Tacal O

General

Full name : Tacal Ozden

First name : Ozden

Mail : Hacettepe University\; School of Pharmacy\; Department of Biochemistry\; 06100\; Ankara

Zip Code :

City :

Country : Turkey

Email : tacal@hacettepe.edu.tr

Phone : +903123054233

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References (24)

Title : Chlorpyrifos oxon crosslinking of amyloid beta 42 peptides is a new route for generation of self-aggregating amyloidogenic oligomers that promote Alzheimer's disease - Onder_2022_Chem.Biol.Interact_363_110029
Author(s) : Onder S , Biberoglu K , Tacal O , Schopfer LM
Ref : Chemico-Biological Interactions , 363 :110029 , 2022
Abstract : Epidemiological evidence suggests that people chronically exposed to organophosphorus pesticides are at increased risk of neurodegenerative disease. Covalently linked amyloid beta dimers have been isolated from the brains of Alzheimer's patients. The toxic forms of amyloid beta are amyloid dimers that spontaneously oligomerize. In the present report we treated recombinant and synthetic amyloid beta (1-42) with 1 mM chlorpyrifos oxon or 1 mM paraoxon. The trypsin-digested samples were analyzed by liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer. Data were searched with Protein Prospector software. We found two new types of crosslinks in amyloid dimers. An isopeptide Asp-Asp link occurred between the N-terminal amine of Asp 1 in one peptide and the beta carboxyl group of Asp 1 in another peptide. An Asp-Arg link occurred between the guanidino group of Arg 5 in one peptide and the beta carboxyl group of Asp 1 in another peptide. These results show that the active metabolites of the pesticides chlorpyrifos and parathion catalyze the crosslinking of amyloid beta (1-42) into toxic dimers. It was concluded that the increased risk of neurodegenerative disease in people exposed to organophosphorus pesticides could be explained by the crosslinking activity of these chemicals. Data are available via ProteomeXchange with identifier PXD034163.
ESTHER : Onder_2022_Chem.Biol.Interact_363_110029
PubMedSearch : Onder_2022_Chem.Biol.Interact_363_110029
PubMedID: 35779611

Title : Toluidine blue O attenuates tau phosphorylation in N2a-APPSwe cells - Onder_2022_Chem.Biol.Interact_366_110126
Author(s) : Onder S , Biberoglu K , Yuksel M , Tacal O
Ref : Chemico-Biological Interactions , 366 :110126 , 2022
Abstract : Alzheimer's disease (AD) is characterized by extracellular amyloid plaques composed of amyloid-beta peptide (Abeta), intracellular neurofibrillary tangles containing hyperphosphorylated tau protein and neuronal loss. Most of the FDA-approved AD drugs currently on the market are cholinesterase inhibitors, which are only effective in relieving the symptoms of AD. However, recent studies in AD drug discovery focus on multi-targeted strategies, including anti-amyloid and anti-tau therapy. In the current study, we have investigated the effects of toluidine blue O (TBO), a cholinesterase inhibitor, on amyloid precursor protein (APP) processing, tau phosphorylation, and tau kinases/phosphatase in N2a mouse neuroblastoma cells stably expressing the Swedish mutation of human APP695 (N2a-APPSwe). The results demonstrated that TBO reduces Abeta40/42 levels by decreasing expression levels of beta-secretase 1 (BACE1), presenilin 1 (PS1) and total APP without causing cytotoxic effects in N2a-APPSwe cells. TBO also decreased the levels of both total tau and phosphorylated tau at residues Ser202/Thr205, Thr181, Ser396 and Ser 396/Ser404. Moreover, when the possible mechanisms underlying its effects on tau pathology were explored, TBO was found to decrease tau phosphorylation at those sites by reducing the expression levels of Akt, GSK-3beta, Cdk5, inactive p-PP2A and increasing the expression levels of p-Akt Ser473 and inactive p-GSK-3beta Ser9. Our new data support the idea that TBO may be a promising multi-target drug candidate for the treatment of AD.
ESTHER : Onder_2022_Chem.Biol.Interact_366_110126
PubMedSearch : Onder_2022_Chem.Biol.Interact_366_110126
PubMedID: 36027949

