Nuclear location of PLA2-I in proliferative cells

1998 ◽  
Vol 111 (7) ◽  
pp. 985-994 ◽  
Author(s):  
J.M. Fayard ◽  
C. Tessier ◽  
J.F. Pageaux ◽  
M. Lagarde ◽  
C. Laugier
Keyword(s):  
Binding Sites ◽  
Culture Medium ◽  
Aristolochic Acid ◽  
Direct Interaction ◽  
Nuclear Level ◽  
Proliferative Effect ◽  
Stromal Cell Line ◽  
Normal Rat ◽  
Nuclear Location ◽  
Insight Into

We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.

scholarly journals Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jothi K. Yuvaraj ◽  
Rebecca E. Roberts ◽  
Yonathan Sonntag ◽  
Xiao-Qing Hou ◽  
Ewald Grosse-Wilde ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Bark Beetle ◽  
Pest Control ◽  
Binding Sites ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Odorant Receptors ◽  
Functional Evolution ◽  
General Importance ◽  
Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

scholarly journals Nucleus-specific expression in the multinuclear mushroom-forming fungusAgaricus bisporusreveals different nuclear regulatory programs

2018 ◽  
Vol 115 (17) ◽  
pp. 4429-4434 ◽  
Author(s):  
Thies Gehrmann ◽  
Jordi F. Pelkmans ◽  
Robin A. Ohm ◽  
Aurin M. Vos ◽  
Anton S. M. Sonnenberg ◽  
...  
Keyword(s):  
Agaricus Bisporus ◽  
Sequencing Data ◽  
Carbohydrate Active Enzymes ◽  
Nuclear Level ◽  
Specific Expression ◽  
Phenotypic Differences ◽  
Gene Level ◽  
Nuclear Separation ◽  
Insight Into ◽  
Specific Mrna

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroomAgaricus bisporuscontains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types ofA. bisporus. Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation.

Mapping of ligand binding sites provides insight into the function of factor H-related protein 5

2017 ◽  
Vol 89 ◽  
pp. 145
Author(s):  
Alexandra Papp ◽  
Marcell Cserhalmi ◽  
Ádám I. Csincsi ◽  
Barbara Uzonyi ◽  
David Ermert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Factor H ◽  
Related Protein ◽  
Ligand Binding Sites ◽  
Insight Into

scholarly journals Effects of cross-linked dimers of ribonuclease A or of lysozyme on the processing of endocytosed peroxidase by hepatoma cells

1982 ◽  
Vol 202 (2) ◽  
pp. 543-550
Author(s):  
J Bartholeyns ◽  
P Baudhuin
Keyword(s):  
Mechanism Of Action ◽  
Binding Sites ◽  
Culture Medium ◽  
Basic Protein ◽  
Intracellular Localization ◽  
Ribonuclease A ◽  
Protein Processing ◽  
Intracellular Protein ◽  
Intracellular Degradation ◽  
Htc Cells

Cross-linked dimers of ribonuclease, added at a concentration of 0.05 mg/ml to the culture medium of hepatoma (HTC) cells, were previously shown to inhibit intracellular degradation of peroxidase taken up by endocytosis. Intracellular localization showed that endocytosed peroxidase does not reach lysosomes in dimer-treated cells. The present study shows that preloading of lysosomes with fluorescent anti-peroxidase IgG, obtained by exposing HTC cells for 48 h to 0.1 mg of antibody/ml, restores intracellular degradation of endocytosed peroxidase. Moreover, accumulation of peroxidase into lysosomes, which no longer occurs in dimer-treated cells, occurs again under these conditions. We conclude that inhibition of transfer of peroxidase from phagosomes to lysosomes is most likely to be the alteration resulting from the exposure of the cells to ribonuclease dimer, rather than inhibition of fusion between phagosomes and lysosomes. The dimer of another basic protein, lysozyme added at a concentration of 0.2 mg/ml to the culture medium, is shown to induce the same type of effects as does the dimer of ribonuclease; the half-life of endocytosed peroxidase increased from 5 to 15 h after 2 h exposure of HTC cells to dimerized lysozyme. The effect of both dimers on intracellular protein processing can be reversed by addition of 100 mm-galactose to the culture medium, up to 5 h after pretreatment of the cells. The dimers of ribonuclease A or of lysozyme have thus probably the same mechanism of action. Evidence that the two dimers share the same binding sites on the cells is presented.

scholarly journals Indomethacin Increases Quercetin Affinity for Human Serum Albumin: A Combined Experimental and Computational Study and Its Broader Implications

