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Sample records for guanine nucleotide-binding protein-regulated

  1. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

    1989-01-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125 I-labeled melatonin ( 125 I-Mel), a potent melatonin agonist. 125 I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K d of 2.3 ± 1.0 x 10 -11 M and 2.06 ± 0.43 x 10 -10 M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[γ-thio]triphosphate (GTP[γS]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125 I-Mel from its receptor. Furthermore, GTP[γS] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125 I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M r > 400,000 and M r ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[γS] before solubilization; only the M r 110,000 peak was present in GTP[γS]-pretreated membranes. The results strongly suggest that 125 I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000

  2. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  3. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  4. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

  5. Guanine nucleotide binding protein-like 3 is a potential prognosis indicator of gastric cancer.

    Chen, Jing; Dong, Shuang; Hu, Jiangfeng; Duan, Bensong; Yao, Jian; Zhang, Ruiyun; Zhou, Hongmei; Sheng, Haihui; Gao, Hengjun; Li, Shunlong; Zhang, Xianwen

    2015-01-01

    Guanine nucleotide binding protein-like 3 (GNL3) is a GIP-binding nuclear protein that has been reported to be involved in various biological processes, including cell proliferation, cellular senescence and tumorigenesis. This study aimed to investigate the expression level of GNL3 in gastric cancer and to evaluate the relationship between its expression and clinical variables and overall survival of gastric cancer patients. The expression level of GNL3 was examined in 89 human gastric cancer samples using immunohistochemistry (IHC) staining. GNL3 in gastric cancer tissues was significantly upregulated compared with paracancerous tissues. GNL3 expression in adjacent non-cancerous tissues was associated with sex and tumor size. Survival analyses showed that GNL3 expression in both gastric cancer and adjacent non-cancerous tissues were not related to overall survival. However, in the subgroup of patients with larger tumor size (≥ 6 cm), a close association was found between GNL3 expression in gastric cancer tissues and overall survival. GNL3-positive patients had a shorter survival than GNL3-negative patients. Our study suggests that GNL3 might play an important role in the progression of gastric cancer and serve as a biomarker for poor prognosis in gastric cancer patients.

  6. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  7. Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

    Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu

    2014-01-01

    Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro

  8. Role of a guanine nucleotide-binding protein in α1-adrenergic receptor-mediated Ca2+ mobilization in DDT1 MF-2 cells

    Cornett, L.E.; Norris, J.S.

    1987-01-01

    In this study the mechanisms involved in α 1 -adrenergic receptor-mediated Ca 2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45 Ca 2+ efflux from saponin-permeabilized DDT 1 MF-2 cells was observed with the addition of either the α 1 -adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [ 32 P] NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT 1 , MF-2 cells, possibly the α-subunit of N/sub i/. However, stimulation of unidirectional 45 Ca 2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the α 1 -adrenergic receptor to Ca 2+ mobilization in DDT 1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family

  9. Mutation analysis of inhibitory guanine nucleotide binding protein alpha (GNAI) loci in young and familial pituitary adenomas.

    Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli

    2014-01-01

    Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15-20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.

  10. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  11. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

    1988-01-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

  12. A novel missense variant (Gln220Arg) of GNB4 encoding guanine nucleotide-binding protein, subunit beta-4 in a Japanese family with autosomal dominant motor and sensory neuropathy.

    Miura, Shiroh; Morikawa, Takuya; Fujioka, Ryuta; Noda, Kazuhito; Kosaka, Kengo; Taniwaki, Takayuki; Shibata, Hiroki

    2017-09-01

    Dominant intermediate Charcot-Marie-Tooth disease F (CMTDIF) is an autosomal dominant hereditary form of Charcot-Marie-Tooth disease (CMT) caused by variations in the guanine nucleotide-binding protein, subunit beta-4 gene (GNB4). We examined two Japanese familial cases with CMT. Case 1 was a 49-year-old male whose chief complaint was slowly progressive gait disturbance and limb dysesthesia that appeared at the age of 47. On neurological examination, he showed hyporeflexia or areflexia, distal limb muscle weakness, and distal sensory impairment with lower dominancy. Nerve conduction studies demonstrated demyelinating sensorimotor neuropathy with reduced action potentials in the lower limbs. Case 2 was an 80-year-old man, Case 1's father, who reported difficulty in riding a bicycle at the age of 76. On neurological examination, he showed areflexia in the upper and lower limbs. Distal sensory impairment in the lower limbs was also observed. Nerve conduction studies revealed mainly axonal involvement. Exome sequencing identified a novel heterozygous nonsynonymous variant (NM_021629.3:c.659T > C [p.Gln220Arg]) in GNB4 exon 8, which is known to be responsible for CMT. Sanger sequencing confirmed that both patients are heterozygous for the variation, which causes an amino acid substitution, Gln220Arg, in the highly conserved region of the WD40 domain of GNB4. The frequency of this variant in the Exome Aggregation Consortium Database was 0.000008247, and we confirmed its absence in 502 Japanese control subjects. We conclude that this novel GNB4 variant is causative for CMTDIF in these patients, who represent the first record of the disease in the Japanese population. Copyright © 2017. Published by Elsevier Masson SAS.

  13. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    Grasso, P.; Reichert, L.E. Jr.

    1990-01-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel

  14. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  15. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

    Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

    2013-12-19

    The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  17. Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

    Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

    2015-11-01

    The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet G s protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al +3 and F - agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF - 4 strongly inhibited 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  19. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-01-01

    Prostaglandin E 2 (PEG 2 ) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its α subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G 0 ) purified from bovine brain. The molecular weight of the α subunit was 40,000, which is between those of G/sub i/ and G 0 . The purified protein was also distinguished immunologically from G/sub i/ and G 0 and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G 0 in phospholipid vesicles resulted in a remarkable restoration of [ 3 H]PGE 2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G 0 in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla

  20. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    Jiang, M; Pandey, S; Tran, V T; Fong, H K

    1991-01-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

  1. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  2. Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

    Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2013-07-01

    Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

  3. The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity

    Meissner, Torsten B.; Li, Amy; Liu, Yuen-Joyce; Gagnon, Etienne; Kobayashi, Koichi S.

    2012-01-01

    Highlights: ► NLRC5 requires an intact NLS for its function as MHC class I transactivator. ► Nuclear presence of NLRC5 is required for MHC class I induction. ► Nucleotide-binding controls nuclear import and transactivation activity of NLRC5. -- Abstract: Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class I and related genes. While a ‘master regulator’ of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine.

  4. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  5. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

    Brewer, Frances K; Follit, Courtney A; Vogel, Pia D; Wise, John G

    2014-12-01

    Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

    Teruel, J.A.; Inesi, G.

    1988-01-01

    The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca 2+ -ATPase of sarcoplasmic reticulum vesicles. The authors found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in their preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon back inhibited by the rise of Ca 2+ concentration in the lumen of the vesicles. When the back inhibition is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain. On the other hand, occupancy of the FITC-sensitive nucleotide site is involved in kinetic regulation not only with respect to utilization of substrate for the phosphoryl transfer reaction but also for subsequent steps related to calcium translocation and phosphoenzyme turnover

  8. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  9. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  10. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

  11. Simulation of the coupling between nucleotide binding and transmembrane domains in the ABC transporter BtuCD

    Sonne, Jacob; Kandt, C.; Peters, Günther H.j.

    2007-01-01

    The nucleotide-induced structural rearrangements in ATP binding cassette (ABC) transporters, leading to substrate translocation, are largely unknown. We have modeled nucleotide binding and release in the vitamin B12 importer BtuCD using perturbed elastic network calculations and biased molecular...

  12. Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release

    Leonardsen, L; DeClue, J E; Lybaek, H

    1996-01-01

    The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino ac...... and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive....

  13. Random mutagenesis of the nucleotide-binding domain of NRC1 (NB-LRR Required for Hypersensitive Response-Associated Cell Death-1), a downstream signalling nucleotide-binding, leucine-rich repeat (NB-LRR) protein, identifies gain-of-function mutations in the nucleotide-binding pocket

    Sueldo, D.J.; Shimels, M.Z.; Spiridon, L.N.; Caldararu, O.; Petrescu, A.J.; Joosten, M.H.A.J.; Tameling, W.I.L.

    2015-01-01

    •Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. •NRC1 (NB-LRR

  14. Genome Wide Analysis of Nucleotide-Binding Site Disease Resistance Genes in Brachypodium distachyon

    Shenglong Tan

    2012-01-01

    Full Text Available Nucleotide-binding site (NBS disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Many R-genes have been identified in various plant species. However, little is known about the NBS-encoding genes in Brachypodium distachyon. In this study, using computational analysis of the B. distachyon genome, we identified 126 regular NBS-encoding genes and characterized them on the bases of structural diversity, conserved protein motifs, chromosomal locations, gene duplications, promoter region, and phylogenetic relationships. EST hits and full-length cDNA sequences (from Brachypodium database of 126 R-like candidates supported their existence. Based on the occurrence of conserved protein motifs such as coiled-coil (CC, NBS, leucine-rich repeat (LRR, these regular NBS-LRR genes were classified into four subgroups: CC-NBS-LRR, NBS-LRR, CC-NBS, and X-NBS. Further expression analysis of the regular NBS-encoding genes in Brachypodium database revealed that these genes are expressed in a wide range of libraries, including those constructed from various developmental stages, tissue types, and drought challenged or nonchallenged tissue.

  15. Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1).

    Li, Huilin; Jin, Hui; Li, Yaqian; Liu, Dejian; Foda, Mohamed Frahat; Jiang, Yunbo; Luo, Rui

    2017-09-01

    Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

    Asita Elengoe

    2015-01-01

    Full Text Available Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD of heat shock 70 kDa protein (PDB: 1HJO with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD simulation. Human DNA binding domain of p53 motif (SCMGGMNR retrieved from UniProt (UniProtKB: P04637 was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

  17. A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

    Alexandr P Kornev

    2008-04-01

    Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

  18. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  19. Association of Nucleotide-binding Oligomerization Domain Receptors with Peptic Ulcer and Gastric Cancer.

    Mohammadian Amiri, Rajeeh; Tehrani, Mohsen; Taghizadeh, Shirin; Shokri-Shirvani, Javad; Fakheri, Hafez; Ajami, Abolghasem

    2016-10-01

    Host innate immunity can affect the clinical outcomes of Helicobacter pylori infection, including gastritis, gastric ulcer, gastric adenocarcinoma, and MALT lymphoma. Nucleotide binding oligomerization domain (NOD)-1 and -2 are two molecules of innate immunity which are involved in the host defense against H. pylori. This study aimed to evaluate the effect of the expression level of NOD1 and NOD2 on the susceptibility to gastric cancer as well as peptic ulcer in individuals with H. pylori infection. The gene expression levels of these molecules were compared in three groups of non-ulcer dyspepsia (NUD) as a control group (n=52); peptic ulcer disease (PUD), (n=53); and gastric cancer (GC), (n=39). Relative expression levels of NOD1 in patients with GC were higher than those of NUD and PUD (p<0.001 and P<0.001, respectively). Similarly in case of NOD1, PUD group showed higher level of expression than NUD group (p<0.01). However, there was no significant difference between H. pylori -positive and -negative patients in NUD, PUD, or GC groups. Moreover, the expression levels of NOD2 showed no significant difference among NUD, PUD, or GC groups, while among H. pylori-positive patients, it was higher in GC group than NUD  and PUD groups (p<0.05 and p<0.01, respectively). In addition, positive correlation coefficients were attained between NOD1 and NOD2 expressions in patients with NUD (R2 Linear=0.349, p<0.001), PUD (R2 Linear=0.695, p<0.001), and GC (R2 Linear=0.385, p<0.001). Collectively, the results suggest that the chronic activation of NOD1 and NOD2 receptors might play a role in the development of gastric cancer.

  20. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    Wang, Zhongshan [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom); College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Xiang, Quanju [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Department of Microbiology, College of Resource and Environment Science, Sichuan Agriculture University, Yaan 625000 (China); Zhu, Xiaofeng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Dong, Haohao [Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); He, Chuan [School of Electronics and Information, Wuhan Technical College of Communications, No. 6 Huangjiahu West Road, Hongshan District, Wuhan, Hubei 430065 (China); Wang, Haiyan; Zhang, Yizheng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Wang, Wenjian, E-mail: Wenjian166@gmail.com [Laboratory of Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080 (China); Dong, Changjiang, E-mail: C.Dong@uea.ac.uk [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom)

    2014-09-26

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

  1. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    Wang, Zhongshan; Xiang, Quanju; Zhu, Xiaofeng; Dong, Haohao; He, Chuan; Wang, Haiyan; Zhang, Yizheng; Wang, Wenjian; Dong, Changjiang

    2014-01-01

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg 2+ . • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg 2+ , which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics

  2. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  3. Somatotropin has no effect on the quantity of guanine nucleotide binding proteins Gq alpha/G11 alpha in goat adipose tissue in vivo

    Krbeček, Vlastimil; Kovářů, H.; Škarda, Josef; Barth, Tomislav; Velek, Jiří; Žižkovský, V.

    2000-01-01

    Roč. 49, - (2000), s. 673-678 ISSN 0862-8408 R&D Projects: GA ČR GA523/99/0843; GA AV ČR IAA7045608; GA AV ČR KSK2020602 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.366, year: 2000

  4. Genetic deficiency of the α subunit of the guanine nucleotide-binding protein G/sub s/ as the molecular basis for Albright hereditary osteodystrophy

    Levine, M.A.; Ahn, T.G.; Klupt, S.F.; Kaufman, K.D.; Smallwood, P.M.; Bourne, H.R.; Sullivan, K.A.; Van Dop, C.

    1988-01-01

    Patients who have pseudohypoparathyroidism type I associated with Albright hereditary osteodystrophy commonly have a genetic deficiency of the α subunit of the G protein that stimulated adenylyl cyclase αG/sub s/. To discover the molecular mechanism that causes αG/sub s/ deficiency in these patients, the authors examined eight kindreds with one or more members affected with Albright hereditary osteodystrophy or pseudohypoparathyroidism and αG/sub s/ deficiency. In these families, αG/sub s/, deficiency and the Albright hereditary osteodystrophy phenotype were transmitted together in a dominant inheritance pattern. Using a cDNA hybridization probe for αG/sub s/, restriction analysis with several analysis with several endonucleases showed no abnormalities of restriction fragments or gene dosage. RNA blot and dot blot analysis of total RNA from cultured fibroblasts obtained from the patients revealed ∼ 50% reduced mRNA levels for αG/sub s/ in affected members of six of the pedigrees but normal levels in affected members of the two other pedigrees, compared to mRNA levels in fibroblasts from unaffected individuals. By contrast, mRNA levels encoding the α subunit of the G protein that inhibits adenylyl cyclase were not altered. These findings suggest that several molecular mechanisms produce αG/sub s/ deficiency in patients with pseudohypoparathyroidism type Ia and that major gene rearrangements or deletions are not a common cause for αG/sub s/ deficiency in pseudohypoparathyroidism type I

  5. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha1-adrenergic system

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha 1 -adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha 1 -adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha 1 -selective radioligand [ 125 I]-I-2-[β-(4-hydroxphenyl)ethylaminomethyl]tetralone ([ 125 I]-IBE 2254) demonstrated that the alpha 1 -adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha 1 -adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha 1 -adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha 1 -mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1 -mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha 1 -adrenergic receptor to its effector under certain circumstances such as high potassium (K + ) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha 1 -response

  6. Supramolecular Properties of Triazole-containing Two Armed Peptidomimetics: From Organogelators to Nucleotide-binding Tweezers

    Chui, Tin Ki

    obtain a clearer picture on the mode of association of these two series of branched peptidomimetics, the length of the tripeptidomimetic arms was truncated to a dipeptide, and the amino acid, valine, was used for further studies. Both the two new candidates, 88-K-V2 and 89-B-V2, were shown to dimerize in chloroform as shown from vapor pressure osmometry (VPO) studies. 1H NMR titration experiments indicated a better dimerization strength for the latter candidate due to the intermolecular pi-pi interactions offered by its benzene ring in addition to the intermolecular hydrogen bonding by the amides and triazole units. H/D exchange and 2D NMR experiments, and molecular modeling revealed that 88-K-V2 dimerized through the formation of antiparallel beta-strands whereas formation of parallel beta-strands took place in 89-B-V2. Compound 88-K-V2 was found to form 1:1 complexes with chloride (Ka 640 M-1) and monobasic diethyl phosphate (DEP) ion (Ka 810 M-1) in chloroform. Interestingly, 89-B-V 2 was shown to form the usual 1:1 complex with the former ion (Ka 970 M-1) while forming an unexpected 2:1 complex with the latter with positive cooperativity. It was observed that both the amides and triazole protons were involved in anion-binding. In the 88-K-V2-DEP complex, the host formed a helix-like structure that wrapped around the anion located at the center of the complex as determined by 2D NMR and molecular modeling studies. Finally, further structural modification of 88-K-V2 gave a water-soluble nucleotide-binding tweezer 93-K-R2·4TFA . This tweezer consisted of four arginines (R), two triazole units, two pyrene probes and a small hydrophilic ethanolamine tail. Fluorescence study showed that this tweezer was able to form 1:1 complexes with different nucleotides in water with similar binding strength regardless of the number of phosphate groups present in the nucleotides. Moleular modeling suggested that such a charge-independent binding behavior was due to the similar number

  7. Direct demonstration of guanine nucleotide sensitive receptors for vasoactive intestinal peptide in the anterior lobe of the rat pituitary gland

    Agui, T.; Matsumoto, K.

    1990-01-01

    The vasoactive intestinal peptide (VIP) receptors were identified on the membranes from the rat anterior pituitary gland with [ 125 I]VIP. The dissociation constant (Kd) and the maximal binding capacity (Bmax) values were estimated from the competitive inhibition data. The Kd and Bmax values were 1.05 +/- 0.75 nM and 103 +/- 11 fmol/mg protein, respectively. The order of molar potency of related peptides to inhibit [ 125 I]VIP binding was VIP greater than peptide histidine isoleucine (PHI) greater than secretin greater than glucagon. Glucagon was not effective to inhibit the binding. [ 125 I]VIP binding was effectively inhibited by the addition of guanine nucleotides. The order of molar potency to inhibit the binding was Gpp(NH)p greater than GTP greater than GDP greater than GMP greater than ATP. These results directly suggest the coupling of VIP receptors with guanine nucleotide binding proteins in the anterior pituitary gland

  8. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M.

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A) + RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A) + RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs

  9. Nucleotide-Binding Oligomerization Domain-1 and -2 Play No Role in Controlling Brucella abortus Infection in Mice

    Fernanda S. Oliveira

    2012-01-01

    Full Text Available Nucleotide-binding oligomerization domain proteins (NODs are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.

  10. Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter.

    Dye, Natalie A; Pincus, Zachary; Fisher, Isabelle C; Shapiro, Lucy; Theriot, Julie A

    2011-07-01

    The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape. © 2011 Blackwell Publishing Ltd.

  11. Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

    Yoon, Hyo-Eun; Ahn, Mee-Young; Kwon, Seong-Min; Kim, Dong-Jae; Lee, Jun; Yoon, Jung-Hoon

    2016-04-01

    Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD 2 in Host Defense during Pneumococcal Pneumonia.

    Tijmen J Hommes

    Full Text Available Streptococcus (S. pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2-/- and wild-type (Wt alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39, an isogenic capsule locus deletion mutant (D39Δcps or serotype 3 S. pneumoniae (6303 via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.

  13. Guanine nucleotide regulation of α1-adrenergic receptors of muscle and kidney eptihelial cells

    Terman, B.I.; Hughes, R.J.; Slivka, S.R.; Insel, P.A.

    1986-01-01

    The authors have examined the effect of guanine nucleotides on the interaction of adrenergic agents with α 1 -adrenergic receptors of two cell lines, the Madin-Darby Canine Kidney (MDCK) and BC3H-1 muscle cells. While gaunylylimidodiphosphoate (Gpp(NH)p) had no effect on the affinity or the total number of [ -3 H]prazosin binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonist epinephrine in competing for [ 3 H]prazosin binding sites in both cell types. The EC 50 of Gpp(NH)p was ∼100 μM, and a maximal effect was seen at 500 μM. In contrast, 100 μM Gpp(NH)p yielding maximal shifts in binding of epinephrine to β-adrenergic receptors in BC3H-1 cell membranes. Guanine nucleotides were significantly more effective than adenine nucleotides in shifting agonist affinity for the α 1 -receptor and Mg ++ was required to observe a maximal effect. α 1 -receptor agonists activated phosphatidylinositol (PI) hydrolysis in both cell types, but have no direct effect on membrane adenylate cyclase activity. In intact BC3H-1 cells, α 1 -agonists inhibited β-adrenergic cAMP production, an effect which appears in preliminary studies not to result from enhanced phosphodieterase activity. These results show that agonist binding to α 1 -adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides. This regulation and inturn transmembrane signalling (PI hydrolysis) by these receptors appear to involve a guanine nucleotide binding (G) protein, which may be different than G/sub s/ and G/sub i/

  14. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-08

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  15. Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

    Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

    2013-01-01

    Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

  16. A primary survey on bryophyte species reveals two novel classes of nucleotide-binding site (NBS genes.

    Jia-Yu Xue

    Full Text Available Due to their potential roles in pathogen defense, genes encoding nucleotide-binding site (NBS domain have been particularly surveyed in many angiosperm genomes. Two typical classes were found: one is the TIR-NBS-LRR (TNL class and the other is the CC-NBS-LRR (CNL class. It is seldom known, however, what kind of NBS-encoding genes are mainly present in other plant groups, especially the most ancient groups of land plants, that is, bryophytes. To fill this gap of knowledge, in this study, we mainly focused on two bryophyte species: the moss Physcomitrella patens and the liverwort Marchantia polymorpha, to survey their NBS-encoding genes. Surprisingly, two novel classes of NBS-encoding genes were discovered. The first novel class is identified from the P. patens genome and a typical member of this class has a protein kinase (PK domain at the N-terminus and a LRR domain at the C-terminus, forming a complete structure of PK-NBS-LRR (PNL, reminiscent of TNL and CNL classes in angiosperms. The second class is found from the liverwort genome and a typical member of this class possesses an α/β-hydrolase domain at the N-terminus and also a LRR domain at the C-terminus (Hydrolase-NBS-LRR, HNL. Analysis on intron positions and phases also confirmed the novelty of HNL and PNL classes, as reflected by their specific intron locations or phase characteristics. Phylogenetic analysis covering all four classes of NBS-encoding genes revealed a closer relationship among the HNL, PNL and TNL classes, suggesting the CNL class having a more divergent status from the others. The presence of specific introns highlights the chimerical structures of HNL, PNL and TNL genes, and implies their possible origin via exon-shuffling during the quick lineage separation processes of early land plants.

  17. Efficacy of the nucleotide-binding oligomerzation domain 1 inhibitor Nodinhibit-1 on corneal alkali burns in rats

    Xu Huang

    2015-10-01

    Full Text Available AIM:To evaluate the therapeutic effect of Nodinhibit-1 on alkali-burn-induced corneal neovascularization (CNV and inflammation.The nucleotide-binding oligomerzation domain 1 (NOD1 is a potent angiogenic gene.METHODS:The alkali-burned rat corneas (32 right eyes were treated with eye drops containing Nodinhibit-1 or phosphate buffered solution (PBS, PH 7.4 only, four times per day. CNV and inflammation were monitored using slit lamp microscopy, and the area of CNV was measured by formula. Vascular endothelial growth factor (VEGF and pigment epithelium-derived factor (PEDF was determined by Western blot analysis. The TUNEL assay was used to assess the corneal apoptosis cells.RESULTS:Alkali-burn-induced progressive CNV and inflammation in the cornea. After treatment for 7d and 14d, there were statistically significant differences in the CNV areas and inflammatory index on that between two group(P<0.05, respectively. Epithelial defect quantification showed a significant difference between the two groups at days 4 and 7 after the alkali burns (P<0.05. The apoptotic cells on days 1, 4, and 7 between the two groups showed significant differences at all time points (P<0.05, respectively. Compared to that in control group, the protein level of VEGF expression was significantly reduced whereas the PEDF expression was increase in the Nodinhibit-1 groups on day 14 (P<0.05, respectively=.CONCLUSION:Topical application of 10.0 μg/mL Nodinhibit-1 may have potential effect for the alkali burn-induced CNV and inflammation. The effect of Nodinhibit-1 on CNV may be by regulation the equilibrium of VEGF and PEDF in the wounded cornea.

  18. Bioinformatic analysis of the nucleotide binding site-encoding disease-resistance genes in foxtail millet (Setaria italica (L.) Beauv.).

    Zhu, Y B; Xie, X Q; Li, Z Y; Bai, H; Dong, L; Dong, Z P; Dong, J G

    2014-08-28

    The nucleotide-binding site (NBS) disease-resistance genes are the largest category of plant disease-resistance gene analogs. The complete set of disease-resistant candidate genes, which encode the NBS sequence, was filtered in the genomes of two varieties of foxtail millet (Yugu1 and 'Zhang gu'). This study investigated a number of characteristics of the putative NBS genes, such as structural diversity and phylogenetic relationships. A total of 269 and 281 NBS-coding sequences were identified in Yugu1 and 'Zhang gu', respectively. When the two databases were compared, 72 genes were found to be identical and 164 genes showed more than 90% similarity. Physical positioning and gene family analysis of the NBS disease-resistance genes in the genome revealed that the number of genes on each chromosome was similar in both varieties. The eighth chromosome contained the largest number of genes and the ninth chromosome contained the lowest number of genes. Exactly 34 gene clusters containing the 161 genes were found in the Yugu1 genome, with each cluster containing 4.7 genes on average. In comparison, the 'Zhang gu' genome possessed 28 gene clusters, which had 151 genes, with an average of 5.4 genes in each cluster. The largest gene cluster, located on the eighth chromosome, contained 12 genes in the Yugu1 database, whereas it contained 16 genes in the 'Zhang gu' database. The classification results showed that the CC-NBS-LRR gene made up the largest part of each chromosome in the two databases. Two TIR-NBS genes were also found in the Yugu1 genome.

  19. The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence

    Jones, J.S.; Prakash, L. (Univ. of Rochester School of Medicine, NY (USA)); Weber, S. (Kodak Research Park, Rochester, NY (USA))

    1988-07-25

    The RAD18 gene of Saccharomyces cerevisiae is required for postreplication repair of UV damaged DNA. The authors have isolated the RAD18 gene, determined its nucleotide sequence and examined if deletion mutations of this gene show different or more pronounced phenotypic effects than the previously described point mutations. The RAD18 gene open reading frame encodes a protein of 487 amino acids, with a calculated molecular weight of 55,512. The RAD18 protein contains three potential zinc finger domains for nucleic acid binding, and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. The DNA binding and nucleotide binding activities could enable the RAD18 protein to bind damaged sites in the template DNA with high affinity. Alternatively, or in addition, RAD18 protein may be a transcriptional regulator. The RAD18 deletion mutation resembles the previously described point mutations in its effects on viability, DNA repair, UV mutagenesis, and sporulation.

  20. Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

    Marques-Carvalho, Maria João; Morais-Cabral, João Henrique

    2012-01-01

    The crystallization conditions and preliminary crystal characterization of the cytoplasmic cyclic nucleotide-binding homology domain from the mouse EAG potassium channel are reported. The members of the family of voltage-gated KCNH potassium channels play important roles in cardiac and neuronal repolarization, tumour proliferation and hormone secretion. These channels have a C-terminal cytoplasmic domain which is homologous to cyclic nucleotide-binding domains (CNB-homology domains), but it has been demonstrated that channel function is not affected by cyclic nucleotides and that the domain does not bind nucleotides in vitro. Here, the crystallization and preliminary crystallographic analysis of a CNB-homology domain from a member of the KCNH family, the mouse EAG channel, is reported. X-ray diffraction data were collected to 2.2 Å resolution and the crystal belonged to the hexagonal space group P3 1 21

  1. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

    Fenyk, Stepan; Townsend, Philip D; Dixon, Christopher H; Spies, Gerhard B; de San Eustaquio Campillo, Alba; Slootweg, Erik J; Westerhof, Lotte B; Gawehns, Fleur K K; Knight, Marc R; Sharples, Gary J; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2015-10-09

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein*

    Fenyk, Stepan; Townsend, Philip D.; Dixon, Christopher H.; Spies, Gerhard B.; de San Eustaquio Campillo, Alba; Slootweg, Erik J.; Westerhof, Lotte B.; Gawehns, Fleur K. K.; Knight, Marc R.; Sharples, Gary J.; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2015-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. PMID:26306038

  3. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  4. C-terminal β9-strand of the cyclic nucleotide-binding homology domain stabilizes activated states of Kv11.1 channels.

    Chai Ann Ng

    Full Text Available 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.

  5. Structural determination of functional units of the nucleotide binding domain (NBD94 of the reticulocyte binding protein Py235 of Plasmodium yoelii.

    Ardina Grüber

    2010-02-01

    Full Text Available Invasion of the red blood cells (RBC by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94 of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547, NBD94(566-663 and NBD94(674-793, respectively. Using fluorescence correlation spectroscopy NBD94(444-547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663 and NBD94(674-793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663 consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793 in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the

  6. 21 CFR 73.1329 - Guanine.

    2010-04-01

    ... in this subpart as safe and suitable for use in color additive mixtures for coloring externally... ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1329 Guanine. (a) Identity. (1) The color additive guanine is the crystalline material obtained from fish scales and consists principally of the two purines...

  7. Synthesis of Lipophilic Guanine N-9 Derivatives

    Wamberg, Michael C; Pedersen, Pernille L; Löffler, Philipp M G

    2017-01-01

    the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid...... bilayer structures, in which both alkyl chain length and attachment chemistry determined their interaction with the membrane. Incubation of these guanine conjugates, as solids, with a decanoic acid vesicle suspension, showed that ether- and triazole-linked C10 anchors yielded an increased partitioning...... of the guanine derivative into the membranous phase compared to directly N-9-linked saturated alkyl anchors. Decanoic acid vesicle membranes could be loaded with up to 5.5 mol % guanine derivative, a 6-fold increase over previous limits. Thus, anchor chemistries exhibiting favorable interactions with a bilayer...

  8. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  9. FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

    Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

    2017-09-01

    The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

  10. ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

    Shah, Abdul Haseeb; Banerjee, Atanu; Rawal, Manpreet Kaur; Saxena, Ajay Kumar; Mondal, Alok Kumar; Prasad, Rajendra

    2015-08-01

    The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

    Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

    1987-01-01

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

  12. Discovery of Nanomolar Desmuramylpeptide Agonists of the Innate Immune Receptor Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Possessing Immunostimulatory Properties.

    Gobec, Martina; Tomašič, Tihomir; Štimac, Adela; Frkanec, Ruža; Trontelj, Jurij; Anderluh, Marko; Mlinarič-Raščan, Irena; Jakopin, Žiga

    2018-04-12

    Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants, and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis, and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.

  13. Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

    Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

    2017-08-25

    Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. The wheat homolog of putative nucleotide-binding site-leucine-rich repeat resistance gene TaRGA contributes to resistance against powdery mildew.

    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.

  15. Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding.

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Callaghan, Richard; Higgins, Christopher F; Ford, Robert C

    2003-03-07

    P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.

  16. Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

    Li, Xiaolu; Zhuo, Wei; Yu, Jie; Ge, Jingpeng; Gu, Jinke; Feng, Yue; Yang, Maojun; Wang, Linfang; Wang, Na

    2013-02-01

    Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

  17. Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

    Saeki, Ayumi; Suzuki, Toshihiko; Hasebe, Akira; Kamezaki, Ryousuke; Fujita, Mari; Nakazawa, Futoshi; Shibata, Ken-Ichiro

    2017-03-01

    Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1β responsible for the development of the disease. However, the mechanism of IL-1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1β and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1β production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1β. The inhibitors for ATP receptor reduced IL-1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity. © 2016 John Wiley & Sons Ltd.

  18. Genome-wide identification and tissue-specific expression analysis of nucleotide binding site-leucine rich repeat gene family in Cicer arietinum (kabuli chickpea).

    Sharma, Ranu; Rawat, Vimal; Suresh, C G

    2017-12-01

    The nucleotide binding site-leucine rich repeat (NBS-LRR) proteins play an important role in the defense mechanisms against pathogens. Using bioinformatics approach, we identified and annotated 104 NBS-LRR genes in chickpea. Phylogenetic analysis points to their diversification into two families namely TIR-NBS-LRR and non-TIR-NBS-LRR. Gene architecture revealed intron gain/loss events in this resistance gene family during their independent evolution into two families. Comparative genomics analysis elucidated its evolutionary relationship with other fabaceae species. Around 50% NBS-LRRs reside in macro-syntenic blocks underlining positional conservation along with sequence conservation of NBS-LRR genes in chickpea. Transcriptome sequencing data provided evidence for their transcription and tissue-specific expression. Four cis -regulatory elements namely WBOX, DRE, CBF, and GCC boxes, that commonly occur in resistance genes, were present in the promoter regions of these genes. Further, the findings will provide a strong background to use candidate disease resistance NBS-encoding genes and identify their specific roles in chickpea.

