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Sample records for 5-lipoxygenase activating protein

  1. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

    PubMed

    Gerstmeier, Jana; Newcomer, Marcia E; Dennhardt, Sophie; Romp, Erik; Fischer, Jana; Werz, Oliver; Garscha, Ulrike

    2016-05-01

    Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. PMID:26842853

  2. Expression, purification and crystallization of human 5-lipoxygenase-activating protein with leukotriene-biosynthesis inhibitors

    SciTech Connect

    Xu, Shihua; McKeever, Brian M.; Wisniewski, Douglas; Miller, Douglas K.; Spencer, Robert H.; Chu, Lin; Ujjainwalla, Feroze; Yamin, Ting-Ting; Evans, Jilly F.; Becker, Joseph W.; Ferguson, Andrew D.

    2007-12-01

    The expression, purification and crystallization of human 5-lipoxygenase-activating protein in complex with two leukotriene-biosynthesis inhibitors is decribed. The processes that were used to generate diffraction quality crystals are presented in detail. The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P42{sub 1}2) and diffracted to a resolution limit of 4 Å.

  3. Myeloid Cell 5-Lipoxygenase Activating Protein Modulates the Response to Vascular Injury

    PubMed Central

    Yu, Zhou; Ricciotti, Emanuela; Miwa, Takashi; Liu, Shulin; Ihida-Stansbury, Kaori; Landersberg, Gavin; Jones, Peter L.; Scalia, Rosario; Song, Wenchao; Assoian, Richard K.; FitzGerald, Garret A.

    2013-01-01

    Rationale Human genetics have implicated the 5- lipoxygenase (5-LO) enzyme in the pathogenesis of cardiovascular disease and an inhibitor of the 5-LO activating protein (FLAP) is in clinical development for asthma. Objective Here we determined whether FLAP deletion modifies the response to vascular injury. Methods and Results Vascular remodeling was characterized 4 weeks after femoral arterial injury in FLAP knockout (FLAP KO) mice and wild type (WT) controls. Both neointimal hyperplasia and the intima/media ratio of the injured artery were significantly reduced in the FLAP KOs while endothelial integrity was preserved. Lesional myeloid cells were depleted and vascular smooth muscle cell (VSMC) proliferation, as reflected by bromodeoxyuridine (BrdU) incorporation, was markedly attenuated by FLAP deletion. Inflammatory cytokine release from FLAP KO macrophages was depressed and their restricted ability to induce VSMC migration ex vivo was rescued with leukotriene B4 (LTB4). FLAP deletion restrained injury and attenuated upregulation of the extracellular matrix protein, tenascin C (TNC), which affords a scaffold for VSMC migration. Correspondingly, the phenotypic modulation of VSMC to a more synthetic phenotype, reflected by morphological change, loss of α-smooth muscle cell actin and upregulation of vascular cell adhesion molecule (VCAM) -1 was also suppressed in FLAP KO mice. Transplantation of FLAP replete myeloid cells rescued the proliferative response to vascular injury. Conclusion Expression of lesional FLAP in myeloid cells promotes LTB4 dependent VSMC phenotypic modulation, intimal migration and proliferation. PMID:23250985

  4. Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice.

    PubMed

    Uz, Tolga; Dimitrijevic, Nikola; Imbesi, Marta; Manev, Hari; Manev, Radmila

    2008-05-01

    A common biological pathway may contribute to the comorbidity of atherosclerosis and depression. Increased activity of the enzymatic 5-lipoxygenase (5-LOX, 5LO) pathway is a contributing factor in atherosclerosis and a 5-LOX inhibitor, MK-886, is beneficial in animal models of atherosclerosis. In the brain, MK-886 increases phosphorylation of the glutamate receptor subunit GluR1, and the increased phosphorylation of this receptor has been associated with antidepressant treatment. In this work, we evaluated the behavioral effects of MK-886 in an automated assay of mouse forced swimming, which identifies antidepressant activity as increased climbing behavior and/or decreased rest time. Whereas a single injection of MK-886 (3 and 10 mg/kg) did not affect forced swimming behaviors assayed 30 min later, six daily injections of 3 mg/kg MK-886 slightly increased climbing and significantly reduced rest time in wild-type mice but not in 5-LOX-deficient mice. A diet delivery of MK-886, 4 micro/(100 mg(body-weight)day), required 3 weeks to affect forced swimming; it increased climbing behavior. Climbing behavior was also increased in naive 5-LOX-deficient mice compared to naive wild-type controls. These results suggest that 5-LOX inhibition and deficiency may be associated with antidepressant activity. Increased climbing in a forced swimming assay is a typical outcome of antidepressants that increase noradrenergic and dopaminergic activity. Interestingly, 5-LOX deficiency and MK-886 treatment have been shown to be capable of increasing the behavioral effects of a noradrenaline/dopamine-potentiating drug, cocaine. Future research is needed to evaluate the clinical relevance of our findings. PMID:18403121

  5. Association between arachidonate 5-lipoxygenase-activating protein (ALOX5AP) and lung function in a Korean population.

    PubMed

    Ro, M; Kim, S; Pyun, J-A; Shin, C; Cho, N H; Lee, J-Y; Koh, I; Kwack, K

    2012-08-01

    Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) plays a role in the 5-lipoxygenase (LO) pathway, which includes the LTC(4), LTD(4), LTE(4) and LTB(4). These leukotrienes are known causative factors of asthma, allergy, atopy and cardiovascular diseases. ALOX5AP lacks enzyme activity and acts by helping 5-LO function. In this study, healthy and general subjects who live in rural and urban areas of Korea were tested for the association of ALOX5AP polymorphisms with lung function. Lung function was also estimated by calculating the predicted values for forced expiratory volume in one second (FEV(1) _%PRED) and the proportion of the forced vital capacity exhaled in the first second (FEV(1) /FVC_PRED). The linear regression was adjusted for residence area, gender, age, height and smoking status. The analysis revealed associations between FEV(1) and the single-nucleotide polymorphism (SNP) rs9506352 and the haplotype TCAC (permuted P-value < 0.05). The linkage disequilibrium block that included the significant SNPs overlapped with SNPs that were revealed previously to associate with myocardial infarction and asthma and to affect lung function. This study is the first to demonstrate the association between lung function and ALOX5AP polymorphisms in a healthy and general population. PMID:22537113

  6. 4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP).

    PubMed

    Banoglu, Erden; Çelikoğlu, Erşan; Völker, Susanna; Olgaç, Abdurrahman; Gerstmeier, Jana; Garscha, Ulrike; Çalışkan, Burcu; Schubert, Ulrich S; Carotti, Andrea; Macchiarulo, Antonio; Werz, Oliver

    2016-05-01

    In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis (IC50 = 0.24 μM, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC50 ≥ 8 μM). Docking studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis. PMID:26922224

  7. A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors.

    PubMed

    Blevitt, Jonathan M; Hack, Michael D; Herman, Krystal; Chang, Leon; Keith, John M; Mirzadegan, Tara; Rao, Navin L; Lebsack, Alec D; Milla, Marcos E

    2016-06-10

    5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4 Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds. PMID:27129215

  8. Phospholipid Ozonation Products Activate the 5-Lipoxygenase Pathway in Macrophages.

    PubMed

    Zemski Berry, Karin A; Murphy, Robert C

    2016-08-15

    Ozone is a highly reactive environmental toxicant that can react with the double bonds of lipids in pulmonary surfactant. This study was undertaken to investigate the proinflammatory properties of the major lipid-ozone product in pulmonary surfactant, 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0/9al-PC), with respect to eicosanoid production. A dose-dependent increase in the formation of 5-lipoxygenase (5-LO) products was observed in murine resident peritoneal macrophages (RPM) and alveolar macrophages (AM) upon treatment with 16:0/9al-PC. In contrast, the production of cyclooxygenase (COX) derived eicosanoids did not change from basal levels in the presence of 16:0/9al-PC. When 16:0/9al-PC and the TLR2 ligand, zymosan, were added to RPM or AM, an enhancement of 5-LO product formation along with a concomitant decrease in COX product formation was observed. Neither intracellular calcium levels nor arachidonic acid release was influenced by the addition of 16:0/9al-PC to RPM. Results from mitogen-activated protein kinase (MAPK) inhibitor studies and direct measurement of phosphorylation of MAPKs revealed that 16:0/9al-PC activates the p38 MAPK pathway in RPM, which results in the activation of 5-LO. Our results indicate that 16:0/9al-PC has a profound effect on the eicosanoid pathway, which may have implications in inflammatory pulmonary disease states where eicosanoids have been shown to play a role. PMID:27448436

  9. Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model.

    PubMed

    Liu, Jun-Yan; Yang, Jun; Inceoglu, Bora; Qiu, Hong; Ulu, Arzu; Hwang, Sung-Hee; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2010-03-15

    Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects. PMID:19896470

  10. The novel benzimidazole derivative BRP-7 inhibits leukotriene biosynthesis in vitro and in vivo by targeting 5-lipoxygenase-activating protein (FLAP)

    PubMed Central

    Pergola, C; Gerstmeier, J; Mönch, B; Çalışkan, B; Luderer, S; Weinigel, C; Barz, D; Maczewsky, J; Pace, S; Rossi, A; Sautebin, L; Banoglu, E; Werz, O

    2014-01-01

    BACKGROUND AND PURPOSE Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis. KEY RESULTS BRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels. CONCLUSIONS AND IMPLICATIONS BRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development. PMID:24641614

  11. The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein.

    PubMed

    Maxis, Kelitha; Delalandre, Aline; Martel-Pelletier, Johanne; Pelletier, Jean-Pierre; Duval, Nicolas; Lajeunesse, Daniel

    2006-01-01

    Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-beta (TGF-beta), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-beta also modulated PGE2 production. TGF-beta stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-beta in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH

  12. Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

    PubMed Central

    Chen, Hsiang-Chin; Xie, Jia; Zhang, Zheng; Su, Li-Ting; Yue, Lixia; Runnels, Loren W.

    2010-01-01

    TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein's expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE's downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel

  13. 5-Lipoxygenase Activity Increases Susceptibility to Experimental Paracoccidioides brasiliensis Infection

    PubMed Central

    Tristão, Fabrine Sales Massafera; Rocha, Fernanda Agostini; Moreira, Ana Paula; Cunha, Fernando Queiroz; Rossi, Marcos Antonio

    2013-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermodimorphic fungus Paracoccidioides brasiliensis. Leukotrienes and lipoxins are lipid mediators produced after 5-lipoxygenase (5-LO) activation that exhibit pro- and anti-inflammatory roles, respectively. Here, we have investigated the contribution of 5-LO enzymatic activity in PCM using an experimental model of P. brasiliensis infection. B6.129 wild-type (B6.129) and 5-LO-deficient (5-LO−/−) mice were intravenously inoculated with a virulent strain of P. brasiliensis (Pb18), and the survival rate of the infected mice was investigated on different days after yeast infection. 5-LO−/− mice exhibited an increased survival rate associated with a decreased number of CFU. The resistance of 5-LO−/− during PCM was associated with augmented nitric oxide (NO) production and the formation of compact granulomas. In addition, the absence of 5-LO was associated with a diminished number of CD4+ CD25+ regulatory T cells, higher levels of gamma interferon and interleukin-12, and increased T-bet (a T-box transcription factor that directs Th1 lineage commitment) mRNA levels in the lungs. Taken together, our results show for the first time that 5-LO enzymatic activity increases susceptibility to P. brasiliensis, suggesting that this pathway may be a potential target for therapeutic intervention during PCM. PMID:23381993

  14. Efficacy, safety and tolerability of GSK2190915, a 5-lipoxygenase activating protein inhibitor, in adults and adolescents with persistent asthma: a randomised dose-ranging study

    PubMed Central

    2013-01-01

    Background GSK2190915 is a high affinity 5-lipoxygenase-activating protein inhibitor being developed for the treatment of asthma. The objective of this study was to evaluate GSK2190915 efficacy, dose–response and safety in subjects with persistent asthma treated with short-acting beta2-agonists (SABAs) only. Methods Eight-week multicentre, randomised, double-blind, double-dummy, stratified (by age and smoking status), parallel-group, placebo-controlled study in subjects aged ≥12 years with a forced expiratory volume in 1 second (FEV1) of 50–85% predicted. Subjects (n = 700) were randomised to receive once-daily (QD) oral GSK2190915 (10–300 mg), twice-daily inhaled fluticasone propionate 100 μg, oral montelukast 10 mg QD or placebo. The primary endpoint was mean change from baseline (randomisation) in trough (morning pre-dose and pre-rescue bronchodilator) FEV1 at the end of the 8-week treatment period. Secondary endpoints included morning and evening peak expiratory flow, symptom-free days and nights, rescue-free days and nights, day and night-time symptom scores, day and night-time rescue medication use, withdrawals due to lack of efficacy, Asthma Control Questionnaire and Asthma Quality of Life Questionnaire scores. Results For the primary endpoint, there was no statistically significant difference between any dose of GSK2190915 QD and placebo. However, repeated measures sensitivity analysis demonstrated nominal statistical significance for GSK2190915 30 mg QD compared with placebo (mean difference: 0.115 L [95% confidence interval: 0.00, 0.23], p = 0.044); no nominally statistically significant differences were observed with any of the other doses. For the secondary endpoints, decreases were observed in day-time symptom scores and day-time SABA use for GSK2190915 30 mg QD versus placebo (p ≤ 0.05). No dose–response relationship was observed for the primary and secondary endpoints across the GSK2190915 dose range studied; the 10

  15. Increased activity of 5-lipoxygenase in polymorphonuclear leukocytes from asthmatic patients

    SciTech Connect

    Mita, H.; Yui, Y.; Taniguchi, N.; Yasueda, H.; Shida, T.

    1985-09-09

    The formation of 5-lipoxygenase products of arachidonic acid, 5-HETE and 5,12-diHETE, was determined in 100,000 x g supernatant of polymorphonuclear leukocytes from 17 healthy subjects, 17 patients with extrinsic asthma and 15 patients with intrinsic asthma. After the supernatant was incubated with /sup 14/C-arachidonic acid in the presence of calcium and indomethacin, the lipoxygenase products of arachidonic acid were separated by thin layer chromatography. The results were expressed as the percentage conversion of /sup 14/C-arachidonic acid into the product per 10/sup 7/ cells. The formation of 5,12-diHETE, but not of the 5-HETE, was significantly increased in the cells from the group of patients with extrinsic asthma (4.38 +/- 0.78%, mean +/- S.E.; p < 0.01) and intrinsic asthma (6.09 +/- 1.11%; p < 0.01), when compared to normal subjects (1.74 +/- 0.30%). Both extrinsic and intrinsic asthmatics had significantly enhanced 5-lipoxygenase activity, which was expressed as the sum of percentage conversion of /sup 14/C-arachidonic acid into 5-HETE and 5,12-diHETE. The percentage conversion in normal subjects was 4.19 +/- 0.39%, 6.24 +/- 0.84% for 17 patients with extrinsic asthma (p < 0.05), and 8.59 +/- 1.29% for 15 patients with intrinsic asthma (p < 0.01). There was no significant difference between these asthmatic groups. These results indicate that 5-lipoxygenase activity is increased in patients with bronchial asthma. 22 references, 3 figures.

  16. Mesenteric lymph diversion abrogates 5-lipoxygenase activation in the kidney following trauma and hemorrhagic shock

    PubMed Central

    Stringham, John R.; Moore, Ernest E.; Gamboni, Fabia; Harr, Jeffrey N.; Fragoso, Miguel; Chin, Theresa L.; Carr, Caitlin E.; Silliman, Christopher C.; Banerjee, Anirban

    2014-01-01

    BACKGROUND Early acute kidney injury (AKI) following trauma is associated with multiorgan failure and mortality. Leukotrienes have been implicated both in AKI and in acute lung injury. Activated 5-lipoxygenase (5-LO) colocalizes with 5-LO–activating protein (FLAP) in the first step of leukotriene production following trauma and hemorrhagic shock (T/HS). Diversion of postshock mesenteric lymph, which is rich in the 5-LO substrate of arachidonate, attenuates lung injury and decreases 5-LO/FLAP associations in the lung after T/HS. We hypothesized that mesenteric lymph diversion (MLD) will also attenuate postshock 5-LO–mediated AKI. METHODS Rats underwent T/HS (laparotomy, hemorrhagic shock to a mean arterial pressure of 30 mm Hg for 45 minutes, and resuscitation), and MLD was accomplished via cannulation of the mesenteric duct. Extent of kidney injury was determined via histology score and verified by urinary neutrophil gelatinase-associated lipocalin assay. Kidney sections were immunostained for 5-LO and FLAP, and colocalization was determined by fluorescence resonance energy transfer signal intensity. The end leukotriene products of 5-LO were determined in urine. RESULTS AKI was evident in the T/HS group by derangement in kidney tubule architecture and confirmed by neutrophil gelatinase-associated lipocalin assay, whereas MLD during T/HS preserved renal tubule morphology at a sham level. MLD during T/HS decreased the associations between 5-LO and FLAP demonstrated by fluorescence resonance energy transfer microscopy and decreased leukotriene production in urine. CONCLUSION 5-LO and FLAP colocalize in the interstitium of the renal medulla following T/HS. MLD attenuates this phenomenon, which coincides with pathologic changes seen in tubules during kidney injury and biochemical evidence of AKI. These data suggest that gut-derived leukotriene substrate predisposes the kidney and the lung to subsequent injury. PMID:24747451

  17. 5-Lipoxygenase and cyclooxygenase-1 inhibitory active compounds from Atractylodes lancea.

    PubMed

    Resch, M; Steigel, A; Chen, Z L; Bauer, R

    1998-03-01

    Lipophilic extracts of Atractylodes lancea rhizomes exhibited potent inhibitory activities in 5-lipoxygenase [IC50 (5-LOX) = 2.9 micrograms/mL (n-hexane extract)] and cyclooxygenase-1 [IC50 (COX-1) = 30.5 micrograms/mL (n-hexane extract)] enzymatic assays. Bioactivity-guided fractionation of the n-hexane extract led to the isolation of a new compound atractylochromene (1), a potent inhibitor in both test systems [IC50 (5-LOX) = 0.6 microM, IC50 (COX-1) = 3.3 microM]. Also obtained was 2-[(2E)-3,7-dimethyl-2,6-octadienyl]-6-methyl-2,5-cyclohexadiene-1 ,4-dione (2), which showed a selective inhibitory activity against 5-LOX [IC50 (5-LOX) 0.2 microM, IC50 (COX-1) 64.3 microM]. The sesquiterpene atractylon (3) and the coumarin osthol (4) turned out to be moderate but selective 5-lipoxygenase inhibitors. Atractylenolides I (5), II (6), and III (7) showed no significant inhibitory effects for either enzyme. Structures were established by spectral data interpretation. PMID:9544564

  18. Impact of Wines and Wine Constituents on Cyclooxygenase-1, Cyclooxygenase-2, and 5-Lipoxygenase Catalytic Activity

    PubMed Central

    Temml, Veronika; Maghradze, David; Vanek, Tomas

    2014-01-01

    Cyclooxygenases and lipoxygenases are proinflammatory enzymes; the former affects platelet aggregation, vasoconstriction, vasodilatation and later the development of atherosclerosis. Red wines from Georgia and central and western Europe inhibited cyclooxygenase-1 (COX-1) activity in the range of 63–94%, cyclooxygenase-2 (COX-2) activity in the range of 20–44% (tested at a concentration of 5 mL/L), and 5-lipoxygenase (5-LOX) activity in the range of 72–84% (at a concentration of 18.87 mL/L). White wines inhibited 5-LOX in the range of 41–68% at a concentration of 18.87 mL/L and did not inhibit COX-1 and COX-2. Piceatannol (IC50 = 0.76 μM) was identified as a strong inhibitor of 5-LOX followed by luteolin (IC50 = 2.25 μM), quercetin (IC50 = 3.29 μM), and myricetin (IC50 = 4.02 μM). trans-Resveratrol was identified as an inhibitor of COX-1 (IC50 = 2.27 μM) and COX-2 (IC50 = 3.40 μM). Red wine as a complex mixture is a powerful inhibitor of COX-1, COX-2, and 5-LOX, the enzymes involved in eicosanoid biosynthetic pathway. PMID:24976682

  19. Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

    SciTech Connect

    Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Bae, Jin U.; Bae, Sun S.; Kim, Chi D.

    2010-05-15

    Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.

  20. Synthesis, SAR, and series evolution of novel oxadiazole-containing 5-lipoxygenase activating protein inhibitors: discovery of 2-[4-(3-{(r)-1-[4-(2-amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915).

