Federation of European Biochemical Societies.

Identification and characterization of gamma-glutamylamine cyclotransferase, an enzyme responsible for gamma-glutamyl-epsilon-lysine catabolism.

PubMed

Oakley, Aaron J; Coggan, Marjorie; Board, Philip G

2010-03-26

Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.

Identification and Characterization of γ-Glutamylamine Cyclotransferase, an Enzyme Responsible for γ-Glutamyl-ϵ-lysine Catabolism*

PubMed Central

Oakley, Aaron J.; Coggan, Marjorie; Board, Philip G.

2010-01-01

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu82). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity. PMID:20110353

An extracellular disulfide bond forming protein (DsbF) from Mycobacterium tuberculosis: Structural, biochemical and gene expression analysis

PubMed Central

Chim, Nicholas; Riley, Robert; The, Juliana; Im, Soyeon; Segelke, Brent; Lekin, Tim; Yu, Minmin; Hung, Li Wei; Terwilliger, Tom; Whitelegge, Julian P.; Goulding, Celia W.

2010-01-01

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here we present structural, biochemical and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pKa for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein co-expression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicate anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions. PMID:20060836

Estrogen-induced transcription factor EGR1 regulates c-Kit transcription in the mouse uterus to maintain uterine receptivity for embryo implantation.

PubMed

Park, Mira; Kim, Hye-Ryun; Kim, Yeon Sun; Yang, Seung Chel; Yoon, Jung Ah; Lyu, Sang Woo; Lim, Hyunjung Jade; Hong, Seok-Ho; Song, Haengseok

2018-07-15

Early growth response 1 (Egr1) is a key transcription factor that mediates the action of estrogen (E 2 ) to establish uterine receptivity for embryo implantation. However, few direct target genes of EGR1 have been identified in the uterus. Here, we demonstrated that E 2 induced EGR1-regulated transcription of c-Kit, which plays a crucial role in cell fate decisions. Spatiotemporal expression of c-Kit followed that of EGR1 in uteri of ovariectomized mice at various time points after E 2 treatment. E 2 activated ERK1/2 and p38 to induce EGR1, which then activated c-Kit expression in the uterus. EGR1 transfection produced rapid and transient induction of c-KIT in a time- and dose-dependent manner. Furthermore, luciferase assays to measure c-Kit promoter activity confirmed that a functional EGR1 binding site(s) (EBS) was located within -1 kb of the c-Kit promoter. Site-directed mutagenesis and chromatin immunoprecipitation-PCR for three putative EBS within -1 kb demonstrated that the EBS at -818/-805 was critical for EGR1-dependent c-Kit transcription. c-Kit expression was significantly increased in the uterus on day 4 and administration of Masitinib, a c-Kit inhibitor, effectively interfered with embryo implantation. Collectively, our results showed that estrogen induces transcription factor EGR1 to regulate c-Kit transcription for uterine receptivity for embryo implantation in the mouse uterus. Copyright © 2017 Elsevier B.V. All rights reserved.

Biochemical investigations and mapping of the calcium-binding sites of heparinase I from Flavobacterium heparinum.

PubMed

Shriver, Z; Liu, D; Hu, Y; Sasisekharan, R

1999-02-12

The heparinases from Flavobacterium heparinum are lyases that specifically cleave heparin-like glycosaminoglycans. Previously, amino acids located in the active site of heparinase I have been identified and mapped. In an effort to further understand the mechanism by which heparinase I cleaves its polymer substrate, we sought to understand the role of calcium, as a necessary cofactor, in the enzymatic activity of heparinase I. Specifically, we undertook a series of biochemical and biophysical experiments to answer the question of whether heparinase I binds to calcium and, if so, which regions of the protein are involved in calcium binding. Using the fluorescent calcium analog terbium, we found that heparinase I tightly bound divalent and trivalent cations. Furthermore, we established that this interaction was specific for ions that closely approximate the ionic radius of calcium. Through the use of the modification reagents N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, we showed that the interaction between heparinase I and calcium was essential for proper functioning of the enzyme. Preincubation with either calcium alone or calcium in the presence of heparin was able to protect the enzyme from inactivation by these modifying reagents. In addition, through mapping studies of Woodward's reagent K-modified heparinase I, we identified two putative calcium-binding sites, CB-1 (Glu207-Ala219) and CB-2 (Thr373-Arg384), in heparinase I that not only are specifically modified by Woodward's reagent K, leading to loss of enzymatic activity, but also conform to the calcium-coordinating consensus motif.

Role of Specificity Protein-1 and Activating Protein-2 Transcription Factors in the Regulation of the Gap Junction Protein Beta-2 Gene in the Epididymis of the Rat.

PubMed

Adam, Cécile; Cyr, Daniel G

2016-06-01

In prepubertal rats, connexin 26 (GJB2) is expressed between adjacent columnar cells of the epididymis. At 28 days of age, when columnar cells differentiate into adult epithelial cell types, Gjb2 mRNA levels decrease to barely detectable levels. There is no information on the regulation of GJB2 in the epididymis. The present study characterized regulation of the Gjb2 gene promoter in the epididymis. A single transcription start site at position -3829 bp relative to the ATG was identified. Computational analysis revealed several TFAP2A, SP1, and KLF4 putative binding sites. A 1.5-kb fragment of the Gjb2 promoter was cloned into a vector containing a luciferase reporter gene. Transfection of the construct into immortalized rat caput epididymal (RCE-1) cells indicated that the promoter contained sufficient information to drive expression of the reporter gene. Deletion constructs showed that the basal activity of the promoter resides in the first -230 bp of the transcriptional start site. Two response elements necessary for GJB2 expression were identified: an overlapping TFAP2A/SP1 site (-136 to -126 bp) and an SP1 site (-50 bp). Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays confirmed that SP1 and TFAP2A were bound to the promoter. ChIP analysis of chromatin from young and pubertal rats indicated that TFAP2A and SP1 binding decreased with age. SP1 and TFAP2A knockdown indicated that SP1 is necessary for Gjb2 expression. DNA methylation did not appear to be involved in the regulation of Gjb2 expression. Results indicate that SP1 and TFAP2A regulate Gjb2 promoter activity during epididymal differentiation in rat. © 2016 by the Society for the Study of Reproduction, Inc.

PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

PubMed

Gunawardana, Dilantha

2016-01-01

Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A.

1992-01-01

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

PubMed

Byrne, C; Miclet, E; Broutin, I; Gallo, D; Pelekanou, V; Kampa, M; Castanas, E; Leclercq, G; Jacquot, Y

2013-10-01

Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation. © 2013 Wiley Periodicals, Inc.

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri.

PubMed

Sergueev, Kirill; Dabrazhynetskaya, Alena; Austin, Stuart

2005-05-01

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

PubMed Central

Naing, Swe-Htet; Kalyoncu, Sibel; Smalley, David M.; Kim, Hyojung; Tao, Xingjian; George, Josh B.; Jonke, Alex P.; Oliver, Ryan C.; Urban, Volker S.; Torres, Matthew P.; Lieberman, Raquel L.

2018-01-01

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His–Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr–Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala–Thr) and the other to the non-pathogenic Aβ48 (Thr–Leu). For the former site, we observed more favorable kinetics in lipid bilayer–mimicking bicelles than in detergent solution, indicating that substrate–lipid and substrate–enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions. PMID:29382721

Listeria monocytogenes PdeE, a phosphodiesterase that contributes to virulence and has hydrolytic activity against cyclic mononucleotides and cyclic dinucleotides

USDA-ARS?s Scientific Manuscript database

We have identified and partially characterized a putative HD domain hydrolase, LMOf2365_2464, which is highly expressed during listerial intracellular replication. LMOf2365_2464 is annotated as a putative HD domain-containing hydrolase. The ability of an isogenic mutant strain, F2365'2464, to adhere...

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family.

PubMed

Pacheco-Alvarez, Diana; Vázquez, Norma; Castañeda-Bueno, María; de-Los-Heros, Paola; Cortes-González, César; Moreno, Erika; Meade, Patricia; Bobadilla, Norma A; Gamba, Gerardo

2012-01-01

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation. Copyright © 2012 S. Karger AG, Basel.

Molecular scaffold reorganization at the transmitter release site with vesicle exocytosis or botulinum toxin C1.

PubMed

Stanley, Elise F; Reese, Tom S; Wang, Gary Z

2003-10-01

Neurotransmitter release sites at the freeze-fractured frog neuromuscular junction are composed of inner and outer paired rows of large membrane particles, the putative calcium channels, anchored by the ribs of an underlying protein scaffold. We analysed the locations of the release site particles as a reflection of the scaffold structure, comparing particle distributions in secreting terminals with those where secretion was blocked with botulinum toxin A, which cleaves a small segment off SNAP-25, or botulinum toxin C1, which cleaves the cytoplasmic domain of syntaxin. In the idle terminal the inner and outer paired rows were located approximately 25 and approximately 44 nm, respectively, from the release site midline. However, adjacent to vesicular fusion sites both particle rows were displaced towards the midline by approximately 25%. The intervals between the particles along each row were examined by a nearest-neighbour approach. In control terminals the peak interval along the inner row was approximately 17 nm, consistent with previous reports and the spacing of the scaffold ribs. While the average distance between particles in the outer row was also approximately 17 nm, a detailed analysis revealed short 'linear clusters' with a approximately 14 nm interval. These clusters were enriched at vesicle fusion sites, suggesting an association with the docking sites, and were eliminated by botulinum C1, but not A. Our findings suggest, first, that the release site scaffold ribs undergo a predictable, and possibly active, shortening during exocytosis and, second, that at the vesicle docking site syntaxin plays a role in the cross-linking of the rib tips to form the vesicle docking sites.

The genetic and functional basis of isolated 17,20-lyase deficiency.

PubMed

Geller, D H; Auchus, R J; Mendonça, B B; Miller, W L

1997-10-01

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. Patients with putative isolated 17,20-lyase deficiency have been reported. The existence of true isolated 17,20-lyase deficiency, however, has been questioned because 17 alpha-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme, microsomal cytochrome P450c17, and because the index case of apparent isolated 17,20-lyase deficiency had combined deficiencies of both activities. We studied two patients with clinical and hormonal findings suggestive of isolated 17,20-lyase deficiency. We found two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17. When expressed in COS-1 cells, the mutants retained 17 alpha-hydroxylase activity but had minimal 17,20-lyase activity. Substrate competition experiments suggested that the mutations did not alter the enzyme's substrate-binding capacity, but co-transfection of cells with P450 oxidoreductase, the electron donor used by P450c17, indicated that the mutants had a diminished ability to interact with redox partners. Computer-graphic modelling of P450c17 suggests that both mutations lie in or near the redox-partner binding site, on the opposite side of the haem from the substrate-binding pocket. These mutations alter electrostatic charge distribution in the redox-partner binding site, so that electron transfer for the 17,20-lyase reaction is selectively lost or diverted to uncoupling reactions. These are the first proven cases of isolated 17,20-lyase deficiency, and they demonstrate a novel mechanism for loss of enzymatic activity.