  19. Genome-wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

    Eunyoung Seo

    2016-08-01

    Full Text Available Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR immune receptors are known play critical roles in effector-triggered immunity (ETI plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analyses and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analyses of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

  20. BAR domain proteins regulate Rho GTPase signaling.

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

  1. Invasive Streptococcus mutans induces inflammatory cytokine production in human aortic endothelial cells via regulation of intracellular toll-like receptor 2 and nucleotide-binding oligomerization domain 2.

    Nagata, E; Oho, T

    2017-04-01

    Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll-like receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD) -like receptors in HAECs. Cytokine production by HAECs was determined using enzyme-linked immunosorbent assays, and the expression of TLRs and NOD-like receptors was evaluated by real-time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  3. Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

    Kasimsetty, Sashi G; Shigeoka, Alana A; Scheinok, Andrew A; Gavin, Amanda L; Ulevitch, Richard J; McKay, Dianne B

    2017-08-01

    Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2 -/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2 -/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2 -/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation. Copyright © 2017 by The American Association of Immunologists, Inc.

  4. ATP-induced conformational changes of nucleotide-binding domains in an ABC transporter. Importance of the water-mediated entropic force.

    Hayashi, Tomohiko; Chiba, Shuntaro; Kaneta, Yusuke; Furuta, Tadaomi; Sakurai, Minoru

    2014-11-06

    ATP binding cassette (ABC) proteins belong to a superfamily of active transporters. Recent experimental and computational studies have shown that binding of ATP to the nucleotide binding domains (NBDs) of ABC proteins drives the dimerization of NBDs, which, in turn, causes large conformational changes within the transmembrane domains (TMDs). To elucidate the active substrate transport mechanism of ABC proteins, it is first necessary to understand how the NBD dimerization is driven by ATP binding. In this study, we selected MalKs (NBDs of a maltose transporter) as a representative NBD and calculated the free-energy change upon dimerization using molecular mechanics calculations combined with a statistical thermodynamic theory of liquids, as well as a method to calculate the translational, rotational, and vibrational entropy change. This combined method is applied to a large number of snapshot structures obtained from molecular dynamics simulations containing explicit water molecules. The results suggest that the NBD dimerization proceeds with a large gain of water entropy when ATP molecules bind to the NBDs. The energetic gain arising from direct NBD-NBD interactions is canceled by the dehydration penalty and the configurational-entropy loss. ATP hydrolysis induces a loss of the shape complementarity between the NBDs, which leads to the dissociation of the dimer, due to a decrease in the water-entropy gain and an increase in the configurational-entropy loss. This interpretation of the NBD dimerization mechanism in concert with ATP, especially focused on the water-mediated entropy force, is potentially applicable to a wide variety of the ABC transporters.

  5. Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

    Kurashima-Ito, Kaori [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Ikeya, Teppei [National Institute of Advanced Industrial Science and Technology (AIST), (Japan); Senbongi, Hiroshi [Mitochondrial Diseases Group, MRC Dunn Human NutritionUnit (United Kingdom); Tochio, Hidehito [International Graduate School of Arts and Sciences, Supramolecular Biology, Yokohama City University, Molecular Biophysics Laboratory (Japan); Mikawa, Tsutomu [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Shibata, Takehiko [RIKEN, Shibata Distinguished Senior Scientist Laboratory (Japan); Ito, Yutaka [RIKEN, Cellular and Molecular Biology Laboratory (Japan)], E-mail: ito-yutaka@center.tmu.ac.jp

    2006-05-15

    Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired {sup 13}C and {sup 15}N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and {alpha}/{beta}-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.

  6. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  7. Reactivity of chitosan derivatives and their interaction with guanine ...

    Density functional theory; hydrogen bonding; chitosan derivative; guanine; solvent effect. 1. Introduction .... Out of different models for accounting the solva- tion energies ..... Authors thank DST, New Delhi for financial support. (Grant No.

  8. Mutagenic and Cytotoxic Properties of 6-Thioguanine, S6-Methylthioguanine, and Guanine-S6-sulfonic Acid*S⃞

    Yuan, Bifeng; Wang, Yinsheng

    2008-01-01

    Thiopurine drugs, including 6-thioguanine (SG), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of SG nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. SG in DNA can be methylated by S-adenosyl-l-methionine to give S6-methylthioguanine (S6mG) and oxidized by UVA light to render guanine-S6-sulfonic acid ...

  9. [Triplet expansion cytosine-guanine-guanine: Three cases of OMIM syndrome in the same family].

    González-Pérez, Jesús; Izquierdo-Álvarez, Silvia; Fuertes-Rodrigo, Cristina; Monge-Galindo, Lorena; Peña-Segura, José Luis; López-Pisón, Francisco Javier

    2016-04-01

    The dynamic increase in the number of triplet repeats of cytosine-guanine-guanine (CGG) in the FMR1 gene mutation is responsible for three OMIM syndromes with a distinct clinical phenotype: Fragile X syndrome (FXS) and two pathologies in adult carriers of the premutation (55-200 CGG repeats): Primary ovarian insufficiency (FXPOI) and tremor-ataxia syndrome (FXTAS) associated with FXS. CGG mutation dynamics of the FMR1 gene were studied in DNA samples from peripheral blood from the index case and other relatives of first, second and third degree by TP-PCR, and the percentage methylation. Diagnosis of FXS was confirmed in three patients (21.4%), eight patients (57.1%) were confirmed in the premutation range transmitters, one male patient with full mutation/permutation mosaicism (7.1%) and two patients (14.3%) with normal study. Of the eight permutated patients, three had FXPOI and one male patient had FXTAS. Our study suggests the importance of making an early diagnosis of SXF in order to carry out a family study and genetic counselling, which allow the identification of new cases or premutated patients with FMR1 gene- associated syndromes (FXTAS, FXPOI). Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  10. Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

    Ahn, Jinhi; Beharry, Seelochan; Molday, Laurie L; Molday, Robert S

    2003-10-10

    ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

  11. Characterization of oxidative guanine damage and repair in mammalian telomeres.

    Zhilong Wang

    2010-05-01

    Full Text Available 8-oxo-7,8-dihydroguanine (8-oxoG and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1-initiated DNA base excision repair (BER. Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH, by chromosome orientation-FISH (CO-FISH, and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/- mouse tissues and primary embryonic fibroblasts (MEFs cultivated in hypoxia condition (3% oxygen, whereas telomere shortening was detected in Ogg1(-/- mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/- mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/- mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/- MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity

  12. Endogenous melatonin and oxidatively damaged guanine in DNA

    Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan

    2009-01-01

    overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were...... attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. METHODS: Mother...

  13. Endogenous melatonin and oxidatively damaged guanine in DNA

    Poulsen Henrik E

    2009-10-01

    Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

  14. In Silico Molecular Modeling and Docking Studies on Novel Mutants (E229V, H225P and D230C) of the Nucleotide-Binding Domain of Homo sapiens Hsp70.

    Elengoe, Asita; Hamdan, Salehhuddin

    2017-12-01

    In this study, we explored the possibility of determining the synergistic interactions between nucleotide-binding domain (NBD) of Homo sapiens heat-shock 70 kDa protein (Hsp70) and E1A 32 kDa of adenovirus serotype 5 motif (PNLVP) in the efficiency of killing of tumor cells in cancer treatment. At present, the protein interaction between NBD and PNLVP motif is still unknown, but believed to enhance the rate of virus replication in tumor cells. Three mutant models (E229V, H225P and D230C) were built and simulated, and their interactions with PNLVP motif were studied. The PNLVP motif showed the binding energy and intermolecular energy values with the novel E229V mutant at -7.32 and -11.2 kcal/mol. The E229V mutant had the highest number of hydrogen bonds (7). Based on the root mean square deviation, root mean square fluctuation, hydrogen bonds, salt bridge, secondary structure, surface-accessible solvent area, potential energy and distance matrices analyses, it was proved that the E229V had the strongest and most stable interaction with the PNLVP motif among all the four protein-ligand complex structures. The knowledge of this protein-ligand complex model would help in designing Hsp70 structure-based drug for cancer therapy.

  15. Nucleotide binding to Na+/K+-ATPase

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  16. Guanine base stacking in G-quadruplex nucleic acids

    Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2013-01-01

    G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5′–5′ manner based on different accessible tetrad stacking modes at the stacking interfaces of 5′–5′ and 3′–3′ stacked G-quadruplexes. PMID:23268444

  17. The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM.

    Bischof, Lisa Franziska; Friedrich, Carmen; Harms, Andrea; Søgaard-Andersen, Lotte; van der Does, Chris

    2016-03-25

    Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

    Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

    2014-10-10

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact, Head-to-Tail Configuration of the Nucleotide-Binding Domains.

    Peter M Jones

    Full Text Available ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs, which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration 'sandwich' dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD 'Switch' mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.

  20. Scambio, a novel guanine nucleotide exchange factor for Rho

    Groffen John

    2004-04-01

    Full Text Available Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

  1. Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release.

    Guilfoyle, Amy P; Deshpande, Chandrika N; Schenk, Gerhard; Maher, Megan J; Jormakka, Mika

    2014-12-12

    GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

  2. Electron detachment of the hydrogen-bonded amino acid side-chain guanine complexes

    Wang, Jing; Gu, Jiande; Leszczynski, Jerzy

    2007-07-01

    The photoelectron spectra of the hydrogen-bonded amino acid side-chain-guanine complexes has been studied at the partial third order (P3) self-energy approximation of the electron propagator theory. The correlation between the vertical electron detachment energy and the charge distributions on the guanine moiety reveals that the vertical electron detachment energy (VDE) increases as the positive charge distribution on the guanine increases. The low VDE values determined for the negatively charged complexes of the guanine-side-chain-group of Asp/Glu suggest that the influence of the H-bonded anionic groups on the VDE of guanine could be more important than that of the anionic backbone structure. The even lower vertical electron detachment energy for guanine is thus can be expected in the H-bonded protein-DNA systems.

  3. Quantum molecular modeling of the interaction between guanine and alkylating agents--2--nitrogen mustard.

    Hamza, A; Broch, H; Vasilescu, D

    1996-06-01

    The alkylation mechanism of guanine by nitrogen mustard (HN2) was studied by using a supermolecular modeling at the ab initio 6-31G level. Our computations show that interaction of guanine with the aziridinium form of HN2 necessitates a transition state for the N7 alkylation route. The pathway of N7-guanine alkylation by nitrogen and sulfur mustards is discussed on the basis of the Molecular Electrostatic Potential and HOMO-LUMO properties of these molecules.

  4. Flexibility of the myosin heavy chain: direct evidence that the region containing SH/sub 1/ and SH/sub 2/ can move 10 /Angstrom/ under the influence of nucleotide binding

    Huston, E.E.; Grammer, J.C.; Yount, R.G.

    1988-12-13

    Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF/sub 1/) could be oxidized to a disulfide bond by treatment with a 2-fold excess of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in the presence of MgADP. The resulting characteristic changes in the ATPase activities of SF/sub 1/ and the fact that MgADP was stably trapped at the active site, suggested that the two thiols cross-linked were SH/sub 1/ (Cys-707) and SH/sub 2/ (Cys-697) from the myosin heavy chain. To verify this suggestion, SF/sub 1/, after DTNB treatment as above, was treated with an excess of N-ethylmaleimide to block all accessible thiols. The single protein disulfide produced by DTNB oxidation was reduced with dithioerythritol and the modified SF/sub 1/ internally cross-linked with equimolar (/sup 14/C)p-phenylenedimaleimide (pPDM) in the presence of MgADP. After extensive trypsinization, the major /sup 14/C-labeled peptide was isolated, characterized, and shown to be Cys-Asn-Gly-Val-Leu-Gly-Ile-Arg-Ile-Cys-Arg, in which the two cysteines were cross-linked by pPDM. This peptide is known to contain SH/sub 2/ and SH/sub 1/ in this order and to come from residues 697-708 in the rabbit skeletal myosin heavy chain. Parallel experiments with (/sup 14/C)pPDM and unmodified SF/sub 1/ similar to those above gave an identical SH/sub 1/, SH/sub 2/ tryptic peptide, verifying earlier labeling results. These combined results demonstrate that SH/sub 1/ and SH/sub 2/ cross-linked by pPDM (12-13 /Angstrom/, S to S) or by oxidation with DTNB (2 /Angstrom/, S to S) can move a minimum of 10 /Angstrom/ under the influence of nucleotide binding. Because these residues are separated by only nine amino acids in the primary sequence, this small section of the heavy chain must possess extraordinary flexibility.

  5. Nucleotide-binding oligomerization domain 1 regulates Porphyromonas gingivalis-induced vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression in endothelial cells through NF-κB pathway.

    Wan, M; Liu, J; Ouyang, X

    2015-04-01

    Porphyromonas gingivalis has been shown to actively invade endothelial cells and induce vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) overexpression. Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition reporter, and its involvement in this process was unknown. This study focused on endothelial cells infected with P. gingivalis, the detection of NOD1 expression and the role that NOD1 plays in the upregulation of VCAM-1 and ICAM-1. The human umbilical vein endothelial cell line (ECV-304) was intruded by P. gingivalis W83, and cells without any treatment were the control group. Expression levels of NOD1, VCAM-1, ICAM-1, phosphorylated P65 between cells with and without treatment on both mRNA and protein levels were compared. Then we examined whether mesodiaminopimelic acid (NOD1 agonist) could increase VCAM-1 and ICAM-1 expression, meanwhile, NOD1 gene silence by RNA interference could reduce VCAM-1, ICAM-1 and phosphorylated P65 release. At last, we examined whether inhibition of NF-κB by Bay117082 could reduce VCAM-1 and ICAM- 1 expression. The mRNA levels were measured by real-time polymerase chain reaction, and protein levels by western blot or electrophoretic mobility shift assays (for phosphorylated P65). P. gingivalis invasion showed significant upregulation of NOD1, VCAM-1 and ICAM-1. NOD1 activation by meso-diaminopimelic acid increased VCAM-1 and ICAM-1 expression, and NOD1 gene silence reduced VCAM-1 and ICAM-1 release markedly. The NF-κB signaling pathway was activated by P. gingivalis, while NOD1 gene silence decreased the activation of NF-κB. Moreover, inhibition of NF-κB reduced VCAM-1 and ICAM-1 expression induced by P. gingivalis in endothelial cells. The results revealed that P. gingivalis induced NOD1 overexpression in endothelial cells and that NOD1 played an important role in the process of VCAM-1 and ICAM-1 expression in endothelial cells infected with P

  6. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Zhang AL

    2017-02-01

    Full Text Available Ailiang Zhang, Kun Wang, Lianghua Ding, Xinnan Bao, Xuan Wang, Xubin Qiu, Jinbo Liu Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China Abstract: Lumbar disc herniation (LDH is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB and nucleotide-binding domain-like receptor protein 3 (NLRP3 inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP was implanted in the left L5 dorsal root ganglion (DRG to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC, caspase-1, interleukin (IL-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. Keywords: pain, NLRP3, NF-κB, dorsal root ganglion, nucleus pulposus

  7. Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid.

    Yuan, Bifeng; Wang, Yinsheng

    2008-08-29

    Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

  8. Quantum molecular modeling of the interaction between guanine and alkylating agents--1--sulfur mustard.

    Broch, H; Hamza, A; Vasilescu, D

    1996-06-01

    Interaction between Guanine and the episulfonium form of Sulfur mustard (HD) was studied using the ab initio LCAO-MO method at the HF/6-31G level. The alkylation mechanism on guanine-N7 was analyzed by using a supermolecular modeling. Our stereostructural results associated with the molecular electrostatic potentials and HOMO-LUMO properties, show that in vacuum the alkylation of the N7 of guanine by HD in the aggressive episulfonium form is a direct process without transition state and of which the pathway is determined.

  9. Application of Ammonium Persulfate for Selective Oxidation of Guanines for Nucleic Acid Sequencing

    Yafen Wang

    2017-07-01

    Full Text Available Nucleic acids can be sequenced by a chemical procedure that partially damages the nucleotide positions at their base repetition. Many methods have been reported for the selective recognition of guanine. The accurate identification of guanine in both single and double regions of DNA and RNA remains a challenging task. Herein, we present a new, non-toxic and simple method for the selective recognition of guanine in both DNA and RNA sequences via ammonium persulfate modification. This strategy can be further successfully applied to the detection of 5-methylcytosine by using PCR.

  10. Purine nucleotide synthesis from exogenous adenine and guanine in rodent small intestine

    Gross, C.J.; Karlberg, P.K.; Savaiano, D.A.

    1986-01-01

    14 C-Adenine and 14 C-guanine uptake was studied in isolated guinea pig enterocytes. Cells were incubated in Hank's buffer and separated from the medium by centrifugation through silicone oil into 1M PCA. Uptake was temperature and concentration dependent. Both compounds were incorporated into nucleotides as measured by HPLC and HVE. Adenine was more extensively incorporated into nucleotides than was guanine. Adenine nucleotides accounted for about 70% of the intracellular label after 30 min with a majority being ADP and ATP (medium concentration = 10 μM). Guanine nucleotides accounted for only 30% of the intracellular label after 30 min. Labeled intracellular free adenine or guanine were not detected. Significantly more guanine vs. adenine was converted to uric acid. After 30 min, 11.5 +/- 3.9% (n=3) and 83.0 +/- 8.4% (n=4) of the label was present as uric acid in the medium when adenine and guanine, respectively, were the substrate. After 1 min, 34.8 +/- 3.4% (n=4) of the label in the medium was present as uric acid when guanine was the substrate. Decreasing the concentration of adenine resulted in an increase in the percent of uric acid in the medium. 14 C-adenine (75 nmol) was injected into 1 gm segments of rat jejunum. After 5 min., segments were quickly flushed and the tissue homogenized in 1M PCA. Only uric acid was present after 5 min (n=6). In contrast, in animals fasted 3 to 5 days, less conversion to uric acid was observed in the intestinal content (50-80% of the same dose was still present as adenine after 5 min) and nucleotide formation was observed in the tissue. The results indicate that uric acid and nucleotide synthesis from exogenous adenine and guanine are concentration dependent and affected by nutritional state

  11. Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

    Boulias, C.; Moscarello, M.A.

    1989-01-01

    Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides

  12. Charge splitters and charge transport junctions based on guanine quadruplexes

    Sha, Ruojie; Xiang, Limin; Liu, Chaoren; Balaeff, Alexander; Zhang, Yuqi; Zhang, Peng; Li, Yueqi; Beratan, David N.; Tao, Nongjian; Seeman, Nadrian C.

    2018-04-01

    Self-assembling circuit elements, such as current splitters or combiners at the molecular scale, require the design of building blocks with three or more terminals. A promising material for such building blocks is DNA, wherein multiple strands can self-assemble into multi-ended junctions, and nucleobase stacks can transport charge over long distances. However, nucleobase stacking is often disrupted at junction points, hindering electric charge transport between the two terminals of the junction. Here, we show that a guanine-quadruplex (G4) motif can be used as a connector element for a multi-ended DNA junction. By attaching specific terminal groups to the motif, we demonstrate that charges can enter the structure from one terminal at one end of a three-way G4 motif, and can exit from one of two terminals at the other end with minimal carrier transport attenuation. Moreover, we study four-way G4 junction structures by performing theoretical calculations to assist in the design and optimization of these connectors.

  13. Active site labeling of the guanine-7-methyltransferase

    Streaker, E.; Sitz, T.O.

    1992-01-01

    Studies on the guanine-7-methyltransferase have defined three domains in the active site: the S-adenosylmethionine (SAM) region, the cap region (GpppG), and the RNA binding domain (--NpNpNpNpNp---). The authors attempted to label the SAM binding domain by a photoaffinity label using 8-azido-SAM and another method using 3 H-SAM and long exposures to uv-light. Neither method was successful. The next approach was to attempt to label the cap-RNA binding domain (GpppGpNpNpNpNpN) by synthesizing RNA containing 8-azido-Ap using an in vitro transcription system and T7 RNA polymerase. The 8-azido-ATP inhibited the T7 RNA polymerase preventing the synthesis of RNA. As they were unable to synthesize the photoaffinity label, they next tried to synthesize an end labeled RNA and directly label by long exposures to uv-light. When the enzyme was incubated with 32 P-labeled RNA for 15 min at 37 degrees and then exposed to a germicidal lamp for various times at O degrees, optimal labeling occurred after 45 min. Various enzyme preparations were labeled by this method and two polypeptides were found to specifically bind the non-methylated mRNA analog. This labeling method should allow characterization of the subunit structure and generate information about the nature of the RNA binding domain

  14. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  15. Fluorescence enhancement of DNA-silver nanoclusters from guanine proximity

    Yeh, Hsin-chih [Los Alamos National Laboratory; Sharma, Jaswinder [Los Alamos National Laboratory; Yoo, Hyojong [Los Alamos National Laboratory; Martinez, Jennifer S [Los Alamos National Laboratory

    2010-01-01

    Oligonucleotide-templated, silver nanoclusters (DNA/Ag NCs) are a versatile set of fluorophores and have already been used for live cell imaging, detection of specific metal ions, and single-nucleotide variation identification. Compared to commonly used organic dyes, these fluorescent nanoclusters have much better photostability and are often a few times brighter. Owing to their small size, simple preparation, and biocompatibility (i.e. made of nontoxic metals), DNA/Ag NCs should find more applications in biological imaging and chemical detection in the years to come. While clearly promising as new fluorophores, DNA/Ag NCs possess a unique and poorly understood dynamic process not shared by organic dyes or photoluminescent nanocrystals - the conversion among different NC species due to silver oxidation/reduction or NC regrouping. While this environmental sensitivity can be viewed as a drawback, in the appropriate context, it can be used as a sensor or reporter. Often reversible, conversions among different NC species have been found to depend upon a number of factors, including time, temperature, oxygen and salt content. In this communication, we report significant fluorescence enhancement of DNA/Ag NCs via interactions with guanine-rich DNA sequences. Moreover, we demonstrated this property can be used for sensitive detection of specific target DNA from a human oncogene (i.e. Braf gene).

  16. Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation

    Mizuta, Ryushin; Mizuta, Midori; Kitamura, Daisuke

    2005-01-01

    It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribes RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine. (author)

  17. High pressure {sup 31}P NMR spectroscopy on guanine nucleotides

    Spoerner, Michael; Karl, Matthias; Lopes, Pedro; Hoering, Marcus; Loeffel, Karoline; Nuehs, Andrea; Adelsberger, Joseph; Kremer, Werner; Kalbitzer, Hans Robert, E-mail: hans-robert.kalbitzer@ur.de [University of Regensburg, Centre of Magnetic Resonance in Chemistry and Biomedicine, Institute of Biophysics and Physical Biochemistry (Germany)

    2017-01-15

    The {sup 31}P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the {sup 31}P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH{sub 2}p and GTPγS was measured in the absence and presence of Mg{sup 2+}-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B{sub 1} was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg{sup 2+}·GMP and Mg{sup 2+}·GppNHp the second order pressure coefficients are positive. To describe the data of Mg{sup 2+}·GppCH{sub 2}p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg{sup 2+} ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure {sup 31}P NMR data on free Mg{sup 2+}-GDP and Mg{sup 2+}-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.

  18. Behavior of the guanine base in G-quadruplexes probed by the fluorescent guanine analog, 6-methyl isozanthopterin

    Han, Ji Hoon; Chitrapriya, Nataraj; Lee, Hyun Suk; Lee, Young Ae; Kim, Seog K. [Dept. of Chemistry, Yeungnam University, Gyeongsan (Korea, Republic of); Jung, Maeng Joon [Dept. of Chemistry, Kyungpook National University, Daegu (Korea, Republic of)

    2017-02-15

    In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G{sub 3}T{sub 2}G{sub 3}TGTG{sub 3}T{sub 2}G{sub 3} sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

  19. Highly sensitive and selective fluorescent assay for guanine based on the Cu(2+)/eosin Y system.

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-15

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu(2+)/eosin Y. Cu(2+) interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu(2+)/eosin Y system, guanine reacted with Cu(2+) to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L(-1) and a linear range of 3.3-116 nmol L(-1). The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Highly sensitive and selective fluorescent assay for guanine based on the Cu2 +/eosin Y system

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-01

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu2 +/eosin Y. Cu2 + interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu2 +/eosin Y system, guanine reacted with Cu2 + to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L- 1 and a linear range of 3.3-116 nmol L- 1. The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe.

  1. Preparation and bioevaluation of 99mTc-carbonyl complex of guanine

    Cigdem Ichedef; Serap Teksoez; Kamile Senocak; Eser Ucar; Ayfer Yurt Kilcar

    2011-01-01

    The aim of this study is to prepare radiolabeled guanine with 99m Tc(CO) 3 + core. For this purpose, guanine has been radiolabeled with 99m Tc(CO) 3 + core. Quality control study of radiolabeled guanine molecule with 99m Tc(CO) 3 + core was performed by thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC). The results showed that the radiolabeling yield was quite high (94 ± 3%). Beside that 99m Tc(CO) 3 -Gua complex has showed good in vitro stability during the 24 h period. Radiopharmaceutical potential of this complex was evaluated in Wistar Albino Rats. It was concluded that 99m Tc(CO) 3 -Gua could be used as a nucleotide radiopharmaceutical for in vivo applications. (author)

  2. Synthesis of a Pseudodisaccharide α-C-Glycosidically Linked to an 8-Alkylated Guanine

    Jan Duchek

    2013-04-01

    Full Text Available The synthesis of stable guanofosfocin analogues has attracted considerable attention in the past 15 years. Several guanofosfocin analogues mimicking the three constitutional elements of mannose, ribose, and guanine were designed and synthesized. Interest in ether-linked pseudodisaccharides and 8-alkylated guanines is increasing, due to their potential applications in life science. In this article, a novel guanofosfocin analogue 6, an ether-linked pseudodisaccharide connected α-C-glycosidically to an 8-alkylated guanine, was synthesized in a 10-longest linear step sequence from known diol 13, resulting in an overall yield of 26%. The key steps involve the ring-opening of cyclic sulfate 8 by alkoxide generated from 7 and a reductive cyclization of 4-N-acyl-2,4-diamino-5-nitrosopyrimidine 19 to form compound 6.

  3. A DFT investigation on interactions between asymmetric derivatives of cisplatin and nucleobase guanine

    Tai, Truong Ba; Nhat, Pham Vu

    2017-07-01

    The interactions of hydrolysis products of cisplatin and its asymmetric derivatives cis- and trans-[PtCl2(iPram)(Mepz)] with guanine were studied using DFT methods. These interactions are dominated by electrostatic effects, namely hydrogen bond contributions and there exists a charge flow from H-atoms of ligands to the O-atoms of guanine. The replacement of NH3 moieties by larger functional groups accompanies with a moderate reaction between PtII and guanine molecule, diminishing the cytotoxicity of the drug. The asymmetric and symmetric NH2 stretching modes of complexes having strong hydrogen bond interactions are red shifted importantly as compared to complexes without presence of hydrogen bond interactions.

  4. Guanine as a hygienic index for allergological relevant mite infestation in mattress dust

    Bronswijk, van J.E.M.H.

    1986-01-01

    Since guanine is not only an essential constituent of vital nucleic acids, but also the main end product of nitrogenous waste excretion in arachnids, it is a potential candidate for a hygienic index for mite activity in house dust. The public health significance of these mites is based on their

  5. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-01-01

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

  6. Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNA

    Kejnovská, Iva; Kypr, Jaroslav; Vorlíčková, Michaela

    2003-01-01

    Roč. 15, č. 7 (2003), s. 584-592 ISSN 0899-0042 R&D Projects: GA AV ČR IAA4004201; GA ČR GA204/01/0561 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA conformation * (guanine + adenine) repeats * homoduplexes Subject RIV: BO - Biophysics Impact factor: 1.793, year: 2003

  7. Primary overproduction of urate caused by a partial deficiency of hypoxanthine-guanine phosphoribosyl transferase

    Cassidy, M.; Gregory, M.C.; Harley, E.H.

    1980-01-01

    Inherited enzyme deficiencies are found in a small proportion of patients with gout who produce an excess of uric acid. The clinical, biochemical and therapeutic aspects of a case of hyperuricaemia caused by an atypical mutant hypoxanthine-guanine phophoribosyl transferase are presented. Urate overproduction was moderate and controlled by allopurinol therapy

  8. The Study of Adsorption of Patulin by Nanocellulose Conjugated with Poly Guanine in Contaminated Apple juice

    M Ghafori Bidakhavidi

    2016-07-01

    Full Text Available Abstract Introdction: Patulin is a dangerous toxin produced by various fungi. Hence, the current study aimed to evaluate adsorption of Patulin by nanocellulose conjugated with Poly-guanine in contaminated apple juice. Methods: Firstly, nanocellulose was synthesized, and then it was bonded to poly-guanine by a cross-linker. Then, concentration serial of Patulin was prepared in the apple juice, conjugated nanoparticles were added to them, and all were incubated at 37 ºC. After incubation, the Patulin concentration was measured by HPLC, and finally the adsorption percentage was calculated for each tube. Regarding molecular simulation, the initial structures of Patulin and nanocellulose conjugated with Poly-guanine were inserted into Hyperchem software, and their intermolecular energy was calculated during 50 picoseconds. Results: The results of the present study demonstrated that there was a significant direct correlation between the initial concentration of Patulin and the adsorption percentage of toxin. In addition, the adsorption maximum was reported 70±5 %, and the intermolecular energy between two structures was -20.3 Kcal/mol based on the computational simulation. Conclusions: It can be concluded that nanocellulose conjugated with Poly-guanine seems to be a good adsorbent for Patulin, which is demanded to be used in the future studies in regard with its application.

  9. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    Sakamoto, C.; Matozaki, T.; Nagao, M.; Baba, S.

    1987-01-01

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125 I-[Tyr 1 ]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg 2+ . When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125 I-[Tyr 1 ]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

  10. Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

    Pissarnitski, Dmitri A.; Zhao, Zhiqiang; Cole, David; Wu, Wen-Lian; Domalski, Martin; Clader, John W.; Scapin, Giovanna; Voigt, Johannes; Soriano, Aileen; Kelly, Theresa; Powles, Mary Ann; Yao, Zuliang; Burnett, Duane A. (Merck)

    2016-11-01

    Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

  11. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    Abelson, H.T.; Gorka, C.

    1983-01-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no [3H]hypoxanthine uptake into tumor or tissue culture cells, no conversion of [3H]hypoxanthine to [3H]IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol

  12. Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

    Usha, S; Selvaraj, S

    2015-01-01

    We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.

  13. Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

    Wenlin An

    Full Text Available Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI with a destabilizing domain (DD specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

  14. Diversity and evolutionary relationship of nucleotide binding site ...

    PRAKASH KUMAR

    site-encoding disease-resistance gene analogues in sweet potato. (Ipomoea batatas Lam.) ... terminal domain of the protein, this class of R-genes can be subdivided into TIR ... from young leaflets using the modified 2.0% (w/v) cetyl trimethyl ...

  15. Benchmark Theoretical and Experimental Study on N-15 NMR Shifts of Oxidatively Damaged Guanine

    Dračínský, Martin; Šála, Michal; Klepetářová, Blanka; Šebera, Jakub; Fukal, Jiří; Holečková, Veronika; Tanaka, Y.; Nencka, Radim; Sychrovský, Vladimír

    2016-01-01

    Roč. 120, č. 5 (2016), s. 915-925 ISSN 1520-6106 R&D Projects: GA ČR GA13-27676S; GA ČR GA15-11223S Institutional support: RVO:61388963 Keywords : NMR spectroscopy * DFT calculations * oxidatively damaged guanine * hOGG1 Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.177, year: 2016

  16. Voltammetric Determination of Guanine on the Electrode Modified by Gold Deposit and Nafion Film

    L.G. Shaidarova

    2016-09-01

    Full Text Available Electrodeposited gold and Nafion-gold composite on the surface of glassy carbon electrodes (GCE have shown electrocatalytic activity during guanine oxidation. In comparison with the unmodified electrode, decreasing of the oxidation potential by 100 mV and increasing of the current of organic compound oxidation have been observed. When the Nafion (NF film is applied to the surface of the glassy carbon electrode with electrodeposited gold, a five-fold increase of guanine oxidation current has been achieved compared to its oxidation on the modified electrode without the NF film. Conditions have been found for electrodeposition of gold on the surface of the glassy carbon electrode, including that one covered with the NF film, as well as for registration of the maximum catalytic current on these electrodes. Linear dependence of the electrocatalytic response of the modified electrode from the guanine concentration has been observed in the range from 5·10–6 to 5·10–3 mol·L–1 (for Au GCE and from 5·10–7 to 5·10–3 mol·L–1 (for NF-Au GCE.