2020 ◽  
Vol 21 (16) ◽  
pp. 5740
Author(s):  
Hrvoje Rimac ◽  
Tana Tandarić ◽  
Robert Vianello ◽  
Mirza Bojić
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Binding Site ◽  
Binding Sites ◽  
Quantum Chemical ◽  
Computational Study ◽  
The Body ◽  
Active Concentration ◽  
Insight Into

Human serum albumin (HSA) is the most abundant carrier protein in the human body. Competition for the same binding site between different ligands can lead to an increased active concentration or a faster elimination of one or both ligands. Indomethacin and quercetin both bind to the binding site located in the IIA subdomain. To determine the nature of the HSA-indomethacin-quercetin interactions, spectrofluorometric, docking, molecular dynamics studies, and quantum chemical calculations were performed. The results show that the indomethacin and quercetin binding sites do not overlap. Moreover, the presence of quercetin does not influence the binding constant and position of indomethacin in the pocket. However, binding of quercetin is much more favorable in the presence of indomethacin, with its position and interactions with HSA significantly changed. These results provide a new insight into drug-drug interactions, which can be important in situations when displacement from HSA or other proteins is undesirable or even desirable. This principle could also be used to deliberately prolong or shorten the xenobiotics’ half-life in the body, depending on the desired outcomes.

Epithelial repair is inhibited by an α1,6-fucose binding lectin

2007 ◽  
Vol 292 (2) ◽  
pp. L462-L468 ◽  
Author(s):  
Elizabeth C. Adam ◽  
Stephen T. Holgate ◽  
Peter M. Lackie
Keyword(s):  
Binding Sites ◽  
Repair Process ◽  
Airway Epithelial ◽  
Free Medium ◽  
Epithelial Repair ◽  
Aleuria Aurantia Lectin ◽  
Cell Substrate ◽  
Binding Lectin ◽  
Aleuria Aurantia ◽  
Insight Into

The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o− cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to α1,6-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that α1,6-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.

scholarly journals Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

2001 ◽  
Vol 355 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Chang Hoon LEE ◽  
Patrick Y. UM ◽  
Myung Hee PARK
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Binding Pocket ◽  
Complete Loss ◽  
Amino Acid Residues ◽  
Deoxyhypusine Synthase ◽  
Positive Identification ◽  
Insight Into

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

Clopidogrel: An Antithrombotic Drug Acting on the ADP-dependent Activation Pathway of Human Platelets

1996 ◽  
Vol 2 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Pierre Savi ◽  
Eric Heilmann ◽  
Paquita Nurden ◽  
Marie-Claude Laplace ◽  
Claude Bihour ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Binding Sites ◽  
Ex Vivo ◽  
Shape Change ◽  
Day Treatment ◽  
Antiplatelet Agent ◽  
Direct Interaction ◽  
Human Platelets ◽  
Human Volunteers ◽  
Induced Aggregation

The aim of the study was to determine the effect of clopidogrel on adenosine diphosphate (ADP)- induced platelet activation in human volunteers. Platelets from human volunteers before and after a 7-day treatment with clopidogrel (75 mg/kg), were tested for their sensi tivity to ADP by measuring ADP-induced aggregation, adenylyl cyclase downregulation, and [3H]-2-MeS-ADP binding. Platelet membrane glycoprotein (GP IIb-IIIa; GP Ib, GMP-140) expression was measured by flow cy tometry using fluorescent-labeled antibodies or fibrino gen. After oral administration to human volunteers (75 mg/day for 7 days), clopidogrel, a novel ADP-selective antiplatelet agent, inhibited ADP-induced aggregation of platelets ex vivo. This effect was irreversible in nature, and no activity could be detected in the plasma of treated subjects. Although clopidogrel did not modify ADP- induced shape change, it prevented the inhibitory effect of ADP (but not that of epinephrine) on the prostoglandin- E 1 (PGE1)-induced increase in platelet cAMP. The num ber of binding sites for [ 3H]-2-MeS-ADP, a stable ana logue of ADP that labels ADP binding sites linked to the inhibition of stimulated adenylyl cyclase, was reduced from 525 ± 62 sites/cell in the controls to 32 ± 5 sites/cell after treatment with clopidogrel (p < 0.001). This effect occurred with no consistent change in the binding affinity of [3H]-2-MeS-ADP, indicating that inhibition of platelet functions by clopidogrel was mainly due to a selective and irreversible reduction of ADP binding sites on plate lets. Flow cytometry experiments showed that clopi dogrel selectively inhibited ADP-inducing binding of fi brinogen to platelets. This effect occurred through a ma jor reduction of the ADP-induced activation of the GP IIb-IIIa complex. These findings therefore indicate that clopidogrel downregulates platelet responses via a selec tive and direct interaction with the ADP receptors, me diating the inhibition of stimulated adenylyl cyclase ac tivity in human platelets.

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