Identification of a putative binding site critical for general anesthetic activation of TRPA1.

PubMed

Ton, Hoai T; Phan, Thieu X; Abramyan, Ara M; Shi, Lei; Ahern, Gerard P

2017-04-04

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABA A receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

The search for sand fly adults in a village in southern Egypt

USDA-ARS?s Scientific Manuscript database

There are several good papers in the literature describing methods for collecting adult phlebotomine sand flies from habitats putatively used for resting sites. The published data from such searches demonstrate that finding adult sand flies can be quite difficult even when using established methods....

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

PubMed

Ueyama, T; Zhu, C; Valenzuela, Y M; Suzow, J G; Stewart, A F

2000-06-09

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

The properties, distribution and function of Na+–Ca2+ exchanger isoforms in rat cutaneous sensory neurons

PubMed Central

Scheff, N N; Yilmaz, E; Gold, M S

2014-01-01

The Na+–Ca2+ exchanger (NCX) appears to play an important role in the regulation of the high K+-evoked Ca2+ transient in putative nociceptive dorsal root ganglion (DRG) neurons. The purpose of the present study was to (1) characterize the properties of NCX activity in subpopulations of DRG neurons, (2) identify the isoform(s) underlying NCX activity, and (3) begin to assess the function of the isoform(s) in vivo. In retrogradely labelled neurons from the glabrous skin of adult male Sprague–Dawley rats, NCX activity, as assessed with fura-2-based microfluorimetry, was only detected in putative nociceptive IB4+ neurons. There were two modes of NCX activity: one was evoked in response to relatively large and long lasting (∼325 nm for >12 s) increases in the concentration of intracellular Ca2+ ([Ca2+]i), and a second was active at resting [Ca2+]i > ∼150 nm. There also were two modes of evoked activity: one that decayed relatively rapidly (<5 min) and a second that persisted (>10 min). Whereas mRNA encoding all three NCX isoforms (NCX1–3) was detected in putative nociceptive cutaneous neurons with single cell PCR, pharmacological analysis and small interfering RNA (siRNA) knockdown of each isoform in vivo suggested that NCX2 and 3 were responsible for NCX activity. Western blot analyses suggested that NCX isoforms were differentially distributed within sensory neurons. Functional assays of excitability, action potential propagation, and nociceptive behaviour suggest NCX activity has little influence on excitability per se, but instead influences axonal conduction velocity, resting membrane potential, and nociceptive threshold. Together these results indicate that the function of NCX in the regulation of [Ca2+]i in putative nociceptive neurons may be unique relative to other cells in which these exchanger isoforms have been characterized and it has the potential to influence sensory neuron properties at multiple levels. PMID:25239455

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

PubMed

Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

2013-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

In silico Prediction, in vitro Antibacterial Spectrum, and Physicochemical Properties of a Putative Bacteriocin Produced by Lactobacillus rhamnosus Strain L156.4

PubMed Central

Oliveira, Letícia de C.; Silveira, Aline M. M.; Monteiro, Andréa de S.; dos Santos, Vera L.; Nicoli, Jacques R.; Azevedo, Vasco A. de C.; Soares, Siomar de C.; Dias-Souza, Marcus V.; Nardi, Regina M. D.

2017-01-01

A bacteriocinogenic Lactobacillus rhamnosus L156.4 strain isolated from the feces of NIH mice was identified by 16S rRNA gene sequencing and MALDI-TOF mass spectrometry. The entire genome was sequenced using Illumina, annotated in the PGAAP, and RAST servers, and deposited. Conserved genes associated with bacteriocin synthesis were predicted using BAGEL3, leading to the identification of an open reading frame (ORF) that shows homology with the L. rhamnosus GG (ATCC 53103) prebacteriocin gene. The encoded protein contains a conserved protein motif associated a structural gene of the Enterocin A superfamily. We found ORFs related to the prebacteriocin, immunity protein, ABC transporter proteins, and regulatory genes with 100% identity to those of L. rhamnosus HN001. In this study, we provide evidence of a putative bacteriocin produced by L. rhamnosus L156.4 that was further confirmed by in vitro assays. The antibacterial activity of the substances produced by this strain was evaluated using the deferred agar-spot and spot-on-the lawn assays, and a wide antimicrobial activity spectrum against human and foodborne pathogens was observed. The physicochemical characterization of the putative bacteriocin indicated that it was sensitive to proteolytic enzymes, heat stable and maintained its antibacterial activity in a pH ranging from 3 to 9. The activity against Lactobacillus fermentum, which was used as an indicator strain, was detected during bacterial logarithmic growth phase, and a positive correlation was confirmed between bacterial growth and production of the putative bacteriocin. After a partial purification from cell-free supernatant by salt precipitation, the putative bacteriocin migrated as a diffuse band of approximately 1.0–3.0 kDa by SDS-PAGE. Additional studies are being conducted to explore its use in the food industry for controlling bacterial growth and for probiotic applications. PMID:28579977

[Preliminary proteomics analysis of the total proteins of HL Type cytoplasmic male sterility rice anther].

PubMed

Wen, Li; Liu, Gai; Zhang, Zai-Jun; Tao, Jun; Wan, Cui-Xiang; Zhu, Ying-Guo

2006-03-01

The proteins of HL type cytoplasmic male sterility rice anther of YTA (CMS) and YTB (maintenance line) were separated by two-dimensional electrophoresis with immobilized ph (3-10 non-linear) gradients as the first dimension and SDS-PAGE as the second. The silver-stained proteins spots were analyzed using Image Master 2D software, there were about 1800 detectable spots on each 2D-gel, and about 85 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching, 9 protein spots out of 16 were identified. Among those proteins, there were Putative nucleic acid binding protein, glucose-1-phosphate adenylyltransferase (ADP-glucose pyrophosphorylase, AGPase) (EC: 2.7.7.27) large chain, UDP-glucuronic acid decarboxylase, putative calcium-binding protein annexin, putative acetyl-CoA synthetase and putative lipoamide dehydrogenase etc. They were closely associated with metabolism, protein biosynthesis, transcription, signal transduction and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins, i.e. AGPase, putative lipoamide dehydrogenase and putative acetyl-CoA synthetase were deeply discussed on the relationship to CMS. AGPase catalyzes a very important step in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants: synthesis of the activated glucosyl donor, ADP-glucose, from glucose-1-phosphate and ATP. The lack of the AGPase in male sterile line might directly result in the reduction of starch, and the synthesis of starch was the most important processes during the development of pollen. In present research, the descent or reduction of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase seemed involved in pollen sterility in rice. The degeneration and formation of various tissues during pollen development may impose high demands for energy and key biosynthetic intermediates. Under such conditions, the TCA cycle needs to operate fully, because the TCA cycle is an important source for many intermediates required for biosynthetic pathways, in addition to performing an oxidative, energy-producing role. Thus, it seemed reasonable to infer that the decrease of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase in anther might prevent the conversion of pyruvate into acetyl-CoA, and as a result, the TCA cycle could no longer operate at a sufficient rate to meet all requirements in anther cells, leading to pollen sterility. This study gave new insights into the mechanism of CMS in rice and demonstrated the power of the proteomic approach in plant biology studies.

Ubiquinol-binding site in the alternative oxidase: mutagenesis reveals features important for substrate binding and inhibition.

PubMed

Albury, Mary S; Elliott, Catherine; Moore, Anthony L

2010-12-01

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in all plants, some fungi, green algae, bacteria and pathogenic protozoa. The lack of AOX in the mammalian host renders this protein an important potential therapeutic target in the treatment of pathogenic protozoan infections. Bioinformatic searches revealed that, within a putative ubiquinol-binding crevice in AOX, Gln242, Asn247, Tyr253, Ser256, His261 and Arg262 were highly conserved. To confirm that these amino-acid residues are important for ubiquinol-binding and hence activity substitution mutations were generated and characterised. Assessment of AOX activity in isolated Schizosaccharomyces pombe mitochondria revealed that mutation of either Gln242, Ser256, His261 and Arg262 resulted in >90% inhibition of antimycin A-insensitive respiration suggesting that hydroxyl, guanidino, imidazole groups, polar and charged residues in addition to the size of the amino-acid chain are important for ubiquinone-binding. Substitution of Asn247 with glutamine or Tyr253 with phenylalanine had little effect upon the respiratory rate indicating that these residues are not critical for AOX activity. However replacement of Tyr253 by alanine resulted in a 72% loss of activity suggesting that the benzoquinone group and not hydroxyl group is important for quinol binding. These results provide important new insights into the ubiquinol-binding site of the alternative oxidase, the identity of which maybe important for future rational drug design. Copyright © 2010 Elsevier B.V. All rights reserved.

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Y.W.; Robinson, H.; Yeung, N.

2010-05-11

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu hasmore » a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.« less

Roles of Glutamates and Metal ions in a Rationally Designed Nitric Oxide Reductase Based on Myoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Lin; N Yeung; Y Gao

2011-12-31

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu hasmore » a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.« less

Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells.

PubMed

Hahn, Young-Il; Kim, Su-Jung; Choi, Bu-Young; Cho, Kyung-Cho; Bandu, Raju; Kim, Kwang Pyo; Kim, Do-Hee; Kim, Wonki; Park, Joon Sung; Han, Byung Woo; Lee, Jeewoo; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

2018-04-23

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin is an anti-inflammatory natural compound isolated from the turmeric (Curcuma longa L., Zingiberaceae) that has been extensively used in a traditional medicine over the centuries. In the present study, we have found that curcumin inhibits STAT3 signaling that is persistently overactivated in H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 of STAT3 as a putative site for curcumin binding. Site-directed mutation of this cysteine residue abolished curcumin-induced inactivation of STAT3 and apoptosis in H-Ras MCF10A cells. The α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its binding to STAT3 in H-Ras MCF10A cells. Tetrahydrocurcumin that lacks such electrophilic moiety failed to interact with STAT3 and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate aberrant activation of STAT3 through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis.

STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

PubMed Central

Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

2014-01-01

Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus*

PubMed Central

Forsberg, Zarah; Nelson, Cassandra E.; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S. M.; Crouch, Lucy I.; Røhr, Åsmund K.; Gardner, Jeffrey G.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

2016-01-01

Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of the CjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles. PMID:26858252

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus.

PubMed

Forsberg, Zarah; Nelson, Cassandra E; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S M; Crouch, Lucy I; Røhr, Åsmund K; Gardner, Jeffrey G; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

2016-04-01

Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of gene expression mediating indeterminate muscle growth in teleosts.