  17. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

    Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

    2016-01-01

    Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

  18. Photoluminescence properties of a novel conjugate of water-soluble CdTe quantum dots to guanine

    Feng Xuejiao [North-East Normal University, Changchun 130024 (China); Shang, Qingkun, E-mail: shangqk995@nenu.edu.c [North-East Normal University, Changchun 130024 (China); Liu Hongjian [Relia Diagnostic Systems, Burlingame, CA 94010 (United States); Wang Wenlan; Wang Zhidan; Liu Junyu [North-East Normal University, Changchun 130024 (China)

    2010-04-15

    A novel conjugate of water-soluble CdTe quantum dots to a small biomolecule guanine has been obtained in aqueous phase. The photoluminescence property and the stability of the conjugate increased comparing to CdTe QDs. The interaction between CdTe QDs and guanine was studied by TEM, fluorescence microscope and photoluminescence (PL), IR, UV-Vis spectra. The effects of reflux time, pH value, ionic strength, and the ratio of CdTe QDs to guanine on the photoluminescence properties of conjugate were investigated in detail. The results show that guanine has a great influence on both the photoluminescence property and stability of thioglycolic acid-stabilized CdTe QDs. The formation of coordination and hydrogen bond between guanine molecules and CdTe including thioglycolic acid on its surface may effectively enhance the PL intensity and stability of CdTe QDs. The maximum PL intensity of the conjugate was obtained on the condition with lower ionic strength, less than 30 min reflux time, neutral pH value and 6/1 as molar ratio of guanine to CdTe.

  19. Increased mobility and on/off ratio in organic field-effect transistors using low-cost guanine-pentacene multilayers

    Shi, Wei; Zheng, Yifan; Taylor, André D.; Yu, Junsheng; Katz, Howard E.

    2017-07-01

    Layer-by-layer deposited guanine and pentacene in organic field-effect transistors (OFETs) is introduced. Through adjusting the layer thickness ratio of guanine and pentacene, the tradeoff of two electronic parameters in OFETs, charge carrier mobility and current on/off ratio, was controlled. The charge mobility was enhanced by depositing pentacene over and between guanine layers and by increasing the proportion of pentacene in the layer-by-layer system, while the current on/off ratio was increased via the decreased off current induced by the guanine layers. The tunable device performance was mainly ascribed to the trap and dopant neutralizing properties of the guanine layers, which would decrease the density of free hydroxyl groups in the OFETs. Furthermore, the cost of the devices could be reduced remarkably via the adoption of low-cost guanine.

  20. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

    Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini; Scott-Browne, James P; Togher, Susan; Pastor, William A; Rothenberg, Ellen V; Chavez, Lukas; Lähdesmäki, Harri; Rao, Anjana

    2017-01-01

    TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4 + CD8 + double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

  2. Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

    Moriel-Carretero, María; Ovejero, Sara; Gérus-Durand, Marie; Vryzas, Dimos; Constantinou, Angelos

    2017-12-04

    Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes. © 2017 Moriel-Carretero et al.

  3. Protein regulation of induced pluripotent stem cells by transplanting in a Huntington's animal model.

    Mu, S; Han, L; Zhou, G; Mo, C; Duan, J; He, Z; Wang, Z; Ren, L; Zhang, J

    2016-10-01

    The purpose of this study was to determine the functional recovery and protein regulation by transplanted induced pluripotent stem cells in a rat model of Huntington's disease (HD). In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle 10 days after the quinolinic acid injection. At 8 weeks after transplantation, fluorodeoxyglucose-PET/CT scan and balance-beam test were performed to evaluate the functional recovery of experimental rats. In addition, immunofluorescence and protein array analysis were used to investigate the regulation of stimulated protein expression in the striatum. At 8 weeks after induced pluripotent stem cell transplantation, motor function was improved in comparison with the quinolinic acid-treated rats. High fluorodeoxyglucose accumulation in the injured striatum was also observed by PET/CT scans. In addition, immunofluorescence analysis demonstrated that implanted cells migrated from the lateral ventricle into the lesioned striatum and differentiated into striatal projection neurons. Array analysis showed a significant upregulation of GFR (Glial cell line-derived neurotrophic factor receptor) alpha-1, Adiponectin/Acrp30, basic-fibroblast growth factors, MIP-1 (Macrophage-inflammatory protein) alpha and leptin, as well as downregulation of cytokine-induced neutrophil chemoattractant-3 in striatum after transplantatation of induced pluripotent stem cells in comparison with the quinolinic acid -treated rats. The findings in this work indicate that transplantation of induced pluripotent stem cells is a promising therapeutic candidate for HD. © 2016 British Neuropathological Society.

  4. Effect O6-guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations.

    Kara, Mahmut; Drsata, Tomas; Lankas, Filip; Zacharias, Martin

    2015-01-01

    Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O(6)-alkylguanin-DNA-Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O(6)-alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O(6)-methyl-guanine (O(6)-MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O(6)-MeG:C or O(6)-MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O(6)-MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O(6)-MeG:C or O(6)-MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc.

  5. Adenine and guanine nucleotide metabolism during platelet storage at 22 degree C

    Edenbrandt, C.M.; Murphy, S.

    1990-01-01

    Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation

  6. Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

    Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

    2015-01-01

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science. PMID:25973536

  7. Silver (I) as DNA glue: Ag(+)-mediated guanine pairing revealed by removing Watson-Crick constraints.

    Swasey, Steven M; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G

    2015-05-14

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag(+) is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg(2+). In contrast to prior studies of Ag(+) incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag(+)-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag(+) bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag(+)-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

  8. Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

    Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

    2015-05-01

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

  9. CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes for electrochemical determination of guanine and adenine

    Wei Yan [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Huang Qinan [Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Li Maoguo [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Huang Xingjiu [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei 230031 (China); Fang Bin, E-mail: binfang_47@yahoo.com.cn [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Wang Lun, E-mail: wanglun@mail.ahnu.edu.cn [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China)

    2011-10-01

    Sub-10 nm CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes has been constructed for electrochemial determination of guanine and adenine. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to characterize the nanoparticles CeO{sub 2}/MWCNTs. Electrochemical impedance spectroscopy (EIS) was used to characterize the electrode modifying process. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to study the electrocatalytic activity toward the electrochemical oxidation of guanine and adenine. The detection limit (S/N = 3) for adenine and guanine was found to be 20 and 10 nM, respectively. The obtained sensitivity toward guanine and adenine was 1.26 and 1.13 {mu}A/{mu}M in the linear concentration range 5-50 {mu}M and 5-35 {mu}M, respectively. These results demonstrate that the carbon nanotubes could provide huge locations and facilitate the adsorptive accumulation of the guanine and adenine, and the CeO{sub 2} nanoparticles are promising substrates for the development of high-performance electrocatalysts for biosensing.

  10. First-Principles Vibrational Electron Energy Loss Spectroscopy of β -Guanine

    Radtke, G.; Taverna, D.; Lazzeri, M.; Balan, E.

    2017-07-01

    A general approach to model vibrational electron energy loss spectra obtained using an electron beam positioned away from the specimen is presented. The energy-loss probability of the fast electron is evaluated using first-principles quantum mechanical calculations (density functional theory) of the dielectric response of the specimen. The validity of the method is assessed using recently measured anhydrous β -guanine, an important molecular solid used by animals to produce structural colors. The good agreement between theory and experiments lays the basis for a quantitative interpretation of this spectroscopy in complex systems.

  11. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  12. The Emerging Role of Guanine Exchange Factors in ALS and other neurodegenerative diseases

    Cristian eDroppelmann

    2014-09-01

    Full Text Available Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs, of which two classes: Dbl-related exchange factors and the more recently described Dock family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF in the pathogenesis of amyotrophic lateral sclerosis (ALS. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament (NEFL mRNA 3’UTR to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.

  13. New investigations of the guanine trichloro cuprate(II) complex crystal

    Fabijanić, Ivana; Matković-Čalogović, Dubravka; Pilepić, Viktor; Ivanišević, Irena; Mohaček-Grošev, Vlasta; Sanković, Krešimir

    2017-01-01

    Crystals of the guanine trichloro cuprate(II) complex, (HGua)2[Cu2Cl6]·2H2O (HGua = protonated guanine), were prepared and analysed by spectroscopic (IR, Raman) and computational methods. A new single-crystal X-ray diffraction analysis was conducted to obtain data with lower standard uncertainties than those in the previously published structure. Raman and IR spectroscopy and quantum-mechanical analysis gave us new insight into the vibrational states of the (HGua)2[Cu2Cl6]·2H2O crystal. The vibrational spectra of the crystal were assigned by performing a normal coordinate analysis for a free dimer with a centre of inversion as the only symmetry element. The stretching vibration observed at 279 cm-1 in the infrared spectrum corresponds to the N-Cu bond. The noncovalent interaction (NCI) plots and quantum theory of atoms in molecules (QTAIM) analysis of the electron density obtained from periodic DFT calculations elucidated the interactions that exist within the crystal structure. Closed-shell ionic attractions, as well as weak and medium strength hydrogen bonds, prevailed in the crystal packing.

  14. A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

    Ishizuka, Takumi; Zhao, Pei-Yan; Bao, Hong-Liang; Xu, Yan

    2017-10-23

    In the present study, we developed a multi-functional guanine derivative, 8F G, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19 F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19 F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

  15. Polymerase recognition of 2-thio-iso-guanine·5-methyl-4-pyrimidinone (iGs·P)--A new DD/AA base pair.

    Lee, Dong-Kye; Switzer, Christopher

    2016-02-15

    Polymerase specificity is reported for a previously unknown base pair with a non-standard DD/AA hydrogen bonding pattern: 2-thio-iso-guanine·5-methyl-4-pyrimidinone. Our findings suggest that atomic substitution may provide a solution for low fidelity previously associated with enzymatic copying of iso-guanine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Electrochemical behaviors and simultaneous determination of guanine and adenine based on graphene–ionic liquid–chitosan composite film modified glassy carbon electrode

    Niu Xiuli; Yang Wu; Ren Jie; Guo Hao; Long Shijia; Chen Jiaojiao; Gao Jinzhang

    2012-01-01

    Highlights: ► This work developed a novel electrochemical biosensors for guanine and adenine detection simultaneously. ► A disposable electrode based on graphene sheets, ionic liquid and chitosan was proposed. ► The presented method was also applied to simultaneous determination of guanine and adenine in denatured DNA samples with satisfying results. ► Easy fabrication, high sensitivity, excellent reproducibility and long-term stability. - Abstract: A graphene sheets (GS), ionic liquid (IL) and chitosan (CS) modified electrode was fabricated and the modified electrode displayed excellent electrochemical catalytic activities toward guanine and adenine. The transfer electron number (n) and the charge transfer coefficient (α) were calculated with the result as n = 2, α = 0.58 for guanine, and n = 2, α = 0.51 for adenine, which indicated the electrochemical oxidation of guanine and adenine on GS/IL/CS modified electrode was a two-electron and two-proton process. The oxidation overpotentials of guanine and adenine were decreased significantly compared with those obtained at the bare glassy carbon electrode and multi-walled carbon nanotubes modified electrode. The modified electrode exhibited good analytical performance and was successfully applied for individual and simultaneous determination of guanine and adenine. Low detection limits of 0.75 μM for guanine and 0.45 μM for adenine were obtained, with the linear calibration curves over the concentration range 2.5–150 μM and 1.5–350 μM, respectively. At the same time, the proposed method was successfully applied for the determination of guanine and adenine in denatured DNA samples with satisfying results. Moreover, the GS/IL/CS modified electrode exhibited good sensitivity, long-term stability and reproducibility for the determination of guanine and adenine.

  17. Synthesis and Evaluation of Asymmetric Acyclic Nucleoside Bisphosphonates as Inhibitors of Plasmodium falciparum and Human Hypoxanthine-Guanine-(Xanthine) Phosphoribosyltransferase

    Špaček, Petr; Keough, D. T.; Chavchich, M.; Dračínský, Martin; Janeba, Zlatko; Naesens, L.; Edstein, M. D.; Guddat, L. W.; Hocková, Dana

    2017-01-01

    Roč. 60, č. 17 (2017), s. 7539-7554 ISSN 0022-2623 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : hypoxanthine-guanine phosphoribosyltransferase * 2nd phosphonate group * 6-oxopurine phosphoribosyltransferases Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 6.259, year: 2016

  18. Nanoswitches based on DNA base pairs: why adenine-thymine is less suitable than guanine-cytosine

    Fonseca Guerra, C.; van der Wijst, T.; Bickelhaupt, F.M.

    2006-01-01

    Substituted Watson-Crick guanine-cytosine (GC) base pairs were recently shown to yield robust three-state nanoswitches. Here, we address the question: Can such supramolecular switches also be based on Watson-Crick adenine-thymine (AT) base pairs? We have theoretically analyzed AT pairs in which

  19. Higher order structural effects stabilizing the reverse watson-crick guanine-cytosine base pair in functional RNAs

    Chawla, Mohit; Abdel-Azeim, Safwat; Oliva, Romina; Cavallo, Luigi

    2013-01-01

    of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G

  20. Examination of the effect of the annealing cation on higher order structures containing guanine or isoguanine repeats

    Pierce, Sarah E.; Wang, Junmei; Jayawickramarajah, Janarthanan; Hamilton, Andrew D.; Brodbelt, Jennifer S.

    2010-01-01

    Isoguanine (2-oxo-6-amino-guanine), a natural but non-standard base, exhibits unique self-association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. The guanine-containing strand (G9) consistently formed quadruplexes upon annealing, whereas the isoguanine strand (Ig9) formed both pentaplexes and quadruplexes depending on the annealing cation. Quadruplex formation with G9 showed some dependence on the identity of the cation present during annealing with high relative quadruplex formation detected with six of ten cations. Analogous annealing experiments with Ig9 resulted in complex formation with all ten cations, and the majority of the resulting complexes were pentaplexes. CD results indicated most of the original complexes survived the desalting process necessary for ESI-MS analysis. In addition, several complexes, especially the pentaplexes, were found to be capable of cation exchange with ammonium ions. Ab initio calculations were conducted for isoguanine tetrads and pentads coordinated with all ten cations to predict the most energetically stable structures of the complexes in the gas phase. The observed preference of forming quadruplexes versus pentaplexes as a function of the coordinated cation can be interpreted by the calculated reaction energies of both the tetrads and pentads in combination with the distortion energies of tetrads. PMID:19746468

  1. The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best.

    Zaccaria, F; Paragi, G; Fonseca Guerra, C

    2016-08-21

    The alkali metal ion affinity of guanine quadruplexes has been studied using dispersion-corrected density functional theory (DFT-D). We have done computational investigations in aqueous solution that mimics artificial supramolecular conditions where guanine bases assemble into stacked quartets as well as biological environments in which telomeric quadruplexes are formed. In both cases, an alkali metal cation is needed to assist self-assembly. Our quantum chemical computations on these supramolecular systems are able to reproduce the experimental order of affinity of the guanine quadruplexes for the cations Li(+), Na(+), K(+), Rb(+), and Cs(+). The strongest binding is computed between the potassium cation and the quadruplex as it occurs in nature. The desolvation and the size of alkali metal cations are thought to be responsible for the order of affinity. Until now, the relative importance of these two factors has remained unclear and debated. By assessing the quantum chemical 'size' of the cation, determining the amount of deformation of the quadruplex needed to accommodate the cation and through the energy decomposition analysis (EDA) of the interaction energy between the cation and the guanines, we reveal that the desolvation and size of the alkali metal cation are both almost equally responsible for the order of affinity.

  2. Structural study and investigation of NMR tensors in interaction of dopamine with Adenine and guanine

    Lingjia Xu

    2007-04-01

    Full Text Available The interaction of dopamine with adenine and guanine were studied at the Hartree-Fock level theory. The structural and vibrational properties of dopamine-4-N7GUA and dopamine-4-N3ADE were studied at level of HF/6-31G*. Interaction energies (ΔE were calculated to be -11.49 and -11.92 kcal/mol, respectively. Some of bond lengths, angels and tortions are compared. NBO studies were performed to the second-order and perturbative estimates of donor-acceptor interaction have been done. The procedures of gauge-invariant atomic orbital (GIAO and continuous-set-of-gauge-transformation (CSGT were employed to calculate isotropic shielding, chemical shifts anisotropy and chemical shifts anisotropy asymmetry and effective anisotropy using 6-31G* basis set. These calculations yielded molecular geometries in good agreement with available experimental data.

  3. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

  4. Modified gold electrodes based on thiocytosine/guanine-gold nanoparticles for uric and ascorbic acid determination

    Vulcu, Adriana; Grosan, Camelia; Muresan, Liana Maria; Pruneanu, Stela; Olenic, Liliana

    2013-01-01

    The present paper describes the preparation of new modified surfaces for electrodes based on guanine/thiocytosine and gold nanoparticles. The gold nanoparticles were analyzed by UV–vis spectroscopy and transmission electron microscopy (TEM) and it was found that they have diameters between 30 and 40 nm. The layers were characterized by specular reflectance infrared spectroscopy (FTIR-RAS) and by atomic force microscopy (AFM). The thickness of layers was found to be approximately 30 nm for TC layers and 300 nm for GU layers. Every layer was characterized as electrochemical sensor (by cyclic voltammetry) both for uric acid and ascorbic acid determinations, separately and in their mixture. The modified sensors have good calibration functions with good sensitivity (between 1.145 and 1.406 mA cm −2 /decade), reproducibility ( t hiocytosine (Au T C) and gold g uanine (Au G U) layers

  5. Research Update: Density functional theory investigation of the interactions of silver nanoclusters with guanine

    Brandon B. Dale

    2017-05-01

    Full Text Available Bare and guanine-complexed silver clusters Ag n z (n = 2-6; z = 0-2 are examined using density functional theory to elucidate the geometries and binding motifs that are present experimentally. Whereas the neutral systems remain planar in this size range, a 2D-3D transition occurs at Ag 5 + for the cationic system and at Ag 4 2 + for the dicationic system. Neutral silver clusters can bind with nitrogen 3 or with the pi system of the base. However, positively charged clusters interact with nitrogen 7 and the neighboring carbonyl group. Thus, the cationic silver-DNA clusters present experimentally may preferentially interact at these sites.

  6. Guanine holes are prominent targets for mutation in cancer and inherited disease.

    Albino Bacolla

    Full Text Available Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials. Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

  7. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  8. Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine.

    Grimme, Stefan; Bauer, Christopher Alexander

    2015-01-01

    The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

  9. Structure of Radicals from X-irradiated Guanine Derivatives: An Experimental and Computational Study of Sodium Guanosine Dihydrate Single Crystals

    Jayatilaka, Nayana; Nelson, William H.

    2008-01-01

    In sodium guanosine dihydrate single crystals, the guanine moiety is deprotonated at N1 due to growth from high-pH (>12) solutions. EPR and ENDOR study of crystals x-irradiated at 10 K detected evidence for three radical forms. Radical R1,characterized by two proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the N1-deprotonated guanine unit. R1 exhibited an unusually distorted structure leading to net positive isotropic components of the hydrogen couplings. Radical R2, characterized by one proton and one nitrogen hyperfine coupling was identified as the primary electron loss product. This product is equivalent to that of deprotonation at N1 by the guanine cation and represents the first ENDOR characterization of that product. Radical R3, characterized by a single hydrogen hyperfine coupling, was identified as the product of net dehydrogenation at C1 of the ribose moiety. The identification of radicals R1-R3 was supported by DFT calculations on several possible structures using the B3LYP/6-311G(2df,p)//6-31G(d,p) approach. Radical R4, detected after warming the crystals to room temperature, was identified as the well-known product of net hydrogenation of C8 of the (N1-deprotonated) guanine component. Radical R1, evidently formed by protonation of the primary electron addition product, was present as roughly 60% of the total radicals detected at 10 K. Radical R2 was present as roughly 27% of the total yield, and the concentration of R3 contributed the remaining 13%. R3 is evidently the product of oneelectron oxidation followed by deprotonation; thus, the balance of oxidation and reduction products is approximately equal within experimental uncertainty. PMID:17249824

  10. Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

    Lech, Christopher Jacques; Phan, Anh Tuân

    2017-06-20

    Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. QuadBase2: web server for multiplexed guanine quadruplex mining and visualization

    Dhapola, Parashar; Chowdhury, Shantanu

    2016-01-01

    DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way. PMID:27185890

  12. WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

    Guorui Huang

    2017-07-01

    Full Text Available Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF. Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

  13. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

    Marei, Hadir; Carpy, Alejandro; Macek, Boris; Malliri, Angeliki

    2016-08-02

    The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.

  14. In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

    Côté, Jean-François; Vuori, Kristiina

    2006-01-01

    Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

  15. Quenching of light flickering in synthetic guanine crystals in aqueous solutions under strong static magnetic fields

    Mootha, A.; Takanezawa, Y.; Iwasaka, M.

    2018-05-01

    The present study focused on the vibration of micro crystal particles of guanine due to Brownian motion. The organic particle has a refractive index of 1.83 and caused a flickering of light. To test the possibility of using magnetic properties under wet conditions, changes in the frequency of particle vibration by applying magnetic fields were investigated. At first, we found that the exposure at 5 T inhibited the flickering light intensities and the particle vibration slightly decreased. Next, we carried out a high speed camera measurement of the Brownian motion of the particle with a time resolution of 100 flame per second (fps) with and without magnetic field exposures. It was revealed that the vibrational speed of synthetic particles was enhanced at 500 mT. Detailed analyses of the particle vibration by changing the direction of magnetic fields versus the light source revealed that the Brownian motion's vibrational frequency was entrained under magnetic fields at 500 mT, and an increase in vibration speed to 20Hz was observed. Additional measurements of light scattering fluctuation using photo-detector and analyses on auto-correlation also confirmed this speculation. The studied Brownian vibration may be influenced by the change in mechanical interactions between the vibration particles and surrounding medium. The discovered phenomena can be applied for molecular and biological interactions in future studies.

  16. Expression of a Rho guanine nucleotide exchange factor, Ect2, in the developing mouse pituitary.

    Islam, M S; Tsuji, T; Higashida, C; Takahashi, M; Higashida, H; Koizumi, K

    2010-05-01

    The pituitary gland is a highly mitotically active tissue after birth. Various cell types are known to undergo proliferation in the anterior pituitary. However, little is known about the mechanisms regulating mitotic activity in this tissue. When searching for genes specifically expressed in the pituitary gland among those that we previously screened in Drosophila, we found epithelial cell-transforming gene 2 (Ect2). Ect2 is a guanine nucleotide exchange factor for Rho GTPases, which is known to play an essential role in cytokinesis. Although there have been many cellular studies regarding the function of Ect2, the temporal and spatial expression patterns of Ect2 in vivo have not been determined. In the present study, we examined the postnatal developmental expression of Ect2 in the mouse pituitary. Enhanced Ect2 expression was detected in the mouse pituitary gland during the first 3 weeks after birth, which coincided well with the period of rapid pituitary expansion associated with increased growth rate. Immunostaining analysis showed that Ect2-expressing cells were distributed in the anterior and intermediate lobes, but not the posterior lobe, of the pituitary. These Ect2-expressing cells frequently incorporated the thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), indicating that these cells were mitotically active. Taken together, the results demonstrate the functional role of Ect2 in postnatal proliferating cells in the two lobes of the pituitary, thereby suggesting roles in developmental growth of the mammalian pituitary.

  17. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  18. Characterization of a mimivirus RNA cap guanine-N2 methyltransferase.

    Benarroch, Delphine; Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

    2009-04-01

    A 2,2,7-trimethylguanosine (TMG) cap is a signature feature of eukaryal snRNAs, telomerase RNAs, and trans-spliced nematode mRNAs. TMG and 2,7-dimethylguanosine (DMG) caps are also present on mRNAs of two species of alphaviruses (positive strand RNA viruses of the Togaviridae family). It is presently not known how viral mRNAs might acquire a hypermethylated cap. Mimivirus, a giant DNA virus that infects amoeba, encodes many putative enzymes and proteins implicated in RNA transactions, including the synthesis and capping of viral mRNAs and the promotion of cap-dependent translation. Here we report the identification, purification, and characterization of a mimivirus cap-specific guanine-N2 methyltransferase (MimiTgs), a monomeric enzyme that catalyzes a single round of methyl transfer from AdoMet to an m(7)G cap substrate to form a DMG cap product. MimiTgs, is apparently unable to convert a DMG cap to a TMG cap, and is thereby distinguished from the structurally homologous yeast and human Tgs1 enzymes. Nonetheless, we show genetically that MimiTgs is a true ortholog of Saccharomyces cerevisiae Tgs1. Our results hint that DMG caps can satisfy many of the functions of TMG caps in vivo. We speculate that DMG capping of mimivirus mRNAs might favor viral protein synthesis in the infected host.

  19. Guanine limitation results in CodY-dependent and -independent alteration of Staphylococcus aureus physiology and gene expression.

    King, Alyssa N; Borkar, Samiksha; Samuels, David J; Batz, Zachary; Bulock, Logan; Sadykov, Marat R; Bayles, Kenneth W; Brinsmade, Shaun R

    2018-04-30

    In Staphylococcus aureus , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on S. aureus physiology and CodY activity by constructing a guaA null mutant (Δ guaA ). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ guaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ guaA cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. Importance Staphylococcus aureus infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing

  20. Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

    Ezekiel Crenshaw

    Full Text Available A central event in Alzheimer's disease is the accumulation of amyloid β (Aβ peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP. APP overexpression leads to increased Aβ generation and Alzheimer's disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes, non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3'UTR (untranslated region at residues 3008-3027 (NM_201414.2. This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.

  1. Haploinsufficiency of the Sec7 guanine nucleotide exchange factor gea1 impairs septation in fission yeast.

    Alan M Eckler

    Full Text Available Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs. Activated Arfs recruit coat protein complex 1 (COP-I to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/-, the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/- strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/- cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/- mutant strain. Importantly, gea1+/- cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/- cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

  2. Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

    Merly Saare

    Full Text Available Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8, a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility.

  3. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

    Carla J Eaton

    Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

  4. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    Jett, J. [Lawrence Livermore National Lab., CA (United States)

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  5. The influence of N-7 guanine modifications on the strength of Watson-Crick base pairing and guanine N-1 acidity: Comparison of gas-phase and condensed-phase trends

    Burda, J. V.; Šponer, Jiří; Hrabáková, J.; Zeizinger, M.; Leszczynski, J.

    2003-01-01

    Roč. 107, č. 22 (2003), s. 5349-5356 ISSN 1520-6106 R&D Projects: GA MŠk ME 517; GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF; ONR(US) N00034-03-1-0116; National Science Foundation(US) CREST 9805465 Institutional research plan: CEZ:AV0Z5004920 Keywords : Watson-Crick base pairing * guanines * gas-phase and condensed-phase trends Subject RIV: BO - Biophysics Impact factor: 3.679, year: 2003

  6. Electron attachment to the guanine-cytosine nucleic acid base pair and the effects of monohydration and proton transfer.

    Gupta, Ashutosh; Jaeger, Heather M; Compaan, Katherine R; Schaefer, Henry F

    2012-05-17

    The guanine-cytosine (GC) radical anion and its interaction with a single water molecule is studied using ab initio and density functional methods. Z-averaged second-order perturbation theory (ZAPT2) was applied to GC radical anion for the first time. Predicted spin densities show that the radical character is localized on cytosine. The Watson-Crick monohydrated GC anion is compared to neutral GC·H2O, as well as to the proton-transferred analogue on the basis of structural and energetic properties. In all three systems, local minima are identified that correspond to water positioned in the major and minor grooves of macromolecular DNA. On the anionic surface, two novel structures have water positioned above or below the GC plane. On the neutral and anionic surfaces, the global minimum can be described as water interacting with the minor groove. These structures are predicted to have hydration energies of 9.7 and 11.8 kcal mol(-1), respectively. Upon interbase proton-transfer (PT), the anionic global minimum has water positioned in the major groove, and the hydration energy increases to 13.4 kcal mol(-1). PT GC·H2O(•-) has distonic character; the radical character resides on cytosine, while the negative charge is localized on guanine. The effects of proton transfer are further investigated through the computed adiabatic electron affinities (AEA) of GC and monohydrated GC, and the vertical detachment energies (VDE) of the corresponding anions. Monohydration increases the AEAs and VDEs by only 0.1 eV, while proton-transfer increases the VDEs substantially (0.8 eV). The molecular charge distribution of monohydrated guanine-cytosine radical anion depends heavily on interbase proton transfer.

  7. The electrochemical reduction of the purines guanine and adenine at platinum electrodes in several room temperature ionic liquids

    Zanoni, Maria Valnice Boldrin; Rogers, Emma I.; Hardacre, Christopher; Compton, Richard G.

    2010-01-01

    The reduction of guanine was studied by microelectrode voltammetry in the room temperature ionic liquids (RTILs) N-hexyltriethylammonium bis (trifluoromethanesulfonyl) imide [N 6,2,2,2 ][N(Tf) 2 ], 1-butyl-3-methylimidazolium hexafluorosphosphate [C 4 mim][PF 6 ], N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide [C 4 mpyrr][N(Tf) 2 ], 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C 4 mim][N(Tf) 2 ], N-butyl-N-methyl-pyrrolidinium dicyanamide [C 4 mpyrr][N(NC) 2 ] and tris(P-hexyl)-tetradecylphosphonium trifluorotris(pentafluoroethyl)phosphate [P 14,6,6,6 ][FAP] on a platinum microelectrode. In [N 6,2,2,2 ][NTf 2 ] and [P 14,6,6,6 ][FAP], but not in the other ionic liquids studied, guanine reduction involves a one-electron, diffusion-controlled process at very negative potential to produce an unstable radical anion, which is thought to undergo a dimerization reaction, probably after proton abstraction from the cation of the ionic liquid. The rate of this subsequent reaction depends on the nature of the ionic liquid, and it is faster in the ionic liquid [P 14,6,6,6 ][FAP], in which the formation of the resulting dimer can be voltammetrically monitored at less negative potentials than required for the reduction of the parent molecule. Adenine showed similar behaviour to guanine but the pyrimidines thymine and cytosine did not; thymine was not reduced at potentials less negative than required for solvent (RTIL) decomposition while only a poorly defined wave was seen for cytosine. The possibility for proton abstraction from the cation in [N 6,2,2,2 ][NTf 2 ] and [P 14,6,6,6 ][FAP] is noted and this is thought to aid the electrochemical dimerization process. The resulting rapid reaction is thought to shift the reduction potentials for guanine and adenine to lower values than observed in RTILs where the scope for proton abstraction is not present. Such shifts are characteristic of so-called EC processes where reversible electron transfer

  8. Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

    Shukla, P. K.; Mishra, P. C.; Suhai, S.

    Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the

  9. Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

    Gromadski, Kirill B; Schümmer, Tobias; Strømgaard, Anne

    2007-01-01

    of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows...... no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. Udgivelsesdato: 2007-Dec-7...

  10. Regulation of mitotic spindle formation by the RhoA guanine nucleotide exchange factor ARHGEF10

    Satoh Takaya

    2009-07-01

    Full Text Available Abstract Background The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains. Results Here we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference. Conclusion Collectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in

  11. Simultaneous protection of organic p- and n-channels in complementary inverter from aging and bias-stress by DNA-base guanine/Al2O3 double layer.

    Lee, Junyeong; Hwang, Hyuncheol; Min, Sung-Wook; Shin, Jae Min; Kim, Jin Sung; Jeon, Pyo Jin; Lee, Hee Sung; Im, Seongil

    2015-01-28

    Although organic field-effect transistors (OFETs) have various advantages of lightweight, low-cost, mechanical flexibility, and nowadays even higher mobility than amorphous Si-based FET, stability issue under bias and ambient condition critically hinder its practical application. One of the most detrimental effects on organic layer comes from penetrated atmospheric species such as oxygen and water. To solve such degradation problems, several molecular engineering tactics are introduced: forming a kinetic barrier, lowering the level of molecule orbitals, and increasing the band gap. However, direct passivation of organic channels, the most promising strategy, has not been reported as often as other methods. Here, we resolved the ambient stability issues of p-type (heptazole)/or n-type (PTCDI-C13) OFETs and their bias-stability issues at once, using DNA-base small molecule guanine (C5H5N5O)/Al2O3 bilayer. The guanine protects the organic channels as buffer/and H getter layer between the channels and capping Al2O3, whereas the oxide capping resists ambient molecules. As a result, both p-type and n-type OFETs are simultaneously protected from gate-bias stress and 30 days-long ambient aging, finally demonstrating a highly stable, high-gain complementary-type logic inverter.

  12. ARHGEF7 (Beta-PIX acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2.

    Karina Haebig

    Full Text Available BACKGROUND: Mutations within the leucine-rich repeat kinase 2 (LRRK2 gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. CONCLUSIONS/SIGNIFICANCE: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i a feedback control mechanism for LRRK2 activity as well as (ii an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis.