Title : Butyrylcholinesterase in SH-SY5Y human neuroblastoma cells - Onder_2022_Neurotoxicol__
Author(s) : Onder S , Schopfer LM , Jiang W , Tacal O , Lockridge O
Ref : Neurotoxicology , : , 2022
Abstract : Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
ESTHER : Onder_2022_Neurotoxicol__
PubMedSearch : Onder_2022_Neurotoxicol__
PubMedID: 35189179

Title : Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry - Onder_2021_J.Proteome.Res__
Author(s) : Onder S , van Grol M , Fidder A , Xiao G , Noort D , Yerramalla U , Tacal O , Schopfer LM , Lockridge O
Ref : J Proteome Res , : , 2021
Abstract : Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (K(d) 3.2 x 10(-8) M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 microM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.
ESTHER : Onder_2021_J.Proteome.Res__
PubMedSearch : Onder_2021_J.Proteome.Res__
PubMedID: 34469172

Title : Inhibition of cholinesterases by safranin O: Integration of inhibition kinetics with molecular docking simulations - Onder_2020_Arch.Biochem.Biophys__108728
Author(s) : Onder S , Sari S , Tacal O
Ref : Archives of Biochemistry & Biophysics , :108728 , 2020
Abstract : In the present study, the inhibitory mechanisms and effects of a synthetic phenazine dye, safranin O (SO) on human plasma butyrylcholinesterase (BChE), human erythrocyte acetylcholinesterase (AChE) and recombinant BChE mutants were investigated. Kinetic studies showed the following information: SO leaded to linear competitive inhibition of human plasma BChE with K(i) = 0.44 +/- 0.085 M; alpha = . It acted as a hyperbolic noncompetitive inhibitor of human erythrocyte AChE with K(i) = 0.69 +/- 0.13; alpha = 1; beta = 0.08 +/- 0.02. On the other hand, the inhibitory effects of SO on two BChE mutants, where A328 was modified to either F or Y, revealed differences in terms of inhibitory patterns and K(i) values, compared to the obtained results with recombinant wild type BChE. SO was found to act as linear competitive inhibitors of A328F and A328Y BChE mutants. Compared to recombinant wild type BChE, A328Y and A328F BChE mutants caused a 4- and 10-fold decrease in K(i) value for SO, respectively. These findings were supported by molecular modelling studies. In conclusion, SO is a potent inhibitor of human cholinesterases and may be useful in the design and development of new drugs for the treatment of AD.
ESTHER : Onder_2020_Arch.Biochem.Biophys__108728
PubMedSearch : Onder_2020_Arch.Biochem.Biophys__108728
PubMedID: 33345803

Title : Characteristic fragment ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine - Biberoglu_2020_Anal.Biochem__113718
Author(s) : Biberoglu K , Schopfer LM , Tacal O , Lockridge O
Ref : Analytical Biochemistry , :113718 , 2020
Abstract : Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.
ESTHER : Biberoglu_2020_Anal.Biochem__113718
PubMedSearch : Biberoglu_2020_Anal.Biochem__113718
PubMedID: 32335065

Title : Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase - Biberoglu_2020_Molecules_25_
Author(s) : Biberoglu K , Tacal O , Schopfer LM , Lockridge O
Ref : Molecules , 25 : , 2020
Abstract : A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 A, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.
ESTHER : Biberoglu_2020_Molecules_25_
PubMedSearch : Biberoglu_2020_Molecules_25_
PubMedID: 31991818