    PubMed

    Takahashi, Hidenori; Riether, Doris; Bartolozzi, Alessandra; Bosanac, Todd; Berger, Valentina; Binetti, Ralph; Broadwater, John; Chen, Zhidong; Crux, Rebecca; De Lombaert, Stéphane; Dave, Rajvee; Dines, Jonathon A; Fadra-Khan, Tazmeen; Flegg, Adam; Garrigou, Michael; Hao, Ming-Hong; Huber, John; Hutzler, J Matthew; Kerr, Steven; Kotey, Adrian; Liu, Weimin; Lo, Ho Yin; Loke, Pui Leng; Mahaney, Paige E; Morwick, Tina M; Napier, Spencer; Olague, Alan; Pack, Edward; Padyana, Anil K; Thomson, David S; Tye, Heather; Wu, Lifen; Zindell, Renee M; Abeywardane, Asitha; Simpson, Thomas

    2015-02-26

    The synthesis, structure-activity relationship (SAR), and evolution of a novel series of oxadiazole-containing 5-lipoxygenase-activating protein (FLAP) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (IC50 < 100 nM). Optimization of binding and functional potencies, as well as physicochemical properties resulted in the identification of compound 69 (BI 665915) that demonstrated an excellent cross-species drug metabolism and pharmacokinetics (DMPK) profile and was predicted to have low human clearance. In addition, 69 was predicted to have a low risk for potential drug-drug interactions due to its cytochrome P450 3A4 profile. In a murine ex vivo whole blood study, 69 demonstrated a linear dose-exposure relationship and a dose-dependent inhibition of LTB4 production. PMID:25671290

  1. Minocycline protects PC12 cells against NMDA-induced injury via inhibiting 5-lipoxygenase activation.

    PubMed

    Song, Ying; Wei, Er-Qing; Zhang, Wei-Ping; Ge, Qiu-Fu; Liu, Jian-Ren; Wang, Meng-Ling; Huang, Xiao-Jia; Hu, Xin; Chen, Zhong

    2006-04-26

    Recently, we have reported that minocycline, a semi-synthetic tetracycline with neuroprotective effects, inhibits the in vitro ischemic-like injury and 5-lipoxygenase (5-LOX) activation in PC12 cells. In the present study, we further determined whether minocycline protects PC12 cells from excitotoxicity via inhibiting 5-LOX activation. We used N-methyl-d-aspartate (NMDA, 200 microM) to induce early (exposure for 6 h) and delayed (exposure for 6 h followed by 24 h recovery) injuries. We found that NMDA receptor antagonist ketamine, 5-LOX inhibitor caffeic acid and minocycline concentration dependently attenuated NMDA-induced early and delayed cell injuries (viability reduction and cell death). However, only ketamine (1 microM) inhibited NMDA-evoked elevation of intracellular calcium. In addition, immunohistochemical analysis showed that NMDA induced 5-LOX translocation to the nuclear membrane after 1- to 6-h exposure which was confirmed by Western blotting, indicating that 5-LOX was activated. Ketamine, caffeic acid and minocycline (each at 1 microM) inhibited 5-LOX translocation after early injury. After delayed injury, PC12 cells were shrunk, and 5-LOX was translocated to the nuclei and nuclear membrane; ketamine, caffeic acid and minocycline inhibited both cell shrinking and 5-LOX translocation. As a control, 12-LOX inhibitor baicalein showed a weak effect on cell viability and death, but no effect on 5-LOX translocation. Therefore, we conclude that the protective effect of minocycline on NMDA-induced injury is partly mediated by inhibiting 5-LOX activation. PMID:16574083

  2. Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon

    PubMed Central

    Sarveswaran, Sivalokanathan; Thamilselvan, Vijayalakshmi; Brodie, Chaya; Ghosh, Jagadananda

    2012-01-01

    Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase. PMID:21824498

  3. Regulation of rotenone-induced microglial activation by 5-lipoxygenase and cysteinyl leukotriene receptor 1.

    PubMed

    Zhang, Xiao-Yan; Chen, Lu; Yang, Yi; Xu, Dong-Min; Zhang, Si-Ran; Li, Chen-Tan; Zheng, Wei; Yu, Shu-Ying; Wei, Er-Qing; Zhang, Li-Hui

    2014-07-14

    The 5-lipoxygenase (5-LOX) products cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators. CysLTs mediate their biological actions through activating CysLT receptors (CysLT(1)R and CysLT(2)R). We have recently reported that 5-LOX and CysLT(1)R mediated PC12 cell injury induced by high concentrations of rotenone (0.3-10 μM), which was reduced by the selective 5-LOX inhibitor zileuton and CysLT(1)R antagonist montelukast. The purpose of this study was to examine the regulatory roles of the 5-LOX/CysLT(1)R pathway in microglial activation induced by low concentration rotenone. After mouse microglial BV2 cells were stimulated with rotenone (0.3-3 nM), phagocytosis and release of pro-inflammatory cytokine were assayed as indicators of microglial activation. We found that rotenone (1 and 3 nM) increased BV2 microglial phagocytosis and the release of the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Zileuton and montelukast prevented rotenone (3 nM)-induced phagocytosis and cytokine release. Furthermore, rotenone significantly up-regulated 5-LOX expression, induced 5-LOX translocation to the nuclear envelope, and increased the production of CysLTs. These responses were inhibited by zileuton. Rotenone also increased CysLT(1)R expression and induced nuclear translocation of CysLT(1)R. In primary rat microglia, rotenone (10 nM) increased release of IL-1β and TNF-α, whereas zileuton (0.1 μΜ) and montelukast (0.01 μΜ) significantly inhibited this response. These results indicated that 5-LOX and CysLT(1)R might be key regulators of microglial activation induced by low concentration of rotenone. Interference of 5-LOX/CysLT(1)R pathway may be an effective therapeutic strategy for microglial inflammation. PMID:24858057

  4. Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

    PubMed Central

    Altavilla, D; Squadrito, F; Bitto, A; Polito, F; Burnett, BP; Di Stefano, V; Minutoli, L

    2009-01-01

    Background and purpose: The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. Experimental approach: LPS-stimulated (1 µg·mL−1) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL−1) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. Key results: LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL−1) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE2 and LTB4 levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the

  5. Synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-LOX) inhibition activities, and molecular docking study of 7-substituted coumarin derivatives.

    PubMed

    Srivastava, Pavan; Vyas, Vivek K; Variya, Bhavesh; Patel, Palak; Qureshi, Gulamnizami; Ghate, Manjunath

    2016-08-01

    In the present study, 7-subsituted coumarin derivatives were synthesized using various aromatic and heterocyclic amines, and evaluated in vivo for anti-inflammatory and analgesic activity, and for ulcerogenic risk. The most active compounds were evaluated in vitro for 5-lipoxygenase (5-LOX) inhibition. Docking study was performed to predict the binding affinity, and orientation at the active site of the enzyme. In vivo anti-inflammatory and analgesic activity, and in vitro 5-LOX enzyme inhibition study revealed that compound 33 and 35 are the most potent compounds in all the screening methods. In vitro kinetic study of 35 showed mixed or non-competitive type of inhibition with 5-LOX enzyme. Presence of OCH3 group in 35 and Cl in 33 at C6-position of benzothiazole ring were found very important substitutions for potent activity. PMID:27376460

  6. 5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.

    PubMed Central

    Woods, J W; Coffey, M J; Brock, T G; Singer, I I; Peters-Golden, M

    1995-01-01

    5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme. Images PMID:7738170

  7. Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells.

    PubMed

    Raghavenra, H; Diwakr, B T; Lokesh, B R; Naidu, K A

    2006-01-01

    Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells. PMID:16216483

  8. 5-lipoxygenase activation is involved in the mechanisms of chronic hepatic injury in a rat model of chronic aluminum overload exposure.

    PubMed

    Mai, Shaoshan; He, Qin; Wang, Hong; Hu, Xinyue; Luo, Ying; Yang, Yang; Kuang, Shengnan; Tian, Xiaoyan; Ma, Jie; Yang, Junqing

    2016-08-15

    We previously confirmed that rats overloaded with aluminum exhibited hepatic function damage and increased susceptibility to hepatic inflammation. However, the mechanism of liver toxicity by chronic aluminum overload is poorly understood. In this study, we investigated changes in the 5-lipoxygenase (5-LO) signaling pathway and its effect on liver injury in aluminum-overloaded rats. A rat hepatic injury model of chronic aluminum injury was established via the intragastric administration of aluminum gluconate (Al(3+) 200mg/kg per day, 5days a week for 20weeks). The 5-LO inhibitor, caffeic acid (10 and 30mg/kg), was intragastrically administered 1h after aluminum administration. Hematoxylin and eosin staining was used to visualize pathological changes in rat liver tissue. A series of biochemical indicators were measured with biochemistry assay or ELISAs. Immunochemistry and RT-PCR methods were used to detect 5-LO protein and mRNA expression in the liver, respectively. Caffeic acid administration protected livers against histopathological injury, decreased plasma ALT, AST, and ALP levels, decreased TNF-α, IL-6, IL-1β and LTs levels, increased the reactive oxygen species content, and down-regulated the mRNA and protein expressions of 5-LO in aluminum overloaded rats. Our results indicate that 5-lipoxygenase activation is mechanistically involved in chronic hepatic injury in a rat model of chronic aluminum overload exposure and that the 5-LO signaling pathway, which associated with inflammation and oxidative stress, is a potential therapeutic target for chronic non-infection liver diseases. PMID:27368151

  9. Rapid Stimulation of 5-Lipoxygenase Activity in Potato by the Fungal Elicitor Arachidonic Acid 1

    PubMed Central

    Bostock, Richard M.; Yamamoto, Hiroyuki; Choi, Doil; Ricker, Karin E.; Ward, Bernard L.

    1992-01-01

    The activity of lipoxygenase (LOX) in aged potato tuber discs increased by almost 2-fold following treatment of the discs with the fungal elicitor arachidonic acid (AA). Enzyme activity increased above that in untreated discs within 30 min after AA treatment, peaked at 1 to 3 h, and returned to near control levels by 6 h. The majority of the activity was detected in a soluble fraction (105,000g supernatant), but a minor portion was also associated with a particulate fraction enriched in microsomal membranes (105,000g pellet); both activities were similarly induced. 5-Hydroperoxyeicosatetraenoic acid was the principal product following incubation of these extracts with AA. Antibodies to soybean LOX strongly reacted with a protein with a molecular mass of approximately 95-kD present in both soluble and particulate fractions whose abundance generally corresponded with LOX activity in extracts. LOX activity was not enhanced by treatment of the discs with nonelicitor fatty acids or by branched β-glucans from the mycelium of Phytophthora infestans. Prior treatment of the discs with abscisic acid, salicylhydroxamic acid, or n-propyl gallate, all of which have been shown to suppress AA induction of the hypersensitive response, inhibited the AA-induced increment in LOX activity. Cycloheximide pretreatment, which abolishes AA elicitor activity for other responses such as phytoalexin induction, did not inhibit LOX activity in water- or elicitor-treated discs but enhanced activity similar to that observed by AA treatment. The elicitor-induced increase in 5-LOX activity preceded or temporally paralleled the induction of other studied responses to AA, including the accumulation of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase and phenylalanine ammonia lyase reported here. The results are discussed in relation to the proposed role of the 5-LOX in signal-response coupling of arachidonate elicitation of the hypersensitive response. Images Figure 4 Figure 7 PMID

  10. Active site characterization and structure based 3D-QSAR studies on non-redox type 5-lipoxygenase inhibitors.

    PubMed

    Ul-Haq, Zaheer; Khan, Naveed; Zafar, Syed Kashif; Moin, Syed Tarique

    2016-06-10

    Structure-based 3D-QSAR study was performed on a class of 5-benzylidene-2-phenylthiazolinones non-redox type 5-LOX inhibitors. In this study, binding pocket of 5-Lipoxygenase (pdb id 3o8y) was identified by manual docking using 15-LOX (pdb id 2p0m) as a reference structure. Additionally, most of the binding site residues were found conserved in both structures. These non-redox inhibitors were then docked into the binding site of 5-LOX. To generate reliable CoMFA and CoMSIA models, atom fit data base alignment method using docked conformation of the most active compound was employed. The q(2)cv and r(2)ncv values for CoMFA model were found to be 0.549 and 0.702, respectively. The q(2)cv and r(2)ncv values for the selected CoMSIA model comprised four descriptors steric, electrostatic, hydrophobic and hydrogen bond donor fields were found to be 0.535 and 0.951, respectively. Obtained results showed that our generated model was statistically reliable. Furthermore, an external test set validates the reliability of the predicted model by calculating r(2)pred i.e.0.787 and 0.571 for CoMFA and CoMSIA model, respectively. 3D contour maps generated from CoMFA and CoMSIA models were utilized to determine the key structural features of ligands responsible for biological activities. The applied protocol will be helpful to design more potent and selective inhibitors of 5-LOX. PMID:27044904

  11. Alveolar lining fluid regulates mononuclear phagocyte 5-lipoxygenase metabolism.

    PubMed

    Phare, S M; Peters-Golden, M; Coffey, M J

    1998-11-01

    The enzyme 5-lipoxygenase (5-LO) catalyses the synthesis of leukotrienes (LT), which are important in phagocytosis and killing of microorganisms. The alveolar macrophage (AM), the primary resident defender of the alveolar space, has a greater capacity for LT synthesis than its precursor, the peripheral blood monocyte (PBM). This study investigated whether the alveolar lining fluid (ALF) upregulates LT synthetic capacity in mononuclear phagocytes. Rat AM, peritoneal macrophages (PM) and ALF were obtained by lavage from pathogen-free animals. Human PBM were isolated from normal subjects. 5-LO metabolism and expression were measured with and without ALF. Rat ALF increased 5-LO metabolism (136.4+/-15.1% of control) in cultured PBM. This was associated with increased 5-LO activating protein (FLAP) (357+/-29.5 %), and 5-LO expression (188+/-31.3%). Culture of AM for 3 days resulted in a greater decrement in LTB4 synthesis (LTB4 15.4+/-6.9% of day 1) than in PM (54.7+/-8.3% of day 1), suggesting a greater dependence of AM 5-LO metabolism on ALF. 5-LO and FLAP expression decreased to a greater degree in AM than PM in culture. Furthermore, AM cultured with ALF maintained their LT synthetic capacity, FLAP and 5-LO expression compared with control cells cultured in medium alone. In conclusion, alveolar lining fluid increased 5-lipoxygenase metabolism in peripheral blood monocytes and maintained it in cultured alveolar macrophages, by a mechanism of increased 5-lipoxygenase and 5-lipoxygenase activating protein expression. This may boost host defence capabilities. PMID:9864011

  12. Suppression of Oxidative Stress and 5-Lipoxygenase Activation by Edaravone Improves Depressive-Like Behavior after Concussion

    PubMed Central

    Hoshijima, Michihiro; Yawata, Toshio; Nobumoto, Atsuya; Tsuda, Masayuki; Shimizu, Takahiro; Saito, Motoaki; Ueba, Tetuya

    2014-01-01

    Abstract Brain concussions are a serious public concern and are associated with neuropsychiatric disorders, such as depression. Patients with concussion who suffer from depression often experience distress. Nevertheless, few pre-clinical studies have examined concussion-induced depression, and there is little information regarding its pharmacological management. Edaravone, a free radical scavenger, can exert neuroprotective effects in several animal models of neurological disorders. However, the effectiveness of edaravone in animal models of concussion-induced depression remains unclear. In this study, we examined whether edaravone could prevent concussion-induced depression. Mice were subjected to a weight-drop injury and intravenously administered edaravone (3.0 mg/kg) or vehicle immediately after impact. Serial magnetic resonance imaging showed no abnormalities of the cerebrum on diffusion T1- and T2-weighted images. We found that edaravone suppressed concussion-induced depressive-like behavior in the forced swim test, which was accompanied by inhibition of increased hippocampal and cortical oxidative stress (OS) and suppression of 5-lipoxygenase (5-LOX) translocation to the nuclear envelope in hippocampal astrocytes. Hippocampal OS in concussed mice was also prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin, and administration of BWB70C, a 5-LOX inhibitor, immediately and 24 h after injury prevented depressive-like behaviors in concussed mice. Further, antidepressant effects of edaravone were observed in mice receiving 1.0 or 3.0 mg/kg of edaravone immediately after impact, but not at a lower dose of 0.1 mg/kg. This antidepressant effect persisted up to 1 h after impact, whereas edaravone treatment at 3 h after impact had no effect on concussion-induced depressive-like behavior. These results suggest that edaravone protects against concussion-induced depression, and this protection is mediated by suppression of

  13. ATP Allosterically Activates the Human 5-Lipoxygenase Molecular Mechanism of Arachidonic Acid and 5(S)-Hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic Acid

    PubMed Central

    2015-01-01

    5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation. PMID:24893149

  14. Characterization of the human 5-lipoxygenase gene promoter

    SciTech Connect

    Hoshiko, S.; Radmark, O.; Samuelsson, B. )

    1990-12-01

    Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5{prime}-flanking sequences up to 5.9 kilobase pairs 5{prime} of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence ({minus}179 to {minus}56 from ATG), which includes five repeated GC boxes (the putative Spl binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment {minus}212 to {minus}88) revealed that the transcriptional factor Spl could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts.

  15. 4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

    SciTech Connect

    Lee, Seung J.; Kim, Chae E.; Yun, Mi R.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Shin, Hwa K.; Bae, Sun S.; Kim, Chi D.

    2010-01-15

    Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B{sub 4} (LTB{sub 4}) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT{sub 1} (cysLT{sub 1}) receptor antagonist, REV-5901 as well as a BLT{sub 1} receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB{sub 4} and cysLT (LTC{sub 4} and LTD{sub 4}) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB{sub 4} and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.

  16. Molecular cloning and amino acid sequence of human 5-lipoxygenase

    SciTech Connect

    Matsumoto, T.; Funk, C.D.; Radmark, O.; Hoeoeg, J.O.; Joernvall, H.; Samuelsson, B.

    1988-01-01

    5-Lipoxygenase (EC 1.13.11.34), a Ca/sup 2 +/- and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B/sub 4/. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the /sup 32/P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite DNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A/sub 4/ hydrolase or Ca/sup 2 +/-binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approx. = 2700 nucleotides in leukocytes, lung, and placenta.

  17. Inhibition of 5-lipoxygenase by vitamin E.

    PubMed

    Reddanna, P; Rao, M K; Reddy, C C

    1985-11-25

    Purified 5-lipoxygenase from potato tubers was inhibited strongly by vitamin E and its analogs. The inhibition by d-alpha-tocopherol was found to be irreversible and non-competitive with respect to arachidonic acid. An IC50 of 5 microM was calculated for d-alpha-tocopherol. The inhibition appears to be unrelated to its antioxidant function. Binding studies with 14C-labelled d-alpha-tocopherol revealed that there is a strong interaction between vitamin E and 5-lipoxygenase. Tryptic digestion and peptide mapping of 5-lipoxygenase-vitamin E complex indicate that vitamin E binds strongly to a single peptide. These studies suggest that cellular vitamin E levels may have profound influence on the formation of leukotrienes. PMID:3934003

  18. Exogenous action of 5-lipoxygenase by its metabolites on luteinizing hormone release in rat pituitary cells.

    PubMed

    Przylipiak, A; Kiesel, L; Habenicht, A J; Przylipiak, M; Runnebaum, B

    1990-02-12

    The stimulatory effect of exogenously administered potato 5-lipoxygenase (0.1-0.3 U/2 ml) on luteinizing hormone (LH) release was demonstrated in rat anterior pituitary cells in a superfusion system. Nordihydroguaiaretic acid (NDGA), an inhibitor of 5-lipoxygenase, abolished the effect of the enzyme on LH secretion. The secretory effect on LH after 5-lipoxygenase administration was biphasic and dependent on Ca2+ indicating that 5-lipoxygenase affects LH release through its oxygenation reaction. Another series of experiments demonstrated that activation of 5-lipoxygenase, expressed as production of leukotriene (LT) B4 and C4 (728 +/- 127 pg/10(6) cells and 178 +/- 23 pg/10(6) cells, respectively) occurs in rat pituitary cells after addition of Ca2+ ionophore A23187. However, LTB4 and LTC4 were not formed by pituitary cells that had previously been desensitized by gonadotropin-releasing hormone (GnRH), the physiological ligand of LH release. These results are consistent with a role of 5-lipoxygenase metabolites in the mechanism of GnRH-induced LH secretion. PMID:2157615

  19. Synthesis and biological evaluation of salicylic acid and N-acetyl-2-carboxybenzenesulfonamide regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore: dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity.