  13. Higher order structural effects stabilizing the reverse watson-crick guanine-cytosine base pair in functional RNAs

    Chawla, Mohit

    2013-10-10

    The G:C reverse Watson-Crick (W:W trans) base pair, also known as Levitt base pair in the context of tRNAs, is a structurally and functionally important base pair that contributes to tertiary interactions joining distant domains in functional RNA molecules and also participates in metabolite binding in riboswitches. We previously indicated that the isolated G:C W:W trans base pair is a rather unstable geometry, and that dicationic metal binding to the Guanine base or posttranscriptional modification of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G:C trans base pairs. Interestingly, 66% of the G:C trans base pairs in the PDB are engaged in additional H-bonding interactions with other bases, the RNA backbone or structured water molecules. High level quantum mechanical calculations on a data set of representative crystal structures were performed to shed light on the structural stability and energetics of the various crystallographic motifs. This analysis was extended to the binding of the preQ1 metabolite to a preQ1-II riboswitch. 2013 The Author(s).

  14. Multiscale QM/MM molecular dynamics study on the first steps of guanine damage by free hydroxyl radicals in solution.

    Abolfath, Ramin M; Biswas, P K; Rajnarayanam, R; Brabec, Thomas; Kodym, Reinhard; Papiez, Lech

    2012-04-19

    Understanding the damage of DNA bases from hydrogen abstraction by free OH radicals is of particular importance to understanding the indirect effect of ionizing radiation. Previous studies address the problem with truncated DNA bases as ab initio quantum simulations required to study such electronic-spin-dependent processes are computationally expensive. Here, for the first time, we employ a multiscale and hybrid quantum mechanical-molecular mechanical simulation to study the interaction of OH radicals with a guanine-deoxyribose-phosphate DNA molecular unit in the presence of water, where all of the water molecules and the deoxyribose-phosphate fragment are treated with the simplistic classical molecular mechanical scheme. Our result illustrates that the presence of water strongly alters the hydrogen-abstraction reaction as the hydrogen bonding of OH radicals with water restricts the relative orientation of the OH radicals with respect to the DNA base (here, guanine). This results in an angular anisotropy in the chemical pathway and a lower efficiency in the hydrogen-abstraction mechanisms than previously anticipated for identical systems in vacuum. The method can easily be extended to single- and double-stranded DNA without any appreciable computational cost as these molecular units can be treated in the classical subsystem, as has been demonstrated here. © 2012 American Chemical Society

  15. Free terminal amines in DNA-binding peptides alter the product distribution from guanine radicals produced by single electron oxidation.

    Konigsfeld, Katie M; Lee, Melissa; Urata, Sarah M; Aguilera, Joe A; Milligan, Jamie R

    2012-03-01

    Electron deficient guanine radical species are major intermediates produced in DNA by the direct effect of ionizing irradiation. There is evidence that they react with amine groups in closely bound ligands to form covalent crosslinks. Crosslink formation is very poorly characterized in terms of quantitative rate and yield data. We sought to address this issue by using oligo-arginine ligands to model the close association of DNA and its binding proteins in chromatin. Guanine radicals were prepared in plasmid DNA by single electron oxidation. The product distribution derived from them was assayed by strand break formation after four different post-irradiation incubations. We compared the yields of DNA damage produced in the presence of four ligands in which neither, one, or both of the amino and carboxylate termini were blocked with amides. Free carboxylate groups were unreactive. Significantly higher yields of heat labile sites were observed when the amino terminus was unblocked. The rate of the reaction was characterized by diluting the unblocked amino group with its amide blocked derivative. These observations provide a means to develop quantitative estimates for the yields in which these labile sites are formed in chromatin by exposure to ionizing irradiation.

  16. Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

    Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

    2015-02-25

    In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. HIV1 V3 loop hypermutability is enhanced by the guanine usage bias in the part of env gene coding for it.

    Khrustalev, Vladislav Victorovich

    2009-01-01

    Guanine is the most mutable nucleotide in HIV genes because of frequently occurring G to A transitions, which are caused by cytosine deamination in viral DNA minus strands catalyzed by APOBEC enzymes. Distribution of guanine between three codon positions should influence the probability for G to A mutation to be nonsynonymous (to occur in first or second codon position). We discovered that nucleotide sequences of env genes coding for third variable regions (V3 loops) of gp120 from HIV1 and HIV2 have different kinds of guanine usage biases. In the HIV1 reference strain and 100 additionally analyzed HIV1 strains the guanine usage bias in V3 loop coding regions (2G>1G>3G) should lead to elevated nonsynonymous G to A transitions occurrence rates. In the HIV2 reference strain and 100 other HIV2 strains guanine usage bias in V3 loop coding regions (3G>2G>1G) should protect V3 loops from hypermutability. According to the HIV1 and HIV2 V3 alignment, insertion of the sequence enriched with 2G (21 codons in length) occurred during the evolution of HIV1 predecessor, while insertion of the different sequence enriched with 3G (19 codons in length) occurred during the evolution of HIV2 predecessor. The higher is the level of 3G in the V3 coding region, the lower should be the immune escaping mutation occurrence rates. This hypothesis was tested in this study by comparing the guanine usage in V3 loop coding regions from HIV1 fast and slow progressors. All calculations have been performed by our algorithms "VVK In length", "VVK Dinucleotides" and "VVK Consensus" (www.barkovsky.hotmail.ru).

  18. Control of Excitation/Inhibition Balance in a Hippocampal Circuit by Calcium Sensor Protein Regulation of Presynaptic Calcium Channels.

    Nanou, Evanthia; Lee, Amy; Catterall, William A

    2018-05-02

    Activity-dependent regulation controls the balance of synaptic excitation to inhibition in neural circuits, and disruption of this regulation impairs learning and memory and causes many neurological disorders. The molecular mechanisms underlying short-term synaptic plasticity are incompletely understood, and their role in inhibitory synapses remains uncertain. Here we show that regulation of voltage-gated calcium (Ca 2+ ) channel type 2.1 (Ca V 2.1) by neuronal Ca 2+ sensor (CaS) proteins controls synaptic plasticity and excitation/inhibition balance in a hippocampal circuit. Prevention of CaS protein regulation by introducing the IM-AA mutation in Ca V 2.1 channels in male and female mice impairs short-term synaptic facilitation at excitatory synapses of CA3 pyramidal neurons onto parvalbumin (PV)-expressing basket cells. In sharp contrast, the IM-AA mutation abolishes rapid synaptic depression in the inhibitory synapses of PV basket cells onto CA1 pyramidal neurons. These results show that CaS protein regulation of facilitation and inactivation of Ca V 2.1 channels controls the direction of short-term plasticity at these two synapses. Deletion of the CaS protein CaBP1/caldendrin also blocks rapid depression at PV-CA1 synapses, implicating its upregulation of inactivation of Ca V 2.1 channels in control of short-term synaptic plasticity at this inhibitory synapse. Studies of local-circuit function revealed reduced inhibition of CA1 pyramidal neurons by the disynaptic pathway from CA3 pyramidal cells via PV basket cells and greatly increased excitation/inhibition ratio of the direct excitatory input versus indirect inhibitory input from CA3 pyramidal neurons to CA1 pyramidal neurons. This striking defect in local-circuit function may contribute to the dramatic impairment of spatial learning and memory in IM-AA mice. SIGNIFICANCE STATEMENT Many forms of short-term synaptic plasticity in neuronal circuits rely on regulation of presynaptic voltage-gated Ca 2+ (Ca V

  19. CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

    Christenson, L K; Johnson, P F; McAllister, J M; Strauss, J F

    1999-09-10

    Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

  20. Jasmonate ZIM-domain (JAZ protein regulates host and nonhost pathogen-induced cell death in tomato and Nicotiana benthamiana.

    Yasuhiro Ishiga

    Full Text Available The nonhost-specific phytotoxin coronatine (COR produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1 is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000, and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS, we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP, INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

  1. Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

    Yoshiaki Okada

    Full Text Available In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4 is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

  2. Fingerprints of Both Watson-Crick and Hoogsteen Isomers of the Isolated (Cytosine-Guanine)H+ Pair.

    Cruz-Ortiz, Andrés F; Rossa, Maximiliano; Berthias, Francis; Berdakin, Matías; Maitre, Philippe; Pino, Gustavo A

    2017-11-16

     Gas phase protonated guanine-cytosine (CGH + ) pair was generated using an electrospray ionization source from solutions at two different pH (5.8 and 3.2). Consistent evidence from MS/MS fragmentation patterns and differential ion mobility spectra (DIMS) point toward the presence of two isomers of the CGH + pair, whose relative populations depend strongly on the pH of the solution. Gas phase infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1900 cm -1 spectral range further confirms that the Watson-Crick isomer is preferentially produced (91%) at pH = 5.8, while the Hoogsteen isomer predominates (66%) at pH = 3.2). These fingerprint signatures are expected to be useful for the development of new analytical methodologies and to trigger isomer selective photochemical studies of protonated DNA base pairs.

  3. Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

    Marei, Hadir; Carpy, Alejandro; Woroniuk, Anna; Vennin, Claire; White, Gavin; Timpson, Paul; Macek, Boris; Malliri, Angeliki

    2016-01-01

    The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner. PMID:26887924

  4. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  5. Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Seyedeh Maryam Banihashemian

    Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  6. Electron transfer from nucleobase electron adducts to 5-bromouracil. Is guanine an ultimate sink for the electron in irradiated DNA?

    Nese, C.; Yuan, Z.; Schuchmann, M.N.; Sonntag, C. von

    1992-01-01

    Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after γ-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatom-protonated forms transfer electrons quantitatively to 5-BrU. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct. (Author)

  7. The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

    Sarah V Consonni

    Full Text Available PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

  8. Estrogen Repression of MicroRNAs Is Associated with High Guanine Content in the Terminal Loop Sequences of Their Precursors

    Amit Cohen

    2017-08-01

    Full Text Available Widespread microRNA (miRNA repression is a phenomenon observed in mammals after exposure to cigarette smoke and in many types of cancer. A comprehensive reduction in miRNA expression after treatment with the hormone estrogen has also previously been described. Here, we reveal a conserved association of miRNA downregulation after estrogen exposure in zebrafish, mouse, and human breast cancer cell line, with a high guanine content in the terminal loop sequences of their precursors, and offer a possible link between estrogen-related miRNA-adducts formation and carcinogenesis. We also show common gene expression patterns shared by breast cancer tumors and estrogen-treated zebrafish, suggesting that this organism can be used as a powerful model system for the study of human breast cancer.

  9. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G. H.; Fukuhara, Shigetomo; Taylor, Susan S.; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-01-01

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

  10. Magnetically-assembled micro/mesopixels exhibiting light intensity enhancement in the (012) planes of fish guanine crystals

    Chikashige, T.; Iwasaka, M.

    2018-05-01

    In this study, a new method was investigated to form light-reflecting dots at the micrometer scale using the magnetic orientations of biogenic guanine crystals obtained from fish skin and scales. The crystal platelets, possessing average dimensions of 5 μm×20 μm×100 nm, were dispersed in water and observed during exposure to vertical magnetic fields up to 5 T. The magnetic field direction was parallel to Earth's gravity, and allowed the narrowest edges of the crystals to be observed at the micrometer scale for the first time. The magnetic orientation process was initiated under conditions where the crystal platelets in water were laid on a glass substrate or where the platelets had random orientations. In the former case, the crystal platelets followed a two-stage magnetic orientation process where, in the first step, the platelet widths were aligned in the magnetic field direction. The second step required rotation of the ˜20-μm-long plates with respect to the Earth's gravity, where application of a 5 T magnetic field enabled their orientation. Real-time images of the magnetically aligning platelets provided new evidence that the crystal platelets also emitted reflected light from a very narrow window at two crystal planes (i.e., (0 1 ¯ 2 ¯ ) and (0 1 ¯ 2 )). In the latter case with random platelet orientation, spatially-condensed light-reflecting dots appeared while the guanine crystal platelets were floating and maintaining their orientation. The technique developed for controlling light-reflecting microscale objects in an aqueous medium can be applied to produce a type of microfluidic optical tool.

  11. Magnetically-assembled micro/mesopixels exhibiting light intensity enhancement in the (012 planes of fish guanine crystals

    T. Chikashige

    2018-05-01

    Full Text Available In this study, a new method was investigated to form light-reflecting dots at the micrometer scale using the magnetic orientations of biogenic guanine crystals obtained from fish skin and scales. The crystal platelets, possessing average dimensions of 5 μm×20 μm×100 nm, were dispersed in water and observed during exposure to vertical magnetic fields up to 5 T. The magnetic field direction was parallel to Earth’s gravity, and allowed the narrowest edges of the crystals to be observed at the micrometer scale for the first time. The magnetic orientation process was initiated under conditions where the crystal platelets in water were laid on a glass substrate or where the platelets had random orientations. In the former case, the crystal platelets followed a two-stage magnetic orientation process where, in the first step, the platelet widths were aligned in the magnetic field direction. The second step required rotation of the ∼20-μm-long plates with respect to the Earth’s gravity, where application of a 5 T magnetic field enabled their orientation. Real-time images of the magnetically aligning platelets provided new evidence that the crystal platelets also emitted reflected light from a very narrow window at two crystal planes (i.e., (01¯2¯ and (01¯2. In the latter case with random platelet orientation, spatially-condensed light-reflecting dots appeared while the guanine crystal platelets were floating and maintaining their orientation. The technique developed for controlling light-reflecting microscale objects in an aqueous medium can be applied to produce a type of microfluidic optical tool.

  12. Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.

    Rajesh, Mathur; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2005-02-15

    The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene

  13. Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT in acute malaria

    Woodberry Tonia

    2009-06-01

    Full Text Available Abstract Background The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. Methods PBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNγ ELISPOT assay and ELISA. Results HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-γ production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. Conclusion The prevalence of acute proliferative and convalescent IFNγ responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

  14. BLOC-3 mutated in Hermansky-Pudlak syndrome is a Rab32/38 guanine nucleotide exchange factor.

    Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A

    2012-11-20

    Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

    2015-12-01

    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

  16. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  17. The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-06-27

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

    Hartley, J.A.; Forrow, S.M.; Souhami, R.L.

    1990-01-01

    Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increase ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. The result differed with both the nitrogen mustard and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove

  19. Bioinformatical Analysis of Organ-Related (Heart, Brain, Liver, and Kidney and Serum Proteomic Data to Identify Protein Regulation Patterns and Potential Sepsis Biomarkers

    Andreas Hohn

    2018-01-01

    Full Text Available During the last years, proteomic studies have revealed several interesting findings in experimental sepsis models and septic patients. However, most studies investigated protein alterations only in single organs or in whole blood. To identify possible sepsis biomarkers and to evaluate the relationship between protein alteration in sepsis affected organs and blood, proteomics data from the heart, brain, liver, kidney, and serum were analysed. Using functional network analyses in combination with hierarchical cluster analysis, we found that protein regulation patterns in organ tissues as well as in serum are highly dynamic. In the tissue proteome, the main functions and pathways affected were the oxidoreductive activity, cell energy generation, or metabolism, whereas in the serum proteome, functions were associated with lipoproteins metabolism and, to a minor extent, with coagulation, inflammatory response, and organ regeneration. Proteins from network analyses of organ tissue did not correlate with statistically significantly regulated serum proteins or with predicted proteins of serum functions. In this study, the combination of proteomic network analyses with cluster analyses is introduced as an approach to deal with high-throughput proteomics data to evaluate the dynamics of protein regulation during sepsis.

  20. Interaction between nucleotide binding sites on chloroplast coupling factor 1 during ATP hydrolysis

    Leckband, D.; Hammes, G.G.

    1987-04-21

    The initial hydrolysis of radioactively-labelled CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rate constant for the first-order process is independent of substrate concentration but increased hyperbolically to a limiting value of 0.43 s/sup -1/ with increasing concentrations of free Ca/sup 2 +/. A mechanism involving a Ca/sup 2 +/-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approx. 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s/sup -1/ as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changes. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.

  1. Role for nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation

    Navarro, Rocio; Delgado-Wicke, Pablo; Nuñez-Prado, Natalia

    2016-01-01

    We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through TLR4. However, gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously ...

  2. Critical role of nucleotide-binding oligomerization domain-like receptor 3 in vascular repair

    Schlaweck, Sebastian; Zimmer, Sebastian; Struck, Rafael [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Bartok, Eva [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Bauernfeind, Franz [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Latz, Eicke [Institute of Innate Immunity, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Hornung, Veit [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Ghanem, Alexander, E-mail: ghanem@uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2011-08-05

    Highlights: {yields} NLRP3 is not required for systemic cardiovascular function in healthy mice. {yields} NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and that it does not alter peripheral differential blood counts. {yields} NLRP3 is critical in neointima formation following vascular injury. -- Abstract: Vascular remodeling characterized by hyperproliferative neointima formation is an unfavorable repair process that is triggered by vascular damage. This process is characterized by an increased local inflammatory and proliferative response that critically involves the pro-inflammatory cytokine interleukin-1{beta} (IL-1{beta}). IL-1{beta} is expressed and cytosolically retained as a procytokine that requires additional processing prior to exerting its pro-inflammatory function. Maturation and release of pro IL-1{beta} is governed by a cytosolic protein scaffold that is known as the inflammasome. Here we show that NLRP3 (NOD-like receptor family, pryin domain containing 3), an important activating component of the inflammasome, is involved in neointima formation after vascular injury. NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and does not alter peripheral differential blood counts. However, neointima development following wire injury of the carotid artery was significantly decreased in NLRP3-deficient mice as compared to wild-type controls. In all, NLRP3 plays a non-redundant role in vascular damage mediated neointima formation. Our data establish NLRP3 as a key player in the response to vascular damage, which could open new avenues to therapeutic intervention.

  3. Nucleotide binding induces conformational changes apparent on the Na+/K+ -ATPase large cytoplasmic loop

    Šimunová, Lenka; Kubala, M.; Lánský, Zdeněk; Teisinger, Jan; Amler, Evžen

    Roč.13, č.1 (2006), s. 37-37 ISSN 1211-5894. [Discussions on Structural Molecular Biology /5./. 16.03.2006-18.03.2006, Nové Hrady] R&D Projects: GA MŠk(CZ) LC554; GA AV ČR 1ET400110403; GA ČR GD305/03/H148 Grant - others:Grantová agentura UK(CZ) 200053 Keywords : Na+/K+ - ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  4. Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

    Thymark, Majbritt; Johansson, Eva; Larsen, Sine

    2007-01-01

    detectable activity with a 30- and 140-fold reduction in k(cat), respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of d...... of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide...

  5. 2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

    Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S

    2015-12-01

    Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.

  6. Critical role of nucleotide-binding oligomerization domain-like receptor 3 in vascular repair

    Schlaweck, Sebastian; Zimmer, Sebastian; Struck, Rafael; Bartok, Eva; Werner, Nikos; Bauernfeind, Franz; Latz, Eicke; Nickenig, Georg; Hornung, Veit; Ghanem, Alexander

    2011-01-01

    Highlights: → NLRP3 is not required for systemic cardiovascular function in healthy mice. → NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and that it does not alter peripheral differential blood counts. → NLRP3 is critical in neointima formation following vascular injury. -- Abstract: Vascular remodeling characterized by hyperproliferative neointima formation is an unfavorable repair process that is triggered by vascular damage. This process is characterized by an increased local inflammatory and proliferative response that critically involves the pro-inflammatory cytokine interleukin-1β (IL-1β). IL-1β is expressed and cytosolically retained as a procytokine that requires additional processing prior to exerting its pro-inflammatory function. Maturation and release of pro IL-1β is governed by a cytosolic protein scaffold that is known as the inflammasome. Here we show that NLRP3 (NOD-like receptor family, pryin domain containing 3), an important activating component of the inflammasome, is involved in neointima formation after vascular injury. NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and does not alter peripheral differential blood counts. However, neointima development following wire injury of the carotid artery was significantly decreased in NLRP3-deficient mice as compared to wild-type controls. In all, NLRP3 plays a non-redundant role in vascular damage mediated neointima formation. Our data establish NLRP3 as a key player in the response to vascular damage, which could open new avenues to therapeutic intervention.

  7. Oxidative generation of guanine radicals by carbonate radicals and their reactions with nitrogen dioxide to form site specific 5-guanidino-4-nitroimidazole lesions in oligodeoxynucleotides.

    Joffe, Avrum; Mock, Steven; Yun, Byeong Hwa; Kolbanovskiy, Alexander; Geacintov, Nicholas E; Shafirovich, Vladimir

    2003-08-01

    A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5'-d(ACC CG(1)C G(2)TC CG(3)C G(4)CC) and 5'-d(ACC CG(1)C G(2)TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G(2)) and 158 (G(3)) mutation hot spots in the former sequence with four Gs and the codon 157 (G(2)) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20-40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2',3',5'-tri-O-acetylguanosine, to 2.1-2.6 in the single-stranded oligodeoxynucleotides and to 0.8-1.1 in the duplexes. The adduct of the 5'-d(ACC CG(1)C G(2)TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G(2)) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion

  8. Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences.

    Matter, Brock; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2004-06-01

    G --> T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligation-mediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (+/-)-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2'-deoxynucleosides. Capillary HPLC-ESI(+)-MS/MS was used to establish the amounts of (-)-trans-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N(2)-BPDE-dG, 5.6 and 16.7% for

  9. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    Liao, Wei; Sharma, Sanjai

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells

  10. Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

    Keough, D T; Emmerson, B T; de Jersey, J

    1991-02-22

    Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

  11. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  12. Guanine nucleotide regulation of muscarinic receptor-mediated inositol phosphate formation in permeabilized 1321N1 cells

    Orellana, S.A.; Trilivas, I.; Brown, J.H.

    1986-01-01

    Carbachol and guanine nucleotides stimulate formation of the ( 3 H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with ( 3 H) inositol. Carbachol alone has little effect on formation of the ( 3 H) inositol phosphates (IPs), but GTPγS causes synergistic accumulation of ( 3 H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTPγS all support formation of the ( 3 H)IPs, with or without hormone, but GTPγS is the most effective. In the presence of GTPγS, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the ( 3 H)IPs occur at ∼ 5 μM and ∼ 100 μM carbachol, respectively; values close to those seen in intact cells. GTPγS alone stimulates formation of the ( 3 H)IPs after a brief lag time. The combination of GTPγS and carbachol both increases the rate of, and decreases the lag in, formation of the ( 3 H)IPs. LiCl increases ( 3 H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of ( 3 H)IP3. GTPγS and carbachol cause formation of ( 3 H)IPs in the absence of Ca ++ , but formation induced by GTPγS with or without carbachol is Ca ++ -sensitive over a range of physiological concentrations. Although carbachol, Ca ++ , and GTPγS all have effects on formation of ( 3 H)IPs, GTPγS appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell

  13. Overexpression of GEFT, a Rho family guanine nucleotide exchange factor, predicts poor prognosis in patients with rhabdomyosarcoma.

    Sun, Chao; Liu, Chunxia; Li, Shugang; Li, Hongan; Wang, Yuanyuan; Xie, Yuwen; Li, Bingcheng; Cui, Xiaobin; Chen, Yunzhao; Zhang, Wenjie; Li, Feng

    2014-01-01

    Rhabdomyosarcoma (RMS) is one of the most common soft-tissue sarcomas in children and adolescents with poor prognosis. Yet, there is lack of effective prognostic biomarkers for RMS. The present study, therefore, aimed to explore potential biomarkers for RMS based on our previous findings using array comparative genomic hybridization. We investigated guanine nucleotide exchange factor, GEFT, at expression level in 45 RMS patients and 36 normal striated muscle controls using immunohistochemistry using tissue microarrays. The expression rate of GEFT in RMS samples (42/45, 93.33%) was significantly higher (Prate of GEFT in RMS (31/45, 68.89%) was also significantly higher (P<0.05) than that in normal controls (0/36, 0.00%). Increased expression of GEFT correlated significantly with advanced disease stages (stages III/IV) (P=0.001), lymph node metastasis (P=0.019), and distant metastasis (P=0.004), respectively, in RMS patients. In addition, RMS patients having overexpressed GEFT experienced worse overall survival (OS) than those having low levels of GEFT (P=0.001). GEFT overexpression was determined to be an independent prognostic factor for poor OS in RMS patients (hazard ratio: 3.491, 95% confidence interval: 1.121-10.871, P=0.004). In conclusion, these observations provide the first evidence of GEFT overexpression in RMS and its correlations with disease aggressiveness and metastasis. These findings suggest that GEFT may serve as a promising biomarker predicting poor prognosis in RMS patients, thus implying its potential as a therapeutic target.

  14. Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

    Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

    1976-07-01

    Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

  15. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    Feltner, D.E.; Marasco, W.A.

    1989-01-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state

  16. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  17. The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

    Elaine Ann Moore

    Full Text Available The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

  18. Decrease in Survival Rate of Colorectal Cancer Patients Due to Insertion of a Single Guanine Base in Promoter Sequences of Matrix Metalloproteinase-1 Gene (in Tehran Population

    Z Hojati

    2009-01-01

    Full Text Available Introduction: Insertion or deletion of a guanine in -1607 at promoter region of matrix metalloproteinase-1 enzyme creates two allelic types for this gene in the population: 2G and 1G, respectively. 2G allele contains an extra binding site for ETS transcription factors that this may increase the level of gene expression. Therefore, aim of this study was investigation of the single Guanine insertion in the promoter gene and its association with colorectal cancer patient survival rate and tumor progression. Methods: Blood samples from 150 colorectal patients and 100 cases were extracted. The mean follow-up was 25 months (12-36 months. Cases and patients were genotyped using genomic DNA extraction and PCR-RFLP. Results: Colorectal cancer patients were divided in two groups; with activity of metastasis (M+ and without activity of metastasis (M-. 2G allele in metastasis group (55% showed more frequency rather than controls (23%. Survival analyses showed that 3 years survival patients rate in the patients without metastasis activity carrying 1G allele (homo and heterozygote was 81% and for 2G homozygote is 66% (p=0.04. The survival rate dependent to cancer was 90% and 71%, respectively (P=0.01. Conclusion: According to the results, it seems that patients carrying 1G allele show a better survival rate dependent on cancer as compared to patients who do not carry this allele.

  19. Causes and consequences of plant radio-resistance. Formation of DNA basis lesions and self-repairing activity of one of them, the 8-oxo-7,8-dihydro-guanine in Arabidopsis thaliana

    Dany, A.L.

    2001-01-01

    In this research thesis, the author first explains how and why DNA is injured when it is submitted to an oxidizing stress, and describes precisely the formation and the biological consequences of lesions of DNA bases, the 8-oxo-7,8-dihydro-guanine (8-oxoGua). She describes the repairing activities of the oxidized DNA, and more particularly the repairing of 8-oxoGua, in prokaryotes as well as in yeast, mammals and plants. Methodologies used are described, together with the repair activities of the 8-oxo-7,8-dihydro-guanine following a biochemical type approach and a molecular biology approach

  20. Hepatitis C virus core protein regulates p300/CBP co-activation function. Possible role in the regulation of NF-AT1 transcriptional activity

    Gomez-Gonzalo, Marta; Benedicto, Ignacio; Carretero, Marta; Lara-Pezzi, Enrique; Maldonado-Rodriguez, Alejandra; Moreno-Otero, Ricardo; Lai, Michael M.C.; Lopez-Cabrera, Manuel

    2004-01-01

    Hepatitis C virus (HCV) core is a viral structural protein; it also participates in some cellular processes, including transcriptional regulation. However, the mechanisms of core-mediated transcriptional regulation remain poorly understood. Oncogenic virus proteins often target p300/CBP, a known co-activator of a wide variety of transcription factors, to regulate the expression of cellular and viral genes. Here we demonstrate, for the first time, that HCV core protein interacts with p300/CBP and enhances both its acetyl-transferase and transcriptional activities. In addition, we demonstrate that nuclear core protein activates the NH 2 -terminal transcription activation domain (TAD) of NF-AT1 in a p300/CBP-dependent manner. We propose a model in which core protein regulates the co-activation function of p300/CBP and activates NF-AT1, and probably other p300/CBP-regulated transcription factors, by a novel mechanism involving the regulation of the acetylation state of histones and/or components of the transcriptional machinery

  1. Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors.

    Mototani, Yasumasa; Okamura, Tadashi; Goto, Motohito; Shimizu, Yukiko; Yanobu-Takanashi, Rieko; Ito, Aiko; Kawamura, Naoya; Yagisawa, Yuka; Umeki, Daisuke; Nariyama, Megumi; Suita, Kenji; Ohnuki, Yoshiki; Shiozawa, Kouichi; Sahara, Yoshinori; Kozasa, Tohru; Saeki, Yasutake; Okumura, Satoshi

    2018-06-01

    The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

  2. Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells.

    Panneerdoss, Subbarayalu; Viswanadhapalli, Suryavathi; Abdelfattah, Nourhan; Onyeagucha, Benjamin C; Timilsina, Santosh; Mohammad, Tabrez A; Chen, Yidong; Drake, Michael; Vuori, Kristiina; Kumar, T Rajendra; Rao, Manjeet K

    2017-09-19

    Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

  3. Identification of the Structural Features of Guanine Derivatives as MGMT Inhibitors Using 3D-QSAR Modeling Combined with Molecular Docking

    Guohui Sun

    2016-06-01

    Full Text Available DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT, which plays an important role in inducing drug resistance against alkylating agents that modify the O6 position of guanine in DNA, is an attractive target for anti-tumor chemotherapy. A series of MGMT inhibitors have been synthesized over the past decades to improve the chemotherapeutic effects of O6-alkylating agents. In the present study, we performed a three-dimensional quantitative structure activity relationship (3D-QSAR study on 97 guanine derivatives as MGMT inhibitors using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA methods. Three different alignment methods (ligand-based, DFT optimization-based and docking-based alignment were employed to develop reliable 3D-QSAR models. Statistical parameters derived from the models using the above three alignment methods showed that the ligand-based CoMFA (Qcv2 = 0.672 and Rncv2 = 0.997 and CoMSIA (Qcv2 = 0.703 and Rncv2 = 0.946 models were better than the other two alignment methods-based CoMFA and CoMSIA models. The two ligand-based models were further confirmed by an external test-set validation and a Y-randomization examination. The ligand-based CoMFA model (Qext2 = 0.691, Rpred2 = 0.738 and slope k = 0.91 was observed with acceptable external test-set validation values rather than the CoMSIA model (Qext2 = 0.307, Rpred2 = 0.4 and slope k = 0.719. Docking studies were carried out to predict the binding modes of the inhibitors with MGMT. The results indicated that the obtained binding interactions were consistent with the 3D contour maps. Overall, the combined results of the 3D-QSAR and the docking obtained in this study provide an insight into the understanding of the interactions between guanine derivatives and MGMT protein, which will assist in designing novel MGMT inhibitors with desired activity.

  4. Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

    Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2017-09-01

    DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. From lin-Benzoguanines to lin-Benzohypoxanthines as Ligands for Zymomonas mobilis tRNA-Guanine Transglycosylase: Replacement of Protein-Ligand Hydrogen Bonding by Importing Water Clusters.

    Barandun, L.J.; Immekus, F.; Kohler, P.C.; Tonazzi, S.; Wagner, B.; Wendelspiess, S.; Ritschel, T.; Heine, A.; Kansy, M.; Klebe, G.; Diederich, F.

    2012-01-01

    The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of

  6. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. (Fitzsimons Army Medical Center, Aurora, CO (USA))

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  7. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we.......5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase....

  8. Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea

    2015-01-01

    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate...... in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described....... More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation...

  9. Relative Stability of the La and Lb Excited States in Adenine and Guanine: Direct Evidence from TD-DFT Calculations of MCD Spectra.

    Santoro, Fabrizio; Improta, Roberto; Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Coriani, Sonia

    2014-06-05

    The relative position of La and Lb ππ* electronic states in purine nucleobases is a much debated topic, since it can strongly affect our understanding of their photoexcited dynamics. To assess this point, we calculated the absorption and magnetic circular dichroism (MCD) spectra of adenine, guanine, and their nucleosides in gas-phase and aqueous solution, exploiting recent developments in MCD computational technology within time-dependent density functional theory. MCD spectroscopy allows us to resolve the intense S0→ La transition from the weak S0→ Lb transition. The spectra obtained in water solution, by using B3LYP and CAM-B3LYP functionals and describing solvent effect by cluster models and by the polarizable continuum model (PCM), are in very good agreement with the experimental counterparts, thus providing direct and unambiguous evidence that the energy ordering predicted by TD-DFT, La < Lb, is the correct one.

  10. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E.

    1990-01-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G- 3 H] hypoxanthine (Hy) into [ 3 H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [ 3 H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation

  11. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor: EFFECT ON PKA LOCALIZATION AND P-Rex1 SIGNALING.

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G H; Fukuhara, Shigetomo; Taylor, Susan S; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-03-18

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Guanine nucleotide-dependent, pertussis toxin-insensitive, stimulation of inositol phosphate formation by carbachol in a membrane preparation from astrocytoma cells

    Hepler, J.R.; Harden, T.K.