Title : Azure B affects amyloid precursor protein metabolism in PS70cells - Biberoglu_2019_Chem.Biol.Interact_299_88
Author(s) : Biberoglu K , Yuksel M , Tacal O
Ref : Chemico-Biological Interactions , 299 :88 , 2019
Abstract : Alzheimer's disease (AD), the most common form of dementia, is characterized by abundant deposition of amyloid-beta (Abeta) peptide that is the result of sequential cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Several studies have documented that inhibition of Abeta peptide synthesis or facilitating its degradation is one of the attractive therapeutic strategies in AD. Methylene blue (MethB), which has recently been investigated in Phase II clinical trials, is a prominent inhibitor in reducing Abeta oligomers. Herein, we wonder whether the mitigating effects of MethB on amyloid metabolism are related to the activity of its major metabolite, azure B. The goal of this study was to investigate the effects of azure B, which is also a cholinesterase inhibitor, on APP processing by using Chinese hamster ovary cells stably expressing human wild-type APP and presenilin 1 (PS70). Azure B significantly decreased the levels of secreted APPalpha (sAPPalpha) and Abeta40/42 in culture medium with a dose-dependent manner. A significant decrease was also observed in the levels of intracellular APP without affecting the cell viability. In parallel with the decrease of APP and APP metabolites, the activity of beta-secretase 1 (BACE1) was significantly attenuated compared to control. Overall, our results show that azure B has a large contribution for the pharmacological profile of MethB in APP metabolism.
ESTHER : Biberoglu_2019_Chem.Biol.Interact_299_88
PubMedSearch : Biberoglu_2019_Chem.Biol.Interact_299_88
PubMedID: 30500345

Title : The kinetics of inhibition of human acetylcholinesterase and butyrylcholinesterase by methylene violet 3RAX - Onder_2019_Chem.Biol.Interact_314_108845
Author(s) : Onder S , Biberoglu K , Tacal O
Ref : Chemico-Biological Interactions , 314 :108845 , 2019
Abstract : Phenazines, naturally produced by bacteria and archaeal Methanosarcina species are nitrogen-containing tricyclic molecules with antibiotic, antitumoral, and antiparasitic activities. Phenazines are used as electron acceptors-donors in wide range of fields including environmental biosensors. In this study, the inhibitory effects of a synthetic phenazine dye, methylene violet 3RAX (also known as diethyl safranine) on human erythrocyte AChE and human plasma BChE were tested and also its inhibitory mechanisms for both enzymes were studied in detail. Kinetic analyses showed that methylene violet 3RAX acts as a hyperbolic noncompetitive inhibitor of AChE with Ki value of 1.58+/-0.36muM; alpha=1; beta=0.12+/-0.0003. On the other hand, it caused linear competitive inhibition of BChE with Ki value of 0.51+/-0.006muM; alpha=infinity. In conclusion, methylene violet 3RAX which is a highly effective inhibitor of both human AChE and human BChE with Ki values in low micromolar range may be a promising candidate for the treatment of Alzheimer's disease.
ESTHER : Onder_2019_Chem.Biol.Interact_314_108845
PubMedSearch : Onder_2019_Chem.Biol.Interact_314_108845
PubMedID: 31593690

Title : Delipidation of Plasma Has Minimal Effects on Human Butyrylcholinesterase - Onder_2018_Front.Pharmacol_9_117
Author(s) : Onder S , Tacal O , Lockridge O
Ref : Front Pharmacol , 9 :117 , 2018
Abstract : Human butyrylcholinesterase (BChE) is purified in large quantities from Cohn fraction IV-4 to use for protection against the toxicity of chemical warfare agents. Small scale preliminary experiments use outdated plasma from the American Red Cross as the starting material for purifying BChE (P06276). Many of the volunteer donor plasma samples are turbid with fat, the donor having eaten fatty food before the blood draw. The turbid fat interferes with enzyme assays performed in the spectrophotometer and with column chromatography. Our goal was to find a method to remove fat from plasma without loss of BChE activity. Satisfactory delipidation was achieved by adding a solution of 10% dextran sulfate and calcium chloride to fatty plasma, followed by centrifugation, and filtration through a 0.8 mum filter. Treatment with Aerosil also delipidated fatty plasma, but was accompanied by loss of 50% of the plasma volume. BChE activity and the BChE isozyme pattern on nondenaturing gel electrophoresis were unaffected by delipidation. BChE in delipidated plasma was efficiently captured by immobilized monoclonal antibodies B2 18-5 and mAb2. The immunopurified BChE was released from antibody binding with acid and visualized as a highly enriched, denatured BChE preparation by SDS gel electrophoresis. In conclusion, delipidation with dextran sulfate/CaCl2 preserves BChE activity and the tetramer structure of BChE.
ESTHER : Onder_2018_Front.Pharmacol_9_117
PubMedSearch : Onder_2018_Front.Pharmacol_9_117
PubMedID: 29497381