    PubMed

    Chowdhury, Morshed A; Abdellatif, Khaled R A; Dong, Ying; Das, Dipankar; Yu, Gang; Velázquez, Carlos A; Suresh, Mavanur R; Knaus, Edward E

    2009-12-15

    A novel class of salicylic acid and N-acetyl-2-carboxybenzenesulfonamide regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore attached to its C-4 or C-5 position was designed for evaluation as anti-inflammatory (AI) agents. Replacement of the 2,4-difluorophenyl ring in diflunisal by the N-difluoromethyl-1,2-dihydropyrid-2-one moiety provided compounds showing dual selective cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory activities. AI structure-activity studies showed that the C-4 (14a) and C-5 (14b) salicylate regioisomers were 1.4- and 1.6-fold more potent than aspirin, and the C-5 N-acetyl-2-carboxybenzenesulfonamide regioisomer (22b) was 1.3- and 2.8-fold more potent than ibuprofen and aspirin, respectively. In vivo ulcer index (UI) studies showed that the 4- and 5-(N-difluoromethyl-1,2-dihydropyrid-2-one-4-yl)salicylic acids (14a and 14b) were completely non-ulcerogenic since no gastric lesions were present (UI=0) relative to aspirin (UI=57) at an equivalent mumol/kg oral dose. The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel 5-LOX pharmacophore for the design of cyclic hydroxamic mimetics for exploitation in the development of dual COX-2/5-LOX inhibitory AI drugs. PMID:19884005

  20. In vitro inhibition of 5-lipoxygenase by protolichesterinic acid from Cetraria islandica.

    PubMed

    Ingolfsdottir, K; Breu, W; Huneck, S; Gudjonsdottir, G A; Müller-Jakic, B; Wagner, H

    1994-12-01

    The aliphatic α-methylene-γ-lactone (+)-protolichesterinic acid (1), isolated from Cetraria islandica, has been shown to exhibit inhibitory effects on the enzyme 5-lipoxygenase in an in vitro assay in which porcine leucocytes are used as a source of the enzyme system. The isomeric compounds (+)-lichesterinic acid (2) and (-)-lichesterinic acid (4), prepared from (+)-protolichesterinic- and (-)-allo-protolichesterinic acids, respectively, exhibited anti-5-lipoxygenase activity of the same order of magnitude. (+)-Methyl lichesterinate (3) was, however, inactive. It was shown that despite its lipophilic nature, protolichesterinic acid is extractable into an aqueous medium, the concentration being dependent on the length of extraction. PMID:23195937

  1. THE 5-LIPOXYGENASE PATHWAY IS REQUIRED FOR ACUTE LUNG INJURY FOLLOWING HEMORRHAGIC SHOCK

    PubMed Central

    Eun, John C.; Moore, Ernest E.; Mauchley, David C.; Johnson, Chris A.; Meng, Xianzhong; Banerjee, Anirban; Wohlauer, Max V.; Zarini, Simona; Gijón, Miguel A.; Murphy, Robert C.

    2012-01-01

    The cellular and biochemical mechanisms leading to acute lung injury and subsequent multiple organ failure are only partially understood. In order to study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of acute lung injury, we used a murine experimental model of acute lung injury induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration as shown by immunofluorescence, and protein leakage into the alveolar space, were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase pathway, as well as transgenic mice deficient in 5-lipoxygenase, were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared to sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-lipoxygenase, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of acute lung injury induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury. PMID:22392149

  2. Natural Forms of Vitamin E and 13′-Carboxychromanol, a Long-Chain Vitamin E Metabolite, Inhibit Leukotriene Generation from Stimulated Neutrophils by Blocking Calcium Influx and Suppressing 5-Lipoxygenase Activity, Respectively

    PubMed Central

    Jiang, Ziying; Yin, Xinmin; Jiang, Qing

    2014-01-01

    Leukotrienes generated by 5-lipoxygenase (5-LOX)–catalyzed reaction are key regulators of inflammation. In ionophore-stimulated (A23187; 1–2.5 μM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited leukotriene B4 (LTB4) with an IC50 of 5–20 μM for γ-tocopherol, δ-tocopherol (δT), and γ-tocotrienol, but a much higher IC50 for α-tocopherol. 13′-Carboxychromanol, a long-chain metabolite of δT, suppressed neutrophil- and HL-60 cell-generated LTB4 with an IC50 of 4–7 μM and potently inhibited human recombinant 5-LOX activity with an IC50 of 0.5–1 μM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key event for 5-LOX activation. Further investigation showed that δT suppressed cytosolic Ca2+ increase and/or LTB4 formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 13′-carboxychromanol decreased cellular production of LTB4 regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that δT does not likely affect fMLP receptor-mediated signaling or store depletion-induced calcium entry. Instead, we found that δT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inflammatory effects of vitamin E forms in vivo. PMID:21169551

  3. Identification of the substrate access portal of 5-Lipoxygenase

    PubMed Central

    Mitra, Sunayana; Bartlett, Sue G.

    2016-01-01

    The overproduction of inflammatory lipid mediators derived from arachidonic acid contributes to asthma and cardiovascular diseases, among other pathologies. Consequently, the enzyme that initiates the synthesis of pro-inflammatory leukotrienes, 5-lipoxygenase (5-LOX), is a target for drug design. The crystal structure of 5-LOX revealed a fully encapsulated active site, thus the point of substrate entry is not known. We asked whether a structural motif, a “cork” present in 5-LOX but absent in other mammalian lipoxygenases, might be ejected to allow substrate access. Our results indicate that reduction of cork volume facilitates access to the active site. However, if cork entry into the site is obstructed, enzyme activity is significantly compromised. The results support a model in which the “cork” that shields the active site in the absence of substrate serves as the active site portal, but the “corking” amino acid Phe-177 plays a critical role in providing a fully functional active site. Thus the more appropriate metaphor for this structural motif is a “twist-and-pour” cap. Additional mutagenesis data are consistent with a role for His-600, deep in the elongated cavity, in positioning the substrate for catalysis. PMID:26427761

  4. The 5-lipoxygenase pathway: oxidative and inflammatory contributions to the Alzheimer’s disease phenotype

    PubMed Central

    Joshi, Yash B.; Praticò, Domenico

    2015-01-01

    Alzheimer’s disease (AD) is the most common, and, arguably, one of the most-well studied, neurodegenerative conditions. Several decades of investigation have revealed that amyloid-β and tau proteins are critical pathological players in this condition. Genetic analyses have revealed specific mutations in the cellular machinery that produces amyloid-β, but these mutations are found in only a small fraction of patients with the early-onset variant of AD. In addition to development of amyloid-β and tau pathology, oxidative damage and inflammation are consistently found in the brains of these patients. The 5-lipoxygenase protein enzyme (5LO) and its downstream leukotriene metabolites have long been known to be important modulators of oxidation and inflammation in other disease states. Recent in vivo evidence using murine knock-out models has implicated the 5LO pathway, which also requires the 5LO activating protein (FLAP), in the molecular pathology of AD, including the metabolism of amyloid-β and tau. In this manuscript, we will provide an overview of 5LO and FLAP, discussing their involvement in biochemical pathways relevant to AD pathogenesis. We will also discuss how the 5LO pathway contributes to the molecular and behavioral insults seen in AD and provide an assessment of how targeting these proteins could lead to therapeutics relevant not only for AD, but also other related neurodegenerative conditions. PMID:25642165

  5. Arachidonate 5 Lipoxygenase Expression in Papillary Thyroid Carcinoma Promotes Invasion via MMP-9 Induction

    PubMed Central

    Kummer, Nicolas T.; Nowicki, Theodore S; Azzi, Jean Paul; Reyes, Ismael; Iacob, Codrin; Xie, Suqing; Swati, Ismatun; Suslina, Nina; Schantz, Stimson; Tiwari, Raj K.; Geliebter, Jan

    2012-01-01

    Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC. PMID:22253131

  6. 5-Lipoxygenase/cyclooxygenase-2 cross-talk through cysteinyl leukotriene receptor 2 in endothelial cells.

    PubMed

    Lötzer, Katharina; Jahn, Steffen; Kramer, Cornelia; Hildner, Markus; Nüsing, Rolf; Funk, Colin D; Habenicht, Andreas J R

    2007-11-01

    The 5-lipoxygenase (5-LO) pathway generates lipid mediators, i.e. the cysteinyl leukotrienes (cysLTs) LTC(4)/LTD(4) and LTB(4). CysLT receptors are expressed in endothelial cells (EC) and EC cysLT(2)-R activation induces diverse pro-inflammatory genes in vitro. We now report that LTD(4) promotes formation of an atherosclerosis-protective and anti-thrombotic eicosanoid by markedly up-regulating EC cyclooxygenase-2 (COX-2). CysLT-induced COX-2 transcripts were transiently up-regulated as determined by microarray and QRT-PCR analyses though COX-2 protein remained elevated for several hours. Prostacyclin formation, measured as its stable metabolite 6-keto-PGF(1alpha), was increased several fold in LTD(4)-stimulated ECs, and was inhibited by the COX-2-specific inhibitor, NS-398. COX-2 up-regulation was Ca(2+)-dependent and was partially blocked by cyclosporin A indicating that the 5-LO/COX-2 cross-talk involved signaling through a nuclear factor of activated T cells (NFAT) dependent pathway. Since prostacyclin is a major blood vessel-protective and anti-thrombotic eicosanoid, the EC cysLT(2)-R may limit its otherwise pro-inflammatory actions through a protective Ca(2+)/calcineurin/NFAT-dependent COX-2 feedback loop. PMID:17991613

  7. Kinetic investigation of human 5-lipoxygenase with arachidonic acid.

    PubMed

    Mittal, Monica; Kumar, Ramakrishnan B; Balagunaseelan, Navisraj; Hamberg, Mats; Jegerschöld, Caroline; Rådmark, Olof; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

    2016-08-01

    Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A4, a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20±4 on kcat and 17±2 on kcat/KM at 25°C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35°C. Also, we observed low Arrhenius prefactor ratio (AH/AD=0.21) and a small change in activation energy (Ea(D)-Ea(H)=3.6J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)kcat, but not (D)kcat. However the overall kcat/KM is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity. PMID:27363940

  8. 5-Lipoxygenase Deficiency Reduces Acetaminophen-Induced Hepatotoxicity and Lethality

    PubMed Central

    Hohmann, Miriam S. N.; Cardoso, Renato D. R.; Pinho-Ribeiro, Felipe A.; Crespigio, Jefferson; Cunha, Thiago M.; Alves-Filho, José C.; da Silva, Rosiane V.; Pinge-Filho, Phileno; Ferreira, Sergio H.; Cunha, Fernando Q.; Casagrande, Rubia; Verri, Waldiceu A.

    2013-01-01

    5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO−/−) mice and background wild type mice were challenged with APAP (0.3–6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO−/− mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1β, TNF-α, IFN-γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-β-D-glucosaminidase activity, Nrf2 and gp91phox mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO−/− mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage. PMID:24288682

  9. Arachidonate 5-lipoxygenase (ALOX5) gene polymorphism is associated with Alzheimer's disease and body mass index.

    PubMed

    Šerý, Omar; Hlinecká, Lýdia; Povová, Jana; Bonczek, Ondřej; Zeman, Tomáš; Janout, Vladimír; Ambroz, Petr; Khan, Naim A; Balcar, Vladimir J

    2016-03-15

    Dementias of old age, in particular Alzheimer's disease (AD), pose a growing threat to the longevity and quality of life of individuals as well as whole societies world-wide. The risk factors are both genetic and environmental (life-style) and there is an overlap with similar factors predisposing to cardiovascular diseases (CVD). Using a case-control genetic approach, we have identified a SNP (rs10507391) in ALOX5 gene, previously associated with an increased risk of stroke, as a novel genetic risk factor for AD. ALOX5 gene encodes a 5'-lipoxygenase (5'-LO) activating protein (FLAP), a crucial component of the arachidonic acid/leukotriene inflammatory cascade. A-allele of rs4769874 polymorphism increases the risk of AD 1.41-fold (p<0.0001), while AA genotype does so 1.79-fold (p<0.0001). In addition, GG genotype of rs4769874 polymorphism is associated with a modest increase in body mass index (BMI). We discuss potential biochemical mechanisms linking the SNP to AD and suggest possible preventive pharmacotherapies some of which are based on commonly available natural products. Finally, we set the newly identified AD risk factors into a broader context of similar CVD risk factors to generate a more comprehensive picture of interacting genetics and life-style habits potentially leading to the deteriorating mental health in the old age. PMID:26944113

  10. The 5-lipoxygenase inhibitor RF-22c potently suppresses leukotriene biosynthesis in cellulo and blocks bronchoconstriction and inflammation in vivo.

    PubMed

    Schaible, Anja M; Filosa, Rosanna; Krauth, Verena; Temml, Veronika; Pace, Simona; Garscha, Ulrike; Liening, Stefanie; Weinigel, Christina; Rummler, Silke; Schieferdecker, Sebastian; Nett, Markus; Peduto, Antonella; Collarile, Selene; Scuotto, Maria; Roviezzo, Fioretina; Spaziano, Giuseppe; de Rosa, Mario; Stuppner, Hermann; Schuster, Daniela; D'Agostino, Bruno; Werz, Oliver

    2016-07-15

    5-Lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. Because LTs play pivotal roles in allergy and inflammation, 5-LO represents a valuable target for anti-inflammatory drugs. Here, we investigated the molecular mechanism, the pharmacological profile, and the in vivo effectiveness of the novel 1,2-benzoquinone-featured 5-LO inhibitor RF-22c. Compound RF-22c potently inhibited 5-LO product synthesis in neutrophils and monocytes (IC50⩾22nM) and in cell-free assays (IC50⩾140nM) without affecting 12/15-LOs, cyclooxygenase (COX)-1/2, or arachidonic acid release, in a specific and reversible manner, supported by molecular docking data. Antioxidant or iron-chelating properties were not evident for RF-22c and 5-LO-regulatory cofactors like Ca(2+) mobilization, ERK-1/2 activation, and 5-LO nuclear membrane translocation and interaction with 5-LO-activating protein (FLAP) were unaffected. RF-22c (0.1mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. Taken together, RF-22c is a highly selective and potent 5-LO inhibitor in intact human leukocytes with pronounced effectiveness in different models of inflammation that warrants further preclinical analysis of this agent as anti-inflammatory drug. PMID:27157409

  11. Ablation of 5-lipoxygenase mitigates pancreatic lesion development

    PubMed Central

    Knab, Lawrence M.; Schultz, Michelle; Principe, Daniel R.; Mascarinas, Windel E.; Gounaris, Elias; Munshi, Hidayatullah G.; Grippo, Paul J.; Bentrem, David J.

    2016-01-01

    Background Pancreatic ductal adenocarcinoma (PDAC), which continues to have a dismal prognosis, is associated with a pronounced fibro-inflammatory response. Inflammation in vivo can be mediated by 5-lipoxygenase (5LO), an enzyme that converts omega-6 fatty acids to eicosanoids, including leukotriene B4 (LTB4). We have previously shown that diets rich in omega-6 fatty acids (FA) increase pancreatic lesions and mast cell infiltration in EL-Kras mice. In this study, we evaluated the role of 5LO in generating higher levels of LTB4 from human cells and in mediating lesion development and mast cell infiltration in EL-Kras mice. Materials and Methods Human pancreatic ductal epithelial (HPDE) and cancer cells were treated with omega-6 FA in vitro. EL-Kras mice lacking 5LO (EL-Kras/5LO−/−) mice were generated and fed standard chow or omega-6 FA diets. Pancreatic lesion frequency and mast cell infiltration were compared to EL-Kras/5LO+/+ mice. Human PDAC tumors were evaluated for 5LO expression and mast cells. Results HPDE and cancer cells treated with omega-6 FA generated increased LTB4 levels in vitro. EL-Kras/5LO−/− developed fewer pancreatic lesions and had decreased mast cell infiltration when compared to EL-Kras/5LO+/+ mice. Human PDAC tumors with increased 5LO expression demonstrate increased mast cell infiltration. Additionally, diets rich in omega-6 FA failed to increase pancreatic lesion development and mast cell infiltration in EL-Kras/5LO−/− mice. Conclusions The expansion of mutant Kras-induced lesions via omega-6 FA is dependent on 5LO, and 5LO functions downstream of mutant Kras to mediate inflammation, suggesting that 5LO may be a potential chemo-preventive and therapeutic target in pancreatic cancer. PMID:25454978

  12. Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs

    PubMed Central

    Kumar, Ramakrishnan B.; Zhu, Lin; Idborg, Helena; Rådmark, Olof; Jakobsson, Per-Johan; Rinaldo-Matthis, Agnes; Hebert, Hans; Jegerschöld, Caroline

    2016-01-01

    An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane. PMID:27010627

  13. Synthesis and Evaluation of 5-Lipoxygenase Translocation Inhibitors from Acylnitroso Hetero-Diels-Alder Cycloadducts†

    PubMed Central

    Bolger, Joshua K.; Tian, Wen; Wolter, William R.; Cho, Wonhwa; Suckow, Mark A.

    2012-01-01

    Acylnitroso cycloadducts have proven to be valuable intermediates in the syntheses of a plethora of biologically active molecules. Recently, organometallic reagents were shown to open bicyclic acylnitroso cycloadducts and, more interestingly, the prospect of highly regioselective openings was raised. This transformation was employed in the synthesis of a compound with excellent inhibitory activity against 5-lipoxygenase ((±)-4a, IC50 51 nM), an important mediator of inflammation intimately involved in a number of disease states including asthma and cancer. Optimization of the copper-mediated organometallic ring opening reaction was accomplished allowing the further exploration of the biological activity. Synthesis of a number of derivatives with varying affinity for metal binding as well as pendant groups in a range of sizes was accomplished. Analogues were tested in a whole cell assay which revealed a subset of the compounds to be inhibitors of enzyme translocation, a mode of action not previously known and, potentially, extremely important for better understanding of the enzyme and inhibitor development. Additionally, the lead compound was tested in vivo in an established colon cancer model and showed very encouraging anti-tumorogenic properties. PMID:21365098

  14. Inhibitory effects of Angelica pubescens f. biserrata on 5-lipoxygenase and cyclooxygenase.

    PubMed

    Liu, J H; Zschocke, S; Reininger, E; Bauer, R

    1998-08-01

    Linoleic acid, osthol, osthenol and two polyacetylenes, falcarindiol and 11(S),16(R)-dihydroxyoctadeca-9Z,17-diene-12,14-diyn-1 -yl acetate were found to be the most active compounds responsible for the inhibitory activity of the dichloromethane extract of the roots of Angelica pubescens f. biserrata on 5-lipoxygenase (5-LO) and cyclooxygenase (COX-1) in vitro. They showed prominent inhibitory effect on 5-LO with IC50 values of 27.9 microM, 36.2 microM, 43.1 microM, 9.4 microM and 24.0 microM, respectively. Linoleic acid, osthenol, falcarindiol and 11(S), 16(R)-dihydroxyoctadeca-9Z,17-diene-12,14-diyn-1-yl acetate exhibited inhibitory activity on COX-1 with IC50 values of 13.3 microM, 64.3 microM, 66.0 microM and 73.3 microM. PMID:9741298

  15. Penta- and hexadienoic acid derivatives: a novel series of 5-lipoxygenase inhibitors.

    PubMed

    Malleron, J L; Roussel, G; Gueremy, G; Ponsinet, G; Robin, J L; Terlain, B; Tissieres, J M

    1990-10-01

    The synthesis of a series of pentadienoic and hexadienoic acid derivatives is reported. These compounds were tested as inhibitors of 5-lipoxygenase (5 LO) and cyclooxygenase (CO) in vitro and as inhibitors of arachidonic acid (AA) induced ear edema in mice in vivo. Their potency is compared with that of the standard inhibitors nafazatrom, BW 755C, NDGA, KME4, quercetine, and L 652,243. The most potent compound in vivo, diethyl 2-hydroxy-5-(ethylthio)-2(Z),4(Z)-hexadienedioate (20) inhibited AA-induced ear edema when administered topically or orally, with an ED50 value of 0.01 mg/ear and 20 mg/kg, respectively. Among the standard compounds tested, L 652,243 was the most active compound in this test with an ED50 value of 0.01 mg/ear and 1 mg/kg po, but unlike this compound, 20 is a selective inhibitor of 5-LO (IC50 = 2 microM) without any significant activity against CO (IC50 greater than 50 microM). Most of the other compounds in this series are also selective 5-LO inhibitors. PMID:2213827

  16. Synthesis and biological evaluation of novel pyrazolopyrimidines derivatives as anticancer and anti-5-lipoxygenase agents.