    1986-01-01

    Formation of the inositol phosphates (InsP), InsP 3 , InsP 2 , and InsP 1 was increased in a concentration dependent manner (K/sub 0.5/ ∼ 5 μM) by GTPΣS in washed membranes prepared from 3 H-inositol-prelabelled 1321N1 human astrocytoma cells. Both GTPγS and GppNHp stimulated InsP formation by 2-3 fold over control; GTP and GDP were much less efficacious and GMP had no effect. Although the muscarinic cholinergic receptor agonist carbachol had no effect in the absence of guanine nucleotide, in the presence of 10 μM GTPγS, carbachol stimulated (K/sub 0.5/ ∼ 10 μ M) the formation of InsP above the level achieved with GTPγS alone. The effect of carbachol was completely blocked by atropine. The order of potency for a series of nucleotides for stimulation of InsP formation in the presence of 500 μM carbachol was GTPγS > GppNHp > GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate G/sub i/, had no effect on InsP formation in the presence of GTPγS or GTPγS plus carbachol. Histamine and bradykinin also stimulated InsP formation in the presence of GTPγS in washed membranes from 1321N1 cells. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not G/sub i/ is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells

  13. A simplified one-pot synthesis of 9-[(3-[{sup 18}F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine([{sup 18}F]FHPG) and 9-(4-[{sup 18}F]Fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG) for gene therapy

    Shiue, Grace G.; Shiue, Chyng-Yann E-mail: Shiue@rad.upenn.edu; Lee, Roland L.; MacDonald, Douglas; Hustinx, Roland; Eck, Stephen L.; Alavi, Abass A

    2001-10-01

    9-[(3-[{sup 18}F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([{sup 18}F]FHPG, 2) has been synthesized by nucleophilic substitution of N{sup 2}-(p-anisyldiphenylmethyl)-9-{l_brace}[1-(p-anisyldiphenylmethoxy)-3 -toluenesulfonyloxy-2-propoxy]methyl{r_brace}guanine (1) with potassium [{sup 18}F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 deg. C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 deg. C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 deg. C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 deg. C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126{+-}0.022, n=5 and 0.165{+-}0.023, n=5, respectively). Although it contains non-radioactive ganciclovir ({approx}5-30 {mu}g) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains {approx}10-25 {mu}g of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in

  14. A simplified one-pot synthesis of 9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F]FHPG) and 9-(4-[18F]Fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for gene therapy

    Shiue, Grace G.; Shiue, Chyng-Yann; Lee, Roland L.; MacDonald, Douglas; Hustinx, Roland; Eck, Stephen L.; Alavi, Abass A.

    2001-01-01

    9-[(3-[ 18 F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG, 2) has been synthesized by nucleophilic substitution of N 2 -(p-anisyldiphenylmethyl)-9-{[1-(p-anisyldiphenylmethoxy)-3 -toluenesulfonyloxy-2-propoxy]methyl}guanine (1) with potassium [ 18 F]fluoride/Kryptofix 2.2.2 followed by deprotection with 1 N HCl and purification with different methods in variable yields. When both the nucleophilic substitution and deprotection were carried out at 90 deg. C and the product was purified by HPLC (method A), the yield of compound 2 was 5-10% and the synthesis time was 90 min from EOB. However, if both the nucleophilic substitution and deprotection were carried out at 120 deg. C and the product was purified by HPLC, the yield of compound 2 decreased to 2%. When compound 2 was synthesized at 90 deg. C and purified by Silica Sep-Pak (method B), the yield increased to 10-15% and the synthesis time was 60 min from EOB. Similarly, 9-(4-[ 18 F]fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG, 4) was synthesized with method A and method B in 9% and 10-15% yield, respectively, in a synthesis time of 90 and 60 min, respectively, from EOB. Compound 2 was relatively unstable in acidic medium at 120 deg. C while compound 4 was stable under the same condition. Both compound 2 and compound 4 had low lipid/water partition coefficient (0.126±0.022, n=5 and 0.165±0.023, n=5, respectively). Although it contains non-radioactive ganciclovir (∼5-30 μg) as a chemical by-product, compound 2 synthesized by method B has a similar uptake in 9L glioma cells as that synthesized by method A, and is a potential tracer for imaging herpes simplex virus thymidine kinase gene expression in tumors using PET. Similarly, compound 4 synthesized by method B contains ∼10-25 μg of penciclovir as a chemical by-product. Thus, the simplified one pot synthesis (method B) is a useful method for synthesizing both compound 2 and compound 4 in good yield for routine clinical use, and the method is

  15. Multi-level Quantum Mechanics and Molecular Mechanics Study of Ring Opening Process of Guanine Damage by Hydroxyl Radical in Aqueous Solution.

    Liu, Peng; Wang, Qiong; Niu, Meixing; Wang, Dunyou

    2017-08-10

    Combining multi-level quantum mechanics theories and molecular mechanics with an explicit water model, we investigated the ring opening process of guanine damage by hydroxyl radical in aqueous solution. The detailed, atomic-level ring-opening mechanism along the reaction pathway was revealed in aqueous solution at the CCSD(T)/MM levels of theory. The potentials of mean force in aqueous solution were calculated at both the DFT/MM and CCSD(T)/MM levels of the theory. Our study found that the aqueous solution has a significant effect on this reaction in solution. In particular, by comparing the geometries of the stationary points between in gas phase and in aqueous solution, we found that the aqueous solution has a tremendous impact on the torsion angles much more than on the bond lengths and bending angles. Our calculated free-energy barrier height 31.6 kcal/mol at the CCSD(T)/MM level of theory agrees well with the one obtained based on gas-phase reaction profile and free energies of solvation. In addition, the reaction path in gas phase was also mapped using multi-level quantum mechanics theories, which shows a reaction barrier at 19.2 kcal/mol at the CCSD(T) level of theory, agreeing very well with a recent ab initio calculation result at 20.8 kcal/mol.

  16. The G-BHQ synergistic effect: Improved double quenching molecular beacons based on guanine and Black Hole Quencher for sensitive simultaneous detection of two DNAs.

    Xiang, Dongshan; Li, Fengquan; Wu, Chenyi; Shi, Boan; Zhai, Kun

    2017-11-01

    We designed two double quenching molecular beacons (MBs) with simple structure based on guanine (G base) and Black Hole Quencher (BHQ), and developed a new analytical method for sensitive simultaneous detection of two DNAs by synchronous fluorescence analysis. In this analytical method, carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were respectively selected as fluorophore of two MBs, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were respectively selected as organic quencher, and three continuous nucleotides with G base were connected to organic quencher (BHQ-1 and BHQ-2). In the presence of target DNAs, the two MBs hybridize with the corresponding target DNAs, the fluorophores are separated from organic quenchers and G bases, leading to recovery of fluorescence of FAM and TAMRA. Under a certain conditions, the fluorescence intensities of FAM and TAMRA all exhibited good linear dependence on their concentration of target DNAs (T1 and T2) in the range from 4 × 10 -10 to 4 × 10 -8 molL -1 (M). The detection limit (3σ, n = 13) of T1 was 3 × 10 -10 M and that of T2 was 2×10 -10 M, respectively. Compared with the existing analysis methods for multiplex DNA with MBs, this proposed method based on double quenching MBs is not only low fluorescence background, short analytical time and low detection cost, but also easy synthesis and good stability of MB probes. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Solvent effect on the intermolecular proton transfer of the Watson and Crick guanine-cytosine and adenine-thymine base pairs: a polarizable continuum model study.

    Romero, Eduardo E; Hernandez, Florencio E

    2018-01-03

    Herein we present our results on the study of the double proton transfer (DPT) mechanism in the adenine-thymine (AT) and guanine-cytosine (GC) base pairs, both in gas phase and in solution. The latter was modeled using the polarizable continuum method (PCM) in different solvents. According to our DFT calculations, the DPT may occur for both complexes in a stepwise mechanism in condensate phase. In gas phase only the GC base pair exhibits a concerted DPT mechanism. Using the Wigner's tunneling corrections to the transition state theory we demonstrate that such corrections are important for the prediction of the rate constants of both systems in gas and in condensate phase. We also show that (i) as the polarity of the medium decreases the equilibrium constant of the DPT reaction increases in both complexes, and (ii) that the equilibrium constant in the GC complex is four orders of magnitude larger than in AT. This observation suggests that the spontaneous mutations in DNA base pairs are more probable in GC than in AT.

  18. The influence of anharmonic and solvent effects on the theoretical vibrational spectra of the guanine-cytosine base pairs in Watson-Crick and Hoogsteen configurations.

    Bende, Attila; Muntean, Cristina M

    2014-03-01

    The theoretical IR and Raman spectra of the guanine-cytosine DNA base pairs in Watson-Crick and Hoogsteen configurations were computed using DFT method with M06-2X meta-hybrid GGA exchange-correlation functional, including the anharmonic corrections and solvent effects. The results for harmonic frequencies and their anharmonic corrections were compared with our previously calculated values obtained with the B3PW91 hybrid GGA functional. Significant differences were obtained for the anharmonic corrections calculated with the two different DFT functionals, especially for the stretching modes, while the corresponding harmonic frequencies did not differ considerable. For the Hoogtseen case the H⁺ vibration between the G-C base pair can be characterized as an asymmetric Duffing oscillator and therefore unrealistic anharmonic corrections for normal modes where this proton vibration is involved have been obtained. The spectral modification due to the anharmonic corrections, solvent effects and the influence of sugar-phosphate group for the Watson-Crick and Hoogsteen base pair configurations, respectively, were also discussed. For the Watson-Crick case also the influence of the stacking interaction on the theoretical IR and Raman spectra was analyzed. Including the anharmonic correction in our normal mode analysis is essential if one wants to obtain correct assignments of the theoretical frequency values as compared with the experimental spectra.

  19. A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

    Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

    2017-09-27

    RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

  20. Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

    Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

    2014-09-01

    Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

  1. GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

    Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

    2010-11-19

    Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

  2. Effect of Six Sessions of High Intensity Interval Training on Levels of Hypoxanthine, Xanthine, Hypoxanthine-Guanine phosphoribosyltransferase (HGPRT and Serum Uric Acid in active young men

    ROHOULLAH HAGHSHENAS GATABI

    2017-01-01

    Full Text Available Abstract Introduction and objectives: long-term sport and physical activity results in compatibility in maintaining purine derivatives but the compatibility achieved within a few sessions is not well investigated. This study aimed to investigate the effect of a 30-seconds high intensity interval training on Hypoxanthine, xanthine, hypoxanthine-guanine phosphoribosyltransferase (HGPRT and serum uric acid in young college men. Methods: In this study, 18 untrained healthy men were divided into two control and training groups after homogenization based on their personal characteristics. Training included six sessions (every other day for two weeks with different intervals (4, 7, 6, 6, 5 & 4, respectively with a fixed four-minute rest between each interval, and with a constant load of .6 on the cycle-ergometer. Blood samples were taken before and 48 hours after the last training session, and were used to analyze hypoxanthine, xanthine, uric acid, and serum HGPRT. Statistical analysis was performed using analysis of covariance (ANCOVA. Results: The results showed that high-intensity interval training for two weeks did not cause significant changes in serum HGPRT (P = .73; likewise, the increase in serum hypoxanthine (P = .170 and serum xanthine (P = .170 was not statistically significant but significant reduction was observed in serum uric acid (P = .025. Discussion and conclusion: The results of this study indicated that two-week HIIT training is likely to enhance athletic performance and recovery of purine nucleotide cycle.

  3. An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

    McKnight, Kevin L.; Sandefur, Stephanie; Phipps, Krista M.; Heinz, Beverly A.

    2003-01-01

    The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs

  4. Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10).

    Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

    2011-08-26

    The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

  5. Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)*

    Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

    2011-01-01

    The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1–332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant. PMID:21719701

  6. Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

    Ziegel, Rebecca; Shallop, Anthony; Upadhyaya, Pramod; Jones, Roger; Tretyakova, Natalia

    2004-01-20

    All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously

  7. Secondary and tertiary structure of nucleotide-binding domain of alpha subunit of Na+/K+-ATPase

    Hofbauerová, Kateřina; Kopecký ml., Vladimír; Ettrich, Rüdiger; Ettrichová, Olga; Amler, Evžen

    2002-01-01

    Roč. 67, 4-5 (2002), s. 242-246 ISSN 0006-3525 R&D Projects: GA ČR GA204/01/0254; GA ČR GA204/01/1001 Grant - others:Volkswagen Foundation(DE) I/74 679 Institutional research plan: CEZ:AV0Z5011922 Keywords : Na+/K+- ATPase * ATP binding * molecular modeling Subject RIV: BO - Biophysics Impact factor: 2.372, year: 2002

  8. Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

    Glynn, Neil C; Comstock, Jack C; Sood, Sushma G; Dang, Phat M; Chaparro, Jose X

    2008-01-01

    Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

  9. Molecular cloning and functional analysis of nucleotide-binding oligomerization domain-containing protein 1 in rainbow trout, Oncorhynchus mykiss.

    Jang, Ju Hye; Kim, Hyun; Kim, Yu Jin; Cho, Ju Hyun

    2016-04-01

    NOD1 has important roles in innate immunity as sensor of microbial components derived from bacterial peptidoglycan. In this study, we identified genes encoding components of the NOD1 signaling pathway, including NOD1 (OmNOD1) and RIP2 (OmRIP2) from rainbow trout, Oncorhynchus mykiss, and investigated whether OmNOD1 has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 treated with NOD1-specific ligand (iE-DAP). The deduced amino acid sequence of OmNOD1 contained conserved CARD, NOD and LRR domains. Loss-of-function and gain-of-function experiments indicated that OmNOD1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmNOD1 in RTH-149 cells treated with iE-DAP decreased the expression of IL-1β, IL-6, IL-8 and TNF-α. Conversely, overexpression of OmNOD1 resulted in up-regulation of IL-1β, IL-6, IL-8 and TNF-α expression. In addition, RIP2 inhibitor (gefitinib) significantly decreased the expression of these pro-inflammatory cytokines induced by iE-DAP in RTH-149 cells. These findings highlight the important role of NOD1 signaling pathway in fish in eliciting innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2

    Vila-Bedmar, Rocio; Cruces-Sande, Marta; Lucas, Elisa; Willemen, Hanneke L D M; Heijnen, Cobi J; Kavelaars, Annemieke; Mayor, Federico; Murga, Cristina

    2015-01-01

    Insulin resistance is a common feature of obesity and predisposes individuals to various prevalent pathological conditions. G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor kinase 2 (GRK2) integrates several signal transduction pathways and is emerging as a

  11. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A

    2007-01-01

    The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its cr...

  12. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

  13. Progressive osseous heteroplasia (POH): an Egyptian patient | El ...

    Progressive osseous heteroplasia is a rare genetic disorder characterized by cutaneous ossification during infancy and progressive ossification of subcutaneous and deep connective tissue including muscle and fascia during childhood. It is at the severe end of a spectrum of Guanine Nucleotide-binding protein, ...

  14. The Rac1 hypervariable region in targeting and signaling: a tail of many stories

    Lam, B. Daniel; Hordijk, Peter L.

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control

  15. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models

    Huang, Yunhong; Skwarek-Maruszewska, Aneta; Horré, Katrien; Vandewyer, Elke; Wolfs, Leen; Snellinx, An; Saito, Takashi; Radaelli, Enrico; Corthout, Nikky; Colombelli, Julien; Lo, Adrian C; Van Aerschot, Leen; Callaerts-Vegh, Zsuzsanna; Trabzuni, Daniah; Bossers, Koen; Verhaagen, Joost; Ryten, Mina; Munck, Sebastian; D'Hooge, Rudi; Swaab, Dick F; Hardy, John; Saido, Takaomi C; De Strooper, Bart; Thathiah, Amantha

    2015-01-01

    The orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer's disease (AD). However, given the vast

  16. Immunostimulation by cytosine-phosphate-guanine oligodeoxynucleotides in combination with IL-2 can improve the success rate of karyotype analysis in chronic lymphocytic leukaemia.

    Lin, Xiaolan; Chen, Jiadi; Huang, Huifang

    2016-07-01

    To assess whether immunostimulatory cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN) combined with interleukin-2 (IL-2) improves the number of mitotic metaphases and the detection rate of chromosomal abnormalities in chronic lymphocytic leukaemia (CLL). Bone marrow specimens were collected from 36 patients with CLL. CLL cells were cultured with CpG-ODN type DSP30 plus IL-2 for 72 h, following which R-banding analysis was conducted. Conventional culture without the immunostimulant served as the control group. The incidence of genetic abnormalities was measured by fluorescence in situ hybridisation (FISH) using a panel of five specific probes: D13S25 (13q14.3), RB1 (13q14), P53 (17p13), ATM (11q22.3) and CSP12 (trisomy 12, +12). In the control group, chromosome analysis achieved a success rate of only 22.2, and 11.1% of abnormal karyotypes were detected. After immunostimulation with DSP30 plus IL-2, chromosome analysis achieved a success rate of up to 91.6, and 41.6% of abnormal karyotypes were detected. FISH analysis detected 77.7% of abnormalities. FISH combined with CpG-ODN DSP30 plus IL-2 improved the detection rate of chromosomal abnormalities in CLL to 83.3%. CpG-ODN DSP30 combined with IL-2 is effective in improving the detection rate of chromosomal abnormalities in CLL cells. This combination with FISH analysis is conducive to increasing the detection rate of genetic abnormalities in CLL.

  17. Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

    Fujiwara, Sachiko; Matsui, Tsubasa S; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

    2018-01-01

    Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

  18. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

    Nobuyuki Takenaka

    Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

  19. The radioprotector WR-2721 reduces neutron-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in mouse splenocytes when administered prior to or following irradiation

    Grdina, D.J.; Basic, I.

    1992-01-01

    An in vitro T-lymphocyte cloning technique has been applied to study the effects of JANUS fission-spectrum neutron irradiation and the radioprotector S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) on the subsequent development of somatic mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in hybrid B6CF1 male mice. In control studies performed to establish an in vitro cloning technique, the mutant frequencies of splenic T-lymphocytes, as a result of exposure to a 100 cGy dose of neutrons, increased with time from a control level of 9 x 10 -7 to a maximum value of 1.7 x 10 -5 at 56 days following irradiation. Between 56 and 150 days after irradiation, mutant frequencies were observed to plateau and remain stable. All subsequent determinations were performed at 56 days following the experimental treatment of animals. WR-2721 at a dose of 400 mg/kg was effective in protecting against the induction of hprt mutants (i.e. a mutant frequency reduction factor, MFRF) following the largest dose of neutrons used (i.e. 150 cGy). The antimutagenic effectiveness of WR-2721 administered 30 min prior to irradiation was unaffected, even when the dose was reduced to 200 mg/kg. These findings confirm our earlier report using the radioprotector N-(2-mercaptoethyl)-1,2-diaminopropane (WR-1065) under in vitro conditions, and demonstrate that these agents can be used as effective antimutagens even when they are administered up to 3 h following radiation exposure. (Author)

  20. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  1. Simultaneous determination of adenine guanine and thymine at multi-walled carbon nanotubes incorporated with poly(new fuchsin) composite film

    Tang Ching; Yogeswaran, Umasankar [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan (China); Chen, S.-M. [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan (China)], E-mail: smchen78@ms15.hinet.net

    2009-03-16

    A composite film (MWCNTs-PNF) which contains multi-walled carbon nanotubes (MWCNTs) along with the incorporation of poly(new fuchsin) (PNF) has been synthesized on glassy carbon electrode (GCE), gold (Au) and indium tin oxide (ITO) by potentiostatic methods. The presence of MWCNTs in the composite film enhances surface coverage concentration ({gamma}) of PNF to {approx}176.5%, and increases the electron transfer rate constant (k{sub s}) to {approx}346%. The composite film also exhibits promising enhanced electrocatalytic activity towards the mixture of biochemical compounds such as adenine (AD), guanine (GU) and thymine (THY). The surface morphology of the composite film deposited on ITO has been studied using scanning electron microscopy and atomic force microscopy. These two techniques reveal that the PNF incorporated on MWCNTs. Electrochemical quartz crystal microbalance study reveals the enhancement in the functional properties of MWCNTs and PNF. The electrocatalytic responses of analytes at MWCNTs and MWCNTs-PNF films were measured using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). From electrocatalysis studies, well separated voltammetric peaks have been obtained at the composite film for AD, GU and THY, with the peak separation of 320.3 and 132.7 mV between GU-AD and AD-THY respectively. The sensitivity of the composite film towards AD, GU and THY in DPV technique is 218.18, 12.62 and 78.22 mA M{sup -1} cm{sup -2} respectively, which are higher than MWCNTs film. Further, electroanalytical studies of AD, GU and THY present in single-strand deoxyribonucleic acid (ssDNA) have been carried out using semi-derivative CV and DPV.

  2. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  3. Modeling of Toxicity-Relevant Electrophilic Reactivity for Guanine with Epoxides: Estimating the Hard and Soft Acids and Bases (HSAB) Parameter as a Predictor.

    Zhang, Jing; Wang, Chenchen; Ji, Li; Liu, Weiping

    2016-05-16

    According to the electrophilic theory in toxicology, many chemical carcinogens in the environment and/or their active metabolites are electrophiles that exert their effects by forming covalent bonds with nucleophilic DNA centers. The theory of hard and soft acids and bases (HSAB), which states that a toxic electrophile reacts preferentially with a biological macromolecule that has a similar hardness or softness, clarifies the underlying chemistry involved in this critical event. Epoxides are hard electrophiles that are produced endogenously by the enzymatic oxidation of parent chemicals (e.g., alkenes and PAHs). Epoxide ring opening proceeds through a SN2-type mechanism with hard nucleophile DNA sites as the major facilitators of toxic effects. Thus, the quantitative prediction of chemical reactivity would enable a predictive assessment of the molecular potential to exert electrophile-mediated toxicity. In this study, we calculated the activation energies for reactions between epoxides and the guanine N7 site for a diverse set of epoxides, including aliphatic epoxides, substituted styrene oxides, and PAH epoxides, using a state-of-the-art density functional theory (DFT) method. It is worth noting that these activation energies for diverse epoxides can be further predicted by quantum chemically calculated nucleophilic indices from HSAB theory, which is a less computationally demanding method than the exacting procedure for locating the transition state. More importantly, the good qualitative/quantitative correlations between the chemical reactivity of epoxides and their bioactivity suggest that the developed model based on HSAB theory may aid in the predictive hazard evaluation of epoxides, enabling the early identification of mutagenicity/carcinogenicity-relevant SN2 reactivity.

  4. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-01-01

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

  5. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  6. [3H]WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

    Norman, A.B.; Battaglia, G.; Creese, I.

    1985-01-01

    In the presence of a 30 nM prazosin mask, [ 3 H]-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ([ 3 H]WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for [ 3 H] WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at [ 3 H]WB4101-binding sites in the presence of 30 nM prazosin and [ 3 H] lysergic acid diethylamide ([ 3 H]LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of [ 3 H]WB4101 is significantly lower than the Bmax of [ 3 H]LSD in various brain regions. WB4101 competition for [ 3 H] LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of [ 3 H]WB4101 binding derived from saturation experiments. This suggests that [ 3 H]WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by [ 3 H]LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for [ 3 H]WB4101 but compete for multiple [ 3 H]LSD 5-HT1 binding sites. These data indicate that [ 3 H]WB4101 selectively labels the 5-HT1A serotonin receptor, whereas [ 3 H] LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of [ 3 H]WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of [ 3 H]WB4101 binding

  7. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    Krause-Gruszczynska, Malgorzata

    2011-12-28

    Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  8. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    Krause-Gruszczynska Malgorzata

    2011-12-01

    Full Text Available Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  9. pH-Modulated Watson-Crick duplex-quadruplex equilibria of guanine-rich and cytosine-rich DNA sequences 140 base pairs upstream of the c-kit transcription initiation site.

    Bucek, Pavel; Jaumot, Joaquim; Aviñó, Anna; Eritja, Ramon; Gargallo, Raimundo

    2009-11-23

    Guanine-rich regions of DNA are sequences capable of forming G-quadruplex structures. The formation of a G-quadruplex structure in a region 140 base pairs (bp) upstream of the c-kit transcription initiation site was recently proposed (Fernando et al., Biochemistry, 2006, 45, 7854). In the present study, the acid-base equilibria and the thermally induced unfolding of the structures formed by a guanine-rich region and by its complementary cytosine-rich strand in c-kit were studied by means of circular dichroism and molecular absorption spectroscopies. In addition, competition between the Watson-Crick duplex and the isolated structures was studied as a function of pH value and temperature. Multivariate data analysis methods based on both hard and soft modeling were used to allow accurate quantification of the various acid-base species present in the mixtures. Results showed that the G-quadruplex and i-motif coexist with the Watson-Crick duplex over the pH range from 3.0 to 6.5, approximately, under the experimental conditions tested in this study. At pH 7.0, the duplex is practically the only species present.

  10. NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2012-01-01

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

  11. Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2012-12-04

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

  12. On the Formation and Properties of Interstrand DNA-DNA Cross-links Forged by Reaction of an Abasic Site With the Opposing Guanine Residue of 5′-CAp Sequences in Duplex DNA

    Johnson, Kevin M.; Price, Nathan E.; Wang, Jin; Fekry, Mostafa I.; Dutta, Sanjay; Seiner, Derrick R.; Wang, Yinsheng; Gates, Kent S.

    2014-01-01

    We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3. PMID:23215239

  13. ASIC proteins regulate smooth muscle cell migration.

    Grifoni, Samira C; Jernigan, Nikki L; Hamilton, Gina; Drummond, Heather A

    2008-03-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration.

  14. ASIC PROTEINS REGULATE SMOOTH MUSCLE CELL MIGRATION

    Grifoni, Samira C.; Jernigan, Nikki L.; Hamilton, Gina; Drummond, Heather A.

    2007-01-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated Epithelial Na+ Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration, however the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence indi...

  15. High-performance liquid chromatography/electrospray mass spectrometry for the analysis of modified bases in DNA: 7-(2-hydroxyethyl)guanine, the major ethylene oxide-DNA adduct.

    Leclercq, L; Laurent, C; De Pauw, E

    1997-05-15

    A method was developed for the analysis of 7-(2-hydroxyethyl)guanine (7HEG), the major DNA adduct formed after exposure to ethylene oxide (EO). The method is based on DNA neutral thermal hydrolysis, adduct micro-concentration, and final characterization and quantification by HPLC coupled to single-ion monitoring electrospray mass spectrometry (HPLC/SIR-ESMS). The method was found to be selective, sensitive, and easy to handle with no need for enzymatic digestion or previous sample derivatization. Detection limit was found to be close to 1 fmol of adduct injected (10(-10) M), thus allowing the detection of approximately three modified bases on 10(8) intact nucleotides in blood sample analysis. Quantification results are shown for 7HEG after calf thymus DNA and blood exposure to various doses of EO, in both cases obtaining clear dose-response relationships.

  16. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  17. Mixed adenine/guanine quartets with three trans-a2 Pt(II) (a=NH(3) or MeNH(2)) cross-links: linkage and rotational isomerism, base pairing, and loss of NH(3).

    Albertí, Francisca M; Rodríguez-Santiago, Luis; Sodupe, Mariona; Mirats, Andrea; Kaitsiotou, Helena; Sanz Miguel, Pablo J; Lippert, Bernhard

    2014-03-17

    Of the numerous ways in which two adenine and two guanines (N9 positions blocked in each) can be cross-linked by three linear metal moieties such as trans-a2 Pt(II) (with a=NH3 or MeNH2 ) to produce open metalated purine quartets with exclusive metal coordination through N1 and N7 sites, one linkage isomer was studied in detail. The isomer trans,trans,trans-[{Pt(NH3 )2 (N7-9-EtA-N1)2 }{Pt(MeNH2 )2 (N7-9-MeGH)}2 ][(ClO4 )6 ]⋅3H2 O (1) (with 9-EtA=9-ethyladenine and 9-MeGH=9-methylguanine) was crystallized from water and found to adopt a flat Z-shape in the solid state as far as the trinuclear cation is concerned. In the presence of excess 9-MeGH, a meander-like construct, trans,trans,trans-[{Pt(NH3 )2 (N7-9-EtA-N1)2 }{Pt(MeNH2 )2 (N7-9-MeGH)2 }][(ClO4 )6 ]⋅[(9-MeGH)2 ]⋅7 H2 O (2) is formed, in which the two extra 9-MeGH nucleobases are hydrogen bonded to the two terminal platinated guanine ligands of 1. Compound 1, and likewise the analogous complex 1 a (with NH3 ligands only), undergo loss of an ammonia ligand and formation of NH4 (+) when dissolved in [D6 ]DMSO. From the analogy between the behavior of 1 and 1 a it is concluded that a NH3 ligand from the central Pt atom is lost. Addition of 1-methylcytosine (1-MeC) to such a DMSO solution reveals coordination of 1-MeC to the central Pt. In an analogous manner, 9-MeGH can coordinate to the central Pt in [D6 ]DMSO. It is proposed that the proton responsible for formation of NH4 (+) is from one of the exocyclic amino groups of the two adenine bases, and furthermore, that this process is accompanied by a conformational change of the cation from Z-form to U-form. DFT calculations confirm the proposed mechanism and shed light on possible pathways of this process. Calculations show that rotational isomerism is not kinetically hindered and that it would preferably occur previous to the displacement of NH3 by DMSO. This displacement is the most energetically costly step, but it is compensated by the proton

  18. Isotope Dilution nanoLC/ESI+-HRMS3 Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

    Sangaraju, Dewakar; Boldry, Emily J; Patel, Yesha M; Walker, Vernon; Stepanov, Irina; Stram, Daniel; Hatsukami, Dorothy; Tretyakova, Natalia

    2017-02-20

    1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of the general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI + -HRMS 3 methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine; limit of quantitation: 1.0 fmol/mL urine). This new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0-200 ppm of BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm of BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1-2.2 ppm of BD (1.25 ± 0.51 pg/mg of creatinine) as compared to administrative controls exposed to <0.01 ppm of BD (0.22 ± 0.08 and pg/mg of creatinine) (p = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers' urine with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg of creatinine, p = 3.1 × 10 -7 ). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Urinary EB-GII adduct levels and urinary mercapturic acids of BD (MHBMA, DHBMA) were compared

  19. Synthesis and preliminary evaluation of 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) in HSV1-tk gene transduced hepatoma cell

    Moon, Byung Seok; Lee, Tae Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Myoung Keun [Yonsei University, Wonju (Korea, Republic of)] (and others)

    2006-08-15

    The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with K[{sup 18}F]/K2.2.2. in acetonitrile using N2-monomethoxytrityl-9-[4-(tosly)-3-monomethoxytritylmethylbutl] guanine as a precursor, followed by deprotection with 1 N HCI. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of [{sup 18}F]FHBG were performed, and was analyzed correlation between [{sup 18}F]FHBG uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor beating Balb/c-nude mouse model. [{sup 18}F]FHBG was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiochemical yield was about 20-25% (corrected for decay), radiochemical purity was > 95% and specific activity was around > 55.5 GBq/ {mu} mol. Specific accumulation of [{sup 18}F]FHBG was observed in HSV1-tk gene transduced MCA-tk cells but not MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked [{sup 18}F]FHBG was retained inside of cells. The uptake of [{sup 18}F]FHBG was showed a highly significant linear correlation (R{sup 2} = 0.995) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. [{sup 18}F]FHBG appears

  20. Surface amplification of pencil graphite electrode with polypyrrole and reduced graphene oxide for fabrication of a guanine/adenine DNA based electrochemical biosensors for determination of didanosine anticancer drug

    Karimi-Maleh, Hassan; Bananezhad, Asma; Ganjali, Mohammad R.; Norouzi, Parviz; Sadrnia, Abdolhossein

    2018-05-01

    Didanosine is nucleoside analog reverse transcriptase inhibitors with many side effects such as nausea and vomiting, stomach pain, tingling, burning and numbness and determination of this drug is very important in biological samples. This paper presents a DNA biosensor for determination of didanosine (DDI) in pharmaceutical samples. A pencil graphite electrode modified with conductive materials such as polypyrrole (PPy) and reduced graphene oxide (rGO) (PGE/PPy/rGO) was used for this goal. The double-stranded DNA was successfully immobilized on PGE/PPy/rGO. The PGE/PPy/rGO was characterized by microscopic and electrochemical methods. Then, the interaction of DDI with DNA was identified by decreases in the oxidation currents of guanine and adenine by differential pulse voltammetric (DPV) method. The dynamic range of DDI identified in the range of 0.02-50.0 μM and this electrode provided a low limit of detection (LOD = 8.0 nM) for DDI. The PGE/PPy/rGO loaded with ds-DNA was utilized for the measurement of DDI in real samples and obtained data were compared with HPLC method. The statistical tests such as F-test and t-test were used for confirming ability of PGE/PPy/rGO loaded with ds-DNA for analysis of DDI in real samples.

  1. The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

    Fenyk, S.; Townsend, P.D.; Dixon, C.H.; Spies, G.B.; Campillo, A.S.E.; Slootweg, E.J.; Westerhof, L.B.; Gawehns, F.K.K.; Knight, M.R.; Sharples, G.J.; Goverse, A.; Palsson, L.O.; Takken, F.L.W.; Cann, M.J.

    2015-01-01

    Plant NLR proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus, however, conserved nuclear targets that support their role in immunity are unknown. Previously we noted a structural homology between the NB domain of NLRs and DNA replication origin-binding Cdc6/Orc1

  2. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.