PubMed

Ahammad, A K Shakur; Asaduzzaman, Md; Asakawa, Shuichi; Watabe, Shugo; Kinoshita, Shigeharu

2015-08-01

Teleosts are unique among vertebrates due to their indeterminate muscle growth, i.e., continued production of neonatal muscle fibers until death. However, the molecular mechanism(s) underlying this property is unknown. Here, we focused on the torafugu (Takifugu rubripes) myosin heavy chain gene, MYHM2528-1, which is specifically expressed in neonatal muscle fibers produced by indeterminate muscle growth. We examined the flanking region of MYHM2528-1 through an in vivo reporter assay using zebrafish (Danio rerio) and identified a 2100 bp 5'-flanking sequence that contained sufficient promoter activity to allow specific gene expression. The effects of enhanced promoter activity were observed at the outer region of the fast muscle and the dorsal edge of slow muscle in zebrafish larvae. At the juvenile stage, the promoter was specifically activated in small diameter muscle fibers scattered throughout fast muscle and in slow muscle near the septum separating slow and fast muscles. This spatio-temporal promoter activity overlapped with known myogenic zones involved in teleost indeterminate muscle growth. A deletion mutant analysis revealed that the -2100 to -600 bp 5'flanking sequence of MYHM2528-1 is essential for promoter activity. This region contains putative binding sites for several representative myogenesis-related transcription factors and nuclear factor of activated T-cell (NFAT), a transcription activator involved in regeneration of mammalian adult skeletal muscle. A significant reduction in the promoter activity of the MYHM2528-1 deletion constructs was observed in accordance with a reduction in the number of these binding sites, suggesting the involvement of specific transcription factors in indeterminate muscle growth. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

PubMed Central

Wojciechowski, Cheryl L.; Cardia, James P.; Kantrowitz, Evan R.

2002-01-01

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a kcat of 16 s−1 and a Km of 175 μM at a pH optimum of 8.0 when assayed at 25°C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum kcat of 100 s−1, with a Km of 93 μM at 65°C. Activity was stable at 65°C for >24 h and at 90°C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer. PMID:11910033

Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.

PubMed

Mishra, Dipti Ranjan; Chaudhary, Sanjib; Krishna, B Madhu; Mishra, Sandip K

2015-01-01

Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

Regulation of CYBB Gene Expression in Human Phagocytes by a Distant Upstream NF-κB Binding Site.

PubMed

Frazão, Josias B; Thain, Alison; Zhu, Zhiqing; Luengo, Marcos; Condino-Neto, Antonio; Newburger, Peter E

2015-09-01

The human CYBB gene encodes the gp91-phox component of the phagocyte oxidase enzyme complex, which is responsible for generating superoxide and other downstream reactive oxygen species essential to microbial killing. In the present study, we have identified by sequence analysis a putative NF-κB binding site in a DNase I hypersensitive site, termed HS-II, located in the distant 5' flanking region of the CYBB gene. Electrophoretic mobility assays showed binding of the sequence element by recombinant NF-κB protein p50 and by proteins in nuclear extract from the HL-60 myeloid leukemia cell line corresponding to p50 and to p50/p65 heterodimers. Chromatin immunoprecipitation demonstrated NF-κB binding to the site in intact HL-60 cells. Chromosome conformation capture (3C) assays demonstrated physical interaction between the NF-κB binding site and the CYBB promoter region. Inhibition of NF-κB activity by salicylate reduced CYBB expression in peripheral blood neutrophils and differentiated U937 monocytic leukemia cells. U937 cells transfected with a mutant inhibitor of κB "super-repressor" showed markedly diminished CYBB expression. Luciferase reporter analysis of the NF-κB site linked to the CYBB 5' flanking promoter region revealed enhanced expression, augmented by treatment with interferon-γ. These studies indicate a role for this distant, 15 kb upstream, binding site in NF-κB regulation of the CYBB gene, an essential component of phagocyte-mediated host defense. © 2015 Wiley Periodicals, Inc.

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

PubMed

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

A putative placebo analysis of the effects of LCZ696 on clinical outcomes in heart failure

PubMed Central

McMurray, John; Packer, Milton; Desai, Akshay; Gong, Jianjian; Greenlaw, Nicola; Lefkowitz, Martin; Rizkala, Adel; Shi, Victor; Rouleau, Jean; Solomon, Scott; Swedberg, Karl; Zile, Michael R.; Andersen, Karl; Arango, Juan Luis; Arnold, Malcolm; Be˘lohlávek, Jan; Böhm, Michael; Boytsov, Sergey; Burgess, Lesley; Cabrera, Walter; Chen, Chen-Huan; Erglis, Andrejs; Fu, Michael; Gomez, Efrain; Gonzalez, Angel; Hagege, Albert-Alain; Katova, Tzvetana; Kiatchoosakun, Songsak; Kim, Kee-Sik; Bayram, Edmundo; Martinez, Felipe; Merkely, Bela; Mendoza, Iván; Mosterd, Arend; Negrusz-Kawecka, Marta; Peuhkurinen, Keijo; Ramires, Felix; Refsgaard, Jens; Senni, Michele; Sibulo, Antonio S.; Silva-Cardoso, José; Squire, Iain; Starling, Randall C.; Vinereanu, Dragos; Teerlink, John R.; Wong, Raymond

2015-01-01

Aims Although active-controlled trials with renin–angiotensin inhibitors are ethically mandated in heart failure with reduced ejection fraction, clinicians and regulators often want to know how the experimental therapy would perform compared with placebo. The angiotensin receptor-neprilysin inhibitor LCZ696 was compared with enalapril in PARADIGM-HF. We made indirect comparisons of the effects of LCZ696 with putative placebos. Methods and results We used the treatment-arm of the Studies Of Left Ventricular Dysfunction (SOLVD-T) as the reference trial for comparison of an ACE inhibitor to placebo and the Candesartan in Heart failure: Assessment of Reduction in Mortality and morbidity-Alternative trial (CHARM-Alternative) as the reference trial for comparison of an ARB to placebo. The hazard ratio of LCZ696 vs. a putative placebo was estimated through the product of the hazard ratio of LCZ696 vs. enalapril (active-control) and that of the historical active-control (enalapril or candesartan) vs. placebo. For the primary composite outcome of cardiovascular death or heart failure hospitalization in PARADIGM-HF, the relative risk reduction with LCZ696 vs. a putative placebo from SOLVD-T was 43% (95%CI 34–50%; P < 0.0001) with similarly large effects on cardiovascular death (34%, 21–44%; P < 0.0001) and heart failure hospitalization (49%, 39–58%; P < 0.0001). For all-cause mortality, the reduction compared with a putative placebo was 28% (95%CI 15–39%; P < 0.0001). Putative placebo analyses based on CHARM-Alternative gave relative risk reductions of 39% (95%CI 27–48%; P < 0.0001) for the composite outcome of cardiovascular death or heart failure hospitalization, 32% (95%CI 16–45%; P < 0.0001) for cardiovascular death, 46% (33–56%; P < 0.0001) for heart failure hospitalization, and 26% (95%CI 11–39%; P < 0.0001) for all-cause mortality. Conclusion These indirect comparisons of LCZ696 with a putative placebo show that the strategy of combined angiotensin receptor blockade and neprilysin inhibition led to striking reductions in cardiovascular and all-cause mortality, as well as heart failure hospitalization. These benefits were obtained even though LCZ696 was added to comprehensive background beta-blocker and mineralocorticoid receptor antagonist therapy. PMID:25416329

Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR*

PubMed Central

Li, Hao; Redinbo, Matthew R.; Venkatesh, Madhukumar; Ekins, Sean; Chaudhry, Anik; Bloch, Nicolin; Negassa, Abdissa; Mukherjee, Paromita; Kalpana, Ganjam; Mani, Sridhar

2013-01-01

The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications. PMID:23525103

The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself.

PubMed Central

Klomp, Adriana E M; Juijn, Jenneke A; van der Gun, Linda T M; van den Berg, Inge E T; Berger, Ruud; Klomp, Leo W J

2003-01-01

We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper-binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter. PMID:12466020

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Hongnan; Pauff, James M.; Hille, Russ

Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp{sup 2}-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 {angstrom} resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 {angstrom} resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative productmore » not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg880 and Glu802, previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.« less

Comprehensive analysis of genome-wide DNA methylation across human polycystic ovary syndrome ovary granulosa cell.

PubMed

Xu, Jiawei; Bao, Xiao; Peng, Zhaofeng; Wang, Linlin; Du, Linqing; Niu, Wenbin; Sun, Yingpu

2016-05-10

Polycystic ovary syndrome (PCOS) affects approximately 7% of the reproductive-age women. A growing body of evidence indicated that epigenetic mechanisms contributed to the development of PCOS. The role of DNA modification in human PCOS ovary granulosa cell is still unknown in PCOS progression. Global DNA methylation and hydroxymethylation were detected between PCOS' and controls' granulosa cell. Genome-wide DNA methylation was profiled to investigate the putative function of DNA methylaiton. Selected genes expressions were analyzed between PCOS' and controls' granulosa cell. Our results showed that the granulosa cell global DNA methylation of PCOS patients was significant higher than the controls'. The global DNA hydroxymethylation showed low level and no statistical difference between PCOS and control. 6936 differentially methylated CpG sites were identified between control and PCOS-obesity. 12245 differential methylated CpG sites were detected between control and PCOS-nonobesity group. 5202 methylated CpG sites were significantly differential between PCOS-obesity and PCOS-nonobesity group. Our results showed that DNA methylation not hydroxymethylation altered genome-wide in PCOS granulosa cell. The different methylation genes were enriched in development protein, transcription factor activity, alternative splicing, sequence-specific DNA binding and embryonic morphogenesis. YWHAQ, NCF2, DHRS9 and SCNA were up-regulation in PCOS-obesity patients with no significance different between control and PCOS-nonobesity patients, which may be activated by lower DNA methylaiton. Global and genome-wide DNA methylation alteration may contribute to different genes expression and PCOS clinical pathology.

Contrasting spatial patterns in active-fire and fire-suppressed Mediterranean climate old-growth mixed conifer forests.

PubMed

Fry, Danny L; Stephens, Scott L; Collins, Brandon M; North, Malcolm P; Franco-Vizcaíno, Ernesto; Gill, Samantha J

2014-01-01

In Mediterranean environments in western North America, historic fire regimes in frequent-fire conifer forests are highly variable both temporally and spatially. This complexity influenced forest structure and spatial patterns, but some of this diversity has been lost due to anthropogenic disruption of ecosystem processes, including fire. Information from reference forest sites can help management efforts to restore forests conditions that may be more resilient to future changes in disturbance regimes and climate. In this study, we characterize tree spatial patterns using four-ha stem maps from four old-growth, Jeffrey pine-mixed conifer forests, two with active-fire regimes in northwestern Mexico and two that experienced fire exclusion in the southern Sierra Nevada. Most of the trees were in patches, averaging six to 11 trees per patch at 0.007 to 0.014 ha(-1), and occupied 27-46% of the study areas. Average canopy gap sizes (0.04 ha) covering 11-20% of the area were not significantly different among sites. The putative main effects of fire exclusion were higher densities of single trees in smaller size classes, larger proportion of trees (≥ 56%) in large patches (≥ 10 trees), and decreases in spatial complexity. While a homogenization of forest structure has been a typical result from fire exclusion, some similarities in patch, single tree, and gap attributes were maintained at these sites. These within-stand descriptions provide spatially relevant benchmarks from which to manage for structural heterogeneity in frequent-fire forest types.