    PubMed

    Rahmouni, Ameur; Souiei, Sawssen; Belkacem, Mohamed Amine; Romdhane, Anis; Bouajila, Jalloul; Ben Jannet, Hichem

    2016-06-01

    A novel series of 6-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 3a-h were synthesized in a single step via condensation of carboxamide 2 with some aromatic aldehydes (presence of iodine). Treatment of aminopyrazole 1a with acetic anhydride afforded pyrazolopyrimidines 4 which on treatment with ethyl chloroacetate in refluxing dry DMF furnished a single product identified as ethyl 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl) acetate 5. On the other hand, esterification of compound 6 with different alcohol, led to the formation of new esters linked pyrazolo[3,4-d]pyrimidinones hybrids 7a-f. The reaction of compound 2 with 3-propargyl bromide gave the compound 8 used as a dipolarophile to access to triazoles (4- and 5-regioisomers (9a-e) and (10a-e), respectively) via the 1,3-dipoar cycloaddition reaction. Finally, condensation reaction of aminopyrazole 1b with α-cyanocinnamonitiles gave the new pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitriles 11a-e. Structures of compounds were established on the basis of (1)H/(13)C NMR and ESI-HRMS. Compounds were screened for their cytotoxic (HCT-116 and MCF-7) and 5-lipoxygenase inhibition activities. The structure-activity relationship (SAR) was discussed. PMID:27179178

  17. A dual inhibitor of cyclooxygenase and 5-lipoxygenase protects against kainic acid-induced brain injury.

    PubMed

    Minutoli, Letteria; Marini, Herbert; Rinaldi, Mariagrazia; Bitto, Alessandra; Irrera, Natasha; Pizzino, Gabriele; Pallio, Giovanni; Calò, Margherita; Adamo, Elena Bianca; Trichilo, Vincenzo; Interdonato, Monica; Galfo, Federica; Squadrito, Francesco; Altavilla, Domenica

    2015-06-01

    Systemic administration of kainic acid causes inflammation and apoptosis in the brain, resulting in neuronal loss. Dual cyclooxygenase/5-lipoxygenase (COX/5-LOX) inhibitors could represent a possible neuroprotective approach in preventing glutamate excitotoxicity. Consequently, we investigated the effects of a dual inhibitor of COX/5-LOX following intraperitoneal administration of kainic acid (KA, 10 mg/kg) in rats. Animals were randomized to receive either the dual inhibitor of COX/5-LOX (flavocoxid, 20 mg/kg i.p.) or its vehicle (1 ml/kg i.p.) 30 min after KA administration. Sham brain injury rats were used as controls. We evaluated protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) and tumor necrosis factor alpha (TNF-α) as well as levels of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the hippocampus. Animals were also observed for monitoring behavioral changes according to Racine Scale. Finally, histological analysis and brain edema evaluation were carried out. Treatment with the dual inhibitor of COX/5-LOX decreased protein expression of p-ERK1/2 and TNF-α in hippocampus, markedly reduced MDA, LTB4 and PGE2 hippocampal levels, and also ameliorated brain edema. Histological analysis showed a reduction in cell damage in rats treated with the dual inhibitor of COX/5-LOX, particularly in hippocampal subregion CA3c. Moreover, flavocoxid significantly improved behavioral signs following kainic acid administration. Our results suggest that dual inhibition of COX/5-LOX by flavocoxid has neuroprotective effects during kainic acid-induced excitotoxicity. PMID:25893744

  18. Arachidonate 5-lipoxygenase gene variants affect response to fish oil supplementation by healthy African Americans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: To determine the effects of arachidonate 5-lipoxygenase gene (ALOX5) variants on plasma lipid and lipoprotein concentrations and changes in response to fish oil supplementation. We hypothesized that Sp1 variants in the ALOX5 promoter, which have previously been associated with cardiovascu...

  19. Impact of simultaneous stimulation of 5-lipoxygenase and myeloperoxidase in human neutrophils.

    PubMed

    Zschaler, Josefin; Arnhold, Jürgen

    2016-04-01

    Human neutrophil 5-lipoxygenase (5-LOX) oxidizes arachidonic acid (AA) to 5S-hydro(pero)xy-6E,8Z,11Z, 14Z-eicosatetraenoic acid (5-H(p)ETE) and leukotriene (LT)A4, which is further converted to the chemoattractant LTB4. These cells contain also the heme enzyme myeloperoxidase (MPO) producing several potent oxidants such as hypochlorous acid (HOCl). Previously, it was shown that MPO-metabolites influence 5-LOX product formation. Here, we addressed the question, whether a simultaneous activation of MPO and 5-LOX in neutrophils results in comparable changes of 5-LOX activity. Human neutrophils were stimulated with H2O2 or phorbol 12-myristate 13-acetate (PMA) for MPO activation and subsequently treated with calcium ionophore A23187 inducing 5-LOX product formation on endogenous AA. Special attention was drawn to neutrophil vitality, formation of MPO-derived metabolites and redox status. The pre-stimulation with H2O2 resulted in a concentration-dependent increase in the ratio of 5-HETE to the sum of LTB4+6-trans-LTB4 in consequence of MPO activation. Thereby no impairment of cell vitality and only a slightly reduction of total glutathione level was observed. An influence of MPO on 5-LOX product formation could be suggested using an MPO inhibitor. In contrast, the pre-stimulation with PMA resulted in different changes of 5-LOX product formation leading to a reduced amount of 5-HETE unaffected by MPO inhibition. Furthermore, impaired cell vitality and diminished redox status was detected after PMA stimulation. Nevertheless, a MPO-induced diminution of LTB4 was obvious. Further work is necessary to define the type of 5-LOX modification and investigate the effect of physiological MPO activators. PMID:27033421

  20. Pharmacophore modeling and virtual screening for designing potential 5-lipoxygenase inhibitors.

    PubMed

    Aparoy, P; Kumar Reddy, K; Kalangi, Suresh K; Chandramohan Reddy, T; Reddanna, P

    2010-02-01

    Inhibitors of the 5-Lipoxygenase (5-LOX) pathway have a therapeutic potential in a variety of inflammatory disorders such as asthma. In this study, chemical feature based pharmacophore models of inhibitors of 5-LOX have been developed with the aid of HipHop and HypoGen modules within Catalyst program package. The best quantitative pharmacophore model, Hypo1, which has the highest correlation coefficient (0.97), consists of two hydrogen-bond acceptors, one hydrophobic feature and one ring aromatic feature. Hypo1 was further validated by test set and cross validation method. The application of the model shows great success in predicting the activities of 65 known 5-LOX inhibitors in our test set with a correlation coefficient of 0.85 with a cross validation of 95% confidence level, proving that the model is reliable in identifying structurally diverse compounds for inhibitory activity against 5-LOX. Furthermore, Hypo1 was used as a 3D query for screening Maybridge and NCI databases within catalyst and also drug like compounds obtained from Enamine Ltd, which follow Lipinski's rule of five. The hit compounds were subsequently subjected to filtering by docking and visualization, to identify the potential lead molecules. Finally 5 potential lead compounds, identified in the above process, were evaluated for their inhibitory activities. These studies resulted in the identification of two compounds with potent inhibition of 5-LOX activity with IC(50) of 14 microM and 35 microM, respectively. These studies thus validate the pharmacophore model generated and suggest the usefulness of the model in screening of various small molecule libraries and identification of potential lead compounds for 5-LOX inhibition. PMID:20045317

  1. Effect of the 5-lipoxygenase inhibitor ZD2138 on aspirin-induced asthma.

    PubMed Central

    Nasser, S. M.; Bell, G. S.; Foster, S.; Spruce, K. E.; MacMillan, R.; Williams, A. J.; Lee, T. H.; Arm, J. P.

    1994-01-01

    BACKGROUND--The cysteinyl leukotrienes may play a central part in the mechanisms of aspirin-sensitive asthma. Previous work has shown that individuals with aspirin-sensitive asthma have high basal urinary LTE4 levels which increase further upon aspirin ingestion, and that sulphidopeptide leukotriene receptor antagonists attenuate aspirin-induced airflow obstruction. If the cysteinyl leukotrienes cause aspirin-induced asthmatic reactions, inhibition of the 5-lipoxygenase pathway should prevent aspirin-induced bronchospasm. This hypothesis has been tested with ZD2138, a specific non-redox 5-lipoxygenase inhibitor. METHODS--Seven subjects (four men) with aspirin-sensitive asthma with baseline FEV1 values > 67% were studied. ZD2138 (350 mg) or placebo was given on two separate occasions two weeks apart in a randomised double blind fashion. A single dose of aspirin was administered four hours after dosing and FEV1 was measured for six hours. Inhibition of the 5-lipoxygenase pathway by ZD2138 was assessed by measurements of urinary LTE4 levels and ex vivo calcium ionophore stimulated LTB4 generation in whole blood, before administration of drug or placebo and at regular time intervals after dosing and aspirin administration. RESULTS--ZD2138 protected against the aspirin-induced reduction in FEV1 with a 20.3 (4.9)% fall in FEV1 following placebo compared with 4.9 (2.9)% following ZD2138. This was associated with 72% inhibition of ex vivo LTB4 generation in whole blood at 12 hours and a 74% inhibition of the rise in urinary LTE4 excretion at six hours after aspirin ingestion. CONCLUSIONS--In aspirin-sensitive asthma the 5-lipoxygenase inhibitor ZD2138 inhibits the fall in FEV1 induced by aspirin and this is associated with substantial inhibition of 5-lipoxygenase. PMID:8091318

  2. Regulation of tumorigenic Wnt signaling by cyclooxygenase-2, 5-lipoxygenase and their pharmacological inhibitors: A basis for novel drugs targeting cancer cells?

    PubMed

    Roos, Jessica; Grösch, Sabine; Werz, Oliver; Schröder, Peter; Ziegler, Slava; Fulda, Simone; Paulus, Patrick; Urbschat, Anja; Kühn, Benjamin; Maucher, Isabelle; Fettel, Jasmin; Vorup-Jensen, Thomas; Piesche, Matthias; Matrone, Carmela; Steinhilber, Dieter; Parnham, Michael J; Maier, Thorsten J

    2016-01-01

    Canonical Wnt signaling is a highly conserved pathway with a prominent role in embryogenic development, adult tissue homeostasis, cell polarization, stem cell biology, cell differentiation, and proliferation. Furthermore, canonical Wnt signaling is of pivotal importance in the pathogenesis of a number of cancer types and crucially affects tumor initiation, cancer cell proliferation, cancer cell apoptosis, and metastasis. Reports over the last decade have provided strong evidence for a pathophysiological role of Wnt signaling in non-malignant classical inflammatory and neurodegenerative diseases. Although, several agents suppressing the Wnt pathway at different levels have been identified, the development of clinically relevant Wnt-inhibiting agents remains challenging due to selectivity and toxicity issues. Several studies have shown that long-term administration of non-steroidal anti-inflammatory drugs protects against colon cancer and potentially other tumor types by interfering both with the COX and the Wnt pathway. Our own studies have shown that non-steroidal anti-inflammatory drugs suppress Wnt signaling by targeting the pro-inflammatory enzyme 5-lipoxygenase which is the key enzyme pathophysiologically involved in the synthesis of leukotrienes. Furthermore, we found a direct link between the 5-lipoxygenase and Wnt signaling pathways, which is essential for the maintenance of leukemic stem cells. Accordingly, genetic and pharmacological inhibition of 5-lipoxygenase led to an impairment of Wnt-dependent acute and chronic myeloid leukemic stem cells. We believe that 5-lipoxygenase inhibitors might represent a novel type of Wnt inhibitor activating a potentially naturally occurring novel mechanism of suppression of Wnt signaling that is non-toxic, at least in mice, and is potentially well tolerated in patients. PMID:26549540

  3. Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors.

    PubMed

    De Lucia, Daniela; Lucio, Oscar Méndez; Musio, Biagia; Bender, Andreas; Listing, Monika; Dennhardt, Sophie; Koeberle, Andreas; Garscha, Ulrike; Rizzo, Roberta; Manfredini, Stefano; Werz, Oliver; Ley, Steven V

    2015-08-28

    In this work the synthesis, structure-activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of 0.2 and 3.2 μm in cell-based and cell-free assays, respectively. Optimization of binding and functional potencies resulted in the identification of compound 13d, which showed an enhanced activity compared to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15 and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests were performed to exclude a relationship between cytotoxicity and the increased activity observed after structure optimization. PMID:26197161

  4. Effect of 5-lipoxygenase on the development of pulmonary hypertension in rats.

    PubMed

    Jones, John E; Walker, Jennifer L; Song, Yanli; Weiss, Norbert; Cardoso, Wellington V; Tuder, Rubin M; Loscalzo, Joseph; Zhang, Ying-Yi

    2004-05-01

    5-Lipoxygenase (5-LO) and its downstream leukotriene products have been implicated in the development of pulmonary hypertension. In this study, we examined the effects of 5-LO overexpression in rat lungs on pulmonary hypertension using a recombinant adenovirus expressing 5-LO (Ad5-LO). Transthoracic echocardiography and right heart catheterization data showed that 5-LO overexpression in the lung did not cause pulmonary hypertension in normal rats; however, it markedly accelerated the progression of pulmonary hypertension in rats treated with monocrotaline (MCT). An increase in pulmonary artery pressure occurred earlier in the rats treated with MCT + Ad5-LO (7-10 days) compared with those treated with control vector, MCT + adenovirus expressing green fluorescent protein (AdGFP), or MCT alone (15-18 days). The weight ratio of the right ventricle to left ventricle plus septum was higher in the MCT + Ad5-LO group than that of the MCT + AdGFP or MCT group (0.45 +/- 0.08 vs. 0.35 +/- 0.03 or 0.33 +/- 0.06). Lung tissue histological sections from MCT + Ad5-LO rats exhibited more severe inflammatory cell infiltration and pulmonary vascular muscularization than those from MCT + AdGFP- or MCT-treated rats. Administration of 5-LO inhibitors, zileuton or MK-886, to either MCT- or MCT + Ad5-LO-treated rats prevented the development of pulmonary hypertension. These data suggest that 5-LO plays a critical role in the progression of pulmonary hypertension in rats and that the detrimental effect of 5-LO is manifest only in the setting of pulmonary vascular endothelial cell dysfunction. PMID:14726295

  5. Computational insight into the catalytic implication of head/tail-first orientation of arachidonic acid in human 5-lipoxygenase: consequences for the positional specificity of oxygenation.

    PubMed

    Saura, Patricia; Maréchal, Jean-Didier; Masgrau, Laura; Lluch, José M; González-Lafont, Àngels

    2016-08-17

    In the present work we have combined homology modeling, protein-ligand dockings, quantum mechanics/molecular mechanics calculations and molecular dynamics simulations to generate human 5-lipoxygenase (5-LOX):arachidonic acid (AA) complexes consistent with the 5-lipoxygenating activity (which implies hydrogen abstraction at the C7 position). Our results suggest that both the holo and the apo forms of human Stable 5-LOX could accommodate AA in a productive form for 5-lipoxygenation. The former, in a tail-first orientation, with the AA carboxylate end interacting with Lys409, gives the desired structures with C7 close to the Fe-OH(-) cofactor and suitable barrier heights for H7 abstraction. Only when using the apo form structure, a head-first orientation with the AA carboxylate close to His600 (a residue recently proposed as essential for AA positioning) is obtained in the docking calculations. However, the calculated barrier heights for this head-first orientation are in principle consistent with 5-LOX specificity, but also with 12/8 regioselectivity. Finally, long MD simulations give support to the recent hypothesis that the Phe177 + Tyr181 pair needs to close the active site access during the chemical reaction, and suggest that in the case of a head-first orientation Phe177 may be the residue interacting with the AA carboxylate. PMID:27489112

  6. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

    PubMed Central

    Zhang, Y Y; Hammarberg, T; Radmark, O; Samuelsson, B; Ng, C F; Funk, C D; Loscalzo, J

    2000-01-01

    5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO. PMID:11042125

  7. 5-lipoxygenase pathway is essential for the control of granuloma extension induced by Schistosoma mansoni eggs in lung.

    PubMed

    Toffoli da Silva, Gabriel; Espíndola, Milena Sobral; Fontanari, Caroline; Rosada, Rogerio Silva; Faccioli, Lúcia Helena; Ramos, Simone Gusmão; Rodrigues, Vanderlei; Frantz, Fabiani Gai

    2016-08-01

    According to WHO, it is estimated that approximately 2 billion people are infected with intestinal helminths worldwide and the number of people who are cured of these diseases is relatively low, resulting in a large percentage of chronically infected individuals. Schistosomiasis is one of the most important parasitic diseases present in developing countries configuring it as a serious public health problem, directly related to poverty and social disadvantage. Once the parasite infection is established, Schistosoma mansoni eggs fall into the bloodstream and are trapped in the liver microcirculation where a strong granulomatous response and fibrosis formation occurs. In the experimental model, granulomas develop in the mouse lung after intravenous injection of purified eggs. Here we aim to understand how leukotrienes are involved in the granuloma formation. Leukotrienes are lipid mediators derived from arachidonic acid metabolites via 5-lipoxygenase (5LO) enzyme. They are potent proinflammatory agents and induce recruitment, cell activation, regulation of microbicidal activity of polymorphonuclear and mononuclear cells. In this study, 5LO deficient mice (5LO(-/-)) were inoculated with S. mansoni eggs for evaluation of immunopathological parameters involved in the induction of type 2 granulomas. We showed that in the absence of leukotrienes, the size of granulomas were decreased comparing to the wild type mice and the inflammatory compromised areas had a lower extension. In 5LO(-/-) mice granulomas presented extensive areas of fibrosis, detected by α-SMA expression along the lesions, indicating remodeling in attempt to reestablish the normal tissue. Also, comparing to WT mice we detected decrease of IL-4 and IL-13 and increase of TGF-β in the lung of 5LO(-/-), but these mice failed to produce protective IFN-γ and IL-12. These results evidenced 5-Lipoxygenase as an important pathway during lung injury due to Schistosoma-eggs injection. PMID:27262746

  8. Impact of myeloperoxidase-derived oxidants on the product profile of human 5-lipoxygenase.

    PubMed

    Zschaler, Josefin; Dorow, Juliane; Schöpe, Louisa; Ceglarek, Uta; Arnhold, Jürgen

    2015-08-01

    Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6 E,8 Z,11 Z,14 Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl(-) system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in

  9. Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663

    SciTech Connect

    Gilbert, Nathaniel C.; Rui, Zhe; Neau, David B.; Waight, Maria T.; Bartlett, Sue G.; Boeglin, William E.; Brash, Alan R.; Newcomer, Marcia E.

    2012-08-31

    The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca{sup 2+}-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A4 from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.

  10. Structural optimization and biological evaluation of 1,5-disubstituted pyrazole-3-carboxamines as potent inhibitors of human 5-lipoxygenase

    PubMed Central

    Zhou, Yu; Liu, Jun; Zheng, Mingyue; Zheng, Shuli; Jiang, Chunyi; Zhou, Xiaomei; Zhang, Dong; Zhao, Jihui; Ye, Deju; Zheng, Mingfang; Jiang, Hualiang; Liu, Dongxiang; Cheng, Jian; Liu, Hong

    2016-01-01

    Human 5-lipoxygenase (5-LOX) is a well-validated drug target and its inhibitors are potential drugs for treating leukotriene-related disorders. Our previous work on structural optimization of the hit compound 2 from our in-house collection identified two lead compounds, 3a and 3b, exhibiting a potent inhibitory profile against 5-LOX with IC50 values less than 1 µmol/L in cell-based assays. Here, we further optimized these compounds to prepare a class of novel pyrazole derivatives by opening the fused-ring system. Several new compounds exhibited more potent inhibitory activity than the lead compounds against 5-LOX. In particular, compound 4e not only suppressed lipopolysaccharide-induced inflammation in brain inflammatory cells and protected neurons from oxidative toxicity, but also significantly decreased infarct damage in a mouse model of cerebral ischemia. Molecular docking analysis further confirmed the consistency of our theoretical results and experimental data. In conclusion, the excellent in vitro and in vivo inhibitory activities of these compounds against 5-LOX suggested that these novel chemical structures have a promising therapeutic potential to treat leukotriene-related disorders. PMID:26904397

  11. Effect of a 5-lipoxygenase (5-LO)/cyclooxygenase (CO) inhibitor, WY-47, 288, on cutaneous models of inflammation.

    PubMed

    Carlson, R P; O'Neill-Davis, L; Calhoun, W; Datko, L; Musser, J H; Kreft, A F; Chang, J Y

    1989-03-01

    WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline) demonstrated topical antiinflammatory activity in several animal models of skin inflammation. Application of WY-47,288 to mouse ear surfaces inhibited arachidonic acid (ED50 = 0.3 mg/ear) and tetradecanoylphorbol acetate (TPA)-induced inflammation (40% at 1 mg/ear). Administration of WY-47,288 (1 mg/ear) at 30 min and 5 h after TPA reduced ear edema and epidermal proliferation by 50%. WY-47,288 also inhibited oxazolone-induced contact hypersensitivity in mouse ears (ED50 = 0.4 mg/ear) and UVB-induced guinea pig skin erythema (ED50 approximately 0.25 mg/spot). These antiinflammatory effects may be due to inhibition of 5-lipoxygenase (5-LO) and cyclooxygenase (CO) since the synthesis of 5-LO and CO products by rat neutrophils and mouse macrophages was dose-dependently reduced by WY-47,288. By contrast, WY-47,288 demonstrated no appreciable inhibition of 12-LO (rabbit platelet), 15-LO (soybean) or phospholipase A2 (human platelet). Furthermore, no systemic adverse effects were observed after topical, parenteral or oral administration of WY-47,288, suggesting that WY-47,288 is a safe topical 5-LO/CO inhibitor for treating skin inflammation. PMID:2500009

  12. Repeated allergen exposure reduce early phase airway response and leukotriene release despite upregulation of 5-lipoxygenase pathways

    PubMed Central

    2012-01-01

    Background Allergen induced early phase airway response and airway plasma exudation are predominantly mediated by inflammatory mast cell mediators including histamine, cysteinyl leukotrienes (cysLTs) and thromboxane A2 (TXA2). The aim of the present study was to evaluate whether repeated allergen exposure affects early phase airway response to allergen challenge. Methods A trimellitic anhydride (TMA) sensitized guinea pig model was used to investigate the effects of low dose repeated allergen exposure on cholinergic airway responsiveness, early phase airway response and plasma exudation, as well as local airway production of mast cell derived cysteinyl leukotrienes and thromboxane B2 (TXB2) after allergen challenge. Results Repeated low dose allergen exposure increased cholinergic airway responsiveness. In contrast, early phase airway response and plasma exudation in response to a high-dose allergen challenge were strongly attenuated after repeated low dose allergen exposure. Inhibition of the airway response was unspecific to exposed allergen and independent of histamine receptor blocking. Furthermore, a significant reduction of cysteinyl leukotrienes and TXB2 was found in the airways of animals repeatedly exposed to a low dose allergen. However, in vitro stimulation of airway tissue from animals repeatedly exposed to a low dose allergen with arachidonic acid and calcium ionophore (A23187) induced production of cysteinyl leukotrienes and TXB2, suggesting enhanced activity of 5-lipoxygenase and cyclooxygenase pathways. Conclusions The inhibition of the early phase airway response, cysteinyl leukotriene and TXB2 production after repeated allergen exposure may result from unresponsive effector cells. PMID:22439792

  13. Zileuton, 5-Lipoxygenase Inhibitor, Acts as a Chemopreventive Agent in Intestinal Polyposis, by Modulating Polyp and Systemic Inflammation

    PubMed Central

    Heiferman, Jeffrey R.; Shrivastav, Manisha; Vitello, Dominic; Blatner, Nichole R.; Knab, Lawrence M.; Phillips, Joseph D.; Cheon, Eric C.; Grippo, Paul J.; Khazaie, Khashayarsha; Munshi, Hidayatullah G.; Bentrem, David J.