Title : Toluidine blue O modifies hippocampal amyloid pathology in a transgenic mouse model of Alzheimer's disease - Yuksel_2018_Biochimie_146_105
Author(s) : Yuksel M , Biberoglu K , Onder S , Akbulut KG , Tacal O
Ref : Biochimie , 146 :105 , 2018
Abstract : Recently, we have demonstrated that toluidine blue O (TBO), a phenothiazine dye, shows inhibitory effects on both cholinesterases and amyloid pathology in Alzheimer's disease (AD) cellular model. In the present study, we aimed to determine the effects of TBO (in a purity of 85%) on amyloid and tau pathologies in a triple transgenic mouse model of AD (3xTg-AD). Beginning at 7.5 (mild pathology) or 13 (severe pathology) months of age, 3xTg-AD mice were treated intraperitoneally with 4mg/kg TBO or vehicle daily for 30 days. TBO treatment significantly reduced the levels of insoluble Abeta40 and Abeta42 in the hippocampi of mild and severe pathology groups compared to vehicle-treated counterparts. When the levels of full-length amyloid precursor protein (APP) and beta-site APP-cleaving enzyme 1 (BACE1) were assessed in 3xTg-AD mice at late pathological stage, no significant changes were observed after TBO treatment. Similarly, TBO did not recover hyperphosphorylation of tau at residues Thr181 and Ser202/Thr205 significantly in soluble and insoluble hippocampal fractions of 3xTg-AD mice. Taken together, the current study is the first in vivo report, to our knowledge, demonstrating that TBO mitigates amyloid pathology in 3xTg-AD mice with no apparent change on tau phosphorylation. Overall, the preliminary data presented here support the possible use of TBO as a disease-modifying drug for AD treatment.
ESTHER : Yuksel_2018_Biochimie_146_105
PubMedSearch : Yuksel_2018_Biochimie_146_105
PubMedID: 29248542

Title : Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure - Onder_2018_Anal.Chem_90_974
Author(s) : Onder S , Schopfer LM , Cashman JR , Tacal O , Johnson RC , Blake TA , Lockridge O
Ref : Analytical Chemistry , 90 :974 , 2018
Abstract : Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.
ESTHER : Onder_2018_Anal.Chem_90_974
PubMedSearch : Onder_2018_Anal.Chem_90_974
PubMedID: 29172437

Title : Hupresin Retains Binding Capacity for Butyrylcholinesterase and Acetylcholinesterase after Sanitation with Sodium Hydroxide - Onder_2017_Front.Pharmacol_8_713
Author(s) : Onder S , David E , Tacal O , Schopfer LM , Lockridge O
Ref : Front Pharmacol , 8 :713 , 2017
Abstract : Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10-15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.
ESTHER : Onder_2017_Front.Pharmacol_8_713
PubMedSearch : Onder_2017_Front.Pharmacol_8_713
PubMedID: 29066970

Title : Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids - Onder_2017_Chem.Res.Toxicol_30_2218
Author(s) : Onder S , Dafferner AJ , Schopfer LM , Xiao G , Yerramalla U , Tacal O , Blake TA , Johnson RC , Lockridge O
Ref : Chemical Research in Toxicology , 30 :2218 , 2017
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 x 10(-9) M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10(-8) M for OP-peptides and 1 x 10(-12) M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 mug/mL of depY was 0.025 mug of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.
ESTHER : Onder_2017_Chem.Res.Toxicol_30_2218
PubMedSearch : Onder_2017_Chem.Res.Toxicol_30_2218
PubMedID: 29137457