β-Catenin Up-regulates Atoh1 Expression in Neural Progenitor Cells by Interaction with an Atoh1 3′ Enhancer*

PubMed Central

Shi, Fuxin; Cheng, Yen-fu; Wang, Xiaohui L.; Edge, Albert S. B.

2010-01-01

Atoh1, a basic helix-loop-helix transcription factor, plays a critical role in the differentiation of several epithelial and neural cell types. We found that β-catenin, the key mediator of the canonical Wnt pathway, increased expression of Atoh1 in mouse neuroblastoma cells and neural progenitor cells, and baseline Atoh1 expression was decreased by siRNA directed at β-catenin. The up-regulation of Atoh1 was caused by an interaction of β-catenin with the Atoh1 enhancer that could be demonstrated by chromatin immunoprecipitation. We found that two putative Tcf-Lef sites in the 3′ enhancer of the Atoh1 gene displayed an affinity for β-catenin and were critical for the activation of Atoh1 transcription because mutation of either site decreased expression of a reporter gene downstream of the enhancer. Tcf-Lef co-activators were found in the complex that bound to these sites in the DNA together with β-catenin. Inhibition of Notch signaling, which has previously been shown to induce bHLH transcription factor expression, increased β-catenin expression in progenitor cells of the nervous system. Because this could be a mechanism for up-regulation of Atoh1 after inhibition of Notch, we tested whether siRNA to β-catenin prevented the increase in Atoh1 and found that β-catenin expression was required for increased expression of Atoh1 after Notch inhibition. PMID:19864427

A Chemocentric Informatics Approach to Drug Discovery: Identification and Experimental Validation of Selective Estrogen Receptor Modulators as ligands of 5-Hydroxytryptamine-6 Receptors and as Potential Cognition Enhancers

PubMed Central

Hajjo, Rima; Setola, Vincent; Roth, Bryan L.; Tropsha, Alexander

2012-01-01

We have devised a chemocentric informatics methodology for drug discovery integrating independent approaches to mining biomolecular databases. As a proof of concept, we have searched for novel putative cognition enhancers. First, we generated Quantitative Structure- Activity Relationship (QSAR) models of compounds binding to 5-hydroxytryptamine-6 receptor (5HT6R), a known target for cognition enhancers, and employed these models for virtual screening to identify putative 5-HT6R actives. Second, we queried chemogenomics data from the Connectivity Map (http://www.broad.mit.edu/cmap/) with the gene expression profile signatures of Alzheimer’s disease patients to identify compounds putatively linked to the disease. Thirteen common hits were tested in 5-HT6R radioligand binding assays and ten were confirmed as actives. Four of them were known selective estrogen receptor modulators that were never reported as 5-HT6R ligands. Furthermore, nine of the confirmed actives were reported elsewhere to have memory-enhancing effects. The approaches discussed herein can be used broadly to identify novel drug-target-disease associations. PMID:22537153

Cloning, identification, and functional analysis of bone marrow stromal cell antigen-2 from sika deer (Cervus nippon).

PubMed

Wang, Jiawen; Bian, Shuai; Liu, Meichun; Zhang, Xin; Wang, Siming; Bai, Xueyuan; Zhao, Daqing; Zhao, Yu

2018-06-30

BST-2(tetherin/CD317/HM1.24) has been identified as a cellular antiviral factor that inhibits the release of a wide range of enveloped viruses from infected cells. Orthologs of BST-2 have been identified in several species including humans, monkeys, cows, sheep, pigs, and mice. In this study, we cloned the gene and characterized the protein of the BST-2 homolog from sika deer (Cervus nippon). cnBST-2 shares 37.8% and 74.2% identity with the BST-2 homologs from Homo sapiens and Ovis aries, respectively. The extracellular domain of cnBST-2 has two putative N-linked glycosylation sites and three potential dimerization sites. cnBST-2 was shown to be expressed on the cell surface, like human BST-2. Exogenous expression of cnBST-2 resulted in potent inhibition of HIV-1 particle release in 293T cells; however, this activity resisted antagonism by HIV-1 Vpu. Moreover, cnBST-2 was not able to activate nuclear factor-κB, in contrast to human BST-2. This study is the first report of the isolation and characterization of BST-2 from C. nippon. Copyright © 2018 Elsevier B.V. All rights reserved.

Zinc at glutamatergic synapses.

PubMed

Paoletti, P; Vergnano, A M; Barbour, B; Casado, M

2009-01-12

It has long been known that the mammalian forebrain contains a subset of glutamatergic neurons that sequester zinc in their synaptic vesicles. This zinc may be released into the synaptic cleft upon neuronal activity. Extracellular zinc has the potential to interact with and modulate many different synaptic targets, including glutamate receptors and transporters. Among these targets, NMDA receptors appear particularly interesting because certain NMDA receptor subtypes (those containing the NR2A subunit) contain allosteric sites exquisitely sensitive to extracellular zinc. The existence of these high-affinity zinc binding sites raises the possibility that zinc may act both in a phasic and tonic mode. Changes in zinc concentration and subcellular zinc distribution have also been described in several pathological conditions linked to glutamatergic transmission dysfunctions. However, despite intense investigation, the functional significance of vesicular zinc remains largely a mystery. In this review, we present the anatomy and the physiology of the glutamatergic zinc-containing synapse. Particular emphasis is put on the molecular and cellular mechanisms underlying the putative roles of zinc as a messenger involved in excitatory synaptic transmission and plasticity. We also highlight the many controversial issues and unanswered questions. Finally, we present and compare two widely used zinc chelators, CaEDTA and tricine, and show why tricine should be preferred to CaEDTA when studying fast transient zinc elevations as may occur during synaptic activity.

TNF induction of jagged-1 in endothelial cells is NFκB-dependent

PubMed Central

Johnston, Douglas A.; Dong, Bamboo; Hughes, Christopher C.W.

2009-01-01

TNF-α is a potent proinflammatory cytokine that induces endothelial cell (EC) adhesion molecules. In addition, TNF promotes angiogenesis by inducing an EC tip cell phenotype and the expression of jagged-1, a ligand for the notch pathway. Notch signaling is critical for vascular patterning and helps to restrict the proliferation of tip cells. Here we demonstrate that TNF induction of jagged-1 in human EC is rapid and dependent upon signaling through TNFR1, but not TNFR2. A luciferase reporter construct carrying 3.7 kb of 5′ promoter sequence from the human gene was responsive to both TNF and overexpression of NFκB pathway components. TNF-induced promoter activation was blocked by treatment with an NFκB inhibitor or co-expression of dominant-negative IKKβ. Mutations in a putative NFκB-binding site at −3.0 kb, which is conserved across multiple species, resulted in a loss of responsiveness to TNF and NFκB. Electromobility shift and chromatin immunoprecipitation assays revealed binding of both p50 and p65 to the promoter in response to TNF treatment. Full promoter activity also depends on an AP-1 site at −2.0 kb. These results indicate that canonical NFκB signaling is required for TNF induction of the notch ligand jagged-1 in EC. PMID:19393188

Facile O-atom insertion into CC and CH bonds by a trinuclear copper complex designed to harness a singlet oxene

PubMed Central

Chen, Peter P.-Y.; Yang, Richard B.-G.; Lee, Jason C.-M.; Chan, Sunney I.

2007-01-01

Two trinuclear copper [CuICuICuI(L)]1+ complexes have been prepared with the multidentate ligands (L) 3,3′-(1,4-diazepane-1,4-diyl)bis(1-((2-(dimethylamino)ethyl)(methyl)amino)propan-2-ol) (7-Me) and (3,3′-(1,4-diazepane-1,4-diyl)bis(1-((2-(diethylamino) ethyl)(ethyl) amino)propan-2-ol) (7-Et) as models for the active site of the particulate methane monooxygenase (pMMO). The ligands were designed to form the proper spatial and electronic geometry to harness a “singlet oxene,” according to the mechanism previously suggested by our laboratory. Consistent with the design strategy, both [CuICuICuI(L)]1+ reacted with dioxygen to form a putative bis(μ3-oxo)CuIICuIICuIII species, capable of facile O-atom insertion across the central CC bond of benzil and 2,3-butanedione at ambient temperature and pressure. These complexes also catalyze facile O-atom transfer to the CH bond of CH3CN to form glycolonitrile. These results, together with our recent biochemical studies on pMMO, provide support for our hypothesis that the hydroxylation site of pMMO contains a trinuclear copper cluster that mediates CH bond activation by a singlet oxene mechanism. PMID:17804786

Inhibitory Effect of 2,3,5,6-Tetrafluoro-4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamide Derivatives on HIV Reverse Transcriptase Associated RNase H Activities

PubMed Central

Pala, Nicolino; Esposito, Francesca; Rogolino, Dominga; Carcelli, Mauro; Sanna, Vanna; Palomba, Michele; Naesens, Lieve; Corona, Angela; Grandi, Nicole; Tramontano, Enzo; Sechi, Mario

2016-01-01

The HIV-1 ribonuclease H (RNase H) function of the reverse transcriptase (RT) enzyme catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate, and represents a suitable target for drug development. A particularly attractive approach is constituted by the interference with the RNase H metal-dependent catalytic activity, which resides in the active site located at the C-terminus p66 subunit of RT. Herein, we report results of an in-house screening campaign that allowed us to identify 4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamides, prepared by the “click chemistry” approach, as novel potential HIV-1 RNase H inhibitors. Three compounds (9d, 10c, and 10d) demonstrated a selective inhibitory activity against the HIV-1 RNase H enzyme at micromolar concentrations. Drug-likeness, predicted by the calculation of a panel of physicochemical and ADME properties, putative binding modes for the active compounds, assessed by computational molecular docking, as well as a mechanistic hypothesis for this novel chemotype are reported. PMID:27556447

Monitoring Notch Signaling-Associated Activation of Stem Cell Niches within Injured Dental Pulp

PubMed Central

Mitsiadis, Thimios A.; Catón, Javier; Pagella, Pierfrancesco; Orsini, Giovanna; Jimenez-Rojo, Lucia

2017-01-01

Dental pulp stem/progenitor cells guarantee tooth homeostasis, repair and regeneration throughout life. The decision between renewal and differentiation of these cells is influenced by physical and molecular interactions with stromal cells and extracellular matrix molecules forming the specialized microenvironment of dental pulp stem cell niches. Here we study the activation of putative pulp niches after tooth injury through the upregulation of Notch signaling pathway. Notch1, Notch2, and Notch3 molecules were used as markers of dental pulp stem/progenitor cells. Upon dental injury, Notch1 and Notch3 are detected in cells related to vascular structures suggesting a role of these proteins in the activation of specific pulpal perivascular niches. In contrast, a population of Notch2-positive cells that are actively proliferative is observed in the apical part of the pulp. Kinetics of these cells is followed up with a lipophilic DiI labeling, showing that apical pulp cells migrate toward the injury site where dynamic regenerative/repair events occur. The knowledge of the activation and regulation of dental pulp stem/progenitor cells within their niches in pathologic conditions may be helpful for the realization of innovative dental treatments in the near future. PMID:28611689

Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes.