    2015-01-01

    Purpose Leukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice. Experimental Design In the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation. Results Zileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice. Conclusions This study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer. PMID:25747113

  14. Anti-proliferative effects of lichen-derived inhibitors of 5-lipoxygenase on malignant cell-lines and mitogen-stimulated lymphocytes.

    PubMed

    Ogmundsdóttir, H M; Zoëga, G M; Gissurarson, S R; Ingólfsdóttir, K

    1998-01-01

    microg mL(-1) for protolichesterinic acid and 30 microg mL(-1) for lobaric acid. We conclude that the anti-proliferative and cytotoxic effects observed might be related to the 5-lipoxygenase inhibitory activity of protolichesterinic acid and lobaric acid. These results open up the opportunity for future studies of these lichen metabolites with regard to their anti-tumour and anti-inflammatory properties. PMID:9504441

  15. 5-Lipoxygenase metabolite 4-HDHA is a mediator of the antiangiogenic effect of ω-3 polyunsaturated fatty acids.

    PubMed

    Sapieha, Przemyslaw; Stahl, Andreas; Chen, Jing; Seaward, Molly R; Willett, Keirnan L; Krah, Nathan M; Dennison, Roberta J; Connor, Kip M; Aderman, Christopher M; Liclican, Elvira; Carughi, Arianna; Perelman, Dalia; Kanaoka, Yoshihide; Sangiovanni, John Paul; Gronert, Karsten; Smith, Lois E H

    2011-02-01

    Lipid signaling is dysregulated in many diseases with vascular pathology, including cancer, diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. We have previously demonstrated that diets enriched in ω-3 polyunsaturated fatty acids (PUFAs) effectively reduce pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy, in part through metabolic products that suppress microglial-derived tumor necrosis factor-α. To better understand the protective effects of ω-3 PUFAs, we examined the relative importance of major lipid metabolic pathways and their products in contributing to this effect. ω-3 PUFA diets were fed to four lines of mice deficient in each key lipid-processing enzyme (cyclooxygenase 1 or 2, or lipoxygenase 5 or 12/15), retinopathy was induced by oxygen exposure; only loss of 5-lipoxygenase (5-LOX) abrogated the protection against retinopathy of dietary ω-3 PUFAs. This protective effect was due to 5-LOX oxidation of the ω-3 PUFA lipid docosahexaenoic acid to 4-hydroxy-docosahexaenoic acid (4-HDHA). 4-HDHA directly inhibited endothelial cell proliferation and sprouting angiogenesis via peroxisome proliferator-activated receptor γ (PPARγ), independent of 4-HDHA's anti-inflammatory effects. Our study suggests that ω-3 PUFAs may be profitably used as an alternative or supplement to current anti-vascular endothelial growth factor (VEGF) treatment for proliferative retinopathy and points to the therapeutic potential of ω-3 PUFAs and metabolites in other diseases of vasoproliferation. It also suggests that cyclooxygenase inhibitors such as aspirin and ibuprofen (but not lipoxygenase inhibitors such as zileuton) might be used without losing the beneficial effect of dietary ω-3 PUFA. PMID:21307302

  16. Pharmacological characterization of SB 202235, a potent and selective 5-lipoxygenase inhibitor: effects in models of allergic asthma.

    PubMed

    Chabot-Fletcher, M C; Underwood, D C; Breton, J J; Adams, J L; Kagey-Sobotka, A; Griswold, D E; Marshall, L A; Sarau, H M; Winkler, J D; Hay, D W

    1995-06-01

    The peptidoleukotrienes and leukotriene B4, formed from arachidonic acid through the action of 5-lipoxygenase (5-LO), exert a spectrum of biological effects. It has been proposed that potent and selective 5-LO inhibitors will be effective therapy in diseases in which the peptidoleukotrienes and leukotriene B4 have been implicated, such as asthma and arthritis. The novel compound (S)-N-hydroxy-N-(2,3-dihydro-6-phenylmethoxy-3-benzyofuranyl )urea (SB 202235) was evaluated as a selective inhibitor of 5-LO in a cell-free system as well as in various cellular assays. In addition, the potential therapeutic value of SB 202235 was assessed in preclinical models of allergic asthma. The activity of the 5-LO enzyme isolated from rat basophilic leukemia-1 cells was inhibited by SB 202235 in a concentration-dependent manner with an IC50 value of 1.9 microM. Consistent with its ability to inhibit 5-LO, SB 202235 inhibited the production of leukotriene B4 by human monocytes and in human whole blood (IC50 values of 1.5 microM and 1.1 microM, respectively). The selectivity of SB 202235 was confirmed by its lack of effect against several other enzymes and receptors. SB 202235 potently and effectively inhibited the contraction produced by a single concentration of ovalbumin in guinea pig trachea (IC50 = 20 microM) and of anti-IgE in human bronchus (IC50 = 2 microM). SB 202235 (3-30 microM) also inhibited the contraction of guinea pig trachea in response to increasing concentration of ovalbumin. When administered orally (30 mg/kg) to conscious guinea pigs, SB 202235 attenuated antigen-induced broncho-constriction and the subsequent eosinophil influx.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7791085

  17. Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: synthesis, COX/LOX inhibition, molecular modeling, and insights into their cytotoxicities.

    PubMed

    Ghatak, Shibnath; Vyas, Alok; Misra, Suniti; O'Brien, Paul; Zambre, Ajit; Fresco, Victor M; Markwald, Roger R; Swamy, K Venkateshwara; Afrasiabi, Zahra; Choudhury, Amitava; Khetmalas, Madhukar; Padhye, Subhash

    2014-01-01

    Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX-LOX inhibitors in colon cancer cells. PMID:24295787

  18. Nutrigenetic association of the 5-lipoxygenase gene with myocardial infarction123

    PubMed Central

    Allayee, Hooman; Baylin, Ana; Hartiala, Jaana; Wijesuriya, Hemani; Mehrabian, Margarete; Lusis, Aldons J; Campos, Hannia

    2010-01-01

    Background 5-Lipoxygenase (5-LO) catalyzes the rate-limiting step of the biosynthesis of proinflammatory leukotrienes from arachidonic acid (AA) and has been associated with atherosclerosis in animal models and humans. We previously reported that variants of a 5-LO promoter repeat polymorphism were associated with carotid atherosclerosis in humans, an effect that was exacerbated by high dietary AA but mitigated by high dietary n–3 fatty acids. Objective We sought to confirm these initial observations with a more clinically relevant phenotype such as myocardial infarction (MI). Design The 5-LO polymorphism was genotyped in 1885 Costa Rican case-control pairs and tested for association with MI. Functional experiments were carried out to determine whether the associated alleles had differences in mRNA expression. Results The frequency of variant genotype groups did not differ significantly between cases and controls. However, a significant gene × diet interaction was observed, in which, relative to the common 5 repeat allele, the 3 and 4 alleles were associated with a higher MI risk in the high (≥0.25 g/d) dietary AA group (odds ratio: 1.31; 95% CI: 1.07, 1.61) and with a lower risk in the low (<0. 25 g/d)AA group (0.77; 0.63, 0.94) (P for interaction = 0.015). Using allele-specific quantitation, the short alleles had expression approximately twice that of the 5 allele (P < 0.0001). Conclusions The 3 and 4 variants lead to higher 5-LO expression and provide additional evidence that these alleles are associated with greater risks of atherosclerosis and MI in the context of a high-AA diet. PMID:18842779

  19. 5-Lipoxygenase Negatively Regulates Th1 Response during Brucella abortus Infection in Mice

    PubMed Central

    Fahel, Júlia Silveira; de Souza, Mariana Bueno; Gomes, Marco Túlio Ribeiro; Corsetti, Patricia P.; Carvalho, Natalia B.; Marinho, Fabio A. V.; de Almeida, Leonardo A.; Caliari, Marcelo V.; Machado, Fabiana Simão

    2015-01-01

    Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B4 and lipoxin A4 in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen. PMID:25583526

  20. Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced Hamster Cheek Pouch Carcinogenesis by a 5-Lipoxygenase Inhibitor, Garcinol

    PubMed Central

    Shim, Joong-Youn; Sang, Shengmin; Sun, Zheng; Chen, Xiaoxin

    2013-01-01

    Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis, and can be targeted for cancer prevention. In order to develop potent topical agents for oral cancer chemoprevention, five known 5-Lox inhibitors from dietary and synthetic sources, Zileuton, ABT-761, Licofelone, Curcumin and Garcinol, were evaluated in silico for their potential efficacy. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be a promising agent based on the calculation of a theoretical activity index. Computer modeling showed that garcinol well fit the active site of 5-Lox, and potentially inhibited enzyme activity through interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. These results demonstrated that topical application of a 5-Lox inhibitor, garcinol, had chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis. PMID:23137051

  1. Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch carcinogenesis by a 5-lipoxygenase inhibitor, garcinol.

    PubMed

    Chen, Xin; Zhang, Xinyan; Lu, Ye; Shim, Joong-Youn; Sang, Shengmin; Sun, Zheng; Chen, Xiaoxin

    2012-01-01

    Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis and can be targeted for cancer prevention. To develop potent topical agents for oral cancer chemoprevention, 5 known 5-Lox inhibitors from dietary and synthetic sources (Zileuton, ABT-761, licofelone, curcumin, and garcinol) were evaluated in silico for their potential efficacy. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be a promising agent based on the calculation of a theoretical activity index. Computer modeling showed that garcinol well fit the active site of 5-Lox, and potentially inhibited enzyme activity through interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. These results demonstrated that topical application of a 5-Lox inhibitor, garcinol, had chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis. PMID:23137051

  2. Modulation of LPS-induced memory insult, γ-secretase and neuroinflammation in 3xTg mice by 5-Lipoxygenase

    PubMed Central

    Joshi, Yash B.; Giannopoulos, Phillip F.; Chu, Jin; Praticò, Domenico

    2014-01-01

    Besides amyloid and tau pathology, a constant feature of Alzheimer’s disease (AD) is an intense inflammatory response, which is considered an active player in its pathogenesis. The 5-Lipoxygenase (5LO) is a proinflammatory enzyme and an endogenous modulator of AD-like phenotype in mouse models of the disease. To further understand the role of 5LO in AD pathogenesis, we exposed the 3xTg and 3xTg/5LO knockout mice to lipopolysaccharide (LPS), a known inducer of neuroinflammation, and evaluated its effect on their AD-like phenotype. 3xTg mice treated with LPS manifested a worsening of behavior, γ-secretase up-regulation, and increased neuroinflammatory responses. These effects were completely prevented in 3xTg mice genetically deficient for 5LO. By contrast, the absence of 5LO did not protect against increase in tau phosphorylation at specific epitopes that were mediated by the activation of the cyclin-dependent kinase 5. Our data demonstrate that the 5LO pathway affects key neuropathological features of the AD-like phenotype (behavior, Abeta, microgliosis, astrocytosis) but not others (tau pathology) in the LPS-dependent neuroinflammation model. The opposite ways whereby 5LO influences the LPS-dependent effects in vivo supports the complex nature of the neuroinflammatory response in AD and its differential role in modulating amyloid and tau neuropathology. PMID:24332986

  3. Further studies on ethyl 5-hydroxy-indole-3-carboxylate scaffold: design, synthesis and evaluation of 2-phenylthiomethyl-indole derivatives as efficient inhibitors of human 5-lipoxygenase.

    PubMed

    Peduto, Antonella; Bruno, Ferdinando; Dehm, Friedrike; Krauth, Verena; de Caprariis, Paolo; Weinigel, Christina; Barz, Dagmar; Massa, Antonio; De Rosa, Mario; Werz, Oliver; Filosa, Rosanna

    2014-06-23

    5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the design, synthesis and biological evaluation of novel series of ethyl 5-hydroxyindole-3-carboxylate derivatives that efficiently inhibit human 5-LO. SAR analysis revealed that the potency of compounds is closely related to the positioning of the substituents at the phenylthiomethyl ring. The introduction of methyl or chlorine groups in ortho- and ortho/para-position of thiophenol represent the most favorable modifications. Among all tested compounds, ethyl 5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (19) is the most potent derivative which blocks 5-LO activity in cell-free assays with IC50 = 0.7 μM, and suppressed 5-LO product synthesis in polymorphonuclear leukocytes with IC50 = 0.23 μM. PMID:24871899

  4. Elucidation of the molecular mechanism and the efficacy in vivo of a novel 1,4-benzoquinone that inhibits 5-lipoxygenase

    PubMed Central

    Schaible, A M; Filosa, R; Temml, V; Krauth, V; Matteis, M; Peduto, A; Bruno, F; Luderer, S; Roviezzo, F; Di Mola, A; Rosa, M; D'Agostino, B; Weinigel, C; Barz, D; Koeberle, A; Pergola, C; Schuster, D; Werz, O

    2014-01-01

    Background and Purpose 1,4-Benzoquinones are well-known inhibitors of 5-lipoxygenase (5-LOX, the key enzyme in leukotriene biosynthesis), but the molecular mechanisms of 5-LOX inhibition are not completely understood. Here we investigated the molecular mode of action and the pharmacological profile of the novel 1,4-benzoquinone derivative 3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (RF-Id) in vitro and its effectiveness in vivo. Experimental Approach Mechanistic investigations in cell-free assays using 5-LOX and other enzymes associated with eicosanoid biosynthesis were conducted, along with cell-based studies in human leukocytes and whole blood. Molecular docking of RF-Id into the 5-LOX structure was performed to illustrate molecular interference with 5-LOX. The effectiveness of RF-Id in vivo was also evaluated in two murine models of inflammation. Key Results RF-Id consistently suppressed 5-LOX product synthesis in human leukocytes and human whole blood. RF-Id also blocked COX-2 activity but did not significantly inhibit COX-1, microsomal PGE2 synthase-1, cytosolic PLA2 or 12- and 15-LOX. Although RF-Id lacked radical scavenging activity, reducing conditions facilitated its inhibitory effect on 5-LOX whereas cell stress impaired its efficacy. The reduced hydroquinone form of RF-Id (RED-RF-Id) was a more potent inhibitor of 5-LOX as it had more bidirectional hydrogen bonds within the 5-LOX substrate binding site. Finally, RF-Id had marked anti-inflammatory effects in mice in vivo. Conclusions and Implications RF-Id represents a novel anti-inflammatory 1,4-benzoquinone that potently suppresses LT biosynthesis by direct inhibition of 5-LOX with effectiveness in vivo. Mechanistically, RF-Id inhibits 5-LOX in a non-redox manner by forming discrete molecular interactions within the active site of 5-LOX. PMID:24467325

  5. The Medicinal Timber Canarium patentinervium Miq. (Burseraceae Kunth.) Is an Anti-Inflammatory Bioresource of Dual Inhibitors of Cyclooxygenase (COX) and 5-Lipoxygenase (5-LOX)

    PubMed Central

    Mogana, R.; Teng-Jin, K.; Wiart, C.

    2013-01-01

    The barks and leaves extracts of Canarium patentinervium Miq. (Burseraceae Kunth.) were investigated for cyclooxygenase (COX) and 5-lipoxygenase (LOX) inhibition via in vitro models. The corresponding antioxidative power of the plant extract was also tested via nonenzyme and enzyme in vitro assays. The ethanolic extract of leaves inhibited the enzymatic activity of 5-LOX, COX-1, and COX-2 with IC50 equal to 49.66 ± 0.02 μg/mL, 0.60 ± 0.01 μg/mL, and 1.07 ± 0.01 μg/mL, respectively, with selective COX-2 activity noted in ethanolic extract of barks with COX-1/COX-2 ratio of 1.22. The ethanol extract of barks confronted oxidation in the ABTS, DPPH, and FRAP assay with EC50 values equal to 0.93 ± 0.01 μg/mL, 2.33 ± 0.02 μg/mL, and 67.00 ± 0.32 μg/mL, respectively, while the ethanol extract of leaves confronted oxidation in β-carotene bleaching assay and superoxide dismutase (SOD) assay with EC50 value of 6.04 ± 0.02 μg/mL and IC50 value of 3.05 ± 0.01 μg/mL. The ethanol extract acts as a dual inhibitor of LOX and COX enzymes with potent antioxidant capacity. The clinical significance of these data is quite clear that they support a role for Canarium patentinervium Miq. (Burseraceae Kunth.) as a source of lead compounds in the management of inflammatory diseases. PMID:25937987

  6. Failure of the inhibition of rat gastric mucosal 5-lipoxygenase by novel acetohydroxamic acids to prevent ethanol-induced damage.

    PubMed

    Boughton-Smith, N K; Whittle, B J

    1988-09-01

    1. The role of leukotriene B4 (LTB4) and LTC4 as mediators of gastric mucosal damage following ethanol challenge in vivo has been investigated using two selective 5-lipoxygenase inhibitors, BW A4C and BW A137C. 2. Oral administration of ethanol to rats in vivo, induced macroscopic damage to the gastric mucosa and markedly increased the formation of the 5-lipoxygenase products, LTB4 and LTC4, from the mucosa ex vivo. 3. Pretreatment with the acetohydroxamic acids BW A4C and BW A137C (5-50 mg kg-1 p.o.) dose-dependently reduced ethanol-stimulated LTB4 and LTC4 formation by the gastric mucosa, with an ID50 of approximately 5 mg kg-1 p.o. 4. A single oral dose of BW A4C (20 mg kg-1) induced near-maximal inhibition of mucosal LTB4 formation within 30 min, which was well maintained for 5 h, whereas BW A137C (20 mg kg-1 p.o.) induced maximal inhibition between 30 and 60 min after administration, which then diminished over the subsequent 5 h. 5. The mucosal formation of the cyclo-oxygenase product, 6-keto-prostaglandin F1 alpha, which was unaltered following ethanol challenge, was not inhibited by the acetohydroxamic acids. Likewise, the small increase in mucosal thromboxane B2 formation following challenge was not inhibited by BW A4C. 6. Neither BW A4C nor BW A137C, at doses that almost completely inhibited the mucosal synthesis of LTB4 or LTC4, reduced the macroscopic gastric mucosal damage induced by ethanol. 7. Pretreatment with the lipoxygenase inhibitor BW 755C (5-50 mg kg-1 p.o.) did reduce mucosal damage, but there was a dissociation between the degree of protection and the inhibition of leukotriene biosynthesis. 8. Oral administration of high doses of either BW A4C or BW A137C (300mgkg-1) did not induce macroscopic gastric damage over a 3 h period. 9. These findings suggest that the leukotrienes, LTB4 and LTC4 are not the primary mediators of ethanol-induced acute mucosal damage, but do not exclude their role in more chronic gastric damage and inflammation. PMID

  7. Discovery of a Novel Dual Fungal CYP51/Human 5-Lipoxygenase Inhibitor: Implications for Anti-Fungal Therapy

    PubMed Central

    Hoobler, Eric K.; Rai, Ganesha; Warrilow, Andrew G. S.; Perry, Steven C.; Smyrniotis, Christopher J.; Jadhav, Ajit; Simeonov, Anton; Parker, Josie E.; Kelly, Diane E.; Maloney, David J.; Kelly, S. L.; Holman, Theodore R.