    1989-01-01

    : DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6......-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent...

  3. Non-Coding Polymorphisms in Nucleotide Binding Domain 1 in ABCC1 Gene Associate with Transcript Level and Survival of Patients with Breast Cancer

    Kunická, T.; Václavíková, R.; Hlaváč, V.; Vrána, D.; Pecha, V.; Rauš, K.; Trnková, M.; Kubáčková, K.; Ambruš, M.; Vodičková, Ludmila; Vodička, Pavel; Souček, P.

    2014-01-01

    Roč. 9, č. 7 (2014), e101740 E-ISSN 1932-6203 Grant - others:GA MZd(CZ) NT13679; GA ČR(CZ) GBP303/12/G163 Institutional support: RVO:68378041 Keywords : expression * chemotherapy * cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  4. Toll-like receptor 2 and nucleotide-binding oligomerization domain-2 play divergent roles in the recognition of gut-derived lactobacilli and bifidobacteria in dendritic cells

    Zeuthen, Louise; Fink, Lisbeth Nielsen; Frøkiær, Hanne

    2008-01-01

    -marrow-derived DC lacking NOD2 produce higher levels of interleukin-10 (IL-10) and reduced levels of IL-12 and tumour necrosis factor-[alpha] (TNF-[alpha]) in response to LAB. This indicates that peptidoglycan is partly responsible for the T helper type 1 skewing effect of certain LAB. Dendritic cells that are TLR2......-[alpha]-inducing bifidobacteria inhibit the T helper type 1 skewing effect induced by strong immunostimulatory lactobacilli. Here we show that this immunoinhibitory effect of bifidobacteria is dependent on TLR2 and independent of NOD2. Moreover, independently of the cytokine pattern induced by intact LAB, cell wall fractions...

  5. Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation

    Owen, GR

    2014-10-01

    Full Text Available –deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide...

  6. The soybean-Phytophthora resistance locus Rps1-k encompasses coiled coil-nucleotide binding-leucine rich repeat-like genes and repetitive sequences

    Bhattacharyya Madan K

    2008-03-01

    Full Text Available Abstract Background A series of Rps (resistance to Pytophthora sojae genes have been protecting soybean from the root and stem rot disease caused by the Oomycete pathogen, Phytophthora sojae. Five Rps genes were mapped to the Rps1 locus located near the 28 cM map position on molecular linkage group N of the composite genetic soybean map. Among these five genes, Rps1-k was introgressed from the cultivar, Kingwa. Rps1-k has been providing stable and broad-spectrum Phytophthora resistance in the major soybean-producing regions of the United States. Rps1-k has been mapped and isolated. More than one functional Rps1-k gene was identified from the Rps1-k locus. The clustering feature at the Rps1-k locus might have facilitated the expansion of Rps1-k gene numbers and the generation of new recognition specificities. The Rps1-k region was sequenced to understand the possible evolutionary steps that shaped the generation of Phytophthora resistance genes in soybean. Results Here the analyses of sequences of three overlapping BAC clones containing the 184,111 bp Rps1-k region are reported. A shotgun sequencing strategy was applied in sequencing the BAC contig. Sequence analysis predicted a few full-length genes including two Rps1-k genes, Rps1-k-1 and Rps1-k-2. Previously reported Rps1-k-3 from this genomic region 1 was evolved through intramolecular recombination between Rps1-k-1 and Rps1-k-2 in Escherichia coli. The majority of the predicted genes are truncated and therefore most likely they are nonfunctional. A member of a highly abundant retroelement, SIRE1, was identified from the Rps1-k region. The Rps1-k region is primarily composed of repetitive sequences. Sixteen simple repeat and 63 tandem repeat sequences were identified from the locus. Conclusion These data indicate that the Rps1 locus is located in a gene-poor region. The abundance of repetitive sequences in the Rps1-k region suggested that the location of this locus is in or near a heterochromatic region. Poor recombination frequencies combined with presence of two functional Rps genes at this locus has been providing stable Phytophthora resistance in soybean.

  7. Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana).

    Baurens, Franc-Christophe; Bocs, Stéphanie; Rouard, Mathieu; Matsumoto, Takashi; Miller, Robert N G; Rodier-Goud, Marguerite; MBéguié-A-MBéguié, Didier; Yahiaoui, Nabila

    2010-07-16

    Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. A large RGA08 gene cluster identified in wild banana corresponds to a highly variable genomic region between haplotypes surrounded by conserved flanking regions. High level of sequence identity (70 to 99%) of the genic and intergenic regions suggests a recent and rapid evolution of this cluster in M. balbisiana.

  8. Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

    Maeta, Kazuhiro; Hattori, Satoko; Ikutomo, Junji; Edamatsu, Hironori; Bilasy, Shymaa E; Miyakawa, Tsuyoshi; Kataoka, Tohru

    2018-05-10

    Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

  9. How does the long G·G* Watson-Crick DNA base mispair comprising keto and enol tautomers of the guanine tautomerise? The results of a QM/QTAIM investigation.

    Brovarets', Ol'ha O; Hovorun, Dmytro M

    2014-08-14

    The double proton transfer (DPT) in the long G·G* Watson-Crick base mispair (|C6N1(G*)N1C6(G)| = 36.4°; C1 symmetry), involving keto and enol tautomers of the guanine (G) nucleobase, along two intermolecular neighboring O6H···O6 (8.39) and N1···HN1 (6.14 kcal mol(-1)) H-bonds that were established to be slightly anti-cooperative, leads to its transformation into the G*·G base mispair through a single transition state (|C6N1N1C6| = 37.1°; C1), namely to the interconversion into itself. It was shown that the G·G* ↔ G*·G tautomerisation via the DPT is assisted by the third specific contact, that sequentially switches along the intrinsic reaction coordinate (IRC) in an original way: (G)N2H···N2(G*) H-bond (-25.13 to -10.37) → N2···N2 van der Waals contact (-10.37 to -9.23) → (G)N2···HN2(G*) H-bond (-9.23 to 0.79) → (G*)N2···HN2(G) H-bond (0.79 to 7.35 Bohr). The DPT tautomerisation was found to proceed through the asynchronous concerted mechanism by employing the QM/QTAIM approach and the methodology of the scans of the geometric, electron-topological, energetic, polar and NBO properties along the IRC. Nine key points, that can be considered as part of the tautomerisation repertoire, have been established and analyzed in detail. Furthermore, it was shown that the G·G* or G*·G base mispair is a thermodynamically and dynamically stable structure with a lifetime of 8.22 × 10(-10) s and all 6 low-frequency intermolecular vibrations are able to develop during this time span. Lastly, our results highlight the importance of the G·G* ↔ G*·G DPT tautomerisation, which can have implications for biological and chemical sensing applications.

  10. Topoisomerase IB of Deinococcus radiodurans resolves guanine ...

    2015-11-28

    Nov 28, 2015 ... All the oligonucleotides men- tioned here were ... further reactions with DraTopoIB were carried out at 37°C ... of G4 DNA moves faster than unfolded and intermolecular .... for its action on intramolecular G4 DNA structure was.

  11. Is the DPT tautomerization of the long A·G Watson-Crick DNA base mispair a source of the adenine and guanine mutagenic tautomers? A QM and QTAIM response to the biologically important question.

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M

    2014-03-05

    Herein, we first address the question posed in the title by establishing the tautomerization trajectory via the double proton transfer of the adenine·guanine (A·G) DNA base mispair formed by the canonical tautomers of the A and G bases into the A*·G* DNA base mispair, involving mutagenic tautomers, with the use of the quantum-mechanical calculations and quantum theory of atoms in molecules (QTAIM). It was detected that the A·G ↔ A*·G* tautomerization proceeds through the asynchronous concerted mechanism. It was revealed that the A·G base mispair is stabilized by the N6H···O6 (5.68) and N1H···N1 (6.51) hydrogen bonds (H-bonds) and the N2H···HC2 dihydrogen bond (DH-bond) (0.68 kcal·mol(-1) ), whereas the A*·G* base mispair-by the O6H···N6 (10.88), N1H···N1 (7.01) and C2H···N2 H-bonds (0.42 kcal·mol(-1) ). The N2H···HC2 DH-bond smoothly and without bifurcation transforms into the C2H···N2 H-bond at the IRC = -10.07 Bohr in the course of the A·G ↔ A*·G* tautomerization. Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···O6 H-bond is anticooperative to the two others-N1H···N1 and N2H···HC2 in the A·G base mispair, while the latters are significantly cooperative, mutually strengthening each other. In opposite, all three O6H···N6, N1H···N1, and C2H···N2 H-bonds are cooperative in the A*·G* base mispair. All in all, we established the dynamical instability of the А*·G* base mispair with a short lifetime (4.83·10(-14) s), enabling it not to be deemed feasible source of the A* and G* mutagenic tautomers of the DNA bases. The small lifetime of the А*·G* base mispair is predetermined by the negative value of the Gibbs free energy for the A*·G* → A·G transition. Moreover, all of the six low-frequency intermolecular vibrations cannot develop during this lifetime that additionally confirms the aforementioned results. Thus, the A*·G* base mispair cannot be

  12. Amyloid beta precursor protein regulates male sexual behavior.

    Park, Jin Ho; Bonthius, Paul J; Tsai, Houng-Wei; Bekiranov, Stefan; Rissman, Emilie F

    2010-07-28

    Sexual behavior is variable between individuals, ranging from celibacy to sexual addictions. Within normal populations of individual men, ranging from young to middle aged, testosterone levels do not correlate with libido. To study the genetic mechanisms that contribute to individual differences in male sexual behavior, we used hybrid B6D2F1 male mice, which are a cross between two common inbred strains (C57BL/6J and DBA/2J). Unlike most laboratory rodent species in which male sexual behavior is highly dependent upon gonadal steroids, sexual behavior in a large proportion of these hybrid male mice after castration is independent of gonadal steroid hormones and their receptors; thus, we have the ability to discover novel genes involved in this behavior. Gene expression arrays, validation of gene candidates, and transgenic mice that overexpress one of the genes of interest were used to reveal genes involved in maintenance of male sexual behavior. Several genes related to neuroprotection and neurodegeneration were differentially expressed in the hypothalamus of males that continued to mate after castration. Male mice overexpressing the human form of one of these candidate genes, amyloid beta precursor protein (APP), displayed enhanced sexual behavior before castration and maintained sexual activity for a longer duration after castration compared with controls. Our results reveal a novel and unexpected relationship between APP and male sexual behavior. We speculate that declining APP during normal aging in males may contribute to the loss of sexual function.

  13. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

  14. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

  15. Control of striatal signaling by G protein regulators

    Keqiang eXie

    2011-08-01

    Full Text Available Signaling via heterotrimeric G proteins plays a crucial role in modulating the responses of striatal neurons that ultimately shape core behaviors mediated by the basal ganglia circuitry, such as reward valuation, habit formation and movement coordination. Activation of G-protein-coupled receptors (GPCRs by extracellular signals activates heterotrimeric G proteins by promoting the binding of GTP to their α subunits. G proteins exert their effects by influencing the activity of key effector proteins in this region, including ion channels, second messenger enzymes and protein kinases. Striatal neurons express a staggering number of GPCRs whose activation results in the engagement of downstream signaling pathways and cellular responses with unique profiles but common molecular mechanisms. Studies over the last decade have revealed that the extent and duration of GPCR signaling are controlled by a conserved protein family named Regulator of G protein Signaling (RGS. RGS proteins accelerate GTP hydrolysis by the α subunits of G proteins, thus promoting deactivation of GPCR signaling. In this review, we discuss the progress made in understanding the roles of RGS proteins in controlling striatal G protein signaling and providing integration and selectivity of signal transmission. We review evidence on the formation of a macromolecular complex between RGS proteins and other components of striatal signaling pathways, their molecular regulatory mechanisms and impacts on GPCR signaling in the striatum obtained from biochemical studies and experiments involving genetic mouse models. Special emphasis is placed on RGS9-2, a member of the RGS family that is highly enriched in the striatum and plays critical roles in drug addiction and motor control.

  16. Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase

    Singh, Ajay; Haldar, Swati; Horback, Katharine; Tom, Cynthia; Zhou, Lan; Meyerson, Howard; Singh, Neena

    2017-01-01

    Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP−/−). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP−/− mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP−/− mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP−/− mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP−/− BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates. PMID:23478311

  17. Shoc2/Sur8 protein regulates neurite outgrowth.

    Gonzalo Leon

    Full Text Available The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.

  18. Mitochondrial fission proteins regulate programmed cell death in yeast

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J.; Qi, Bing; Pevsner, Jonathan; McCaffery, J. Michael; Hill, R. Blake; Basañez, Gorka; Hardwick, J. Marie

    2004-01-01

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we fo...

  19. Mitochondrial fission proteins regulate programmed cell death in yeast.

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

    2004-11-15

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

  20. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular deve...

  1. Myostatin-like proteins regulate synaptic function and neuronal morphology.

    Augustin, Hrvoje; McGourty, Kieran; Steinert, Joern R; Cochemé, Helena M; Adcott, Jennifer; Cabecinha, Melissa; Vincent, Alec; Halff, Els F; Kittler, Josef T; Boucrot, Emmanuel; Partridge, Linda

    2017-07-01

    Growth factors of the TGFβ superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth. © 2017. Published by The Company of Biologists Ltd.

  2. Identification of proteins regulated by curcumin in cerebral ischemia.

    Shah, Fawad-Ali; Gim, Sang-Ah; Sung, Jin-Hee; Jeon, Seong-Jun; Kim, Myeong-Ok; Koh, Phil-Ok

    2016-03-01

    Curcumin is known to have a neuroprotective effect against cerebral ischemia. The objective of this study was to identify various proteins that are differentially expressed by curcumin treatment in focal cerebral ischemia using a proteomic approach. Adult male rats were treated with vehicle or curcumin 1 h after middle cerebral artery occlusion. Brain tissues were collected 24 h after the onset of middle cerebral artery occlusion, and cerebral cortices proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. We detected several proteins with altered expression levels between vehicle- and curcumin-treated animals. Among these proteins, ubiquitin carboxy-terminal hydrolase L1, isocitrate dehydrogenase, adenosylhomocysteinase, and eukaryotic initiation factor 4A were decreased in the vehicle-treated animal, and curcumin treatment attenuated the injury-induced decreases of these proteins. Conversely, pyridoxal phosphate phosphatase was increased in the vehicle-treated animal, and curcumin treatment prevented decreases in this protein. The identified altered proteins are associated with cellular metabolism and differentiation. The results of this study suggest that curcumin exerts a neuroprotective effect by regulating the expression of various proteins in focal cerebral ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Piezo proteins: regulators of mechanosensation and other cellular processes.

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

  5. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

    Floss, Daniela S; Levy, Julien G; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J

    2013-12-17

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

  6. Caenorhabditis elegans intersectin: a synaptic protein regulating neurotransmission

    Rose, Simon; Malabarba, Maria Grazia; Krag, Claudia

    2007-01-01

    the characterization of intersectin function in Caenorhabditis elegans. Nematode intersectin (ITSN-1) is expressed in the nervous system, and it is enriched in presynaptic regions. The C. elegans intersectin gene (itsn-1) is nonessential for viability. In addition, itsn-1-null worms do not display any evident...

  7. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. © 2016 Federation of European Biochemical Societies.

  8. Fission Yeast Model Study for Dissection of TSC Pathway

    2010-04-01

    cerevisiae;Hs, Homo sapiens ;Dm,Drosophilamelanogaster. (C)Themulticopy suppressors ofDtsc2 cpp1-1cells.Dtsc2 cpp1-1cells carrying each of the five...distribution of their cell length was measured (right pan- els ). For each strain, .100 cells were counted. The experi- ments were performed three times with...dependent GAP activity. Taken together, we specu- late that rhb1-DA4 can stimulate its downstream ele - ments regardless of the guanine-nucleotide-binding

  9. Ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

    Comerford, J.G.; Gibson, J.R.; Dawson, A.P.; Gibson, I.

    1989-01-01

    The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma- 35 S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma- 35 S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines

  10. Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles

    Knudsen, Charlotte Rohde; Kjaersgård, I V; Wiborg, O

    1995-01-01

    All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches. Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism. Gly94 and Gly126...... were separately mutated to alanine residues to study their role in the switch mechanism. The mutant proteins are denoted [G94A]EF-Tu and [G126A]EF-Tu, respectively. Both mutations affect the affinities for guanine nucleotides considerably, resulting in a decrease in the characteristic preference...... for GDP over GTP. Furthermore the [G94A]EF-Tu mutant possesses an increased GTPase activity. The aminoacyl-tRNA affinity is much reduced for [G94A]EF-Tu, as reflected in an increase of the dissociation rate constant for the ternary complex by a factor of 40. Surprisingly, however, both mutants...

  11. A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

    Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi

    2016-01-01

    Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

  12. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

    2015-01-01

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol

  13. Td4IN2: A drought-responsive durum wheat (Triticum durum Desf.) gene coding for a resistance like protein with serine/threonine protein kinase, nucleotide binding site and leucine rich domains.

    Rampino, Patrizia; De Pascali, Mariarosaria; De Caroli, Monica; Luvisi, Andrea; De Bellis, Luigi; Piro, Gabriella; Perrotta, Carla

    2017-11-01

    Wheat, the main food source for a third of world population, appears strongly under threat because of predicted increasing temperatures coupled to drought. Plant complex molecular response to drought stress relies on the gene network controlling cell reactions to abiotic stress. In the natural environment, plants are subjected to the combination of abiotic and biotic stresses. Also the response of plants to biotic stress, to cope with pathogens, involves the activation of a molecular network. Investigations on combination of abiotic and biotic stresses indicate the existence of cross-talk between the two networks and a kind of overlapping can be hypothesized. In this work we describe the isolation and characterization of a drought-related durum wheat (Triticum durum Desf.) gene, identified in a previous study, coding for a protein combining features of NBS-LRR type resistance protein with a S/TPK domain, involved in drought stress response. This is one of the few examples reported where all three domains are present in a single protein and, to our knowledge, it is the first report on a gene specifically induced by drought stress and drought-related conditions, with this particular structure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Hokkaido 062-8517 (Japan)

    2015-10-23

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  15. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-01-01

    Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

  16. Gene expression profiles in relation to tension and dissociation in borderline personality disorder.

    Christian Schmahl

    Full Text Available The biological underpinnings of borderline personality disorder (BPD and its psychopathology including states of aversive tension and dissociation is poorly understood. Our goal was to examine transcriptional changes associated with states of tension or dissociation within individual patients in a pilot study. Dissociation is not only a critical symptom of BPD but has also been associated with higher risk for self-mutilation and depression. We conducted a whole blood gene expression profile analysis using quantitative PCR in 31 female inpatients with BPD. For each individual, two samples were drawn during a state of high tension and dissociation, while two samples were drawn at non-tension states. There was no association between gene expression and tension states. However, we could show that Interleukin-6 was positively correlated to dissociation scores, whereas Guanine nucleotide-binding protein G(s subunit alpha isoforms, Mitogen-activated protein kinase 3 and 8, Guanine nucleotide-binding protein G(i subunit alpha-2, Beta-arrestin-1 and 2, and Cyclic AMP-responsive element-binding protein were negatively correlated to dissociation. Our data point to a potential association of dissociation levels with the expression of genes involved in immune system regulation as well as cellular signalling/second-messenger systems. Major limitations of the study are the the possibly heterogeneous cell proportions in whole blood and the heterogeneous medication.

  17. Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line.

    Kalra, Sukirti; Paul, Manash K; Balaram, Hemalatha; Mukhopadhyay, Anup Kumar

    2007-05-01

    The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.

  18. Photochemical selectivity in guanine-cytosine base-pair structures

    Abo-Riziq, A.; Grace, L.; Nir, E.; Kabeláč, Martin; Hobza, Pavel; Vries de, M. S.

    2005-01-01

    Roč. 102, č. 1 (2005), s. 20-23 ISSN 0027-8424 R&D Projects: GA ČR(CZ) GA203/05/0009 Grant - others:NSF(US) CHE-0244341 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA base pairs * IR-UV spectroscopy * phytochemistry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 10.231, year: 2005

  19. Oligomeric state of hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis

    Eng, W. S.; Keough, D. T.; Hocková, Dana; Winzor, D. J.; Guddat, L. W.

    2017-01-01

    Roč. 135, Apr (2017), s. 6-14 ISSN 0300-9084 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : enzyme inhibitors * acyclic nucleoside phosphonates * 6-oxopurine phosphoribosyltransferase Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.112, year: 2016

  20. Towards understanding of poly-guanine activated fluorescent silver nanoclusters

    Walczak, Sylwia; Morishita, Kiyoshi; Ahmed, Moin; Liu, Juewen

    2014-01-01

    It has been recently reported that the fluorescence of some DNA-templated silver nanoclusters (AgNCs) can be significantly enhanced upon by hybridizing with a partially complementary DNA containing a G-rich overhang near the AgNCs. This discovery has found a number of analytical applications but many fundamental questions remain to be answered. In this work, the photostability of these activated AgNCs is reported. After adding the G-rich DNA activator, the fluorescence intensity peaks in ∼1 h and then starts to decay, where the decaying rate is much faster with light exposure. The lost fluorescence is recovered by adding NaBH 4 , suggesting that the bleaching is an oxidative process. Once activated, the G-rich activator can be removed while the AgNCs still maintain most of their fluorescence intensity. UV–vis spectroscopy suggests that new AgNC species are generated upon hybridization with the activator. The base sequence and length of the template DNA have also been varied, leading to different emission colors and color change after hybridization. G-rich aptamers can also serve as activators. Our results indicate that activation of the fluorescence by G-rich DNA could be a convenient method for biosensor development since the unstable NaBH 4 is not required for the activation step. (paper)

  1. In silico docking of forchlorfenuron (FCF to septins suggests that FCF interferes with GTP binding.

    Dimitrios Angelis

    Full Text Available Septins are GTP-binding proteins that form cytoskeleton-like filaments, which are essential for many functions in eukaryotic organisms. Small molecule compounds that disrupt septin filament assembly are valuable tools for dissecting septin functions with high temporal control. To date, forchlorfenuron (FCF is the only compound known to affect septin assembly and functions. FCF dampens the dynamics of septin assembly inducing the formation of enlarged stable polymers, but the underlying mechanism of action is unknown. To investigate how FCF binds and affects septins, we performed in silico simulations of FCF docking to all available crystal structures of septins. Docking of FCF with SEPT2 and SEPT3 indicated that FCF interacts preferentially with the nucleotide-binding pockets of septins. Strikingly, FCF is predicted to form hydrogen bonds with residues involved in GDP-binding, mimicking nucleotide binding. FCF docking with the structure of SEPT2-GppNHp, a nonhydrolyzable GTP analog, and SEPT7 showed that FCF may assume two alternative non-overlapping conformations deeply into and on the outer side of the nucleotide-binding pocket. Surprisingly, FCF was predicted to interact with the P-loop Walker A motif GxxxxGKS/T, which binds the phosphates of GTP, and the GTP specificity motif AKAD, which interacts with the guanine base of GTP, and highly conserved amino acids including a threonine, which is critical for GTP hydrolysis. Thus, in silico FCF exhibits a conserved mechanism of binding, interacting with septin signature motifs and residues involved in GTP binding and hydrolysis. Taken together, our results suggest that FCF stabilizes septins by locking them into a conformation that mimics a nucleotide-bound state, preventing further GTP binding and hydrolysis. Overall, this study provides the first insight into how FCF may bind and stabilize septins, and offers a blueprint for the rational design of FCF derivatives that could target septins with

  2. Function of mammalian genes regulation cellular growth; Saibo zoshoku wo seigyosuru dobutsu saibo idenshi no kino kaiseki

    Matsumoto, K. [Nagoya University, Nagoya (Japan)

    1995-12-15

    Intracellular signaling from receptor tyrosine kindles in mammalian cells results in activation of a signal cascade that includes the guanine nucleotide binding protein Ras and the protein kinases Raf, MEK [Mitogen activated protein kindle (MAPK) or Extracellular signal regulated kinase (ERK) kinase] and MAPK. MAPK activation that is dependent on the coupling of Ras and Raf was reconstituted in yeast. Yeast genes were isolated that, when overexpressed, enhanced the function of Raf. One of them is identical to BMH 1, which encodes a protein similar to members of the mammalian 14-3-3 family. Bacterially synthesized mammalian 14-3-3 protein stimulated the activity of Raf prepared from yeast cells expressing c-Raf-1. Thus, the 14-3-3 protein may participate in or be required for activation of Raf. 9 refs., 2 figs.

  3. Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

    Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2017-02-22

    Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

  4. Molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor

    Numa, S.; Fukuda, K.; Kubo, T.; Maeda, A.; Akiba, I.; Bujo, H.; Nakai, J.; Mishina, M.; Higashida, H.

    1988-01-01

    The muscarinic acetylcholine receptor (mAChR) mediates a variety of cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides, and modulation of potassium channels, through the action of guanine-nucleotide-binding regulatory proteins (G proteins). The question then arises as to whether multiple mAChR species exist that are responsible for the various biochemical and physiological effects. In fact, pharmacologically distinguishable forms of the mAChR occur in different tissues and have been provisionally classified into M 1 (I), M 2 cardiac (II), and M 2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. Here, the authors have made attempts to understand the molecular basis of the functional heterogeneity of the mAChR, using recombinant DNA technology

  5. Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

    Förster, C; Limmer, S; Zeidler, W; Sprinzl, M

    1994-01-01

    tRNA(Val) from Escherichia coli was aminoacylated with [1-13C]valine and its complex with Thermus thermophilus elongation factor EF-Tu.GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'- or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA. PMID:8183898

  6. Proteins that interact with GTP during sporulation of Bacillus subtilis

    Mitchell, C.; Vary, J.C.

    1989-01-01

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link [α- 32 P]GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either [α- 32 P]GTP or [γ- 32 P]GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming the GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms

  7. Induction of synaptic long-term potentiation after opioid withdrawal.

    Drdla, Ruth; Gassner, Matthias; Gingl, Ewald; Sandkühler, Jürgen

    2009-07-10

    mu-Opioid receptor (MOR) agonists represent the gold standard for the treatment of severe pain but may paradoxically also enhance pain sensitivity, that is, lead to opioid-induced hyperalgesia (OIH). We show that abrupt withdrawal from MOR agonists induces long-term potentiation (LTP) at the first synapse in pain pathways. Induction of opioid withdrawal LTP requires postsynaptic activation of heterotrimeric guanine nucleotide-binding proteins and N-methyl-d-aspartate receptors and a rise of postsynaptic calcium concentrations. In contrast, the acute depression by opioids is induced presynaptically at these synapses. Withdrawal LTP can be prevented by tapered withdrawal and shares pharmacology and signal transduction pathways with OIH. These findings provide a previously unrecognized target to selectively combat pro-nociceptive effects of opioids without compromising opioid analgesia.

  8. Lack of GNAQ and GNA11 germ-line mutations in familial melanoma pedigrees with uveal melanoma or blue nevi

    Jason Ezra Hawkes

    2013-06-01

    Full Text Available Approximately 10% of melanoma cases are familial, but only 25-40% of familial melanoma cases can be attributed to germ-line mutations in the CDKN2A - the most significant high-risk melanoma susceptibility locus identified to date. The pathogenic mutation(s in most of the remaining familial melanoma pedigrees have not yet been identified. The most common mutations in nevi and sporadic melanoma are found in BRAF and NRAS, both of which result in constitutive activation of the MAPK pathway. However, these mutations are not found in uveal melanomas or the intradermal melanocytic proliferations known as blue nevi. Rather, multiple studies report a strong association between these lesions and somatic mutations in Guanine nucleotide-binding protein G(q subunit alpha (GNAQ, Guanine nucleotide-binding protein G(q subunit alpha-11 (GNA11 and BRCA1 associated protein-1 (BAP1. Recently, germ-line mutations in BAP1, the gene encoding a tumor suppressing deubiquitinating enzyme, have been associated with predisposition to a variety of cancers including uveal melanoma, but no studies have examined the association of germ-line mutations in GNAQ and GNA11 with uveal melanoma and blue nevi. We have now done so by sequencing exon 5 of both of these genes in 13 unique familial melanoma pedigrees, members of which have had either uveal or cutaneous melanoma and/or blue nevi. Germ-line DNA from a total of 22 individuals was used for sequencing; however no deleterious mutations were detected. Nevertheless, such candidate gene studies and the discovery of novel germ-line mutations associated with an increased MM susceptibility can lead to a better understanding of the pathways involved in melanocyte transformation, formulation of risk assessment, and the development of specific drug therapies.

  9. Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

    Samuel H. Friedman

    2013-11-01

    Fragile X syndrome (FXS, the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1 gene product (FMRP, an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1 null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs: GPI-anchored glypican Dally-like protein (Dlp and transmembrane Syndecan (Sdc. Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg ligand abundance and downstream Frizzled-2 (Fz2 receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb, and downstream ERK phosphorylation (dpERK are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb and downstream signaling via phosphorylation of the transcription factor MAD (pMAD seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1 Wg and Jeb trans-synaptic signaling, and (2 synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.

  10. Topology and Oligomerization of Mono- and Oligomeric Proteins Regulate Their Half-Lives in the Cell.

    Mallik, Saurav; Kundu, Sudip

    2018-06-05

    To find additional structural constraints (besides disordered segments) that regulate protein half-life in the cell, we herein assess the influence of native topology of monomeric and sequestration of oligomeric proteins into multimeric complexes in yeast, human, and mouse. Native topology acts as a molecular marker of globular protein's mechanical resistance and consequently captures their half-life variations on genome scale. Sequestration into multimeric complexes elongates oligomeric protein half-life in the cell, presumably by burying ubiquitinoylation sites and disordered segments required for proteasomal recognition. The latter effect is stronger for proteins associated with multiple complexes and for those binding early during complex self-assembly, including proteins that oligomerize with large proportions of surface buried. After gene duplication, diversification of topology and sequestration into non-identical sets of complexes alter half-lives of paralogous proteins during the course of evolution. Thus, native topology and sequestration into multimeric complexes reflect designing principles of proteins to regulate their half-lives. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.

    Manford, Andrew G; Stefan, Christopher J; Yuan, Helen L; Macgurn, Jason A; Emr, Scott D

    2012-12-11

    Endoplasmic reticulum-plasma membrane (ER-PM) junctions are conserved structures defined as regions of the ER that tightly associate with the plasma membrane. However, little is known about the mechanisms that tether these organelles together and why such connections are maintained. Using a quantitative proteomic approach, we identified three families of ER-PM tethering proteins in yeast: Ist2 (related to mammalian TMEM16 ion channels), the tricalbins (Tcb1/2/3, orthologs of the extended synaptotagmins), and Scs2 and Scs22 (vesicle-associated membrane protein-associated proteins). Loss of all six tethering proteins results in the separation of the ER from the PM and the accumulation of cytoplasmic ER. Importantly, we find that phosphoinositide signaling is misregulated at the PM, and the unfolded protein response is constitutively activated in the ER in cells lacking ER-PM tether proteins. These results reveal critical roles for ER-PM contacts in cell signaling, organelle morphology, and ER function. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

    Ramsey, Jolene; Renzi, Emily C; Arnold, Randy J; Trinidad, Jonathan C; Mukhopadhyay, Suchetana

    2017-02-01

    Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans. Copyright © 2017 American Society for Microbiology.

  13. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

    Seth, Puneet; Yeowell, Heather N

    2010-04-01

    Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease the levels of fibrosis-associated LH2(long) mRNA in primary scleroderma cells may suggest a novel approach to strategies directed against scleroderma.

  14. Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

    Verweij, W.; Spelt, C.E.; Bliek, M.; de Vries, M.; Wit, N.; Faraco, M.; Koes, R.; Quattrocchio, F.

    2016-01-01

    The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) fromArabidopsis thalianaand associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein

  15. Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

    Knudsen, Kirsten Quyen Nguyen; Olesen, Ping; Ledet, Thomas

    2007-01-01

    The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformat......The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved...... in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL......) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were...

  16. Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.

    Molla, Muriel; Descroix, Vianney; Aïoub, Muhanad; Simon, Stéphane; Castañeda, Beatriz; Hotton, Dominique; Bolaños, Alba; Simon, Yohann; Lezot, Frédéric; Goubin, Gérard; Berdal, Ariane

    2010-11-01

    Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.

  17. Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms

    Kobune, M; Iyama, S; Kikuchi, S; Horiguchi, H; Sato, T; Murase, K; Kawano, Y; Takada, K; Ono, K; Kamihara, Y; Hayashi, T; Miyanishi, K; Sato, Y; Takimoto, R; Kato, J

    2012-01-01

    Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34 + cells, CD34 + blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34 + acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO + leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO + leukemic cell proliferation. The demethylating agent, 5-aza-2′-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells

  18. Secretory leukocyte protease inhibitor protein regulates the penetrance of frontotemporal lobar degeneration in progranulin mutation carriers.