PubMed

Fung, Herman K H; Gadd, Morgan S; Drury, Thomas A; Cheung, Samantha; Guss, J Mitchell; Coleman, Nicholas V; Matthews, Jacqueline M

2015-08-01

Haloalkane dehalogenases (HLDs) catalyse the hydrolysis of haloalkanes to alcohols, offering a biological solution for toxic haloalkane industrial wastes. Hundreds of putative HLD genes have been identified in bacterial genomes, but relatively few enzymes have been characterised. We identified two novel HLDs in the genome of Mycobacterium rhodesiae strain JS60, an isolate from an organochlorine-contaminated site: DmrA and DmrB. Both recombinant enzymes were active against C2-C6 haloalkanes, with a preference for brominated linear substrates. However, DmrA had higher activity against a wider range of substrates. The kinetic parameters of DmrA with 4-bromobutyronitrile as a substrate were Km  = 1.9 ± 0.2 mM, kcat  = 3.1 ± 0.2 s(-1) . DmrB showed the highest activity against 1-bromohexane. DmrA is monomeric, whereas DmrB is tetrameric. We determined the crystal structure of selenomethionyl DmrA to 1.7 Å resolution. A spacious active site and alternate conformations of a methionine side-chain in the slot access tunnel may contribute to the broad substrate activity of DmrA. We show that M. rhodesiae JS60 can utilise 1-iodopropane, 1-iodobutane and 1-bromobutane as sole carbon and energy sources. This ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB. © 2015 John Wiley & Sons Ltd.

Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens

PubMed Central

Rensing, Stefan A; Fritzowsky, Dana; Lang, Daniel; Reski, Ralf

2005-01-01

Background The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. Results We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. Conclusion Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in comparison with the seed plant Arabidopsis. Putative retained genes exhibit possible functions that might explain the peculiar physiological properties of mosses. Both the transcriptome representation (including a BLAST and retrieval service) and splice site prediction have been made available on , setting the basis for assembly and annotation of the Physcomitrella genome, of which draft shotgun sequences will become available in 2005. PMID:15784153

Klotho Up-regulates Renal Calcium Channel Transient Receptor Potential Vanilloid 5 (TRPV5) by Intra- and Extracellular N-glycosylation-dependent Mechanisms*

PubMed Central

Wolf, Matthias T. F.; An, Sung-Wan; Nie, Mingzhu; Bal, Manjot S.; Huang, Chou-Long

2014-01-01

The anti-aging protein Klotho is a type 1 membrane protein produced predominantly in the distal convoluted tubule. The ectodomain of Klotho is cleaved and secreted into the urine to regulate several ion channels and transporters. Secreted Klotho (sKL) up-regulates the TRPV5 calcium channel from the cell exterior by removing sialic acids from N-glycan of the channel and inhibiting its endocytosis. Because TRPV5 and Klotho coexpress in the distal convoluted tubule, we investigated whether Klotho regulates TRPV5 action from inside the cell. Whole-cell TRPV5-mediated channel activity was recorded in HEK cells coexpressing TRPV5 and sKL or membranous Klotho (mKL). Transfection of sKL, but not mKL, produced detectable Klotho protein in cell culture media. As for sKL, mKL increased TRPV5 current density. The role of sialidase activity of mKL acting inside is supported by findings that mutations of putative sialidase activity sites in sKL and mKL abrogated the regulation of TRPV5 but that the extracellular application of a sialidase inhibitor prevented the regulation of TRPV5 by sKL only. Mechanistically, coexpression with a dominant-negative dynamin II prevented the regulation of TRPV5 by sKL but not by mKL. In contrast, blocking forward trafficking by brefeldin A prevented the effect with mKL but not with sKL. Therefore, Klotho up-regulates TRPV5 from both the inside and outside of cells. The intracellular action of Klotho is likely due to enhanced forward trafficking of channel proteins, whereas the extracellular action is due to inhibition of endocytosis. Both effects involve putative Klotho sialidase activity. These effects of Klotho may play important roles regarding calcium reabsorption in the kidney. PMID:25378396

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

PubMed

Khandokar, Yogesh B; Srivastava, Parul; Cowieson, Nathan; Sarker, Subir; Aragao, David; Das, Shubagata; Smith, Kate M; Raidal, Shane R; Forwood, Jade K

2017-12-15

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn 24 and Asp 39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg 93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Distribution and innervation of putative peripheral arterial chemoreceptors in the red-eared slider (Trachemys scripta elegans).

PubMed

Reyes, Catalina; Fong, Angelina Y; Milsom, William K

2015-06-15

Peripheral arterial chemoreceptors have been isolated to the common carotid artery, aorta, and pulmonary artery of turtles. However, the putative neurotransmitters associated with these chemoreceptors have not yet been described. The goal of the present study was to determine the neurochemical content, innervations, and distribution of putative oxygen-sensing cells in the central vasculature of turtles and to derive homologies with peripheral arterial chemoreceptors of other vertebrates. We used tract tracing together with immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine synthesis), and serotonin (5HT) to identify putative oxygen-sensing cells and to determine their anatomical relation to branches of the vagus nerve (Xth cranial nerve). We found potential oxygen-sensing cells in all three chemosensory areas innervated by branches of the Xth cranial nerve. Cells containing either 5HT or VAChT were found in all three sites. The morphology and size of these cells resemble glomus cells found in amphibians, mammals, tortoises, and lizards. Furthermore, we found populations of cholinergic cells located at the base of the aorta and pulmonary artery that are likely involved in efferent regulation of vessel resistance. Catecholamine-containing cells were not found in any of the putative chemosensitive areas. The presence of 5HT- and VAChT-immunoreactive cells in segments of the common carotid artery, aorta, and pulmonary artery appears to reflect a transition between cells containing the major neurotransmitters seen in fish (5HT) and mammals (ACh and adenosine). © 2015 Wiley Periodicals, Inc.

A resource of single-nucleotide polymorphisms for rainbow trout generated by restriction-site associated DNA sequencing of doubled haploids

USDA-ARS?s Scientific Manuscript database

Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of an evolutionarily recent genome duplication event. This situation complicates single nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and ...

The Role of the EGF Receptor First Intron in Its Regulation in Breast Canceer.

DTIC Science & Technology

1997-08-01

of EGFR, to malignantly transform mammary glands in transgenic mice (46). Future work could determine if these putative oncogene factor binding sites...general transcription factors, and the use or overuse of one promoter could monopolize the abundance of these transcription factors within a cell. The ideal

Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

PubMed

Katchman, Alexander; Yang, Lin; Zakharov, Sergey I; Kushner, Jared; Abrams, Jeffrey; Chen, Bi-Xing; Liu, Guoxia; Pitt, Geoffrey S; Colecraft, Henry M; Marx, Steven O

2017-08-22

Calcium influx through the voltage-dependent L-type calcium channel (Ca V 1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca V 1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α 1C and C-terminal proteolytic cleavage of the α 1C subunit. We generated transgenic mice expressing an α 1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca V 1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α 1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α 1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca V 1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α 1C in β-adrenergic stimulation of Ca V 1.2, and show that phosphoregulatory sites on α 1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α 1C is not required for β-adrenergic stimulation of Ca V 1.2.

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the "intermediate" and reaction mechanism.

PubMed

Calhoun, Jennifer R; Bell, Caleb B; Smith, Thomas J; Thamann, Thomas J; DeGrado, William F; Solomon, Edward I

2008-07-23

The DFsc and DFscE11D de novo designed protein scaffolds support biomimetic diiron cofactor sites that react with dioxygen forming a 520 nm "intermediate" species with an apparent pseudo-first-order formation rate constant of 2.2 and 4.8 s-1, respectively. Resonance Raman spectroscopy shows that this absorption feature is due to a phenolate-to-ferric charge transfer transition arising from a single tyrosine residue coordinating terminally to one of the ferric ions in the site. Phenol coordination could provide a proton to promote rapid loss of a putative peroxo species.

Effects of intraguild predators on nest-site selection by prey.

PubMed

Huang, Wen-San; Pike, David A

2012-01-01

Nest-site selection involves tradeoffs between the risk of predation (on females and/or nests) and nest-site quality (microenvironment), and consequently suitable nesting sites are often in limited supply. Interactions with "classical" predators (e.g., those not competing for shared resources) can strongly influence nest-site selection, but whether intraguild predation also influences this behavior is unknown. We tested whether risk of predation from an intraguild predator [the diurnal scincid lizard Eutropis (Mabuya) longicaudata] influences nest-site selection by its prey (the nocturnal gecko Gekko hokouensis) on Orchid Island, Taiwan. These two species putatively compete for shared resources, including invertebrate prey and nesting microhabitat, but the larger E. longicaudata also predates G. hokouensis (but not its hard-shelled eggs). Both species nested within a concrete wall containing a series of drainage holes that have either one ("closed-in") or two openings ("open"). In allopatry, E. longicaudata preferred to nest within holes that were plugged by debris (thereby protecting eggs from water intrusion), whereas G. hokouensis selected holes that were open at both ends (facilitating escape from predators). When we experimentally excluded E. longicaudata from its preferred nesting area, G. hokouensis not only nested in higher abundances, but also modified its nest-site selection, such that communal nesting was more prevalent and both open and closed-in holes were used equally. Egg viability was unaffected by the choice of hole type, but was reduced slightly (by 7%) in the predator exclusion area (presumably due to higher local incubation temperatures). Our field experiment demonstrates that intraguild predators can directly influence the nest density of prey by altering maternal nest-site selection behavior, even when the predator and prey are active at different times of day and the eggs are not at risk of predation.

Comparative Proteomics of Tandem Mass Spectrometry Analyses for Bacterial Strains Identification and Differentiation

DTIC Science & Technology

2012-02-01

risk, bio -terrorism utility, Homeland Security, agricultural monitoring, quality of foodstuffs, environmental monitoring, and biological warfare agents...CAL19717 Putative surface antigen CAL21872 Putative sigma 54 modulation protein NP_395233 Plasminogen activator protease precursor CAL19882 OMP...S. (2005). Chemical and biological weapons : current concepts for future defenses. Johns Hopkins APL Tech. Digest, 26, 321-333. Dworzanski, J.P

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Culturable endophytic bacteria from the salt marsh plant Halimione portulacoides: phylogenetic diversity, functional characterization, and influence of metal(loid) contamination.