Title : Effects of phenothiazine-structured compounds on APP processing in Alzheimer's disease cellular model - Yuksel_2017_Biochimie_138_82
Author(s) : Yuksel M , Biberoglu K , Onder S , Akbulut KG , Tacal O
Ref : Biochimie , 138 :82 , 2017
Abstract : The excess accumulation of amyloid-beta (Abeta) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Abeta, soluble APP-alpha (sAPPalpha), intracellular APP and beta-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Abeta40, Abeta42 and sAPPalpha were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Abeta40, Abeta42 and sAPPalpha in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Abeta pathology by lowering APP levels whereas reduced Abeta caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro.
ESTHER : Yuksel_2017_Biochimie_138_82
PubMedSearch : Yuksel_2017_Biochimie_138_82
PubMedID: 28457944

Title : Toluidine blue O is a potent inhibitor of human cholinesterases - Biberoglu_2016_Arch.Biochem.Biophys_604_57
Author(s) : Biberoglu K , Tek MY , Ghasemi ST , Tacal O
Ref : Archives of Biochemistry & Biophysics , 604 :57 , 2016
Abstract : In this study, the inhibitory effects of three phenothiazines [toluidine blue O (TBO), thionine (TH) and methylene violet (MV)] were tested on human plasma butyrylcholinesterase (BChE) and their inhibitory mechanisms were studied in detail. MV acted as a linear mixed type inhibitor of human BChE with Ki = 0.66 +/- 0.06 muM and alpha = 13.6 +/- 3.5. TBO and TH caused nonlinear inhibition of human BChE, compatible to double occupancy. Ki values estimated by nonlinear regression analysis for TBO and TH were 0.008 +/- 0.003 muM and 2.1 +/- 0.42 muM, respectively. The inhibitory potential of TBO was also tested on human erythrocyte AChE. TBO acted as a linear mixed type inhibitor of human AChE with Ki = 0.041 +/- 0.005 muM and alpha = 1.6 +/- 0.007. Using four site-directed BChE mutants, the role of peripheral anionic site residues of human BChE was also investigated in the binding of TBO to BChE. The peripheral anionic site mutants of BChE caused 16-69-fold increase in Ki value of TBO, compared to recombinant wild-type, suggesting that peripheral anionic site residues are involved in the binding of TBO to human BChE. In conclusion, TBO which is a potent inhibitor of human cholinesterases, may be a potential drug candidate for the treatment of Alzheimer's disease.
ESTHER : Biberoglu_2016_Arch.Biochem.Biophys_604_57
PubMedSearch : Biberoglu_2016_Arch.Biochem.Biophys_604_57
PubMedID: 27296777

Title : Healthy F-16 pilots show no evidence of exposure to tri-ortho-cresyl phosphate through the on-board oxygen generating system - Tacal_2014_Chem.Biol.Interact_215C_69
Author(s) : Tacal O , Schopfer LM
Ref : Chemico-Biological Interactions , 215C :69 , 2014
Abstract : About 18% of fighter pilots complain of ill symptoms that begin during flight and persist for days. A possible source of toxicity is the air supplied through the on board oxygen generating system. The air passes through the jet engine before it is enriched for oxygen and breathed through an oxygen mask. While in the jet engine, the air can become contaminated with jet engine lubricating oil. A potentially toxic component in jet engine oil is tri-ortho-cresyl phosphate (TOCP), which is metabolically activated to the highly reactive cresyl saligenin phosphate. The cresyl saligenin phosphate reacts with butyrylcholinesterase (BChE) to make a covalent adduct on serine 198. The purpose of this work was to determine whether the blood of healthy, active-duty F-16 pilots has measurable levels of the cresyl phosphate adduct. BChE was immunopurified from 0.5ml plasma by binding to immobilized monoclonal mAb2. BChE protein was released with acetic acid, digested with pepsin and analyzed by LC-MS/MS on the 5600 Triple TOF mass spectrometer. Positive controls for quantifying the limit of detection were plasma samples containing known amounts of cresyl saligenin phosphate treated plasma. The cresyl phosphate adduct eluted at 31.3min with an observed parent ion mass of 966.4m/z and characteristic daughter ions 778.3, 673.3, and 602.3m/z. Control experiments demonstrated that as little as 0.1% of the 1-2mug BChE recovered from 0.5ml plasma could be detected as the cresyl phosphate adduct on peptide FGES198AGAAS. Mass spectrometry analysis of plasma from fifteen healthy F-16 pilots showed that none had evidence of exposure to TOCP. It was concluded that the on-board oxygen generating system, when operating properly, did not deliver tri-ortho-cresyl phosphate in the oxygen supply.
ESTHER : Tacal_2014_Chem.Biol.Interact_215C_69
PubMedSearch : Tacal_2014_Chem.Biol.Interact_215C_69
PubMedID: 24661946