    Ghidoni, Roberta; Flocco, Rosa; Paterlini, Anna; Glionna, Michela; Caruana, Loredana; Tonoli, Elisa; Binetti, Giuliano; Benussi, Luisa

    2014-01-01

    The discovery that mutations in the gene encoding for progranulin (GRN) cause frontotemporal lobar degeneration (FTLD) and other neurodegenerative diseases leading to dementia has brought renewed interest in progranulin and its functions in the central nervous system. Full length progranulin is preserved from cleavage by secretory leukocyte protease inhibitor (SLPI), one of the smallest serine protease inhibitor circulating in plasma. Herein, we investigated the relationship between circulating SLPI and progranulin in affected and unaffected subjects belonging to 26 Italian pedigrees carrying GRN null mutations. In GRN null mutation carriers, we demonstrated: i) an increase of circulating SLPI levels in affected subjects; ii) an age-related upregulation of the serine-protease inhibitor in response to lifetime progranulin shortage; and iii) a delay in the age of onset in subjects with the highest SLPI protein levels. The study of SLPI and its relation to progranulin suggests the existence of unexpected molecular players in progranulin-associated neurodegeneration.

  19. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-09-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

  20. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted fro...

  1. Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

    Li, Ping; Shao, Yize; Jin, Hui

    2015-01-01

    Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084

  2. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

    Loll-Krippleber, Raphael; Brown, Grant W

    2017-09-15

    mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

  3. Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

    Norifumi Shioda

    2017-10-01

    Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

  4. Deleted in breast cancer 1 (DBC1) protein regulates hepatic gluconeogenesis.

    Nin, Veronica; Chini, Claudia C S; Escande, Carlos; Capellini, Verena; Chini, Eduardo N

    2014-02-28

    Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.

  5. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  6. Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

    Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan)

    2014-09-26

    Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.

  7. A Novel Family of Cell Wall-Related Proteins Regulated Differently during the Yeast Life Cycle

    Rodríguez-Peña, José Manuel; Cid, Víctor J.; Arroyo, Javier; Nombela, César

    2000-01-01

    The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial β-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Δ crh2Δ strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1–green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle. PMID:10757808

  8. Low- and high-risk human papillomavirus E7 proteins regulate p130 differently

    Barrow-Laing, Lisa; Chen Wei; Roman, Ann

    2010-01-01

    The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.

  9. Dicer-like Proteins Regulate the Growth, Conidiation, and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

    Qiannan Wang

    2018-01-01

    Full Text Available Colletotrichum gloeosporioides from Hevea brasiliensis is the hemibiotrophic fungi which could cause anthracnose in rubber trees. Dicer like proteins (DCL were the core enzymes for generation of small RNAs. In the present study, the knocking-out mutants of two dicer like proteins encoding genes of C. gloeosporioides were constructed; and functions of two proteins were investigated. The results showed that DCL play important roles in regulating the growth, conidiation and pathogenicity of C. gloeosporioides; and there is a functional redundancy between DCL1 and DCL2. Microscopy analysis and DAB staining revealed that loss of penetration ability into the host cells, instead of the decreased growth rate, was the main cause for the impaired pathogenicity of the ΔDcl1ΔDcl2 double mutant. Proteomics analysis suggested that DCL proteins affected the expression of functional proteins to regulating multiple biological processes of C. gloeosporioides. These data lead to a better understanding of the functions of DCL proteins in regulating the development and pathogenesis of C. gloeosporioides.

  10. DNA-binding proteins regulating pIP501 transfer and replication

    Elisabeth Grohmann

    2016-08-01

    Full Text Available pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently found in clinical Enterococcus faecalis and Enterococcus faecium isolates. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual role: It acts as transcriptional repressor at the repR promoter and prevents convergent transcription of RNAIII and repR mRNA (RNAII, thereby indirectly increasing RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII. Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including filamentous streptomycetes and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter, which overlaps with the origin of transfer (oriT. The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling protein and the VirB4-like ATPase, TraE. Both proteins are actively involved in conjugative DNA transport. Moreover, the operon encodes TraN, a small cytoplasmic protein, whose specific binding to a sequence upstream of the oriT nic-site was demonstrated. TraN seems to be an effective repressor of pIP501 transfer, as conjugative transfer rates were significantly increased in an E. faecalis pIP501ΔtraN mutant.

  11. The fragile X mental retardation protein regulates tumor invasiveness-related pathways in melanoma cells.

    Zalfa, Francesca; Panasiti, Vincenzo; Carotti, Simone; Zingariello, Maria; Perrone, Giuseppe; Sancillo, Laura; Pacini, Laura; Luciani, Flavie; Roberti, Vincenzo; D'Amico, Silvia; Coppola, Rosa; Abate, Simona Osella; Rana, Rosa Alba; De Luca, Anastasia; Fiers, Mark; Melocchi, Valentina; Bianchi, Fabrizio; Farace, Maria Giulia; Achsel, Tilmann; Marine, Jean-Christophe; Morini, Sergio; Bagni, Claudia

    2017-11-16

    The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets.

  12. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    Kokusho, Ryuhei; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu

    2016-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

  13. Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

    DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

    2012-12-10

    Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

  14. Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity.

    Bringolf, Fanny; Herren, Michael; Wyss, Marianne; Vidondo, Beatriz; Langedijk, Johannes P; Zurbriggen, Andreas; Plattet, Philippe

    2017-08-15

    Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity. IMPORTANCE Despite the availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that postexposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, the lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here we combined structure-guided mutagenesis with cellular, biochemical, and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provide evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher-order oligomers necessary to promote plasma membrane-budding activity. Copyright © 2017 American Society for Microbiology.

  15. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

    Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

    2014-01-01

    Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

  16. Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli.

    Kato , J; Katayama, T

    2001-08-01

    The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replication.

  17. Neuronal sphingolipidoses: Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism.

    Sandhoff, Konrad

    2016-11-01

    Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.ac.jp; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu, E-mail: katsuma@ss.ab.a.u-tokyo.ac.jp

    2016-11-15

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

  19. A model for variable phytoplankton stoichiometry based on cell protein regulation

    J. A. Bonachela

    2013-06-01

    Full Text Available The elemental ratios of marine phytoplankton emerge from complex interactions between the biotic and abiotic components of the ocean, and reflect the plastic response of individuals to changes in their environment. The stoichiometry of phytoplankton is, thus, dynamic and dependent on the physiological state of the cell. We present a theoretical model for the dynamics of the carbon, nitrogen and phosphorus contents of a phytoplankton population. By representing the regulatory processes controlling nutrient uptake, and focusing on the relation between nutrient content and protein synthesis, our model qualitatively replicates existing experimental observations for nutrient content and ratios. The population described by our model takes up nutrients in proportions that match the input ratios for a broad range of growth conditions. In addition, there are two zones of single-nutrient limitation separated by a wide zone of co-limitation. Within the co-limitation zone, a single point can be identified where nutrients are supplied in an optimal ratio. When different species compete, the existence of a wide co-limitation zone implies a more complex pattern of coexistence and exclusion compared to previous model predictions. However, additional comprehensive laboratory experiments are needed to test our predictions. Our model contributes to the understanding of the global cycles of oceanic nitrogen and phosphorus, as well as the elemental ratios of these nutrients in phytoplankton populations.

  20. HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

    Ryan, Robert P.; Lucey, Jean; O'Donovan, Karen

    2009-01-01

    residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa. Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa, consistent with the predicted activity of the encoded......2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108, PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P...

  1. Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

    Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

    2014-03-01

    During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

  2. Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

    Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

    2016-03-01

    The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

  3. RhoG protein regulates platelet granule secretion and thrombus formation in mice.

    Goggs, Robert; Harper, Matthew T; Pope, Robert J; Savage, Joshua S; Williams, Christopher M; Mundell, Stuart J; Heesom, Kate J; Bass, Mark; Mellor, Harry; Poole, Alastair W

    2013-11-22

    Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

  4. G Protein Regulation of Neuronal Calcium Channels: Back to the Future

    Proft, Juliane; Weiss, Norbert

    2015-01-01

    Roč. 87, č. 6 (2015), s. 890-906 ISSN 0026-895X R&D Projects: GA ČR GA15-13556S Institutional support: RVO:61388963 Keywords : voltage gated calcium channels Cav * G proteins * GPCR Subject RIV: CE - Biochemistry Impact factor: 3.931, year: 2015

  5. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    Zhang, Heyu; Ma, Xi; Shi, Taiping; Song, Quansheng; Zhao, Hongshan; Ma, Dalong

    2010-01-01

    NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

  6. NPR1 protein regulates pathogenic and symbiotic interactions between Rhizobium and legumes and non-legumes.

    Smadar Peleg-Grossman

    Full Text Available BACKGROUND: Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA. SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1. METHODOLOGY/PRINCIPAL FINDINGS: We used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling. CONCLUSIONS/SIGNIFICANCE: We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes.

  7. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-01-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

  8. Fragile x mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells.

    Yuping Luo

    2010-04-01

    Full Text Available Fragile X syndrome (FXS, the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP. FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs. We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3beta. Dysregulation of GSK3beta led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.

  9. A kinetic analysis of kappa-opioid agonist binding using the selective radioligand (/sup 3/H)U69593

    Smith, J.A.; Hunter, J.C.; Hill, R.G.; Hughes, J.

    1989-07-01

    The interaction of the nonselective opioid ligand (3H)bremazocine and of the kappa-opioid (3H)U69593 with the kappa-receptor was investigated in guinea-pig cortical membranes. Each radioligand bound to a single population of high-affinity sites, although (3H)U69593 apparently recognised only 70% of those sites labelled by (3H)bremazocine. Naloxone and the kappa-selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both (3H)bremazocine and (3H)U69593. Detailed analysis of the binding of (3H)U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl-5'-imidodiphosphate (GppNHp) inhibited binding of (3H)U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of (3H)U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high-affinity complex between the kappa-receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower-affinity state of the receptor.

  10. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S. (Merck Sharp and Dohme Research Lab., Rahway, NJ (United States))

    1991-02-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

  11. Changes in expression of a functional Gi protein in cultured rat heart cells

    Allen, I.S.; Gaa, S.T.; Rogers, T.B.

    1988-01-01

    The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G i ) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for G i in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated 32 P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G i is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G i is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G i in heart

  12. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - Gi complex

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S.

    1991-01-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K d of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the α and β subunits of G i , respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects

  13. Link between D1 and D2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain

    Seeman, P.; Niznik, H.B.; Guan, H.C.; Booth, G.; Ulpian, C.

    1989-01-01

    Dopamine receptor types D 1 and D 2 can oppose enhance each other's actions for electrical, biochemical, and psychomotor effects. The authors report a D 1 -D 2 interaction in homogenized tissue as revealed by ligand binding. D 2 agonists lowered the binding of [ 3 H]raclopride to D 2 receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D 1 -selective antagonist SCH 23390 prevented the agonist-induced decrease in [ 3 H]raclopride binding to D 2 sites in the striatum but not in the anterior pituitary, which has no D 1 receptors. Conversely, a dopamine-induced reduction in the binding of [ 3 H]SCH 23390 to D 1 receptors could be prevented by the D 2 -selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D 1 -D 2 interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D 2 receptors in the high-affinity state. Thus, the D 1 -D 2 link may be mediated by guanine nucleotide-binding protein components. The link may underlie D 1 -D 2 interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata

  14. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  15. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes

    Kondo, Jiro; Westhof, Eric

    2011-01-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide–protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson–Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson–Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues. PMID:21737431

  16. Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

    Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

    1988-01-01

    The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated 32 P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32 P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice

  17. The hydrogen bonds between Arg423 and Glu472 and other key residues, Asp443, Ser477, and Pro489, are responsible for the formation and a different positioning of TNP-ATP and ATP within the nucleotide-binding site of Na(+)/K(+)-ATPase

    Lánský, Zdeněk; Kubala, Martin; Ettrich, Rüdiger; Kutý, Michal; Plášek, J.; Teisinger, Jan; Schoner, W.; Amler, Evžen

    2004-01-01

    Roč. 43, č. 26 (2004), s. 8303-8311 ISSN 0006-2960 R&D Projects: GA MŠk LN00A141; GA ČR GP206/03/D082; GA ČR GA309/02/1479; GA ČR GD305/03/H148 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100001; CEZ:MSM 111300002 Keywords : sodium pump * ATP-binding site * TNP-ATP Subject RIV: CE - Biochemistry Impact factor: 4.008, year: 2004

  18. Repair of O6-(2-chloroethyl)guanine mediates the biological effects of chloroethylnitrosoureas.

    Bodell, W J; Aida, T; Berger, M S; Rosenblum, M L

    1985-01-01

    Chloroethylnitrosoureas (CENUs) are alkylating and crosslinking agents used for the treatment of human cancer; they are both mutagenic and carcinogenic. We compared the levels of induction of sister chromatid exchanges (SCEs) and the cytotoxicity of nitrosoureas that alkylate only with CENUs. CENUs are 200-fold more cytotoxic and induce SCEs with 45-fold greater efficiency than agents that do not crosslink; therefore, crosslinking is probably the most important molecular event that leads to c...

  19. A Theoretical Study of the Mechanism of the Alkylation of Guanine by N- Nitroso Compounds.

    1992-01-01

    these chemical agents alkylate DNA, but, as yet, the precise mechanism is unknown. What is known is that the result is a DNA-mutagen adduct with an alkyl ... nitrosoureas , Singer et. al. found that about 25% of the alkylation caused by MNU was on the DNA phospate backbone while, for ENU, phosphate...sites. 1.3 Mutagenicity of N-Nitroso Compounds In early experimental work with agents which alkylate DNA, comparisons of ultraviolet absorption

  20. Theoretical study of the guanine → 6-thioguanine substitution in duplexes, triplexes, and tetraplexes

    Špačková, Naďa; Cubero, E.; Šponer, Jiří; Orozco, M.

    2004-01-01

    Roč. 126, č. 44 (2004), s. 14642-14650 ISSN 0002-7863 R&D Projects: GA MŠk LN00A016 Grant - others:European Training Mobility Program(XE) HPRI-CT-1999-00071; Ministry of Science and Technology (ES) BIO2003-06848; Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : 6-thioguanine * molecular dynamics simulations * thermodynamic integration Subject RIV: BO - Biophysics Impact factor: 6.903, year: 2004

  1. The regulatory G4 motif of the Kirsten ras (KRAS) gene is sensitive to guanine oxidation

    Cogoi, Susanna; Ferino, Annalisa; Miglietta, Giulia

    2018-01-01

    KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. W...

  2. Voltametric Determination of Adenine, Guanine and DNA Using Liquid Mercury Free Polished Silver Solid Amalgam Electrode

    Fadrná, Renata; Josypčuk, Bohdan; Fojta, Miroslav; Navrátil, Tomáš; Novotný, Ladislav

    2004-01-01

    Roč. 37, č. 3 (2004), s. 399-413 ISSN 0003-2719 R&D Projects: GA AV ČR KSK4040110 Grant - others:GIT(AR) 101/02/U111/CZ Keywords : voltammetry * DNA * polished silver solid amalgam electrode Subject RIV: CG - Electrochemistry Impact factor: 1.165, year: 2004

  3. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Di Paolo, T.; Falardeau, P.

    1987-08-31

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p < 0.001). Competition for (/sup 3/H)-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-(..beta..-..gamma..-imino)triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables.

  4. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Di Paolo, T.; Falardeau, P.

    1987-01-01

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p 3 H]-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-[β-γ-imino]triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables

  5. Theoretical study on the detailed repair of O6-methyl guanine to ...

    Living cells are constantly exposed to both exogenous. (chemical pollutants and UV radiation) and endoge- nous (the normal metabolic byproducts, especially in the oxidative deamination process) sources of DNA damaging agents.1–6 Methylating agents which lead to a wide range of DNA damage such as aging, chronic.

  6. Biocatalytic separation of N-7/N-9 guanine nucleosides

    Singh, Sunil K.; Sharma, Vivek K.; Olsen, Carl Erik

    2010-01-01

    Vorbrüggen coupling of trimethylsilylated 2-N-isobutanoylguanine with peracetylated pentofuranose derivatives generally gives inseparable N-7/N-9 glycosyl mixtures. We have shown that the two isomers can be separated biocatalytically by Novozyme-435-mediated selective deacetylation of the 5'-O-a...

  7. Effect O6-Guanine Alkylation on DNA Flexibility Studied by Comparative Molecular Dynamics Simulations

    Kara, M.; Dršata, Tomáš; Lankaš, Filip; Zacharias, M.

    2015-01-01

    Roč. 103, č. 1 (2015), s. 23-32 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA damage * DNA alkylation * DNA repair * molecular simulation * molecular dynamics simulation Subject RIV: BO - Biophysics Impact factor: 2.248, year: 2015

  8. Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2014-01-01

    In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

  9. Molecular dynamics simulations of guanine quadruplex loops: Advances and force field limitations

    Fadrná, E.; Špačková, Naďa; Štefl, R.; Koča, J.; Cheatham III, T. E.; Šponer, Jiří

    2004-01-01

    Roč. 87, č. 1 (2004), s. 227-242 ISSN 0006-3495 R&D Projects: GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : quanine quadruplex * four-thymidine loop * locally enhanced sampling Subject RIV: BO - Biophysics Impact factor: 4.585, year: 2004

  10. Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

    Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

    2018-03-05

    Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

  11. Study of Adenine and Guanine Oxidation Mechanism by Surface-Enhanced Raman Spectroelectrochemistry

    Ibanez, D.; Santidrian, Ana; Heras, A.; Kalbáč, Martin; Colina, A.

    2015-01-01

    Roč. 119, č. 15 (2015), s. 8191-8198 ISSN 1932-7447 R&D Projects: GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : nucleic- acid bases * electrochemical oxidation * silver electrode Subject RIV: CG - Electrochemistry Impact factor: 4.509, year: 2015

  12. Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: A new class of antimalarial therapeutics

    Keough, D. T.; Hocková, Dana; Holý, Antonín; Naesens, L.; Skinner-Adams, T. S.; de Jersey, J.; Guddat, L. W.

    2009-01-01

    Roč. 52, č. 14 (2009), s. 4391-4399 ISSN 0022-2623 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * phosphoribosyltransferase * enzyme inhibitors * Plasmodium falciparum Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2009

  13. Oxidative damage to guanine nucleosides following combination chemotherapy with 5-fluorouracil and oxaliplatin

    Afzal, Shoaib; Jensen, Søren Astrup; Sørensen, Jens Benn

    2011-01-01

    PURPOSE: Recent in vitro and animal studies have suggested that the cytotoxicity of 5-fluorouracil and oxaliplatin is linked to increased formation of reactive oxygen species (ROS). This prospective study was undertaken to examine the generation of oxidative stress, in 106 colorectal cancer patie...... concentrations of 8-oxoGuo and 8-oxodG and the treatment effect and the other variables. RESULTS: The analysis showed that chemotherapy increased the excretion of 8-oxoGuo and 8-oxodG around 15% (P ...

  14. Crystal structures and inhibition of Trypanosoma brucei hypoxanthine-guanine phosphoribosyltransferase

    Terán, D.; Hocková, Dana; Česnek, Michal; Zíková, Alena; Naesens, L.; Keough, D. T.; Guddat, L. W.

    2016-01-01

    Roč. 6, Oct 27 (2016), č. článku 35894. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA16-06049S; GA MŠk LL1205 Institutional support: RVO:61388963 ; RVO:60077344 Keywords : enzyme inhibitors * acyclic nucleoside phosphonates * HGPRT Subject RIV: CC - Organic Chemistry; EE - Microbiology, Virology (BC-A) Impact factor: 4.259, year: 2016 http://www.nature.com/articles/srep35894

  15. Design of Plasmodium vivax Hypoxanthine-Guanine Phosphoribosyltransferase Inhibitors as Potential Antimalarial Therapeutics

    Keough, D. T.; Rejman, Dominik; Pohl, Radek; Zborníková, Eva; Hocková, Dana; Croll, T.; Edstein, M. D.; Birrell, G. W.; Chavchich, M.; Naesens, L. M. J.; Pierens, G. K.; Brereton, I. M.; Guddat, L. W.

    2018-01-01

    Roč. 13, č. 1 (2018), s. 82-90 ISSN 1554-8929 R&D Projects: GA ČR(CZ) GA16-06049S; GA ČR GA15-11711S Institutional support: RVO:61388963 Keywords : plasmodium vivax * inhibitor * pyrrolidine nucleotide bisphosphonate * HXGPRT Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.995, year: 2016

  16. The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes

    Fojtík, Petr; Kejnovská, Iva; Vorlíčková, Michaela

    2004-01-01

    Roč. 32, č. 1 (2004), s. 298-306 ISSN 0305-1048 R&D Projects: GA ČR GA204/01/0561; GA AV ČR IAA4004201 Institutional research plan: CEZ:AV0Z5004920 Keywords : fragile X chromosome syndrom * trinucleotide repeats * DNA polymorphism Subject RIV: BO - Biophysics Impact factor: 7.260, year: 2004

  17. The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans.

    Alvarez, Francisco J; Douglas, Lois M; Rosebrock, Adam; Konopka, James B

    2008-12-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Delta mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Delta mutant are similar to the effects of inhibiting beta-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.

  18. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M

    2013-01-01

    N-MYC down-regulated-like (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  19. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Yashwanti Mudgil

    Full Text Available N-MYC down-regulated-like (NDL proteins interact with the Gβ subunit (AGB1 of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development.Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem.NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  20. N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

    2013-01-01

    Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the G? subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the presen...

  1. RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

    Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

    2015-02-01

    Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. © 2014 The Authors.

  2. PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4.

    Saavedra, Francisco; Rivera, Carlos; Rivas, Elizabeth; Merino, Paola; Garrido, Daniel; Hernández, Sergio; Forné, Ignasi; Vassias, Isabelle; Gurard-Levin, Zachary A; Alfaro, Iván E; Imhof, Axel; Almouzni, Geneviève; Loyola, Alejandra

    2017-11-16

    Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

    Sears, James C.; Broadie, Kendal

    2018-01-01

    Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis. We discuss future directions for these mechanisms, and intersecting research priorities for FMRP in glial and signaling interactions. PMID:29375303

  4. RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

    Saito, Suguru; Kawamura, Toshihiko; Higuchi, Masaya; Kobayashi, Takahiro; Yoshita-Takahashi, Manami; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Kanda, Yasuhiro; Kawamura, Hiroki; Jiang, Shuying; Naito, Makoto; Yoshizaki, Takumi; Takahashi, Masahiko; Fujii, Masahiro

    2015-05-01

    Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts.

    Xu, Wen-Xiao; Liu, Sheng-Zhi; Wu, Di; Qiao, Guo-Fen; Yan, Jinglong

    2015-01-01

    Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel.

  6. Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and β-cell apoptosis

    Berchtold, Lukas Adrian; Størling, Zenia Marian; Ortis, Fernanda

    2011-01-01

    Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease...... genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi...... knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated...

  7. Immediate early gene activity-regulated cytoskeletal-associated protein regulates estradiol-induced lordosis behavior in female rats.

    Christensen, Amy; Dewing, Phoebe; Micevych, Pavel

    2015-01-01

    Sensory feedback is an important component of any behavior, with each instance influencing subsequent activity. Female sexual receptivity is mediated both by the steroid hormone milieu and interaction with the male. We tested the influence of repeated mating on the level of sexual receptivity in ovariectomized rats treated with estradiol benzoate (EB) once every fourth day to mimic the normal phasic changes of circulating estradiol. Females were divided into two groups: naïve, which were tested for lordosis behavior once, and experienced rats, which were tested for lordosis after each EB injection. To monitor the effect of mating, the number of neurons expressing the immediate early gene activity-regulated cytoskeleton-associated protein (Arc) were counted in the mediobasal hypothalamus. Females were unreceptive following the first EB treatment, but the mating induced Arc expression. In naïve rats, each subsequent EB injection increased the levels of sexual receptivity. This ramping was not observed in experienced rats, which achieved only a moderate level of sexual receptivity. However, experienced females treated with EB and progesterone were maximally receptive and did not have Arc expression. To test whether the expression of Arc attenuated lordosis, Arc antisense oligodeoxynucleotides (asODN) were microinjected into experienced females' arcuate nuclei. Arc expression was attenuated, and the experienced EB-treated females achieved maximal sexual receptivity. These results demonstrate that Arc expression in the hypothalamus might influence future sexual receptivity and provides evidence of learning in the arcuate nucleus. The loss of Arc results in unrestrained sexual receptivity. © 2014 Wiley Periodicals, Inc.

  8. Endothelial Plasmalemma Vesicle Associated Protein regulates the homeostasis of splenic immature B cell and B1 B cells

    Elgueta, Raul; Tse, Dan; Deharvengt, Sophie J.; Luciano, Marcus R.; Carriere, Catherine; Noelle, Randolph J.; Stan, Radu V.

    2016-01-01

    Plasmalemma vesicle associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble antigens into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM+IgDlo B cells in both the spleen and peritoneal cavity. Tissue specific deletion of Plvap demonstrates that the defect is B cell extrinsic, as B cell and pan hematopoietic Plvap deletion has no effect on IgM+IgDlo B cell numbers. Endothelial specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype whereas endothelial specific reconstitution of Plvap under the Chd5 promoter rescues the IgM+IgDlo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM+ B cells in the spleen and peritoneal cavity. PMID:27742829

  9. Endothelial Plasmalemma Vesicle-Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells.

    Elgueta, Raul; Tse, Dan; Deharvengt, Sophie J; Luciano, Marcus R; Carriere, Catherine; Noelle, Randolph J; Stan, Radu V

    2016-11-15

    Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM + IgD lo B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM + IgD lo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM + IgD lo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM + B cells in the spleen and peritoneal cavity. Copyright © 2016 by The American Association of Immunologists, Inc.

  10. Identification of proteins regulated by ferulic acid in a middle cerebral artery occlusion animal model-a proteomics approach.

    Sung, Jin-Hee; Cho, Eun-Hae; Cho, Jae-Hyeon; Won, Chung-Kil; Kim, Myeong-Ok; Koh, Phil-Ok

    2012-11-01

    Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

  11. MSH3 mismatch repair protein regulates sensitivity to cytotoxic drugs and a histone deacetylase inhibitor in human colon carcinoma cells.

    Jae Myung Park

    Full Text Available MSH3 is a DNA mismatch repair (MMR gene that undergoes frequent somatic mutation in colorectal cancers (CRCs with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.We utilized isogenic HCT116 (MLH1-/MSH3- cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3 and also MSH3 by chromosome 5 (HCT116+3+5. We generated HCT116+3+5, SW480 (MLH1+/MSH3+ and SW48 (MLH1-/MSH3+ cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU, SN-38, oxaliplatin, or the histone deacetylase (HDAC inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB repair. We then utilized PCI-24781 that interferes with homologous recombination (HR indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory.

  12. ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential

    Carcamo-Orive, Ivan; Tejados, Naiara; Delgado, Jesus; Gaztelumendi, Ainhoa; Otaegui, David; Lang, Valerie; Trigueros, Cesar

    2008-01-01

    Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes

  13. The Tomato Hybrid Proline-Rich Protein regulates the abcission zone competence to respond to ethylene signals

    The Tomato Hybrid Proline-Rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4,significantly inh...

  14. Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages*

    Ben, Jingjing; Zhang, Yan; Zhou, Rongmei; Zhang, Haiyang; Zhu, Xudong; Li, Xiaoyu; Zhang, Hanwen; Li, Nan; Zhou, Xiaodan; Bai, Hui; Yang, Qing; Li, Donghai; Xu, Yong; Chen, Qi

    2013-01-01

    Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis. PMID:23703615

  15. MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

    Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

    2018-02-02

    Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

  16. Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

    Berzina, Zane; Solanko, Lukasz M; Mehadi, Ahmed S

    2018-01-01

    /LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous...... but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM...

  17. Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress.

    Garaigorta, Urtzi; Heim, Markus H; Boyd, Bryan; Wieland, Stefan; Chisari, Francis V

    2012-10-01

    Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

  18. The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

    Labrecque, Mark P.; Takhar, Mandeep K.; Nason, Rebecca; Santacruz, Stephanie; Tam, Kevin J.; Massah, Shabnam; Haegert, Anne; Bell, Robert H.; Altamirano-Dimas, Manuel; Collins, Colin C.; Lee, Frank J.S.; Prefontaine, Gratien G.; Cox, Michael E.; Beischlag, Timothy V.

    2016-01-01

    Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. PMID:27015368

  19. Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis

    van der Horst, M.A.; Stalcup, T.P.; Kaledhonkar, S.; Kumauchi, M.; Hara, M.; Xie, A.; Hellingwerf, K.J.; Hoff, W.D.

    2009-01-01

    Idiomarina loihiensis is a heterotrophic deep sea bacterium with no known photobiology. We show that light suppresses biofilm formation in this organism. The genome of I. loihiensis encodes a single photoreceptor protein: a homologue of photoactive yellow protein (PYP), a blue light receptor with

  20. Analysis of the embryo proteome of sycamore (Acer pseudoplatanus L.) seeds reveals a distinct class of proteins regulating dormancy release.

    Pawłowski, Tomasz Andrzej; Staszak, Aleksandra Maria

    2016-05-20

    Acer pseudoplatanus seeds are characterized by a deep physiological embryo dormancy that requires a few weeks of cold stratification in order to promote germination. Understanding the function of proteins and their related metabolic pathways, in conjunction with the plant hormones implicated in the breaking of seed dormancy, would expand our knowledge pertaining to this process. In this study, a proteomic approach was used to analyze the changes occurring in seeds in response to cold stratification, which leads to dormancy release. In addition, the involvement of abscisic (ABA) and gibberellic acids (GA) was also examined. Fifty-three proteins showing significant changes were identified by mass spectrometry. An effect of ABA on protein variation was observed at the beginning of stratification, while the influence of GA on protein abundance was observed during the middle phase of stratification. The majority of proteins associated with dormancy breaking in the presence of only water, and also ABA or GA, were classified as being involved in metabolism and genetic information processing. For metabolic-related proteins, the effect of ABA on protein abundance was stimulatory for half of the proteins and inhibitory for half of the proteins. On the other hand, the effect on genetic information processing related proteins was stimulatory. GA was found to upregulate both metabolic-related and genetic information processing-related proteins. While seed dormancy breaking depends on proteins involved in a variety of processes, proteins associated with methionine metabolism (adenosine kinase, methionine synthase) and glycine-rich RNA binding proteins appear to be of particular importance. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

    Mitsios, Nick; Saka, Mohamad; Krupinski, Jerzy; Pennucci, Roberta; Sanfeliu, Coral; Wang, Qiuyu; Rubio, Francisco; Gaffney, John; Kumar, Pat; Kumar, Shant; Sullivan, Matthew; Slevin, Mark

    2007-01-01

    Background Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. Results Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. Conclusion Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents. PMID:17997827

  2. Caenorhabditis elegans VEM-1, a novel membrane protein, regulates the guidance of ventral nerve cord-associated axons.

    Runko, Erik; Kaprielian, Zaven

    2004-10-13

    In the developing CNS, pathfinding growth cones use intermediate target- and pioneer axon-associated guidance cues to navigate along stereotypical trajectories. We previously showed that the novel membrane-associated protein Vema is localized to the floor plate and the optic chiasm, intermediate targets located at the ventral midline of the spinal cord and diencephalon in the developing rodent CNS, respectively. Here, we report that the Caenorhabditis elegans ortholog of vema, vem-1, is expressed by the AVG pioneer midline neuron and by several neurons that extend longitudinally projecting axons into the ventral nerve cord (VNC). In vem-1 mutants and vem-1 (RNAi) animals, a subset of posteriorly projecting interneuron axons either fail to extend ventrally to the VNC and, instead, assume aberrant lateral positions or are inappropriately located in the left tract of the VNC. In addition, ventral motor neuron axons exhibit pathfinding errors within the VNC and along the dorsoventral body axis. The conserved UNC-40/DCC and SAX-3-/Robo receptors mediate signaling events that regulate axon guidance in a wide variety of systems. Double-mutant analyses reveal that vem-1 genetically interacts with unc-40 and is likely to function in parallel with sax-3 to regulate the guidance of a subset of VNC-associated interneuron and motor neuron axons. Consistent with these genetic data, we also show that VEM-1 is capable of physically interacting with UNC-40 but not SAX-3.

  3. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Kim, Hyun-Suk; Guo, Chunlu; Thompson, Eric L.; Jiang, Yanlin; Kelley, Mark R.; Vasko, Michael R.; Lee, Suk-Hee

    2015-01-01

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons

  4. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Kim, Hyun-Suk [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Guo, Chunlu; Thompson, Eric L. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Jiang, Yanlin [Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Lee, Suk-Hee, E-mail: slee@iu.edu [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States)

    2015-09-15

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

  5. Transduced PEP-1-PON1 proteins regulate microglial activation and dopaminergic neuronal death in a Parkinson's disease model.