PubMed

Fidalgo, Cátia; Henriques, Isabel; Rocha, Jaqueline; Tacão, Marta; Alves, Artur

2016-05-01

Halimione portulacoides is abundant in salt marshes, accumulates mercury (Hg), and was proposed as useful for phytoremediation and pollution biomonitoring. Endophytic bacteria promote plant growth and provide compounds with industrial applications. Nevertheless, information about endophytic bacteria from H. portulacoides is scarce. Endophytic isolates (n = 665) were obtained from aboveground and belowground plant tissues, from two Hg-contaminated sites (sites E and B) and a noncontaminated site (site C), in the estuary Ria de Aveiro. Representative isolates (n = 467) were identified by 16S rRNA gene sequencing and subjected to functional assays. Isolates affiliated with Proteobacteria (64 %), Actinobacteria (23 %), Firmicutes (10 %), and Bacteroidetes (3 %). Altererythrobacter (7.4 %), Marinilactibacillus (6.4 %), Microbacterium (10.2 %), Salinicola (8.8 %), and Vibrio (7.8 %) were the most abundant genera. Notably, Salinicola (n = 58) were only isolated from site C; Hoeflea (17), Labrenzia (22), and Microbacterium (67) only from belowground tissues. This is the first report of Marinilactibacillus in the endosphere. Principal coordinate analysis showed that community composition changes with the contamination gradient and tissue. Our results suggest that the endosphere of H. portulacoides represents a diverse bacterial hotspot including putative novel species. Many isolates, particularly those affiliated to Altererythrobacter, Marinilactibacillus, Microbacterium, and Vibrio, tested positive for enzymatic activities and plant growth promoters, exposing H. portulacoides as a source of bacteria and compounds with biotechnological applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctionalmore » region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.« less

The hetC Gene Is a Direct Target of the NtcA Transcriptional Regulator in Cyanobacterial Heterocyst Development

PubMed Central

Muro-Pastor, Alicia M.; Valladares, Ana; Flores, Enrique; Herrero, Antonia

1999-01-01

The heterocyst is the site of nitrogen fixation in aerobically grown cultures of some filamentous cyanobacteria. Heterocyst development in Anabaena sp. strain PCC 7120 is dependent on the global nitrogen regulator NtcA and requires, among others, the products of the hetR and hetC genes. Expression of hetC, tested by RNA- DNA hybridization, was impaired in an ntcA mutant. A nitrogen-regulated, NtcA-dependent putative transcription start point was localized at nucleotide −571 with respect to the hetC translational start. Sequences upstream from this transcription start point exhibit the structure of the canonical cyanobacterial promoter activated by NtcA, and purified NtcA protein specifically bound to a DNA fragment containing this promoter. Activation of expression of hetC during heterocyst development appears thus to be directly operated by NtcA. NtcA-mediated activation of hetR expression was not impaired in a hetC mutant, indicating that HetC is not an NtcA-dependent element required for hetR induction. PMID:10542167

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

PubMed

da Costa e Silva, O; Klein, L; Schmelzer, E; Trezzini, G F; Hahlbrock, K

1993-07-01

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region.

PubMed

Zhou, Mingguang; Zhang, Qiang; Zhao, Jianping; Jin, Meilin

2012-01-01

Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. We report here H. parasuis encodes two copies of cytolethal distending toxins (Cdts), which these two Cdts showed the uniform toxin activity in vitro. We demonstrate that three Cdt peptides can form an active tripartite holotoxin that exhibits maximum cellular toxicity, and CdtA and CdtB form a more active toxin than CdtB and CdtC. Moreover, the cellular toxicity is associated with the binding of Cdt subunits to cells. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus (CRAC) region. The mutation of CRAC site resulted in decreased cell toxicity. Finally, western blot analysis show all the 15 H. parasuis reference strains and 109 clinical isolates expressed CdtB subunit, indicating that Cdt is a conservative putative virulence factor for H. parasuis. This is the first report of the molecular and cellular basis of Cdt host interactions in H. parasuis.

Molecular and characterization of NnPPO cDNA from lotus (Nelumbo nucifera) in rhizome browning.

PubMed

Dong, C; Yu, A Q; Yang, M G; Zhou, M Q; Hu, Z L

2016-04-30

The complete cDNA (NnPPO) of polyphenol oxidase in Nelumbo nucifera was successfully isolated, using Rapid amplification cDNA end (RACE) assays. The full-length cDNA of NnPPO was 2069 bp in size, containing a 1791 bp open reading frame coding 597 amino acids. The putative NnPPO possessed the conserved active sites and domains for PPO function. Phylogenetic analysis revealed that NnPPO shared high homology with PPO of high plants, and the homology modeling proved that NnPPO had the typical structure of PPO family. In order to characterize the role of NnPPO, Real-time PCR assay demonstrated that NnPPO mRNA was expressed in different tissues of N. nucifera including young leave, rhizome, flower, root and leafstalk, with the highest expression in rhizome. Patterns of NnPPO expression in rhizome illustrated its mRNA level was significantly elevated, which was consistent with the change of NnPPO activity during rhizome browning. Therefore, transcriptional activation of NnPPO was probably the main reason causing rhizome browning.

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

PubMed

Csermely, P; Szamel, M; Resch, K; Somogyi, J

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

PubMed

Tanaka, Shiho; Endo, Haruka; Adegawa, Satomi; Iizuka, Ami; Imamura, Kazuhiro; Kikuta, Shingo; Sato, Ryoichi

2017-12-01

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770 DYWL 773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770 DYWL 773 of ECL 4 in the ABCC2. Copyright © 2017 Elsevier Ltd. All rights reserved.

Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding deltaEF1.

PubMed

Miller, Myrna M; Jarosinski, Keith W; Schat, Karel A

2008-12-01

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (deltaEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and deltaEF1.

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

PubMed Central

Heyma, P; Harrison, L C

1984-01-01

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor. Images PMID:6088581

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome.

PubMed

Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

2016-10-01

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Posttranslational Modifications of p53 in Replicative Senescence Overlapping but Distinct from Those Induced by DNA Damage

PubMed Central

Webley, Katherine; Bond, Jane A.; Jones, Christopher J.; Blaydes, Jeremy P.; Craig, Ashley; Hupp, Ted; Wynford-Thomas, David

2000-01-01

Replicative senescence in human fibroblasts is absolutely dependent on the function of the phosphoprotein p53 and correlates with activation of p53-dependent transcription. However, no evidence for posttranslational modification of p53 in senescence has been presented, raising the possibility that changes in transcriptional activity result from upregulation of a coactivator. Using a series of antibodies with phosphorylation-sensitive epitopes, we now show that senescence is associated with major changes at putative regulatory sites in the N and C termini of p53 consistent with increased phosphorylation at serine-15, threonine-18, and serine-376 and decreased phosphorylation at serine-392. Ionizing and UV radiation generated overlapping but distinct profiles of response, with increased serine-15 phosphorylation being the only common change. These results support a direct role for p53 in signaling replicative senescence and are consistent with the generation by telomere erosion of a signal which shares some but not all of the features of DNA double-strand breaks. PMID:10733583

Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

PubMed

Krastel, Philipp; Roggo, Silvio; Schirle, Markus; Ross, Nathan T; Perruccio, Francesca; Aspesi, Peter; Aust, Thomas; Buntin, Kathrin; Estoppey, David; Liechty, Brigitta; Mapa, Felipa; Memmert, Klaus; Miller, Howard; Pan, Xuewen; Riedl, Ralph; Thibaut, Christian; Thomas, Jason; Wagner, Trixie; Weber, Eric; Xie, Xiaobing; Schmitt, Esther K; Hoepfner, Dominic

2015-08-24

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cannabidiol Reduces Aβ-Induced Neuroinflammation and Promotes Hippocampal Neurogenesis through PPARγ Involvement

PubMed Central

Valenza, Marta; Togna, Giuseppina Ines; Latina, Valentina; De Filippis, Daniele; Cipriano, Mariateresa; Carratù, Maria Rosaria; Iuvone, Teresa; Steardo, Luca

2011-01-01

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported. PMID:22163051

RhoA Regulation of Cardiomyocyte Differentiation

PubMed Central

Kaarbø, Mari; Crane, Denis I.; Murrell, Wayne G.

2013-01-01

Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α-actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes. PMID:23935420

Amylocyclicin, a Novel Circular Bacteriocin Produced by Bacillus amyloliquefaciens FZB42

PubMed Central

Scholz, Romy; Vater, Joachim; Budiharjo, Anto; Wang, Zhiyuan; He, Yueqiu; Dietel, Kristin; Schwecke, Torsten; Herfort, Stefanie; Lasch, Peter

2014-01-01

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization–time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon. PMID:24610713

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango.

PubMed

Talay, Mustafa; Richman, Ethan B; Snell, Nathaniel J; Hartmann, Griffin G; Fisher, John D; Sorkaç, Altar; Santoyo, Juan F; Chou-Freed, Cambria; Nair, Nived; Johnson, Mark; Szymanski, John R; Barnea, Gilad

2017-11-15

Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

PubMed

Bavaro, Teodora; Tengattini, Sara; Piubelli, Luciano; Mangione, Francesca; Bernardini, Roberta; Monzillo, Vincenzina; Calarota, Sandra; Marone, Piero; Amicosante, Massimo; Pollegioni, Loredano; Temporini, Caterina; Terreni, Marco

2017-06-29

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo- glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

Identification of a novel selD homolog from Eukaryotes, Bacteria, and Archaea: Is there an autoregulatory mechanism in selenocysteine metabolism?

PubMed Central

Guimarães, M. Jorge; Peterson, David; Vicari, Alain; Cocks, Benjamin G.; Copeland, Neal G.; Gilbert, Debra J.; Jenkins, Nancy A.; Ferrick, David A.; Kastelein, Robert A.; Bazan, J. Fernando; Zlotnik, Albert

1996-01-01

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme’s putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life. PMID:8986768

In vitro and ex vivo analysis of CHRNA3 and CHRNA5 haplotype expression.