Title : Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase - Biberoglu_2013_Biochim.Biophys.Acta_1834_745
Author(s) : Biberoglu K , Schopfer LM , Saxena A , Tacal O , Lockridge O
Ref : Biochimica & Biophysica Acta , 1834 :745 , 2013
Abstract : Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.
ESTHER : Biberoglu_2013_Biochim.Biophys.Acta_1834_745
PubMedSearch : Biberoglu_2013_Biochim.Biophys.Acta_1834_745
PubMedID: 23352838

Title : Determination of binding points of methylene blue and cationic phenoxazine dyes on human butyrylcholinesterase - Sezgin_2013_Arch.Biochem.Biophys_532_32
Author(s) : Sezgin Z , Biberoglu K , Chupakhin V , Makhaeva GF , Tacal O
Ref : Archives of Biochemistry & Biophysics , 532 :32 , 2013
Abstract : In this study, the binding points of MethB and two structurally-related cationic phenoxazine dyes [meldola blue (MB) and nile blue (NB)] to human butyrylcholinesterase (BChE) were investigated by molecular docking and site directed mutagenesis. The comparative inhibitory effects of MethB, MB and NB on recombinant wild type BChE and six human BChE mutants were spectrophotometrically studied. Kinetic analyses yielded the following information: MethB and MB were found to cause nonlinear inhibition of all recombinant BChEs except Y332A, compatible with a multi-site binding model. On the other hand, MethB and MB caused linear mixed inhibition of Y332A mutant, compatible with a single binding mode. Comparing the inhibitory effects in aspect of Ki values with recombinant wild type BChE (Ki=0.042 muM), MethB was found to be approximately 30, 80 and 270-fold less effective as an inhibitor of Y332A, F329A and T120F, respectively. NB caused nonlinear inhibition of all recombinant BChEs. The inhibitory effect of NB on Y332A mutant was approximately 370-fold lower, compared to recombinant wild type BChE (Ki=0.006 muM). Considering both kinetic and molecular docking results together, it was concluded that threonine 120, phenylalanine 329 and tyrosine 332 are critical amino acids in binding of cationic phenoxazine/phenothiazine structured ligands to human BChE.
ESTHER : Sezgin_2013_Arch.Biochem.Biophys_532_32
PubMedSearch : Sezgin_2013_Arch.Biochem.Biophys_532_32
PubMedID: 23353050

Title : Resistance of human butyrylcholinesterase to methylene blue-catalyzed photoinactivation\; mass spectrometry analysis of oxidation products - Tacal_2013_Photochem.Photobiol_89_336
Author(s) : Tacal O , Li B , Lockridge O , Schopfer LM
Ref : Photochem Photobiol , 89 :336 , 2013
Abstract : Methylene blue, 3, 7-bis(dimethylamino)-phenothiazin-5-ium chloride, is a reversible inhibitor of human butyrylcholinesterase (BChE) in the absence of light. In the presence of light and oxygen, methylene blue promotes irreversible inhibition of human BChE as a function of time, requiring 3 h irradiation to inhibit 95% activity. Inactivation was accompanied by a progressive loss of Coomassie-stained protein bands on native and denaturing polyacrylamide gels, suggesting backbone fragmentation. Aggregation was not detected. MALDI-TOF/TOF mass spectrometry identified oxidized tryptophan (W52, 56, 231, 376, 412, 490, 522), oxidized methionine (M81, 144, 302, 532, 554, 555), oxidized histidine (H214), oxidized proline (P230), oxidized cysteine (C519) and oxidized serine (S215). A 20 min irradiation in the presence of methylene blue resulted in 17% loss of BChE activity, suggesting that BChE is relatively resistant to methylene blue-catalyzed photoinactivation and that therefore this process could be used to sterilize BChE preparations.
ESTHER : Tacal_2013_Photochem.Photobiol_89_336
PubMedSearch : Tacal_2013_Photochem.Photobiol_89_336
PubMedID: 23136924