    Kim, Mi Jin; Park, Meeyoung; Kim, Dae Won; Shin, Min Jea; Son, Ora; Jo, Hyo Sang; Yeo, Hyeon Ji; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Kim, Duk-Soo; Kwon, Oh-Shin; Kim, Joon; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2015-09-01

    Parkinson's disease (PD) is an oxidative stress-mediated neurodegenerative disorder caused by selective dopaminergic neuronal death in the midbrain substantia nigra. Paraoxonase 1 (PON1) is a potent inhibitor of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) against oxidation by destroying biologically active phospholipids with potential protective effects against oxidative stress-induced inflammatory disorders. In a previous study, we constructed protein transduction domain (PTD) fusion PEP-1-PON1 protein to transduce PON1 into cells and tissue. In this study, we examined the role of transduced PEP-1-PON1 protein in repressing oxidative stress-mediated inflammatory response in microglial BV2 cells after exposure to lipopolysaccharide (LPS). Moreover, we identified the functions of transduced PEP-1-PON1 proteins which include, mitigating mitochondrial damage, decreasing reactive oxidative species (ROS) production, matrix metalloproteinase-9 (MMP-9) expression and protecting against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in SH-SY5Y cells. Furthermore, transduced PEP-1-PON1 protein reduced MMP-9 expression and protected against dopaminergic neuronal cell death in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. Taken together, these results suggest a promising therapeutic application of PEP-1-PON1 proteins against PD and other inflammation and oxidative stress-related neuronal diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

    Sebastian, J.; Sancar, G.B.

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

  7. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    Holowachuk, Eugene W.

    2007-01-01

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3β inhibitors (Li + or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNFα-induced rates of lipolysis by 50%. Adipocytes preincubated with Li + or TZDZ-8 prior to CsA and/or TNFα, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPARγ, ACS and Adn), compared with control or TNFα-treatment, whereas Li + pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPARγ, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li + treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis

  8. CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Maciel, Renata de Moraes; da Costa, Rodrigo Furtado Madeiro; Furtado, Daniel Rodrigues; de Oliveira, Francisco Meirelles Bastos; da Silva-Neto, Mário Alberto Cardoso; Rumjanek, Franklin David; Fantappié, Marcelo Rosado

    2011-01-01

    BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding t...

  9. CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Maciel, Renata de Moraes; da Costa, Rodrigo Furtado Madeiro; Furtado, Daniel Rodrigues; de Oliveira, Francisco Meirelles Bastos; da Silva-Neto, Mário Alberto Cardoso; Rumjanek, Franklin David; Fantappié, Marcelo Rosado

    2011-01-01

    The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

  10. DISC1 Protein Regulates γ-Aminobutyric Acid, Type A (GABAA) Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons.

    Wei, Jing; Graziane, Nicholas M; Gu, Zhenglin; Yan, Zhen

    2015-11-13

    Association studies have suggested that Disrupted-in-Schizophrenia 1 (DISC1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress in understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Because alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABAA receptor (GABAAR). We found that cellular knockdown of DISC1 significantly reduced GABAAR-mediated synaptic and whole-cell current, whereas overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABAAR currents in pyramidal neurons of the prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABAAR expression. Moreover, the regulation of GABAARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin 1 (KIF5). Our results suggest that DISC1 exerts an important effect on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABAAR trafficking in the cortex. The knowledge gained from this study would shed light on how DISC1 and the GABA system are linked mechanistically and how their interactions are critical for maintaining a normal mental state. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

    Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam; Konopka, James B.

    2008-01-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenes...

  12. The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

    Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam

    2008-01-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains. PMID:18799621

  13. CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

    Isabel Caetano de Abreu da Silva

    Full Text Available BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1, a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1 is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

  14. Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

    Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

    2014-11-01

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity.

    Ueno, Akiko; Omori, Yoshihiro; Sugita, Yuko; Watanabe, Satoshi; Chaya, Taro; Kozuka, Takashi; Kon, Tetsuo; Yoshida, Satoyo; Matsushita, Kenji; Kuwahara, Ryusuke; Kajimura, Naoko; Okada, Yasushi; Furukawa, Takahisa

    2018-03-27

    In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity

    Akiko Ueno

    2018-03-01

    Full Text Available Summary: In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. : Ueno et al. finds that Lrit1 plays an important role in regulating the synaptic connection between cone photoreceptors and cone ON-bipolar cells. The Frmpd2-Lrit1-mGluR6 axis is crucial for selective synapse formation in cone photoreceptors and for development of normal visual function. Keywords: retina, circuit, synapse formation, cone photoreceptor cell, ON-bipolar cell, visual acuity

  17. Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar

    Houeix Benoit

    2010-03-01

    Full Text Available Abstract Background The male Atlantic salmon generally matures in fresh water upon returning after one or several years at sea. Some fast-growing male parr develop an alternative life strategy where they sexually mature before migrating to the oceans. These so called 'precocious' parr or 'sneakers' can successfully fertilise adult female eggs and so perpetuate their line. We have used a custom-built cDNA microarray to investigate gene expression changes occurring in the salmon gonad and brain associated with precocious maturation. The microarray has been populated with genes selected specifically for involvement in sexual maturation (precocious and adult and in the parr-smolt transformation. Results Immature and mature parr collected from a hatchery-reared stock in January were significantly different in weight, length and condition factor. Changes in brain expression were small - never more than 2-fold on the microarray, and down-regulation of genes was much more pronounced than up-regulation. Significantly changing genes included isotocin, vasotocin, cathepsin D, anamorsin and apolipoprotein E. Much greater changes in expression were seen in the testes. Among those genes in the testis with the most significant changes in expression were anti-Mullerian hormone, collagen 1A, and zinc finger protein (Zic1, which were down-regulated in precocity and apolipoproteins E and C-1, lipoprotein lipase and anti-leukoproteinase precursor which were up-regulated in precocity. Expression changes of several genes were confirmed in individual fish by quantitative PCR and several genes (anti-Mullerian hormone, collagen 1A, beta-globin and guanine nucleotide binding protein (G protein beta polypeptide 2-like 1 (GNB2L1 were also examined in adult maturing testes. Down-regulation of anti-Mullerian hormone was judged to be greater than 160-fold for precocious males and greater than 230-fold for November adult testes in comparison to July testes by this method. For

  18. Link between D sub 1 and D sub 2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain

    Seeman, P.; Niznik, H.B.; Guan, H.C.; Booth, G.; Ulpian, C. (Univ. of Toronto (Canada))

    1989-12-01

    Dopamine receptor types D{sub 1} and D{sub 2} can oppose enhance each other's actions for electrical, biochemical, and psychomotor effects. The authors report a D{sub 1}-D{sub 2} interaction in homogenized tissue as revealed by ligand binding. D{sub 2} agonists lowered the binding of ({sup 3}H)raclopride to D{sub 2} receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D{sub 1}-selective antagonist SCH 23390 prevented the agonist-induced decrease in ({sup 3}H)raclopride binding to D{sub 2} sites in the striatum but not in the anterior pituitary, which has no D{sub 1} receptors. Conversely, a dopamine-induced reduction in the binding of ({sup 3}H)SCH 23390 to D{sub 1} receptors could be prevented by the D{sub 2}-selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D{sub 1}-D{sub 2} interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D{sub 2} receptors in the high-affinity state. Thus, the D{sub 1}-D{sub 2} link may be mediated by guanine nucleotide-binding protein components. The link may underlie D{sub 1}-D{sub 2} interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata.

  19. Thermodynamics of the GTP-GDP-operated conformational switch of selenocysteine-specific translation factor SelB.

    Paleskava, Alena; Konevega, Andrey L; Rodnina, Marina V

    2012-08-10

    SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

  20. Maintenance of neural activities in torpid Rhinolophus ferrumequinum bats revealed by 2D gel-based proteome analysis.

    Yin, Qiuyuan; Zhang, Yijian; Dong, Dong; Lei, Ming; Zhang, Shuyi; Liao, Chen-Chung; Pan, Yi-Hsuan

    2017-08-01

    Bats are the only mammals capable of self-powered flying. Many bat species hibernate in winter. A reversible control of cerebral activities is critical for bats to accommodate a repeated torpor-arousal cycle during hibernation. Little is known about the molecular mechanisms that regulate neuronal activities in torpid bats. In this study, Rhinolophus ferrumequinum bat brain proteins were fractionated, and their abundance in active and torpid states was compared. Results of 2D gel-based proteomics showed that 38% of identified proteins with a significant change in abundance are involved in synaptic vesicle recycling and cytoskeletal integrity. Changes in the abundance of proteins related to RNA splicing, proteostasis, redox homeostasis, mitochondrial function, and energy metabolism were also detected. In addition, the levels of GNAO1 (guanine nucleotide-binding protein G αo subunit), an important modulator of neuronal transmembrane signaling, were significantly increased in the insoluble protein fraction of torpid bats; this may be due to GNAO1 palmitoylation making it insoluble. Our data provide molecular evidence for the maintenance of neuronal activities in torpid bats and suggest that a reversible palmitoylation of the G protein plays a role in the regulation of neuronal activities during bat hibernation. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Cloning and expression of a human kidney cDNA for an α2-adrenergic receptor subtype

    Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1988-01-01

    An α 2 -adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet α 2 -adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet α 2 -adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the α 2 -adrenergic ligand [ 3 H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the α 2 B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet α 2 -adrenergic receptor (α 2 A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective α-adrenergic ligands

  2. Reconstitution of high-affinity opioid agonist binding in brain membranes

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  3. Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

    Ihnatko, R; Post, C; Blomqvist, A

    2013-10-01

    Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

  4. The Rac1 hypervariable region in targeting and signaling

    Lam, B. Daniel; Hordijk, Peter L.

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control subcellullar localization. The HVR encodes in many small GTPases a polybasic region (PBR) that permits charge-mediated association to the inner leaflet of the plasma membrane or to intracellular organelles. Over the past 15–20 years, evidence has accumulated for specific protein–protein interactions, mediated by the HVR, that control both targeting and signaling specificity of small GTPases. Using the RhoGTPase Rac1 as a paradigm we here review a series of protein partners that require the Rac1 HVR for association and that control various aspects of localized Rac1 signaling. Some of these proteins represent Rac1 activators, whereas others mediate Rac1 inactivation and degradation and yet others potentiate Rac1 downstream signaling. Finally, evidence is discussed which shows that the HVR of Rac1 also contributes to effector interactions, co-operating with the N-terminal effector domain. The complexity of localized Rac1 signaling, reviewed here, is most likely exemplary for many other small GTPases as well, representing a challenge to identify and define similar mechanisms controlling the specific signaling induced by small GTPases. PMID:23354415

  5. Retinoic acid-induced granulocytic differentiation of HL60 human promyelocytic leukemia cells is preceded by downregulation of autonomous generation of inositol lipid-derived second messengers

    Porfiri, E.; Hoffbrand, A.V.; Wickremasinghe, R.G.

    1991-01-01

    Inositol phosphates (InsPs) and diacyglycerol (DAG) are second messengers derived via the breakdown of inositol phospholipids, and which play important signalling roles in the regulation of proliferation of some cell types. The authors have studied the operation of this pathway during the early stages of retionic acid (RA)-induced granulocytic differentiation of HL60 myeloid leukemia cells. The autonomous breakdown of inositol lipids that occurred in HL60 cells labeled with [3H] inositol was completely abolished following 48 hours of RA treatment. The rate of influx of 45Ca2+ was also significantly decreased at 48 hours, consistent with the role of inositol lipid-derived second messengers in regulating Ca2+ entry into cells. The downregulation of inositol lipid metabolism clearly preceded the onset of reduced proliferation induced by RA treatment, and was therefore not a consequence of decreased cell growth. The generation of InsPs in RA-treated cells was reactivated by the fluoroaluminate ion, a direct activator of guanine nucleotide-binding protein(s) (G proteins) that regulate the inositol lipid signalling pathway. Subtle alterations to a regulatory mechanism may therefore mediate the RA-induced downregulation of this pathway. The data are consistent with the hypothesis that the autonomous generation of inositol lipid-derived second messengers may contribute to the continuous proliferation of HL60 cells, and that the RA-induced downregulation of this pathway may, in turn, play a role in signalling the cessation of proliferation that preceedes granulocytic differentiation

  6. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  7. Identification of a GTP-binding protein α subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-01-01

    Recent molecular cloning of cDNA for the α subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein α subunit, which they refer to as G/sub z/α, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ α-subunit cDNA. The deduced amino acid sequence of G/sub z/α is 41-67% identical with those of other known G-protein α subunits. However, the 355-residue G/sub z/α lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein α subunits. They suggest that G/sub z/α, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems

  8. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-01-01

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G i α) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [ 35 S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  9. G-protein α-subunit expression, myristoylation, and membrane association in COS cells

    Mumby, S.M.; Gilman, A.G.; Heukeroth, R.O.; Gordon, J.I.

    1990-01-01

    Myristolyation of seven different α subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that [ 3 H]myristate was incorporated into α i1 , α i2 , α i3 , α 0 , and α 1 , and α z but not α s subunits. The role of myristoylation in the association of α subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of α 0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated α subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein α subunits dissociated from βγ

  10. G protein in stimulation of PI hydrolysis by CCK [cholecystokinin] in isolated rat pancreatic acinar cells

    Matozaki, Takashi; Sakamoto, Choitsu; Nagao, Munehiko; Nishizaki, Hogara; Baba, Shigeaki

    1988-01-01

    To clarify the possible role of a guanine nucleotide-binding protein (G protein) in the signal transducing system activated by cholecystokinin (CCK), actions of CCK on rat pancreatic acini were compared with those of fluoride, a well-known activator of stimulatory (G s ) or inhibitory (G i ) G protein. When acini were incubated with increasing concentrations of either CCK-octapeptide (CCK8) or NaF, a maximal stimulation of amylase release from acini occurred at 100 pM CCK8 or 10 mM NaF, respectively; this secretory rate decreased as CCK8 or NaF concentration was increased. NaF caused an increase in cytoplasmic Ca 2+ concentration from the internal Ca 2+ store and stimulated accumulation of inositol phosphates in acini, as observed with CCK. Guanylimidodiphosphate activated the generation of inositol phosphates in the [ 3 H]inositol-labeled pancreatic acinar cell membrane preparation, with half-maximal and maximal stimulation at 1 and 10 μM, respectively. Furthermore, the effects of submaximal CCK concentrations on inositol phosphate accumulation in membranes were markedly potentiated in the presence of 100 μM GTP, which alone was ineffective. Combined findings of the present study strongly suggest that pancreatic CCK receptors are probably coupled to the activation of polyphosphoinositide (PI) breakdown by a G protein, which appears to be fluoride sensitive but is other than G s - or G i -like protein

  11. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum

    Inouye, Minoru; Yamamura, Hideki; Nakano, Atsuhiro.

    1995-01-01

    Proliferating cells of the external granular layer (EGL) in the developing cerebellum are highly sensitive to ionizing radiation. We examined the effect of lithium, an inhibitor of intracellular signaling, on the manifestation of radiation-induced apoptosis. Newborn mice were exposed to 0.5 Gy gamma-irradiation alone, or first were treated with lithium (10 μmol/g, SC) then given 0.5 Gy irradiation 2 hr later. The EGL was examined histologically for apoptosis at various times after treatment. Apoptotic cells increased rapidly, peaked (about 14%) 6 hr after irradiation, then decreased gradually to the control level by 24 hr. Prior treatment with lithium delayed the manifestation of apoptosis, the peak appearing at 12 hr. The disappearance of dead cells was delayed for about one day. The lithium concentration in the whole brain increased rapidly, being 30 μg/g at the time of irradiation and remaining at more than 40 μg/g for 40 hr. Lithium is reported to inhibit guanine-nucleotide binding to G proteins as well as phosphoinositide turnover. Of the variety of lesions induced by radiation, DNA double strand breaks are the most important source of cell lethality. The present findings, however, suggest that cyclic AMP-mediated and/or phosphoinositide-mediated signaling systems regulate radiation-induced apoptosis. (author)

  12. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum.

    Inouye, M; Yamamura, H; Nakano, A

    1995-09-01

    Proliferating cells of the external granular layer (EGL) in the developing cerebellum are highly sensitive to ionizing radiation. We examined the effect of lithium, an inhibitor of intracellular signaling, on the manifestation of radiation-induced apoptosis. Newborn mice were exposed to 0.5 Gy gamma-irradiation alone, or first were treated with lithium (10 mumol/g, SC) then given 0.5 Gy irradiation 2 hr later. The EGL was examined histologically for apoptosis at various times after treatment. Apoptotic cells increased rapidly, peaked (about 14%) 6 hr after irradiation, then decreased gradually to the control level by 24 hr. Prior treatment with lithium delayed the manifestation of apoptosis, the peak appearing at 12 hr. The disappearance of dead cells was delayed for about one day. The lithium concentration in the whole brain increased rapidly, being 30 micrograms/g at the time of irradiation and remaining at more than 40 micrograms/g for 40 hr. Lithium is reported to inhibit guanine-nucleotide binding to G proteins as well as phosphoinositide turnover. Of the variety of lesions induced by radiation, DNA double strand breaks are the most important source of cell lethality. The present findings, however, suggest that cyclic AMP-mediated and/or phosphoinositidemediated signaling systems regulate radiation-induced apoptosis.

  13. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum

    Inouye, Minoru; Yamamura, Hideki [Nagoya Univ. (Japan). Research Inst. of Environmental Medicine; Nakano, Atsuhiro

    1995-09-01

    Proliferating cells of the external granular layer (EGL) in the developing cerebellum are highly sensitive to ionizing radiation. We examined the effect of lithium, an inhibitor of intracellular signaling, on the manifestation of radiation-induced apoptosis. Newborn mice were exposed to 0.5 Gy gamma-irradiation alone, or first were treated with lithium (10 {mu}mol/g, SC) then given 0.5 Gy irradiation 2 hr later. The EGL was examined histologically for apoptosis at various times after treatment. Apoptotic cells increased rapidly, peaked (about 14%) 6 hr after irradiation, then decreased gradually to the control level by 24 hr. Prior treatment with lithium delayed the manifestation of apoptosis, the peak appearing at 12 hr. The disappearance of dead cells was delayed for about one day. The lithium concentration in the whole brain increased rapidly, being 30 {mu}g/g at the time of irradiation and remaining at more than 40 {mu}g/g for 40 hr. Lithium is reported to inhibit guanine-nucleotide binding to G proteins as well as phosphoinositide turnover. Of the variety of lesions induced by radiation, DNA double strand breaks are the most important source of cell lethality. The present findings, however, suggest that cyclic AMP-mediated and/or phosphoinositide-mediated signaling systems regulate radiation-induced apoptosis. (author).

  14. CNGA3 mutations in two United Arab Emirates families with achromatopsia.

    Ahuja, Yachna; Kohl, Susanne; Traboulsi, Elias I

    2008-07-10

    ACHROMATOPSIA RESULTS FROM MUTATIONS IN ONE OF THREE GENES: cyclic nucleotide-gated channel, alpha-3 (CNGA3); cyclic nucleotide-gated channel, beta-3 (CNGB3); and guanine nucleotide-binding protein, alpha-transducing activity polypeptide 2 (GNAT2). We report the responsible mutations in two United Arab Emirates families who have this autosomal recessive disease. Clinical examinations were performed in seven patients from three nuclear families. Molecular genetic testing for common CNGA3 and CNGB3 mutations was undertaken using standard protocols. All patients were extremely light sensitive and had reduced visual acuity and no color perception. Fundus examinations did not show any visible abnormalities. After further pedigree analysis, two of the families were found to be linked through the paternal line. Two mutations in CNGA3 were identified: Arg283Trp and Gly397Val. Family A, the larger pedigree, had one branch in which two sisters and one brother were homozygous for the Gly397Val mutation and another branch in which a brother and sister were compound heterozygous for both aforenamed mutations. Family B, however, only had two brothers who were homozygous for the Arg283Trp mutation. Achromatopsia in these two United Arab Emirates families results from two different mutations in CNGA3. Two branches of the same pedigree had individuals with both homozygous and compound heterozygous disease, demonstrating a complex molecular pathology in this large family.

  15. Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approach

    Zupancic, Klemen; Blejec, Andrej; Herman, Ana; Veber, Matija; Verbovsek, Urska; Korsic, Marjan; Knezevic, Miomir; Rozman, Primoz; Turnsek, Tamara Lah; Gruden, Kristina; Motaln, Helena

    2014-01-01

    Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients. We used a commercially available antibody array that includes 656 antibodies to analyse blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with GBM. We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM. These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM. Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients

  16. Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-01-01

    Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

  17. Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

    Ecay, T.W.; Valentich, J.D.

    1991-01-01

    Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3 H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells

  18. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  19. TC10 is regulated by caveolin in 3T3-L1 adipocytes.

    Dave Bridges

    Full Text Available TC10 is a small GTPase found in lipid raft microdomains of adipocytes. The protein undergoes activation in response to insulin, and plays a key role in the regulation of glucose uptake by the hormone.TC10 requires high concentrations of magnesium in order to stabilize guanine nucleotide binding. Kinetic analysis of this process revealed that magnesium acutely decreased the nucleotide release and exchange rates of TC10, suggesting that the G protein may behave as a rapidly exchanging, and therefore active protein in vivo. However, in adipocytes, the activity of TC10 is not constitutive, indicating that mechanisms must exist to maintain the G protein in a low activity state in untreated cells. Thus, we searched for proteins that might bind to and stabilize TC10 in the inactive state. We found that Caveolin interacts with TC10 only when GDP-bound and stabilizes GDP binding. Moreover, knockdown of Caveolin 1 in 3T3-L1 adipocytes increased the basal activity state of TC10.Together these data suggest that TC10 is intrinsically active in vivo, but is maintained in the inactive state by binding to Caveolin 1 in 3T3-L1 adipocytes under basal conditions, permitting its activation by insulin.

  20. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  1. PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana.

    Blenau, Wolfgang; Balfanz, Sabine; Baumann, Arnd

    2017-10-30

    The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP] i ) whereas type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . Here; we report that the American cockroach ( Periplaneta americana ) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP] i . Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana ; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

  2. PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana

    Wolfgang Blenau

    2017-10-01

    Full Text Available The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G protein-coupled receptors (GPCRs. Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana expresses a second type 1 tyramine receptor (PeaTAR1B in addition to PeaTAR1A (previously called PeaTYR1. When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

  3. Association between genetic variants of the ADD1 and GNB3 genes and blood pressure response to the cold pressor test in a Chinese Han population: the GenSalt Study.

    Wang, Laiyuan; Chen, Shufeng; Zhao, Qi; Hixson, James E; Rao, Dabeeru C; Jaquish, Cashell E; Huang, Jianfeng; Lu, Xiangfeng; Chen, Jichun; Cao, Jie; Li, Jianxin; Li, Hongfan; He, Jiang; Liu, De-Pei; Gu, Dongfeng

    2012-08-01

    Genetic factors influence blood pressure (BP) response to the cold pressor test (CPT), which is a phenotype related to hypertension risk. We examined the association between variants of the α-adducin (ADD1) and guanine nucleotide binding protein (G protein) β-polypeptide 3 (GNB3) genes and BP response to the CPT. A total of 1998 Han Chinese participants from the Genetic Epidemiology Network of Salt Sensitivity completed the CPT. The area under the curve (AUC) above the baseline BP during the CPT was used to measure the BP response. Twelve single-nucleotide polymorphisms (SNPs) of the ADD1 and GNB3 genes were selected and genotyped. Both single-marker and haplotype association analyses were conducted using linear mixed models. The rs17833172 and rs3775067 SNPs of the ADD1 gene and the rs4963516 SNP of the GNB3 gene were significantly associated with the BP response to CPT, even after adjusting for multiple testing. For the ADD1 gene, the AA genotype of SNP rs17833172 was associated with lower systolic BP (SBP) reactivity (PAUC (P=0.003). Haplotype analysis indicated that the CCGC haplotype of ADD1 constructed by rs1263359, rs3775067, rs4961 and rs4963 was significantly associated with the BP response to CPT. These data suggest that genetic variants of the ADD1 and GNB3 genes may have important roles in BP response to the CPT. Future studies aimed at replicating these novel findings are warranted.

  4. Targeting GTPases in Parkinson’s disease: comparison to the historic path of kinase drug discovery and perspectives

    LIN eHONG

    2014-06-01

    Full Text Available Neurological diseases have placed heavy social and financial burdens on modern society. As the life expectancy of humans is extended, neurological diseases, such as Parkinson’s disease, have become increasingly common among senior populations. Although the enigmas of Parkinson’s diseases await resolution, more vivid pictures on the cause, progression and control of the illness are emerging after years of research. On the molecular level, GTPases are implicated in the etiology of Parkinson’s disease and are rational pharmaceutical targets for their control. However, targeting individual GTPases, which belong to a superfamily of proteins containing multiple members with a conserved guanine nucleotide binding domain, has proven to be challenging. In contrast, pharmaceutical pursuit of inhibition of kinases, which constitute another superfamily of proteins with more than 500 members, has been fairly successful. We reviewed the breakthroughs in the history of kinase drug discovery to provide guidance for the GTPase field. We summarize recent progress made in the regulation of GTPase activity. We also present an efficient and cost effective approach to drug screening, which uses multiplex flow cytometry and mixture-based positional scanning libraries. These methods allow simultaneous measurements of both the activity and the selectivity of the screened library. Several GTPase activator clusters were identified which showed selectivity against different GTPase subfamilies. While the clusters need to be further deconvoluted to identify individual active compounds, the method described here and the structure information gathered create a foundation for further developments to build upon.

  5. Purification and characterization of the V1 vasopressin receptor from rat liver

    Fishman, J.B.; Dickey, B.F.; Attisano, C.; Fine, R.E.

    1987-01-01

    The rat liver V1 vasopressin receptor was purified approximately 21,000-fold from rat liver microsomes. The receptor was solubilized from membranes using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate). Since the V1 receptor loses its ability to bind ligand when solubilized, the authors devised a liposome reconstitution system to assay vasopressin binding activity during purification. The purified receptor exhibits a K/sub d/ of 6 nm, when, prior to solubilization, the membranes were exposed to 1 μm vasopressin. This resulted in the association of a pertussis-toxin insensitive guanine-nucleotide binding protein with the receptor during most of the purification procedure. The authors are further characterizing the V1-associated G-proteins. In the absence of this association, the receptor has a K/sub d/ of 30 nM. Crosslinking of 125 I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under non-reducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g., bradykinin, angiotensin II) and perhaps their associated G-proteins as well

  6. Functional somatostatin receptors on a rat pancreatic acinar cell line

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A.

    1988-01-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125 I-[Tyr 11 ]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/10 6 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125 I-[Tyr 11 ]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N i to inhibit adenylate cyclase

  7. Assay of cyclic nucleotide phosphodiesterase using radiolabeled and fluorescent substrates

    Kincaid, R.L.; Manganiello, V.C.

    1988-01-01

    There are four major classes of phosphodiesterase with different specificities for cAMP and cGMP and different allosteric regulators. Type I phosphodiesterase is activated by calmodulin plus Ca/sup 2+/ and has a higher affinity for cGMP than cAMP. Type II phosphodiesterase likewise has a higher affinity for cGMP than cAMP, but the activity toward one substrate is markedly stimulated by low (micromolar) concentrations of the other nucleotide. Type III phosphodiesterase has a higher affinity for cAMP than cGMP; its activity is increased in responsive cells by certain hormones, e.g., insulin, isoproterenol. Type IV phosphodiesterase is the cGMP-specific enzyme, which also has an allosteric binding site for cGMP. An example of this class of enzyme is the one from retinal rod outer segments, which is activated by light via rhodopsin and the guanine nucleotide-binding protein transducin. There appears to be little structural relatedness among these enzymes based on immunologic analysis, consistent with the possibility that divergent forms evolved from an ancestral enzyme. Determination of the amount of a specific form of phosphodiesterase in crude material is often difficult. Modification of assay conditions by judicious choice of substrate and/or inhibitor concentrations may selectively favor (or reduce) the activity of a particular form; in many instances, however, some fractionation of enzymes may be necessary. This is discussed more fully in the final section of this chapter

  8. RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

    Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

    1990-01-01

    Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

  9. Combined theoretical and computational study of interstrand DNA guanine-guanine cross-linking by trans-[Pt(pyridine)2] derived from the photoactivated prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2

    Tai, H.-Ch.; Brodbeck, R.; Kašpárková, Jana; Farrer, N.J.; Brabec, Viktor; Sadler, P.J.; Deeth, R.J.

    2012-01-01

    Roč. 51, č. 12 (2012), s. 6830-6841 ISSN 0020-1669 R&D Projects: GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : platinum * photoactivation * DNA Subject RIV: BO - Biophysics Impact factor: 4.593, year: 2012

  10. Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

    Jacobsen, Jack Hummeland; Schmiegelow, Kjeld; Nersting, Jakob

    2012-01-01

    was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra...

  11. Relative Stability of Different DNA Guanine Quadruplex Stem Topologies Derived Using Large-Scale Quantum-Chemical Computations

    Šponer, Jiří (ed.); Mládek, Arnošt; Špačková, Naďa; Cang, X.; Cheatham III, Thomas E.; Grimme, S.

    2013-01-01

    Roč. 135, č. 26 (2013), s. 9785-9796 ISSN 0002-7863 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068; GA ČR(CZ) GAP208/11/1822 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : DENSITY -FUNCTIONAL THEORY * MOLECULAR-DYNAMICS SIMULATIONS * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 11.444, year: 2013

  12. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    Kibe, A.; Shimada, K.; Tagaki, Y.

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA + gene function(s) and suggest that ColE1 plasmit itself provides no recA + -like functions. (orig.) [de

  13. [Quantum-chemical investigation of tautomerization ways of Watson-Crick DNA base pair guanine-cytosine].

    Brovarets', O O; Hovorun, D M

    2010-01-01

    A novel physico-chemical mechanism of the Watson-Crick DNA base pair Gua.Cyt tautomerization Gua.Cyt*Gua.CytGua*.Cyt (mutagenic tautomers of bases are marked by asterisks) have been revealed and realized in a pathway of single proton transfer through two mutual isoenergetic transition states with Gibbs free energy of activation 30.4 and 30.6 kcal/mol and they are ion pairs stabilized by three (N2H...N3, N1H...N4- and O6+H...N4-) and five (N2H...O2, N1H...O2, N1H...N3, O6+H...N4- and 06+H...N4-) H-bonds accordingly. Stable base pairs Gua-Cyt* and Gua*.Cyt which dissociate comparably easy into monomers have acceptable relative Gibbs energies--12.9 and 14.3 kcal/mol--for the explanation of the nature of the spontaneous transitions of DNA replication. Results are obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-31 1++G(d,p) level of theory in vacuum approach.

  14. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  15. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  16. Folding of guanine quadruplex molecules-funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies

    Šponer, Jiří; Bussi, G.; Stadlbauer, Petr; Kührová, P.; Banáš, P.; Islam, Barira; Haider, S.; Neidle, S.; Otyepka, M.

    2017-01-01

    Roč. 1861, č. 5 (2017), s. 1246-1263 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : telomeric g-quadruplex * parallel g-quadruplex Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  17. VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

    Garrett, Tiana A.; van Buul, Jaap D.; Burridge, Keith

    2007-01-01

    Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the

  18. Are genes destiny? Have adenine, cytosine, guanine and thymine replaced Lachesis, Clotho and Atropos as the weavers of our fate?

    Eisenberg, Leon

    2005-02-01

    It is as futile to ask how much of the phenotype of an organism is due to nature and how much to its nurture as it is to determine how much of the area of a rectangle is due to its length and how much to its height. Phenotype and area are joint products. The spectacular success of genomics, unfortunately, threatens to re-awaken belief in genes as the principal determinants of human behavior. This paper develops the thesis that gene expression is modified by environmental inputs and that the impact of the environment on a given organism is modified by its genome. Genes set the boundaries of the possible; environments parse out the actual.

  19. Effect of Guanine to Inosine Substitution on Stability of Canonical DNA and RNA Duplexes: Molecular Dynamics Thermodynamics Integration Study

    Krepl, Miroslav; Otyepka, M.; Banáš, Pavel; Šponer, Jiří

    2013-01-01

    Roč. 117, č. 6 (2013), s. 1872-1879 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GBP305/12/G034 Grant - others:GA ČR(CZ) GPPP301/11/P558; GA MŠk(CZ) ED1.1.00/02.0068 Program:ED Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : FREE-ENERGY CALCULATIONS * PARTICLE MESH EWALD * ACID BASE-PAIRS Subject RIV: BO - Biophysics Impact factor: 3.377, year: 2013

  20. Can We Execute Reliable MM-PBSA Free Energy Computations of Relative Stabilities of Different Guanine Quadruplex Folds?

    Islam, B.; Stadlbauer, Petr; Neidle, S.; Haider, S.; Šponer, Jiří

    2016-01-01

    Roč. 120, č. 11 (2016), s. 2899-2912 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * TELOMERIC G-QUADRUPLEX * AMBER FORCE-FIELD Subject RIV: BO - Biophysics Impact factor: 3.177, year: 2016