    2013-01-01

    We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component. PMID:23826084

  8. Structural insights into human 5-lipoxygenase inhibition: combined ligand-based and target-based approach.

    PubMed

    Charlier, Caroline; Hénichart, Jean-Pierre; Durant, François; Wouters, Johan

    2006-01-12

    The human 5-LOX enzyme and its interaction with competitive inhibitors were investigated by means of a combined ligand-based and target-based approach. First, a pharmacophore model was generated for 16 non redox 5-LOX inhibitors with Catalyst (HipHop module). It includes two hydrophobic groups, an aromatic ring, and two hydrogen bond acceptors. The 3D structure of human 5-LOX was then modeled based on the crystal structure of rabbit 15-LOX, and the binding modes of representative ligands were studied by molecular docking. Confrontation of the docking results with the pharmacophore model allowed the weighting of the pharmacophoric features and the integration of structural information. This led to the proposal of an interaction model inside the 5-LOX active site, consisting of four major and two secondary interaction points: on one hand, two hydrophobic groups, an aromatic ring, and a hydrogen bond acceptor, and, on the other hand, an acidic moiety and an additional hydrogen bond acceptor. PMID:16392803

  9. Deletion of 5-Lipoxygenase in the Tumor Microenvironment Promotes Lung Cancer Progression and Metastasis through Regulating T Cell Recruitment.

    PubMed

    Poczobutt, Joanna M; Nguyen, Teresa T; Hanson, Dwight; Li, Howard; Sippel, Trisha R; Weiser-Evans, Mary C M; Gijon, Miguel; Murphy, Robert C; Nemenoff, Raphael A

    2016-01-15

    Eicosanoids, including PGs, produced by cyclooxygenases (COX), and leukotrienes, produced by 5-lipoxygenase (5-LO) have been implicated in cancer progression. These molecules are produced by both cancer cells and the tumor microenvironment (TME). We previously reported that both COX and 5-LO metabolites increase during progression in an orthotopic immunocompetent model of lung cancer. Although PGs in the TME have been well studied, less is known regarding 5-LO products produced by the TME. We examined the role of 5-LO in the TME using a model in which Lewis lung carcinoma cells are directly implanted into the lungs of syngeneic WT mice or mice globally deficient in 5-LO (5-LO-KO). Unexpectedly, primary tumor volume and liver metastases were increased in 5-LO-KO mice. This was associated with an ablation of leukotriene (LT) production, consistent with production mainly mediated by the microenvironment. Increased tumor progression was partially reproduced in global LTC4 synthase KO or mice transplanted with LTA4 hydrolase-deficient bone marrow. Tumor-bearing lungs of 5-LO-KO had decreased numbers of CD4 and CD8 T cells compared with WT controls, as well as fewer dendritic cells. This was associated with lower levels of CCL20 and CXL9, which have been implicated in dendritic and T cell recruitment. Depletion of CD8 cells increased tumor growth and eliminated the differences between WT and 5-LO mice. These data reveal an antitumorigenic role for 5-LO products in the microenvironment during lung cancer progression through regulation of T cells and suggest that caution should be used in targeting this pathway in lung cancer. PMID:26663781

  10. Deletion of 5-Lipoxygenase in the Tumor Microenvironment Promotes Lung Cancer Progression and Metastasis through Regulating T Cell Recruitment

    PubMed Central

    Poczobutt, Joanna M.; Nguyen, Teresa T.; Hanson, Dwight; Li, Howard; Sippel, Trisha R.; Weiser-Evans, Mary C. M.; Gijon, Miguel; Murphy, Robert C.

    2016-01-01

    Eicosanoids, including PGs, produced by cyclooxygenases (COX), and leukotrienes, produced by 5-lipoxygenase (5-LO) have been implicated in cancer progression. These molecules are produced by both cancer cells and the tumor microenvironment (TME). We previously reported that both COX and 5-LO metabolites increase during progression in an orthotopic immunocompetent model of lung cancer. Although PGs in the TME have been well studied, less is known regarding 5-LO products produced by the TME. We examined the role of 5-LO in the TME using a model in which Lewis lung carcinoma cells are directly implanted into the lungs of syngeneic WT mice or mice globally deficient in 5-LO (5-LO-KO). Unexpectedly, primary tumor volume and liver metastases were increased in 5-LO-KO mice. This was associated with an ablation of leukotriene (LT) production, consistent with production mainly mediated by the microenvironment. Increased tumor progression was partially reproduced in global LTC4 synthase KO or mice transplanted with LTA4 hydrolase-deficient bone marrow. Tumor-bearing lungs of 5-LO-KO had decreased numbers of CD4 and CD8 T cells compared with WT controls, as well as fewer dendritic cells. This was associated with lower levels of CCL20 and CXL9, which have been implicated in dendritic and T cell recruitment. Depletion of CD8 cells increased tumor growth and eliminated the differences between WT and 5-LO mice. These data reveal an antitumorigenic role for 5-LO products in the microenvironment during lung cancer progression through regulation of T cells and suggest that caution should be used in targeting this pathway in lung cancer. PMID:26663781

  11. The regulation of human MMP-13 by licofelone, an inhibitor of cyclo-oxygenases and 5-lipoxygenase, in human osteoarthritic chondrocytes is mediated by the inhibition of the p38 MAP kinase signalling pathway

    PubMed Central

    Boileau, C; Pelletier, J; Tardif, G; Fahmi, H; Laufer, S; Lavigne, M; Martel-Pelletier, J

    2005-01-01

    Background: MMP-13 is one of the most important metalloproteases (MMP) involved in osteoarthritis. Licofelone, a novel dual inhibitor of cyclo-oxygenases (COX) and 5-lipoxygenase (5-LOX), can modulate MMP-13 production in human osteoarthritis chondrocytes. Objective: To evaluate the impact of licofelone on MMP-13 expression/production, promoter, and major MAP kinase signalling pathways and transcription factors. Methods: Human osteoarthritis chondrocytes were stimulated by interleukin 1ß (IL1ß) and treated with or without: licofelone (0.3, 1, or 3 µg/ml); NS-398 (10 µM; a specific COX-2 inhibitor); or BayX-1005 (10 µM; a specific 5-LOX inhibitor). MMP-13 synthesis was determined by specific enzyme linked immunosorbent assay, and expression by real time polymerase chain reaction. The effect of licofelone on the MMP-13 promoter was studied through transient transfection; dexamethasone (10–7 M) was used as comparison. The effect on IL1ß induced MMP-13 signalling pathways was determined using specific ELISA for phosphorylated MAP kinases and transcription factors. Results: Licofelone dose dependently inhibited the IL1ß stimulated production and expression of MMP-13. NS-398 and BayX-1005 had very little effect. Licofelone also inhibited MMP-13 transcription on each of the promoter constructs used. The licofelone inhibition was comparable to that obtained with dexamethasone. Licofelone had no effect on phosphorylated p44/42 or JNK1/2; however, it decreased phosphorylated c-jun and inhibited phosphorylated p38, CREB, and AP-1 activity. Conclusions: Licofelone inhibited MMP-13 production under proinflammatory conditions on human osteoarthritis chondrocytes, through inhibition of the p38/AP-1 pathway and the transcription factor CREB. This may explain some of the mechanisms whereby licofelone exerts its positive effect on osteoarthritic changes. PMID:15498796

  12. Evidence that M1 muscarinic receptors enhance noradrenaline release in mouse atria by activating protein kinase C.

    PubMed Central

    Costa, M.; Barrington, M.; Majewski, H.

    1993-01-01

    1. The M1 selective muscarinic agonist, McNeil A 343, enhanced the electrically evoked release of noradrenaline from postganglionic sympathetic nerves in mouse atria. This has been found previously to be due to activation of muscarinic receptors of the M1 subtype, probably located on sympathetic nerve terminals. The present study investigated the signal transduction mechanisms involved in the release-enhancing effects of McNeil A 343. The release of noradrenaline from mouse atria was assessed by measuring the electrically-induced (3 Hz, 60 s) outflow of radioactivity from atria which had been pre-incubated with [3H]-noradrenaline. 2. 8-Bromo cyclic AMP in the presence of IBMX was used to enhance maximally S-I noradrenaline release through cyclic AMP-dependent mechanisms. However, the facilitatory effect of McNeil A 343 (10 microM) was not different from the effect in the absence of these drugs, suggesting that McNeil A 343 enhances noradrenaline release independently of the cyclic AMP system. Furthermore, the release-enhancing effect of McNeil A 343 (10 microM) on noradrenaline release was also not altered by the 5-lipoxygenase inhibitor, BW A4C. 3. The facilitatory effect of McNeil A 343 was not altered in the presence of drugs (trifluoperazine, W7, and calmidazolium) which inhibit calmodulin-dependent processes, suggesting that the mechanisms of action of McNeil A 343 does not depend on calmodulin. 4. It was considered likely that the facilitatory effect of McNeil A 343 on noradrenaline release may be due to activation of protein kinase C, since activators of protein kinase C enhance noradrenaline release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7694761

  13. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  14. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  15. The effects of oral Cardax (disodium disuccinate astaxanthin) on multiple independent oxidative stress markers in a mouse peritoneal inflammation model: influence on 5-lipoxygenase in vitro and in vivo.

    PubMed

    Lockwood, Samuel F; Penn, Marc S; Hazen, Stanley L; Bikádi, Zsolt; Zsila, Ferenc

    2006-06-01

    observed at time points two and five. When normalized to the concentration of the oxidative substrates, statistically significant reductions of 8-isoprostane-F(2alpha) (8-iso-F(2alpha)) at time point three (maximal neutrophil recruitment/activation), and 5-HETE, 5-oxo-EET, 11-HETE, 9-HODE, and PGF(2alpha) at time point five (maximal monocyte/macrophage recruitment/activation) were observed. Subsequently, the direct interaction of the optically inactive stereoisomer of Cardax (meso-dAST) with human 5-lipoxygenase (5-LOX) was evaluated in vitro with circular dichroism (CD) and electronic absorption (UV/Vis) spectroscopy, and subsequent molecular docking calculations were made using mammalian 15-LOX as a surrogate (for which XRC data has been reported). The results suggested that the meso-compound was capable of interaction with, and binding to, the solvent-exposed surface of the enzyme. These preliminary studies provide the foundation for more detailed evaluation of the therapeutic effects of this compound on the 5-LOX enzyme, important in chronic diseases such as atherosclerosis, asthma, and prostate cancer in humans. PMID:16466747

  16. Cysteinyl-leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via cys-LT1 receptors and mitogen-activated protein kinase pathway.

    PubMed

    Ciccarelli, Renata; D'Alimonte, Iolanda; Santavenere, Clara; D'Auro, Mariagrazia; Ballerini, Patrizia; Nargi, Eleonora; Buccella, Silvana; Nicosia, Simonetta; Folco, Giancarlo; Caciagli, Francesco; Di Iorio, Patrizia

    2004-09-01

    Cysteinyl-leukotrienes (cys-LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5'-lipoxygenase and LTC(4) synthase) required for cys-LT production, and release cys-LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen-glucose deprivation or 2-methyl-thioATP, a selective P2Y(1)/ATP receptor agonist. MK-886, a LT synthesis inhibitor, prevented basal and evoked cys-LT release. In addition, 2-methyl-thioATP-induced cys-LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys-LT release. We also showed that astrocytes express cys-LT(1) and not cys-LT(2) receptors. The stimulation of these receptors by LTD(4) activated the mitogen-activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor-coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK-571, a cys-LT(1) selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin-dependent kinase II (KN-93), Ca(2+)-dependent and -independent (GF102903X) or Ca(2+)-dependent (Gö6976) protein kinase C isoforms. LTD(4) also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK-571 or PD98059. Thus, cys-LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar. PMID:15355318

  17. Bacterial Heat Shock Protein Activity

    PubMed Central

    Maleki, Farajollah; Khosravi, Afra; Nasser, Ahmad; Taghinejad, Hamid

    2016-01-01

    Bacteria are exposed to different types of stress in their growth conditions. They have developed appropriate responses, modulated by the re-modeling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive in a variety range of nature. Proteins are essential components for biologic activity in the eukaryotic and prokaryotic cell. Heat Shock Proteins (HSP) have been identified from various organisms and have critical role in cell hemostasis. Chaperone can sense environment and have different potential role in the organism evolution. PMID:27134861

  18. Antiviral activities of whey proteins.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  19. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  20. Western blot expression of 5-lipoxygenase in the brain from striped dolphins (stenella coeruleoalba) and bottlenose dolphins (tursiops truncatus) with or without encephalitis/meningo-encephalitis of infectious nature.

    PubMed

    Di Guardo, G; Falconi, A; Di Francesco, A; Mazzariol, S; Centelleghe, C; Casalone, C; Pautasso, A; Cocumelli, C; Eleni, C; Petrella, A; Di Francesco, C E; Sabatucci, A; Leonardi, L; Serroni, A; Marsili, L; Storelli, M M; Giacominelli-Stuffler, R

    2015-01-01

    Dolphin Morbillivirus (DMV), Toxoplasma gondii and Brucella ceti are pathogens of major concern for wild cetaceans. Although a more or less severe encephalitis/meningo-encephalitis may occur in striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) infected by the aforementioned agents, almost no information is available on the neuropathogenesis of brain lesions, including the neuronal and non-neuronal cells targeted during infection, along with the mechanisms underlying neurodegeneration. We analyzed 5-lipoxygenase (5-LOX) expression in the brain of 11 striped dolphins and 5 bottlenose dolphins, affected or not by encephalitic lesions of various degrees associated with DMV, T. gondii and B. ceti. All the 8 striped dolphins with encephalitis showed a more consistent 5-LOX expression than that observed in the 3 striped dolphins showing no morphologic evidence of brain lesions, with the most prominent band intensity being detected in a B. ceti-infected animal. Similar results were not obtained in T. gondii-infected vs T. gondii-uninfected bottlenose dolphins. Overall, the higher 5-LOX expression found in the brain of the 8 striped dolphins with infectious neuroinflammation is of interest, given that 5-LOX is a putative marker for neurodegeneration in human patients and in experimental animal models. Therefore, further investigation on this challenging issue is also needed in stranded cetaceans affected by central neuropathies. PMID:25864766

  1. Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.

    PubMed

    Kleppe, Rune; Herfindal, Lars; Døskeland, Stein Ove

    2015-10-01

    Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity. PMID:26506362

  2. Cell Death Inducing Microbial Protein Phosphatase Inhibitors—Mechanisms of Action

    PubMed Central

    Kleppe, Rune; Herfindal, Lars; Døskeland, Stein Ove

    2015-01-01

    Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca2+/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity. PMID:26506362

  3. Bioassay-guided identification of an anti-inflammatory prenylated acylphloroglucinol from Melicope ptelefolia and molecular insights into its interaction with 5-lipoxygenase.

    PubMed

    Shaari, Khozirah; Suppaiah, Velan; Wai, Lam Kok; Stanslas, Johnson; Tejo, Bimo Ario; Israf, Daud Ahmad; Abas, Faridah; Ismail, Intan Safinar; Shuaib, Nor Hasifi; Zareen, Seema; Lajis, Nordin Hj

    2011-11-01

    A bioassay-guided investigation of Melicope ptelefolia Champ ex Benth (Rutaceae) resulted in the identification of an acyphloroglucinol, 2,4,6-trihydroxy-3-geranylacetophenone or tHGA, as the active principle inhibiting soybean 15-LOX. The anti-inflammatory action was also demonstrated on human leukocytes, where the compound showed prominent inhibitory activity against human PBML 5-LOX, with an IC(50) value of 0.42 μM, very close to the effect produced by the commonly used standard, NDGA. The compound concentration-dependently inhibited 5-LOX product synthesis, specifically inhibiting cysteinyl leukotriene LTC(4) with an IC(50) value of 1.80 μM, and showed no cell toxicity effects. The anti-inflammatory action does not seem to proceed via redox or metal chelating mechanism since the compound tested negative for these bioactivities. Further tests on cyclooxygenases indicated that the compound acts via a dual LOX/COX inhibitory mechanism, with greater selectivity for 5-LOX and COX-2 (IC(50) value of 0.40 μM). The molecular features that govern the 5-LOX inhibitory activity was thus explored using in silico docking experiments. The residues Ile 553 and Hie 252 were the most important residues in the interaction, each contributing significant energy values of -13.45 (electrostatic) and -5.40 kcal/mol (electrostatic and Van der Waals), respectively. The hydroxyl group of the phloroglucinol core of the compound forms a 2.56Å hydrogen bond with the side chain of the carboxylate group of Ile 553. Both Ile 553 and Hie 252 are crucial amino acid residues which chelate with the metal ion in the active site. Distorting the geometry of these ligands could be the reason for the inhibition activity shown by tHGA. The molecular simulation studies supported the bioassay results and served as a good model for understanding the way tHGA binds in the active site of human 5-LOX enzyme. PMID:21958738

  4. Macelignan inhibits histamine release and inflammatory mediator production in activated rat basophilic leukemia mast cells.

    PubMed

    Han, Young Sun; Kim, Myung-Suk; Hwang, Jae-Kwan

    2012-10-01

    Type I allergy is characterized by the release of granule-associated mediators, lipid-derived substances, cytokines, and chemokines by activated mast cells. To evaluate the anti-allergic effects of macelignan isolated from Myristica fragrans Houtt., we determined its ability to inhibit calcium (Ca(2+)) influx, degranulation, and inflammatory mediator production in RBL-2 H3 cells stimulated with A23187 and phorbol 12-myristate 13-acetate. Macelignan inhibited Ca(2+) influx and the secretion of β-hexosaminidase, histamine, prostaglandin E(2), and leukotriene C(4); decreased mRNA levels of cyclooxygenase-2, 5-lipoxygenase, interleukin-4 (IL-4), IL-13, and tumor necrosis factor-α; and attenuated phosphorylation of Akt and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. These results indicate the potential of macelignan as a type I allergy treatment. PMID:22729280

  5. Protein Needs of Physically Active Children.

    PubMed

    Volterman, Kimberly A; Atkinson, Stephanie A

    2016-05-01

    Current Dietary Reference Intakes (DRI) for protein for children and youth require revision as they were derived primarily on nitrogen balance data in young children or extrapolated from adult values; did not account for the possible influence of above average physical activity; and did not set an upper tolerable level of intake. Revision of the protein DRIs requires new research that investigates: 1) long-term dose-response to identify protein and essential amino acid requirements of both sexes at various pubertal stages and under differing conditions of physical activity; 2) the acute protein needs (quantity and timing) following a single bout of exercise; 3) the potential adverse effects of chronic high intakes of protein; and 4) new measurement techniques (i.e., IAAO or stable isotope methodologies) to improve accuracy of protein needs. While active individuals may require protein in excess of current DRIs, most active Canadian children and youth have habitual protein intakes that exceed current recommendations. PMID:27137165

  6. Investigation for the amorphous state of ER-34122, a dual 5-lipoxygenase/cyclooxygenase inhibitor with poor aqueous solubility, in HPMC solid dispersion prepared by the solvent evaporation method.

    PubMed

    Kushida, Ikuo; Gotoda, Masaharu

    2013-10-01

    ER-34122, a poorly water-soluble dual 5-lipoxygenase/cyclooxygenase inhibitor, exists as a crystalline form. According to an Oak Ridge thermal ellipsoid plot drawing, carbonyl oxygen O (5) makes an intermolecular hydrogen bond with the hydrogen bonded to N (3) in the crystal structure. The FTIR and the solid-state ¹³C NMR spectra suggest that the network is spread out in the amorphous state and the hydrogen bonding gets weaker than that in the crystalline phase, because the carbonyl signals significantly shift in both spectra. When amorphous ER-34122 was heated, crystallization occurred at around 140°C. Similar crystallization happened in the solid dispersion; however, the degree of crystallization was much lower than that observed in the pure amorphous material. Also, the DSC thermogram of the solid dispersion did not show any exothermic peaks implying crystallization. The heat of fusion (ΔHf) determined in the pure amorphous material was nearly equal to that for the crystalline form, whereas the ΔHf value obtained in the solid dispersion was less than a third of them. These data prove that crystallization of the amorphous form is dramatically restrained in the solid dispersion system. The carbonyl wavenumber shifts in the FTIR spectra indicate that the average hydrogen bond in the solid dispersion is lower than that in the pure amorphous material. Therefore, HPMC will suppress formation of the intermolecular network observed in ER-34122 crystal and preserve the amorphous state, which is thermodynamically less stable, in the solid dispersed system. PMID:22519663

  7. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  8. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  9. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  10. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging. PMID:25355192

  11. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  12. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  13. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  14. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  15. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  16. Petunia nectar proteins have ribonuclease activity.