PubMed

Doyle, Glenn A; Wang, Min-Jung; Chou, Andrew D; Oleynick, John U; Arnold, Steven E; Buono, Russell J; Ferraro, Thomas N; Berrettini, Wade H

2011-01-01

Genome-wide association studies implicate variations in CHRNA5 and CHRNA3 as being associated with nicotine addiction (NA). Multiple common haplotypes ("risk", "mixed" and "protective") exist in Europeans; however, high linkage disequilibrium between variations in CHRNA5 and CHRNA3 makes assigning causative allele(s) for NA difficult through genotyping experiments alone. We investigated whether CHRNA5 or CHRNA3 promoter haplotypes, associated previously with NA, might influence allelic expression levels. For in vitro analyses, promoter haplotypes were sub-cloned into a luciferase reporter vector. When assessed in BE(2)-C cells, luciferase expression was equivalent among CHRNA3 haplotypes, but the combination of deletion at rs3841324 and variation at rs503464 decreased CHRNA5 promoter-derived luciferase activity, possibly due to loss of an SP-1 and other site(s). Variation within the CHRNA5 5'UTR at rs55853698 and rs55781567 also altered luciferase expression in BE(2)-C cells. Allelic expression imbalance (AEI) from the "risk" or "protective" haplotypes was assessed in post-mortem brain tissue from individuals heterozygous at coding polymorphisms in CHRNA3 (rs1051730) or CHRNA5 (rs16969968). In most cases, equivalent allelic expression was observed; however, one individual showed CHRNA5 AEI that favored the "protective" allele and that was concordant with heterozygosity at polymorphisms ∼13.5 kb upstream of the CHRNA5 transcription start site. Putative enhancer activity from these distal promoter elements was assessed using heterologous promoter constructs. We observed no differences in promoter activity from the two distal promoter haplotypes examined, but found that the distal promoter region strongly repressed transcription. We conclude that CHRNA5 promoter variants may affect relative risk for NA in some heterozygous individuals.

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.

2008-06-01

The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target formore » tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.« less

Structural analysis of secretory phospholipase A2 from Clonorchis sinensis: therapeutic implications for hepatic fibrosis.

PubMed

Hariprasad, Gururao; Kaur, Punit; Srinivasan, Alagiri; Singh, Tej Pal; Kumar, Manoj

2012-07-01

Hepatic fibrosis is a common complication of the infection by the parasite, Clonorchis sinensis. There is a high incidence of this disease in the Asian countries with an increased risk of conversion to cancer. A secretory phospholipase A(2) (PLA(2)) enzyme from the parasite is implicated in the pathology. This is an attractive drug target in the light of extensive structural characterization of this class of enzyme. In this study, the structure of the enzyme was modeled based on its sequence homology to the group III bee venom PLA(2). On analysis, the overall structure essentially is comprised of three helices, two sets of β-wings and an elongated C-terminal extension. The structure is stabilized by four disulfide bonds. The structure is comprised of a calcium binding loop, active site and a substrate binding hydrophobic channel. The active site of the enzyme shows the classical features of PLA(2) with the participation of the three residues: histidine-aspartic acid-tyrosine in hydrogen bond formation. This is an interesting variation from the house keeping group III PLA(2) enzyme of human which has a histidine-aspartic acid and phenylalanine arrangement at the active site. This difference is therefore an important structural parameter that can be exploited to design specific inhibitor molecules against the pathogen PLA(2). Likewise, there are certain unique structural features in the hydrophobic channel and the putative membrane binding surface of the PLA(2) from Clonorchis sinensis that not only help understand the mechanism of action but also provide knowledge for a targeted therapy of liver fibrosis caused by the parasite.

Life detection at a Mars analogue site of present-day serpentinization in the Tablelands Ophiolite of Newfoundland (Invited)

NASA Astrophysics Data System (ADS)

Morrill, P. L.; Szponar, N.; Brazelton, W. J.; Woodruff, Q.; Schrenk, M. O.; Bower, D. M.; Steele, A.

2010-12-01

The Tableland Ophiolite was created during the collision of Laurentia and Gondwana continents ca. 470 million years ago. Ultramafic mantle rocks, from the ancient sea bed that once separated these continents, were thrusted westward onto the old continental margin, which is now Western Newfoundland. Weathering due to recent glaciations has left large areas of ultramafic rock at the surface and created fissures for fluid flow. As a result serpentinization is occurring as fresh water penetrates the unaltered ultramafic rock. Serpentinization is of particular interest because, through hydration of ultramafic rock, this reaction produces H2 and the reducing conditions necessary for abiogenic hydrocarbon synthesis, while also producing conditions amenable for chemolithotrophic life. Therefore sites of active serpentinization can be the source of either abiogenic or biogenic organics, or both. Serpentinization is a suspected (past or present) source of (detected or putative) hydrocarbons on Mars, Titan and Europa, hence these astrobodies may be potentially habitable or once habitable environments. The Tablelands Ophiolite is an analogue site that is ideal for testing methods of life detection in an extreme environment of high pH and low microbial biomass characteristic of sites of serpentinization. Multiple ultrabasic reducing springs characteristic of present-day serpentinization have been identified and characterized based on their geochemistry and microbiology. Field-based instruments were deployed for the detection of microbial activity (ATP), microbial cell wall material, and mineralogy, in yet untested high pH and low biomass environment. In this talk I will give an overview of the in situ measurements of life detection and put these measurements in context of geochemistry, microbiology, carbon source and reaction pathways, and I will discuss what we have learned that will help us plan for future mission measurements.

Landscape genomics reveal signatures of local adaptation in barley (Hordeum vulgare L.)

PubMed Central

Abebe, Tiegist D.; Naz, Ali A.; Léon, Jens

2015-01-01

Land plants are sessile organisms that cannot escape the adverse climatic conditions of a given environment. Hence, adaptation is one of the solutions to surviving in a challenging environment. This study was aimed at detecting adaptive loci in barley landraces that are affected by selection. To that end, a diverse population of barley landraces was analyzed using the genotyping by sequencing approach. Climatic data for altitude, rainfall and temperature were collected from 61 weather sites near the origin of selected landraces across Ethiopia. Population structure analysis revealed three groups whereas spatial analysis accounted significant similarities at shorter geographic distances (< 40 Km) among barley landraces. Partitioning the variance between climate variables and geographic distances indicated that climate variables accounted for most of the explainable genetic variation. Markers by climatic variables association analysis resulted in altogether 18 and 62 putative adaptive loci using Bayenv and latent factor mixed model (LFMM), respectively. Subsequent analysis of the associated SNPs revealed putative candidate genes for plant adaptation. This study highlights the presence of putative adaptive loci among barley landraces representing original gene pool of the farming communities. PMID:26483825

The Role of Ventral Tegmental Area Gamma-Aminobutyric Acid in Chronic Neuropathic Pain after Spinal Cord Injury in Rats.

PubMed

Ko, Moon Yi; Jang, Eun Young; Lee, June Yeon; Kim, Soo Phil; Whang, Sung Hun; Lee, Bong Hyo; Kim, Hee Young; Yang, Chae Ha; Cho, Hee Jung; Gwak, Young S

2018-04-20

Spinal cord injury (SCI) frequently results in chronic neuropathic pain (CNP). However, the understanding of brain neural circuits in CNP modulation is unclear. The present study examined the changes of ventral tegmental area (VTA) putative GABAergic and dopaminergic neuronal activity with CNP attenuation in rats. SCI was established by T10 clip compression injury (35 g, 1 min) in rats, and neuropathic pain behaviors, in vivo extracellular single-cell recording of putative VTA gamma-aminobutyric acid (GABA)/dopamine neurons, extracellular GABA level, glutamic acid decarboxylase (GAD), and vesicular GABA transporters (VGATs) were measured in the VTA, respectively. The results revealed that extracellular GABA level was significantly increased in the CNP group (50.5 ± 18.9 nM) compared to the sham control group (10.2 ± 1.7 nM). In addition, expression of GAD 65/67 , c-Fos, and VGAT exhibited significant increases in the SCI groups compared to the sham control group. With regard to neuropathic pain behaviors, spontaneous pain measured by ultrasound vocalizations (USVs) and evoked pain measured by paw withdrawal thresholds showed significant alteration, which was reversed by intravenous (i.v.) administration of morphine (0.5-5.0 mg/kg). With regard to in vivo electrophysiology, VTA putative GABAergic neuronal activity (13.6 ± 1.7 spikes/sec) and putative dopaminergic neuronal activity (2.4 ± 0.8 spikes/sec) were increased and decreased, respectively, in the SCI group compared to the sham control group. These neuronal activities were reversed by i.v. administration of morphine. The present study suggests that chronic increase of GABAergic neuronal activity suppresses dopaminergic neuronal activity in the VTA and is responsible for negative emotion and motivation for attenuation of SCI-induced CNP.

Neuronal activity determines distinct gliotransmitter release from a single astrocyte

PubMed Central

Covelo, Ana

2018-01-01

Accumulating evidence indicates that astrocytes are actively involved in brain function by regulating synaptic activity and plasticity. Different gliotransmitters, such as glutamate, ATP, GABA or D-serine, released form astrocytes have been shown to induce different forms of synaptic regulation. However, whether a single astrocyte may release different gliotransmitters is unknown. Here we show that mouse hippocampal astrocytes activated by endogenous (neuron-released endocannabinoids or GABA) or exogenous (single astrocyte Ca2+ uncaging) stimuli modulate putative single CA3-CA1 hippocampal synapses. The astrocyte-mediated synaptic modulation was biphasic and consisted of an initial glutamate-mediated potentiation followed by a purinergic-mediated depression of neurotransmitter release. The temporal dynamic properties of this biphasic synaptic regulation depended on the firing frequency and duration of the neuronal activity that stimulated astrocytes. Present results indicate that single astrocytes can decode neuronal activity and, in response, release distinct gliotransmitters to differentially regulate neurotransmission at putative single synapses. PMID:29380725

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt -1) in the NDUFS4 gene in Leigh syndrome.

PubMed

Bénit, Paule; Steffann, Julie; Lebon, Sophie; Chretien, Dominique; Kadhom, Noman; de Lonlay, Pascale; Goldenberg, Alice; Dumez, Yves; Dommergues, Marc; Rustin, Pierre; Munnich, Arnold; Rötig, Agnès

2003-05-01

Complex I deficiency, the most common cause of mitochondrial disorders, accounts for a variety of clinical symptoms and its genetic heterogeneity makes identification of the disease genes particularly tedious. Indeed, most of the 43 complex I subunits are encoded by nuclear genes, only seven of them being mitochondrially encoded. In order to offer urgent prenatal diagnosis, we have studied an inbred/multiplex family with complex I deficiency by using microsatellite DNA markers flanking the putative disease loci. Microsatellite DNA markers have allowed us to exclude the NDUFS7, NDUFS8, NDUFV1 and NDUFS1 genes and to find homozygosity at the NDUFS4 locus. Direct sequencing has led to identification of a homozygous splice acceptor site mutation in intron 1 of the NDUFS4 gene (IVS1nt -1, G-->A); this was not found in chorion villi of the ongoing pregnancy. We suggest that genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families helps to identify the disease-causing mutation. More generally, we suggest giving consideration to a more systematic microsatellite analysis of putative disease loci for identification of disease genes in inbred/multiplex families affected with genetically heterogeneous conditions.