    PubMed

    Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

    2010-06-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  17. Petunia nectar proteins have ribonuclease activity

    PubMed Central

    Hillwig, Melissa S.; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W.; MacIntosh, Gustavo C.

    2010-01-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  18. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  19. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  20. De Novo Construction of Redox Active Proteins.

    PubMed

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. PMID:27586341

  1. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  2. Detection of protein C activation in humans.

    PubMed Central

    Bauer, K A; Kass, B L; Beeler, D L; Rosenberg, R D

    1984-01-01

    We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the

  3. Crowding Activates Heat Shock Protein 90.

    PubMed

    Halpin, Jackson C; Huang, Bin; Sun, Ming; Street, Timothy O

    2016-03-18

    Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro. PMID:26797120

  4. Heat dissipation guides activation in signaling proteins.

    PubMed

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  5. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  6. Leukotriene activity modulation in asthma.

    PubMed

    Spector, S L

    1997-09-01

    Leukotrienes constitute a class of inflammatory mediators synthesised from arachidonic acid, a product of cell membrane metabolism. Synthesis occurs in the 5-lipoxygenase enzyme pathway, which produces several species of leukotrienes, each with characteristic biological activities. With regard to asthma, the leukotrienes are particularly important because of their ability to directly and potently mediate bronchoconstriction; in addition, they specifically stimulate the secretion of mucus into the airways and the extravasation of fluids and proteins into the airway tissues, both of which contribute to airway obstruction. A number of antileukotriene agents have been developed with the goal of modulating the inflammatory process in various disease states. These agents fall into 2 general classes: leukotriene receptor antagonists and leukotriene synthesis inhibitors. Results of antileukotriene agents in preclinical and clinical trials indicate that antileukotriene agents attenuate the response to challenges with inhaled leukotrienes, cold air, exercise, aspirin and allergen; in addition, they have shown efficacy in clinical asthma and have not been associated with serious adverse effects. Although results to date indicate that these medications are well tolerated and effective in the treatment of asthma, the recent approval by the FDA of 2 antileukotriene agents will give physicians further insight into how patients with asthma respond to them. PMID:9279501

  7. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  8. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of activeprotein. PMID:27124090

  9. Monooxygenase activity of type 3 copper proteins.

    PubMed

    Itoh, Shinobu; Fukuzumi, Shunichi

    2007-07-01

    The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism. PMID:17461541

  10. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  11. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  12. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  13. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  14. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  15. Designing Mimics of Membrane Active Proteins

    PubMed Central

    Sgolastra, Federica; deRonde, Brittany M.; Sarapas, Joel M.; Som, Abhigyan; Tew, Gregory N.

    2014-01-01

    CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian

  16. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-01

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD. PMID:21262823

  17. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    PubMed Central

    LEE, JIN-HAENG; JEONG, JAEHO; JEONG, EUI MAN; CHO, SUNG-YUP; KANG, JEONG WOOK; LIM, JISUN; HEO, JINBEOM; KANG, HYUNSOOK; KIM, IN-GYU; SHIN, DONG-MYUNG

    2014-01-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases. PMID:24481335

  18. Modeling Protein Folding and Applying It to a Relevant Activity

    ERIC Educational Resources Information Center

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  19. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  20. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  1. P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes.

    PubMed

    Ballerini, P; Ciccarelli, R; Caciagli, F; Rathbone, M P; Werstiuk, E S; Traversa, U; Buccella, S; Giuliani, P; Jang, S; Nargi, E; Visini, D; Santavenere, C; Di Iorio, P

    2005-01-01

    Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured

  2. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  3. Synthesis of new isoxazoline derivatives from harmine and evaluation of their anti-Alzheimer, anti-cancer and anti-inflammatory activities.

    PubMed

    Filali, Insaf; Bouajila, Jalloul; Znati, Mansour; Bousejra-El Garah, Fatima; Ben Jannet, Hichem

    2015-06-01

    In our study, a series of new harmine derivatives has been prepared by cycloaddition reaction using various arylnitrile oxides and evaluated in vitro against acetylcholinesterase and 5-lipoxygenase enzymes, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of acetylcholinesterase and MCF7 cell line. The greatest activity against acetylcholinesterase (IC50 = 10.4 µM) was obtained for harmine 1 and cytotoxic activities (IC50 = 0.2 µM) for compound 3a. Two derivatives 3e and 3f with the thiophene and furan systems, respectively, showed good activity against 5- lipoxygenase enzyme (IC50 = 29.2 and 55.5 µM, respectively). PMID:25068731

  4. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  5. Protein determination in seeds by proton activation

    NASA Astrophysics Data System (ADS)

    Morales, J. R.; Dinator, M. I.; Cerda, P.

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce 11C and 13N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm 2. Slices of the seed were radially explored, showing a larger concentration of protein in the center.

  6. Monitoring G protein activation in cells with BRET

    PubMed Central

    Masuho, Ikuo; Martemyanov, Kirill A.; Lambert, Nevin A.

    2016-01-01

    Summary Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using standard laboratory equipment. PMID:26260597

  7. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. PMID:26231325

  8. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-05-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. PMID:4004796

  9. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed Central

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-01-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. Images Fig. 1. Fig. 2. Fig. 7. Fig. 8. PMID:4004796

  10. The kinetics of ER fusion protein activation in vivo

    PubMed Central

    Wilson, Catherine H.; Gamper, Ivonne; Perfetto, Alessandra; Auw, Jeremy; Littlewood, Trevor D.; Evan, Gerard I.

    2014-01-01

    Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified estrogen receptor (ERTAM) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ERTAM fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterised ERTAM fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long term activation of ERTAM fusion proteins in vivo. PMID:24662815

  11. Hemagglutinating activity of proteins from Parkia speciosa seeds.

    PubMed

    Chankhamjon, Kanokwan; Petsom, Amorn; Sawasdipuksa, Narumon; Sangvanich, Polkit

    2010-01-01

    Proteins from Parkia speciosa Hassk. (Fabaceae) seeds were extracted and stepwise precipitated using ammonium sulfate. Proteins precipitated with 25% ammonium sulfate were separated by affinity chromatography on Affi-Gel Blue gel followed by protein liquid chromatography on Superdex 200. The protein Gj, which was identified as a protein similar to putative aristolochene synthase, 3'-partial from Oryza sativa L. (Poaceae), had hemagglutinating activity of 0.39 mug/muL. Moreover, fraction C2 from the proteins precipitated with 60% ammonium sulfate, separated by lectin-specific adsorption chromatography using Con A Sepharose, had hemagglutinating activity of 1.17 mug/muL. Using gel electrophoresis, two proteins C2a and C2b were separated, having molecular weights of 45 kDa and 23 kDa, respectively. From protein identification, C2a was found to be similar to the hypothetical protein B1342F01.11 from Oryza sativa, and C2b was similar to the hypothetical protein At1g51560 from Arabidopsis thaliana (L.) Heynh. (Brassicaceae). PMID:20645760

  12. Modeling the SHG activities of diverse protein crystals

    SciTech Connect

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-11-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.

  13. Evidence for a receptor protein of activated carcinogen

    PubMed Central

    Mainigi, Kumar D.; Sorof, Sam

    1977-01-01

    During carcinogenesis in rat liver by 3′-methyl-4-dimethylaminoazobenzene, the two moieties of the principal liver carcinogen-protein complex have considerably different turnover rates. With continued ingestion of the azocarcinogen by rats, the bound azo dye in the complex has a half-life of 2.5 ± 0.25 (SD) days, while the protein moiety has a half-life of 8.7 ± 1.6 days (probability of identity <0.001). In addition, the interaction of the azocarcinogen with the principal target protein in vivo appears to extend the half-life of the protein itself from 3.3 ± 0.2 days in normal liver to 8.7 ± 1.6 days (P < 0.001). The slowing of the turnover of the protein by the carcinogen appears to be readily reversible, since soon after the cessation of azocarcinogen feeding, the half-life of the protein returns to that of the target protein in normal liver. The considerable difference in turnover rates of the two moieties of the complex and the reversible effects of the carcinogen in slowing the turnover of the protein moiety suggest that the two moieties of the native azoprotein are noncovalently linked and that they have different biological activities. The native complex appears to contain azo dye in an activated state that is capable of yielding a reactive electrophile, because after protein denaturation the bound azo dye was previously found to have properties that are indicative of covalent linkage to the protein. The retardation in the biological turnover rate of the protein moiety, apparently resulting from interaction with azocarcinogen, is in agreement with the known ligand-induced stabilization in vitro and reduced rate of proteolytic degradation in vivo of other proteins that result from conformational change to a more compact configuration. Our evidence is consistent with the hypothesis that the principal liver carcinogen-protein complex contains hydrophobically bound activated azocarcinogen, whose specificity of reaction with critical macromolecule(s) in

  14. Imperatorin Suppresses Degranulation and Eicosanoid Generation in Activated Bone Marrow-Derived Mast Cells

    PubMed Central

    Jeong, Kyu-Tae; Lee, Eujin; Park, Na-Young; Kim, Sun-Gun; Park, Hyo-Hyun; Lee, Jiean; Lee, Youn Ju; Lee, Eunkyung

    2015-01-01

    Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation. PMID:26336581

  15. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana. PMID:10721722

  16. Pestivirus NS3 (p80) protein possesses RNA helicase activity.

    PubMed Central

    Warrener, P; Collett, M S

    1995-01-01

    The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed. PMID:7853509

  17. Activation of an Endoribonuclease by Non-intein Protein Splicing.

    PubMed

    Campbell, Stephen J; Stern, David B

    2016-07-29

    The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a non-conserved linker (NCL) sequence flanked by highly conserved N- and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions. PMID:27311716

  18. Iron-chelating activity of chickpea protein hydrolysate peptides.

    PubMed

    Torres-Fuentes, Cristina; Alaiz, Manuel; Vioque, Javier

    2012-10-01

    Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption. PMID:25005984

  19. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  20. Activators of G Protein Signaling in the Kidney

    PubMed Central

    2015-01-01

    Heterotrimeric G proteins play a crucial role in regulating signal processing to maintain normal cellular homeostasis, and subtle perturbations in its activity can potentially lead to the pathogenesis of renal disorders or diseases. Cell-surface receptors and accessory proteins, which normally modify and organize the coupling of individual G protein subunits, contribute to the regulation of heterotrimeric G protein activity and their convergence and/or divergence of downstream signaling initiated by effector systems. Activators of G protein signaling (AGS) are a family of accessory proteins that intervene at multiple distinct points during the activation–inactivation cycle of G proteins, even in the absence of receptor stimulation. Perturbations in the expression of individual AGS proteins have been reported to modulate signal transduction pathways in a wide array of diseases and disorders within the brain, heart, immune system, and more recently, the kidney. This review will provide an overview of the expression profile, localization, and putative biologic role of the AGS family in the context of normal and diseased states of the kidney. PMID:25628392

  1. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  2. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGESBeta

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; et al

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  3. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  4. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  5. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  6. Activation of a human chromosomal replication origin by protein tethering

    PubMed Central

    Chen, Xiaomi; Liu, Guoqi; Leffak, Michael

    2013-01-01

    The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences. PMID:23658226

  7. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  8. G Protein Activation without a GEF in the Plant Kingdom

    PubMed Central

    Wang, Hao; Matthews, Melissa; Bradford, William; Bennetzen, Jeffrey L.; Jones, Alan M.

    2012-01-01

    Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent

  9. Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

    PubMed

    Yamaguchi, Fuminori; Tsuchiya, Mitsumasa; Shimamoto, Seiko; Fujimoto, Tomohito; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2016-01-01

    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis. PMID:27600583

  10. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  11. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    PubMed

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts. PMID:26689178

  12. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  13. Liposomal packaging generates Wnt protein with in vivo biological activity.

    PubMed

    Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

    2008-01-01

    Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373

  14. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  15. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  16. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  17. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  18. Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

    The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. PMID:9592147

  19. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  20. Probing heterotrimeric G protein activation: applications to biased ligands

    PubMed Central

    Denis, Colette; Saulière, Aude; Galandrin, Ségolène; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting activation of specific signals and thus generating textured responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligand responses. However, considering that a single receptor has pleiotropic signalling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even if some G protein-independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discuss them in the context of G protein biased -ligands. PMID:22229559

  1. Probing heterotrimeric G protein activation: applications to biased ligands.

    PubMed

    Denis, Colette; Saulière, Aude; Galandrin, Segolene; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands. PMID:22229559

  2. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  3. NALP3 inflammasome activation in protein misfolding diseases.

    PubMed

    Shi, Fushan; Kouadir, Mohammed; Yang, Yang

    2015-08-15

    Protein-misfolding diseases, such as Alzheimer's disease, type 2 diabetes, Prion diseases, and Parkinson's disease, are characterized by inflammatory reactions. In all these diseases, IL-1β (Interlukine-1β) has been shown to be an important regulator, and the misfolded proteins are proved to be triggers of the release of IL-1β. Recently, several reports demonstrated that the inflammasome activation is involved in the progress of the misfolded protein diseases, and that the inflammasome can recognize pathogenic proteins leading to the release of IL-1β. In this review, we discuss the role of inflammasome in the pathogenesis of misfolded protein diseases and the potential of inflammasome-targeting therapeutic interventions in the management of these diseases. PMID:26037399

  4. Enhancer-specific modulation of E protein activity.

    PubMed

    Markus, Maurice; Du, Zhimei; Benezra, Robert

    2002-02-22

    Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes (Bain, G., Gruenwald, S., and Murre, C. (1993) Mol. Cell Biol. 13, 3522-3529; Shen, C. P., and Kadesch, T. (1995) Mol. Cell Biol. 15, 4518-4524; Jacobs, Y., et al. (1994) Mol. Cell Biol. 14, 4087-4096; Wilson, R. B., et al. (1991) Mol. Cell Biol. 11, 6185-6191). These homodimers, however, have little activity on myogenic enhancers (Weintraub, H., Genetta, T., and Kadesch, T. (1994) Genes Dev. 8, 2203-2211). We report here the identification of a novel cis-acting transcriptional repression domain in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, we demonstrate that Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes. PMID:11724804

  5. Peptides and proteins with antimicrobial activity

    PubMed Central

    Coutinho, Henrique Douglas Melo; Lôbo, Katiuscia Menezes; Bezerra, Denise Aline Casimiro; Lôbo, Inalzuir

    2008-01-01

    The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants. PMID:21264153

  6. Chemical labelling of active serum thioester proteins for quantification.

    PubMed

    Holm, Lotta; Ackland, Gareth L; Edwards, Mark R; Breckenridge, Ross A; Sim, Robert B; Offer, John

    2012-02-01

    The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum. PMID:21852021

  7. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  8. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  9. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges

    PubMed Central

    Gaestel, Matthias

    2016-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  10. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges.

    PubMed

    Gaestel, Matthias

    2015-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  11. Hydrodynamic collective effects of active proteins in biological membranes.

    PubMed

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015)PNASA60027-842410.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them. PMID:27627343

  12. Proteins of the ETS family with transcriptional repressor activity.

    PubMed

    Mavrothalassitis, G; Ghysdael, J

    2000-12-18

    ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways. PMID:11175368

  13. Signal peptides are allosteric activators of the protein translocase.

    PubMed

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G; Economou, Anastassios

    2009-11-19

    Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping' that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  14. G Protein Activation Stimulates Phospholipase D Signaling in Plants.

    PubMed Central

    Munnik, T.; Arisz, S. A.; De Vrije, T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals. PMID:12242371

  15. A Theileria parva type 1 protein phosphatase activity.

    PubMed

    Cayla, X; Garcia, A; Baumgartner, M; Ozon, R; Langsley, G

    2000-09-01

    The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1. PMID:10989153

  16. Contractions Activate Hormone-Sensitive Lipase in Rat Muscle by Protein Kinase C and Mitogen-Activated Protein Kinase

    PubMed Central

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia; Ploug, Thorkil; Galbo, Henrik

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50 % by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway. PMID:12794177

  17. Antioxidant activity of whey protein hydrolysates in milk beverage system.

    PubMed

    Mann, Bimlesh; Kumari, Anuradha; Kumar, Rajesh; Sharma, Rajan; Prajapati, Kishore; Mahboob, Shaik; Athira, S

    2015-06-01

    The aim of the present study was to evaluate the antioxidant activity of flavoured milk enriched with antioxidative whey protein hydrolysates (WPHs) by radical scavenging method. Whey protein concentrate (WPC) was hydrolyzed by using three commercial proteases; flavouzyme, alcalase and corolase PP and these WPHs were analyzed for degree of hydrolysis and antioxidant activity. The antioxidant activities of these WPHs were evaluated using ABTS method. Trolox equivalent antioxidant activity of all the hydrolysates i.e. flavourzyme (0.81 ± 0.04), alcalase (1.16 ± 0.05) and corolase (1.42 ± 0.12) was higher than the WPC (0.19 ± 0.01). Among these, whey protein hydrolysates prepared using corolase showed maximum antioxidant activity. Total 15 β-lactoglobulin, 1 α-lactoalbumin, and 6 β-casein derived peptide fragments were identified in the WPHs by LC-MS/MS. Due to their size and characteristic amino acid composition, all the identified peptides may contribute for the antioxidant activity. The strawberry and chocolate flavoured milk was supplemented with WPC and WPHs and 2 % addition has shown increase in antioxidant activity upto 42 %. The result suggests that WPH could be used as natural biofunctional ingredients in enhancing antioxidant properties of food products. PMID:26028704

  18. Evaluation of antioxidant activities of zein protein fractions.

    PubMed

    Tang, Ning; Zhuang, Hong

    2014-11-01

    Zein protein was extracted from the by-product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC-ESI-Q-TOF MS/MS as Pro-Phe (263.13 Da) and Leu-Pro-Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry. PMID:25350353

  19. Oxidation of methionine residues in proteins of activated human neutrophils.

    PubMed Central

    Fliss, H; Weissbach, H; Brot, N

    1983-01-01

    A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins. PMID:6580633

  20. DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.

    PubMed Central

    Dong, Q; Ebright, R H

    1992-01-01

    The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2. PMID:1322886

  1. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    PubMed

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  2. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on /sup 32/P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of /sup 32/P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble (/sup 32/P)phosphate.

  3. Ubiquitously expressed transcript is a novel interacting protein of protein inhibitor of activated signal transducer and activator of transcription 2

    PubMed Central

    KONG, XIANG; MA, SHIKUN; GUO, JIAQIAN; MA, YAN; HU, YANQIU; WANG, JIANJUN; ZHENG, YING

    2015-01-01

    Protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is a member of the PIAS protein family. This protein family modulates the activity of several transcription factors and acts as an E3 ubiquitin ligase in the sumoylation pathway. To improve understanding of the physiological roles of PIAS2, the current study used a yeast two-hybrid system to screen mouse stem cell cDNA libraries for proteins that interact with PIAS2. The screening identified an interaction between PIAS2 and ubiquitously expressed transcript (UXT). UXT, also termed androgen receptor trapped clone-27, is an α-class prefoldin-type chaperone that acts as a coregulator for various transcription factors, including nuclear factor-κB and androgen receptor (AR). A direct interaction between PIAS2 and UXT was confirmed by direct yeast two-hybrid analysis. In vitro evidence of the association of UXT with PIAS2 was obtained by co-immunoprecipitation. Colocalization between PIAS2 and UXT was identified in the nucleus and cytoplasm of HEK 293T and human cervical carcinoma HeLa cells. The results of the current study suggested that UXT is a binding protein of PIAS2, and interaction between PIAS2 and UXT may be important for the transcriptional activation of AR. PMID:25434787

  4. Activities at the Universal Protein Resource (UniProt)

    PubMed Central

    2014-01-01

    The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequences and functional annotation. It integrates, interprets and standardizes data from literature and numerous resources to achieve the most comprehensive catalog possible of protein information. The central activities are the biocuration of the UniProt Knowledgebase and the dissemination of these data through our Web site and web services. UniProt is produced by the UniProt Consortium, which consists of groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads. PMID:24253303

  5. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  6. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma.