Unigenic Evolution: A Novel Genetic Method Localizes a Putative Leucine Zipper That Mediates Dimerization of the Saccharomyces Cerevisiae Regulator Gcr1p

PubMed Central

Deminoff, S. J.; Tornow, J.; Santangelo, G. M.

1995-01-01

The GCR1 gene of Saccharomyces cerevisiae encodes a transcriptional activator that complexes with Rap1p and, through UAS(RPG) elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes. To map the functionally important domains in Gcr1p, we combined multiple rounds of random mutagenesis in vitro with in vivo selection of functional genes to locate conserved, or hypomutable, regions. We name this method unigenic evolution, a statistical analysis of mutations in evolutionary variants of a single gene in an otherwise isogenic background. Examination of the distribution of 315 mutations in 24 variant alleles allowed the localization of four hypomutable regions in GCR1 (A, B, C, and D). Dispensable N-terminal (intronic) and C-terminal portions of the evolved region of GCR1 were included in the analysis as controls and were, as expected, not hypomutable. The analysis of several insertion, deletion, and point mutations, combined with a comparison of the hypomutability and hydrophobicity plots of Gcr1p, suggested that some of the hypomutable regions may individually or in combination correspond to functionally important surface domains. In particular, we determined that region D contains a putative leucine zipper and is necessary and sufficient for Gcr1p homodimerization. PMID:8601472

Evolutionary profiles from the QR factorization of multiple sequence alignments

PubMed Central

Sethi, Anurag; O'Donoghue, Patrick; Luthey-Schulten, Zaida

2005-01-01

We present an algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of the homologous group. The method, based on the multidimensional QR factorization of numerically encoded multiple sequence alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. We observe a general trend that these smaller, more evolutionarily balanced profiles have comparable and, in many cases, better performance in database searches than conventional profiles containing hundreds of sequences, constructed in an iterative and computationally intensive procedure. For more diverse families or superfamilies, with sequence identity <30%, structural alignments, based purely on the geometry of the protein structures, provide better alignments than pure sequence-based methods. Merging the structure and sequence information allows the construction of accurate profiles for distantly related groups. These structure-based profiles outperformed other sequence-based methods for finding distant homologs and were used to identify a putative class II cysteinyl-tRNA synthetase (CysRS) in several archaea that eluded previous annotation studies. Phylogenetic analysis showed the putative class II CysRSs to be a monophyletic group and homology modeling revealed a constellation of active site residues similar to that in the known class I CysRS. PMID:15741270

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

PubMed Central

Sogi, Kimberly M.; Gartner, Zev J.; Breidenbach, Mark A.; Appel, Mason J.; Schelle, Michael W.; Bertozzi, Carolyn R.

2013-01-01

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases. PMID:23762287

The glnAntrBC operon of Herbaspirillum seropedicae is transcribed by two oppositely regulated promoters upstream of glnA.

PubMed

Schwab, Stefan; Souza, Emanuel M; Yates, Marshall G; Persuhn, Darlene C; Steffens, M Berenice R; Chubatsu, Leda S; Pedrosa, Fábio O; Rigo, Liu U

2007-01-01

Herbaspirillum seropedicae is an endophytic bacterium that fixes nitrogen under microaerophilic conditions. The putative promoter sequences glnAp1 (sigma70-dependent) and glnAp2 (sigma54), and two NtrC-binding sites were identified upstream from the glnA, ntrB and ntrC genes of this microorganism. To study their transcriptional regulation, we used lacZ fusions to the H. seropedicae glnA gene, and the glnA-ntrB and ntrB-ntrC intergenic regions. Expression of glnA was up-regulated under low ammonium, but no transcription activity was detected from the intergenic regions under any condition tested, suggesting that glnA, ntrB and ntrC are co-transcribed from the promoters upstream of glnA. Ammonium regulation was lost in the ntrC mutant strain. A point mutation was introduced in the conserved -25/-24 dinucleotide (GG-->TT) of the putative sigma54-dependent promoter (glnAp2). Contrary to the wild-type promoter, glnA expression with the mutant glnAp2 promoter was repressed in the wild-type strain under low ammonium levels, but this repression was abolished in an ntrC background. Together our results indicate that the H. seropedicae glnAntrBC operon is regulated from two functional promoters upstream from glnA, which are oppositely regulated by the NtrC protein.

Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.

PubMed

Meng, C X; Wei, W; Su, Z- L; Qin, S

2006-10-01

Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.

Harmane: an atypical neurotransmitter?

PubMed

Abu Ghazaleh, Haya; Lalies, Maggie D; Nutt, David J; Hudson, Alan L

2015-03-17

Harmane is an active component of clonidine displacing substance and a candidate endogenous ligand for imidazoline binding sites. The neurochemistry of tritiated harmane was investigated in the present study examining its uptake and release properties in the rat brain central nervous system (CNS) in vitro. At physiological temperature, [(3)H]harmane was shown to be taken up in rat brain cortex. Further investigations demonstrated that treatment with monoamine uptake blockers (citalopram, nomifensine and nisoxetine) did not alter [(3)H]harmane uptake implicating that the route of [(3)H]harmane transport was distinct from the monoamine uptake systems. Furthermore, imidazoline ligands (rilmenidine, efaroxan, 2-BFI and idazoxan) showed no prominent effect on [(3)H]harmane uptake suggesting the lack of involvement of imidazoline binding sites. Subsequent analyses showed that disruption of the Na(+) gradient using ouabain or choline chloride did not block [(3)H]harmane uptake suggesting a Na(+)-independent transport mechanism. Moreover, higher temperatures (50°C) failed to impede [(3)H]harmane uptake implying a non-physiological transporter. The failure of potassium to evoke the release of preloaded [(3)H]harmane from rat brain cortex indicates that the properties of this putative endogenous ligand for imidazoline binding sites do not resemble that of a conventional neurotransmitter. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor.

PubMed

Wines, Bruce D; Ramsland, Paul A; Trist, Halina M; Gardam, Sandra; Brink, Robert; Fraser, John D; Hogarth, P Mark

2011-09-23

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.

New record of Scedosporium dehoogii from Chile: Phylogeny and susceptibility profiles to classic and novel putative antifungal agents.

PubMed

Alvarez, Eduardo; Sanhueza, Camila

Scedosporium species are considered emerging agents causing illness in immunocompromised patients. In Chile, only Scedosporium apiospermum, Scedosporium boydii and Lomentospora prolificans haven been reported previously. The study aimed to characterize genetically Scedosporium dehoogii strains from Chilean soil samples, and assessed the antifungal susceptibility profile to classic and novel putative antifungal molecules. In 2014, several samples were obtained during a survey of soil fungi in urban areas from Chile. Morphological and phylogenetic analyses of the internal transcribed spacer region (ITS), tubulin (TUB), and calmodulin (CAL) sequences were performed. In addition, the susceptibility profiles to classic antifungal and new putative antifungal molecules were determined. Four strains of Scedosporium dehoogii were isolated from soil samples. The methodology confirmed the species (reported here as a new record for Chile). Antifungal susceptibility testing demonstrates the low activity of terpenes (α-pinene and geraniol) against this species. Voriconazole (VRC), posaconazole (PSC), and the hydroxyquinolines (clioquinol, and 5,7-dibromo-8-hydroxyquinoline) showed the best antifungal activity. Our results demonstrate that Scedosporium dehoogii is present in soil samples from Chile. This study shows also that hydroxyquinolines have potential as putative antifungal molecules. Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas.

PubMed

Sangiolo, Dario; Mesiano, Giulia; Gammaitoni, Loretta; Leuci, Valeria; Todorovic, Maja; Giraudo, Lidia; Cammarata, Cristina; Dell'Aglio, Carmine; D'Ambrosio, Lorenzo; Pisacane, Alberto; Sarotto, Ivana; Miano, Sara; Ferrero, Ivana; Carnevale-Schianca, Fabrizio; Pignochino, Ymera; Sassi, Francesco; Bertotti, Andrea; Piacibello, Wanda; Fagioli, Franca; Aglietta, Massimo; Grignani, Giovanni

2014-01-01

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.

Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells

PubMed Central

Audit, Benjamin; Zaghloul, Lamia; Vaillant, Cédric; Chevereau, Guillaume; d'Aubenton-Carafa, Yves; Thermes, Claude; Arneodo, Alain

2009-01-01

For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions ∼300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as ‘master’ replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these ‘master’ origins are likely to play a key role in genome dynamics during evolution and in pathological situations. PMID:19671527

Distribution of Archaeal Communities along the Coast of the Gulf of Finland and Their Response to Oil Contamination

PubMed Central

Yan, Lijuan; Yu, Dan; Hui, Nan; Naanuri, Eve; Viggor, Signe; Gafarov, Arslan; Sokolov, Sergei L.; Heinaru, Ain; Romantschuk, Martin

2018-01-01

The Baltic Sea is vulnerable to environmental changes. With the increasing shipping activities, the risk of oil spills remains high. Archaea are widely distributed in many environments. However, the distribution and the response of archaeal communities to oil contamination have rarely been investigated in brackish habitats. Hence, we conducted a survey to investigate the distribution, diversity, composition, and species interactions of indigenous archaeal communities at oil-contaminated sites along the coast of the Gulf of Finland (GoF) using high-throughput sequencing. Surface water and littoral sediment samples were collected at presumably oil-contaminated (oil distribution facilities) and clean sites along the coastline of the GoF in the winter 2015 and the summer 2016. Another three samples of open sea surface water were taken as offshore references. Of Archaea, Euryarchaeota dominated in the surface water and the littoral sediment of the coast of the GoF, followed by Crenarchaeota (including Thaumarchaeota, Thermoprotei, and Korarchaeota based on the Greengenes database used). The unclassified sequences accounted for 5.62% of the total archaeal sequences. Our study revealed a strong dependence of the archaeal community composition on environmental variables (e.g., salinity, pH, oil concentration, TOM, electrical conductivity, and total DNA concentration) in both littoral sediment and coastal water in the GoF. The composition of archaeal communities was season and ecosystem dependent. Archaea was highly diverse in the three ecosystems (littoral sediment, coastal water, and open sea water). Littoral sediment harbored the highest diversity of archaea. Oil was often detected in the littoral sediment but rarely detected in water at those presumably contaminated sites. Although the composition of archaeal community in the littoral sediment was sensitive to low-input oil contamination, the unchanged putative functional profiles and increased interconnectivity of the archaeal core species network plausibly revealed resilience and the potential for oil degradation. Halobacteriaceae and putative cytochrome P450 pathways were significantly enriched in the oil-contaminated littoral sediment. The archaeal taxa formed highly interconnected and interactive networks, in which Halobacteriaceae, Thermococcus, and methanogens were the main components, implying a potential relevant trophic connection between hydrocarbon degradation, methanogenesis, sulfate reduction, and/or fermentative growth. PMID:29410652