    PubMed

    Zhou, Weiwei; Wu, Luming; Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations. PMID:27606678

  7. Installing hydrolytic activity into a completely de novo protein framework.

    PubMed

    Burton, Antony J; Thomson, Andrew R; Dawson, William M; Brady, R Leo; Woolfson, Derek N

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis. PMID:27554410

  8. Streptococcal Surface Proteins Activate the Contact System and Control Its Antibacterial Activity*

    PubMed Central

    Wollein Waldetoft, Kristofer; Svensson, Lisbeth; Mörgelin, Matthias; Olin, Anders I.; Nitsche-Schmitz, D. Patric; Björck, Lars; Frick, Inga-Maria

    2012-01-01

    Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects. PMID:22648411

  9. Gi/o proteins: expression for direct activation enquiry.

    PubMed

    Di Cesare Mannelli, Lorenzo; Pacini, Alessandra; Toscano, Annarita; Fortini, Martina; Berti, Debora; Ghelardini, Carla; Galeotti, Nicoletta; Baglioni, Piero; Bartolini, Alessandro

    2006-05-01

    G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the alphai1, alphai3, alphao1, beta1, and gamma2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activation profile of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently purified by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPgammaS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated alpha-subunit and on heterotrimeric alphabetagamma complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies. PMID:16364655

  10. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  11. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

    PubMed Central

    Zinck, R; Cahill, M A; Kracht, M; Sachsenmaier, C; Hipskind, R A; Nordheim, A

    1995-01-01

    Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction. PMID:7651411

  12. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  13. Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression.

    PubMed

    Andruska, Neal D; Zheng, Xiaobin; Yang, Xujuan; Mao, Chengjian; Cherian, Mathew M; Mahapatra, Lily; Helferich, William G; Shapiro, David J

    2015-04-14

    Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration. PMID:25825714

  14. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator. PMID:2514798

  15. Reassessing the Potential Activities of Plant CGI-58 Protein.

    PubMed

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

  16. Reassessing the Potential Activities of Plant CGI-58 Protein

    PubMed Central

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

  17. In vitro antithrombotic activities of peanut protein hydrolysates.

    PubMed

    Zhang, Shao Bing

    2016-07-01

    The antithrombotic activities of peanut protein hydrolysates were investigated using a microplates assay. When peanut proteins were hydrolyzed to a limited extent by various enzymes, their thrombin inhibitory abilities were significantly enhanced. However, the resultant hydrolysates showed significantly different activities even at the same degrees of hydrolysis. The hydrolysates generated by Alcalase 2.4L displayed the best antithrombotic activities and the hydrolysis process was further optimized by response surface methodology. The antithrombotic activities were increased to 86% based on a protein concentration of 50mg/ml under the optimal conditions: pH 8.5, enzyme concentration of 5000IU/g of peanut proteins, and 2h hydrolysis time at 50°C. The Alcalase 2.4L crude hydrolysates were then fractionated successively by preparative and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide fraction collected inhibited thrombin-catalyzed coagulation of fibrinogen completely at a concentration of 0.4mg/ml, with an antithrombotic activity close to that of heparin at quite a low concentration (0.2mg/ml). This peptide fraction was further analyzed by online reverse-phase ultra-performance liquid chromatography (RP-UPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and three new peptides were identified as Ser-Trp-Ala-Gln-Leu, Gly-Asn-His-Glu-Ala-Gly-Glu and Cys-Phe-Asn-Glu-Tyr-Glu, respectively. This research provided an effective way to produce antithrombotic peptides from peanut proteins, and also helped to elucidate the structure-function relationships of peanut peptides. PMID:26920259

  18. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  19. A Novel Method for Assessing the Chaperone Activity of Proteins.

    PubMed

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families-molecules expressed during adverse conditions, infection, and diseases-chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  20. A Novel Method for Assessing the Chaperone Activity of Proteins

    PubMed Central

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families–molecules expressed during adverse conditions, infection, and diseases–chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  1. Design of a Split Intein with Exceptional Protein Splicing Activity.

    PubMed

    Stevens, Adam J; Brown, Zachary Z; Shah, Neel H; Sekar, Giridhar; Cowburn, David; Muir, Tom W

    2016-02-24

    Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques. PMID:26854538

  2. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    PubMed

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  3. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    PubMed

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

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

    PubMed

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

    2015-06-12

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

  5. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  6. Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.

    PubMed

    Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C

    2016-01-01

    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. PMID:26616142

  7. A GTPase-activating protein for the G protein Galphaz. Identification, purification, and mechanism of action.

    PubMed

    Wang, J; Tu, Y; Woodson, J; Song, X; Ross, E M

    1997-02-28

    A GTPase-activating protein (GAP) specific for Galphaz was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. Gz GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of Mr approximately 22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Galphaz, the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min -1 at 15 degrees C, or to >/=20 min-1 at 30 degrees C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric Gz and m2 muscarinic receptor, Gz GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the Km of the GAP for Galphaz-GTP is 2 nM. Its activity is inhibited by Galphaz-guanosine 5'-O-thiotriphosphate (Galphaz-GTPgammaS) or by Galphaz-GDP/AlF4 with Ki approximately 1.5 nM for both species; Galphaz-GDP does not inhibit. G protein betagamma subunits inhibit Gz GAP activity, apparently by forming a GTP-Galphazbetagamma complex that is a poor GAP substrate. Gz GAP displays little GAP activity toward Galphai1 or Galphao, but its activity with Galphaz is competitively inhibited by both Galphai1 and Galphao at nanomolar concentrations when they are bound to GTPgammaS but not to GDP. Neither phospholipase C-beta1 (a Gq GAP) nor several adenylyl cyclase isoforms display Gz GAP activity. PMID:9038185

  8. Current activities of the Yersinia effector protein YopM.

    PubMed

    Höfling, Sabrina; Grabowski, Benjamin; Norkowski, Stefanie; Schmidt, M Alexander; Rüter, Christian

    2015-05-01

    Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed. PMID:25865799

  9. Pharmacological activities in thermal proteins: relationships in molecular evolution

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Hefti, F.; Hartikka, J.; Junard, E.; Przybylski, A. T.; Vaughan, G.

    1987-01-01

    The model of protobiological events that has been presented in these pages has increasing relevance to pharmacological research. The thermal proteins that function as key substances in the proteinoid theory have recently been found to prolong the survival of rat forebrain neurons in culture and to stimulate the growth of neurites. A search for such activity in thermal proteins added to cultures of modern neurons was suggested by the fact that some of the microspheres assembled from proteinoids rich in hydrophobic amino acids themselves generate fibrous outgrowths.

  10. Novel condensation products having high activity to insolubilize proteins and protein-insolubilized products

    SciTech Connect

    Krasnobajew, V.; Boeniger, R.

    1980-01-01

    According to the invention a substantially more active product with respect to the fixing or insolubilization pf proteins, including enzymes, is obtained when 1,3 phenylenediamine is condensed with glutardialdehyde. One application of the process is the enzymatic hydrolysis of lactose in milk products by lactase.

  11. Implications of a peroxisome proliferator-activated receptor alpha (PPARα) ligand clofibrate in breast cancer.

    PubMed

    Chandran, Karthic; Goswami, Sudeshna; Sharma-Walia, Neelam

    2016-03-29

    Inflammatory and invasive breast cancers are aggressive and require better understanding for the development of new treatments and more accurate prognosis. Here, we detected high expression of PPARα in human primary inflammatory (SUM149PT) and highly invasive (SUM1315MO2) breast cancer cells, and tissue sections of human breast cancer. PPARα ligands are clinically used to treat dyslipidemia. Among lipid lowering drugs clofibrate, fenofibrate and WY14643, clofibrate showed high chemo-sensitivity towards breast cancer cells. Clofibrate treatment significantly induced PPARα DNA binding activity, and remarkably reduced cyclooxygenase-2/PGE2 and 5-lipoxygenase/LTB4 inflammatory pathways. Clofibrate treatment reduced the proliferation of breast cancer cells probably by inhibiting NF-κB and ERK1/2 activation, reducing cyclinD1, cyclinA, cyclinE, and inducing pro-apoptotic P21 levels. Surprisingly, the expression of lipogenic pathway genes including SREBP-1c (sterol regulatory element-binding protein-1c), HMG-CoA synthase, SPTLC1 (serine palmitoyltransferase long-chain), and Acyl-CoA oxidase (ACO) decreased with a concurrent increase in fatty acid oxidation genes such as CPT-1a (carnitine palmitoyltransferase 1a) and SREBP-2 (Sterol regulatory element-binding protein-2). Clofibrate treatment induced secretion of free fatty acids and effectively decreased the level of phosphorylated active form of fatty acid synthase (FASN), an enzyme catalyzing de novo synthesis of fatty acids. High level of coactivators steroid receptor coactivator-1 (SRC-1) and histone acetylase CBP-300 (CREB binding protein-300) were observed in the nuclear complexes of clofibrate treated breast cancer cells. These findings implicate that stimulating PPARα by safe, well-tolerated, and clinically approved clofibrate may provide a safer and more effective strategy to target the signaling, lipogenic, and inflammatory pathways in aggressive forms of breast cancer. PMID:26621841

  12. Implications of a peroxisome proliferator-activated receptor alpha (PPARα) ligand clofibrate in breast cancer

    PubMed Central

    Goswami, Sudeshna

    2016-01-01

    Inflammatory and invasive breast cancers are aggressive and require better understanding for the development of new treatments and more accurate prognosis. Here, we detected high expression of PPARα in human primary inflammatory (SUM149PT) and highly invasive (SUM1315MO2) breast cancer cells, and tissue sections of human breast cancer. PPARα ligands are clinically used to treat dyslipidemia. Among lipid lowering drugs clofibrate, fenofibrate and WY14643, clofibrate showed high chemo-sensitivity towards breast cancer cells. Clofibrate treatment significantly induced PPARα DNA binding activity, and remarkably reduced cyclooxygenase-2/PGE2 and 5-lipoxygenase/LTB4 inflammatory pathways. Clofibrate treatment reduced the proliferation of breast cancer cells probably by inhibiting NF-κB and ERK1/2 activation, reducing cyclinD1, cyclinA, cyclinE, and inducing pro-apoptotic P21 levels. Surprisingly, the expression of lipogenic pathway genes including SREBP-1c (sterol regulatory element-binding protein-1c), HMG-CoA synthase, SPTLC1 (serine palmitoyltransferase long-chain), and Acyl-CoA oxidase (ACO) decreased with a concurrent increase in fatty acid oxidation genes such as CPT-1a (carnitine palmitoyltransferase 1a) and SREBP-2 (Sterol regulatory element-binding protein-2). Clofibrate treatment induced secretion of free fatty acids and effectively decreased the level of phosphorylated active form of fatty acid synthase (FASN), an enzyme catalyzing de novo synthesis of fatty acids. High level of coactivators steroid receptor coactivator-1 (SRC-1) and histone acetylase CBP-300 (CREB binding protein-300) were observed in the nuclear complexes of clofibrate treated breast cancer cells. These findings implicate that stimulating PPARα by safe, well-tolerated, and clinically approved clofibrate may provide a safer and more effective strategy to target the signaling, lipogenic, and inflammatory pathways in aggressive forms of breast cancer. PMID:26621841

  13. Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Foster, David A.; Rosner, Marsha Rich

    1999-01-01

    In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cδ (PKCδ), whereas ERK activation in response to the mitogenic EGF is independent of PKCδ. Antisense PKCδ oligonucleotides or the PKCδ-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCδ functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCδ also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCδ in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCδ requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCδ in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCδ contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling. PMID:10330161

  14. Pharmacokinetics of activated protein C in guinea pigs

    SciTech Connect

    Berger, H. Jr.; Kirstein, C.G.; Orthner, C.L. )

    1991-05-15

    Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of {sup 125}I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of {sup 125}I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. (Abstract Truncated)

  15. Functions of AMP-activated protein kinase in adipose tissue

    PubMed Central

    Daval, Marie; Foufelle, Fabienne; Ferré, Pascal

    2006-01-01

    AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Its functions have been extensively studied in muscles and liver. AMPK stimulates pathways which increase energy production (glucose transport, fatty acid oxidation) and switches off pathways which consume energy (lipogenesis, protein synthesis, gluconeogenesis). This has led to the concept that AMPK has an interesting pharmaceutical potential in situations of insulin resistance and it is indeed the target of existing drugs and hormones which improve insulin sensitivity. Adipose tissue is a key player in energy metabolism through the release of substrates and hormones involved in metabolism and insulin sensitivity. Activation of AMPK in adipose tissue can be achieved through situations such as fasting and exercise. Leptin and adiponectin as well as hypoglycaemic drugs are activators of adipose tissue AMPK. This activation probably involves changes in the AMP/ATP ratio and the upstream kinase LKB1. When activated, AMPK limits fatty acid efflux from adipocytes and favours local fatty acid oxidation. Since fatty acids have a key role in insulin resistance, especially in muscles, activating AMPK in adipose tissue might be found to be beneficial in insulin-resistant states, particularly as AMPK activation also reduces cytokine secretion in adipocytes. PMID:16709632

  16. Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

    PubMed Central

    Jones, T. L.; Ort, D. R.

    1997-01-01

    Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state. PMID:12223667

  17. Site–Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function

    PubMed Central

    Baker, Rachael; Lewis, Steven M.; Sasaki, Atsuo T.; Wilkerson, Emily M.; Locasale, Jason W.; Cantley, Lewis C.; Kuhlman, Brian; Dohlman, Henrik G.; Campbell, Sharon L.

    2012-01-01

    SUMMARY Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K–Ras to promote tumor growth. To determine the mechanism of human Ras activation we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling, and biochemical activity measurements. We established that monoubiquitination has little effect on Ras GTP binding, GTP hydrolysis, or exchange factor activation, but severely abrogates the response to GTPase activating proteins in a site–specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation. PMID:23178454

  18. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation. PMID:25837301

  19. Unwinding activity of cold shock proteins and RNA metabolism.

    PubMed

    Phadtare, Sangita

    2011-01-01

    Temperature downshift from 37 °C to 15 °C results in the exertion of cold shock response in Escherichia coli, which induces cold shock proteins, such as CsdA. Previously, we showed that the helicase activity of CsdA is critical for its function in the cold acclimation of cells and its primary role is mRNA degradation. Only RhlE (helicase), CspA (RNA chaperone) and RNase R (exoribonuclease) were found to complement the cold shock function of CsdA. RNase R has two independent activities, helicase and ribonuclease, only helicase being essential for the functional complementation of CsdA. Here, we discuss the significance of above findings as these emphasize the importance of the unwinding activity of cold-shock-inducible proteins in the RNA metabolism at low temperature, which may be different than that at 37 °C. It requires assistance of proteins to destabilize the secondary structures in mRNAs that are stabilized upon temperature downshift, hindering the activity of ribonucleases. PMID:21445001

  20. Analysis of antifreeze protein activity using colorimetric gold nanosensors

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Choi, Ho-seok; Park, Ji-In; Kim, Young-Pil

    2015-07-01

    High activity and long stability of antifreeze proteins (AFPs), also known as ice-binding proteins (IBPs), are necessary for exerting their physiological functions in biotechnology and cryomedicine. Here we report a simple analysis of antifreeze protein activity and stability based on self-assembly of gold nanoparticles (AuNPs) via freezing and thawing cycles. While the mercaptosuccinic acid-capped AuNP (MSA-AuNP) was easily self-assembled after a freezing/thawing cycle, due to the mechanical attack of ice crystal on the MSA-AuNP surface, the presence of AFP impeded the self-assembly of MSA-AuNP via the interaction of AFP with ice crystals via freezing and thawing cycles, which led to a strong color in the MSA-AuNP solution. As a result, the aggregation parameter (E520/E650) of MSA-AuNP showed the rapid detection of both activity and stability of AFPs. We suggest that our newly developed method is very suitable for measuring antifreeze activity and stability in a simple and rapid manner with reliable quantification.

  1. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    NASA Astrophysics Data System (ADS)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  2. Protein kinase activity associated with simian virus 40 T antigen.

    PubMed Central

    Griffin, J D; Spangler, G; Livingston, D M

    1979-01-01

    Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. Images PMID:223152

  3. Dynamical theory of activated processes in globular proteins.

    PubMed Central

    Northrup, S H; Pear, M R; Lee, C Y; McCammon, J A; Karplus, M

    1982-01-01

    A method is described for calculating the reaction rate in globular proteins of activated processes such as ligand binding or enzymatic catalysis. The method is based on the determination of the probability that the system is in the transition state and of the magnitude of the reactive flux for transition-state systems. An "umbrella sampling" simulation procedure is outlined for evaluating the transition-state probability. The reactive flux is obtained from an approach described previously for calculating the dynamics of transition-state trajectories. An application to the rotational isomerization of an aromatic ring in the bovine pancreatic trypsin inhibitor is presented. The results demonstrate the feasibility of calculating rate constants for reactions in proteins and point to the importance of solvent effects for reactions that occur near the protein surface. PMID:6955788

  4. Dynamical Theory of Activated Processes in Globular Proteins

    NASA Astrophysics Data System (ADS)

    Northrup, Scott H.; Pear, Michael R.; Lee, Chyuan-Yih; McCammon, J. Andrew; Karplus, Martin

    1982-07-01

    A methos is described for calculating the reaction rate in globular proteins of activated processes such as ligand binding or enzymatic catalysis. The method is based on the determination of the probability that the system is in the transition state and of the magnitude of the reactive flux for transition-state systems. An ``umbrella sampling'' simulation procedure is outlined for evaluating the transition-state probability. The reactive flux is obtained from an approach described previously for calculating the dynamics of transition-state trajectories. An application to the rotational isomerization of an aromatic ring in the bovine pancreatic trypsin inhibitor is presented. The results demonstrate the feasibility of calculating rate constants for reactions in proteins and point to the importance of solvent effects for reactions that occur near the protein surface.

  5. Receptor Activity-Modifying Proteins (RAMPs): New Insights and Roles.

    PubMed

    Hay, Debbie L; Pioszak, Augen A

    2016-01-01

    It is now recognized that G protein-coupled receptors (GPCRs), once considered largely independent functional units, have a far more diverse molecular architecture. Receptor activity-modifying proteins (RAMPs) provide an important example of proteins that interact with GPCRs to modify their function. RAMPs are able to act as pharmacological switches and chaperones, and they can regulate signaling and/or trafficking in a receptor-dependent manner. This review covers recent discoveries in the RAMP field and summarizes the known GPCR partners and functions of RAMPs. We also discuss the first peptide-bound structures of RAMP-GPCR complexes, which give insight into the molecular mechanisms that enable RAMPs to alter the pharmacology and signaling of GPCRs. PMID:26514202

  6. Antibacterial activity of a lectin-like Burkholderia cenocepacia protein.

    PubMed

    Ghequire, Maarten G K; De Canck, Evelien; Wattiau, Pierre; Van Winge, Iris; Loris, Remy; Coenye, Tom; De Mot, René

    2013-08-01

    Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombinant LlpA-like protein from the human isolate Burkholderia cenocepacia AU1054 displayed narrow-spectrum genus-specific antibacterial activity, thus representing the first functionally characterized bacteriocin within this β-proteobacterial genus. Strain-specific killing was confined to other members of the Bcc, with mostly Burkholderia ambifaria strains being susceptible. In addition to killing planktonic cells, this bacteriocin also acted as an antibiofilm agent. PMID:23737242

  7. Molecular architecture of the active mitochondrial protein gate.

    PubMed

    Shiota, Takuya; Imai, Kenichiro; Qiu, Jian; Hewitt, Victoria L; Tan, Khershing; Shen, Hsin-Hui; Sakiyama, Noriyuki; Fukasawa, Yoshinori; Hayat, Sikander; Kamiya, Megumi; Elofsson, Arne; Tomii, Kentaro; Horton, Paul; Wiedemann, Nils; Pfanner, Nikolaus; Lithgow, Trevor; Endo, Toshiya

    2015-09-25

    Mitochondria fulfill central functions in cellular energetics, metabolism, and signaling. The outer membrane translocator complex (the TOM complex) imports most mitochondrial proteins, but its architecture is unknown. Using a cross-linking approach, we mapped the active translocator down to single amino acid residues, revealing different transport paths for preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel to recruit chaperones from the intermembrane space that guide the transfer of hydrophobic preproteins. The translocator contains three Tom40 β-barrel channels sandwiched between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex exchanges with a dimeric isoform to assemble new TOM complexes. Dynamic coupling of α-helical receptors, β-barrel channels, and chaperones generates a versatile machinery that transports about 1000 different proteins. PMID:26404837

  8. Modulation of the protein kinase activity of mTOR.

    PubMed

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

  9. Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3

    PubMed Central

    Burnier, Laurent

    2013-01-01

    The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC’s cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC’s cytoprotective versus thrombin’s proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects. PMID:23788139

  10. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins

    PubMed Central

    Vallejo, Luis Felipe; Rinas, Ursula

    2004-01-01

    Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures. PMID:15345063