Title : The proline-rich tetramerization peptides in equine serum butyrylcholinesterase - Biberoglu_2012_Febs.J_279_3844
Author(s) : Biberoglu K , Schopfer LM , Tacal O , Lockridge O
Ref : Febs J , 279 :3844 , 2012
Abstract : Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline-rich region of human lamellipodin. Our goal was to examine the tetramer-organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in MALDI-TOF-TOF and linear ion trap quadrupole Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to nine proteins, although not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse butyrylcholinesterase. The function of these polyproline peptides is to serve as tetramer-organizing peptides.
ESTHER : Biberoglu_2012_Febs.J_279_3844
PubMedSearch : Biberoglu_2012_Febs.J_279_3844
PubMedID: 22889087

Title : The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase - Biberoglu_2011_Arch.Biochem.Biophys_511_64
Author(s) : Biberoglu K , Tacal O , Akbulut H
Ref : Archives of Biochemistry & Biophysics , 511 :64 , 2011
Abstract : Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25 degrees C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 muM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.
ESTHER : Biberoglu_2011_Arch.Biochem.Biophys_511_64
PubMedSearch : Biberoglu_2011_Arch.Biochem.Biophys_511_64
PubMedID: 21530486

Title : Methamidophos, dichlorvos, O-methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry - Tacal_2010_J.Appl.Toxicol_30_469
Author(s) : Tacal O , Lockridge O
Ref : J Appl Toxicol , 30 :469 , 2010
Abstract : Organophosphorus pesticides used most commonly in Turkey include methamidophos, dichlorvos, O-methoate and diazinon. These toxic chemicals or their metabolites make a covalent bond with the active site serine of butyrylcholinesterase. Our goal was to identify the adducts that result from the reaction of human butyrylcholinesterase with these pesticides. Highly purified human butyrylcholinesterase was treated with a 20-fold molar excess of pesticide. The protein was denatured by boiling and digested with trypsin. MS and MSMS spectra of HPLC-purified peptides were acquired on a MALDI-TOF-TOF 4800 mass spectrometer. It was found that methamidophos added a mass of +93, consistent with addition of methoxy aminophosphate. A minor amount of adduct with an added mass of +109 was also found. Dichlorvos and O-methoate both made dimethoxyphosphate (+108) and monomethoxyphosphate adducts (+94). Diazinon gave a novel adduct with an added mass of +152 consistent with diethoxythiophosphate. Inhibition of enzyme activity in the presence of diazinon developed slowly (15 h), concomitant with isomerization of diazinon via a thiono-thiolo rearrangement. The isomer of diazinon yielded diethoxyphosphate and monoethoxyphosphate adducts with added masses of +136 and +108. MSMS spectra confirmed that each of the pesticides studied made a covalent bond with serine 198 of butyrylcholinesterase. These results can be used to identify the class of pesticides to which a patient was exposed.
ESTHER : Tacal_2010_J.Appl.Toxicol_30_469
PubMedSearch : Tacal_2010_J.Appl.Toxicol_30_469
PubMedID: 20229498

Title : Comparative effects of cationic triarylmethane, phenoxazine and phenothiazine dyes on horse serum butyrylcholinesterase - Yucel_2008_Arch.Biochem.Biophys_478_201
Author(s) : Yucel YY , Tacal O , Ozer I
Ref : Archives of Biochemistry & Biophysics , 478 :201 , 2008
Abstract : The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25( degrees )C in 50mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K(i) values of 4.5+/-0.50 microM, 0.41+/-0.007 microM, 0.44+/-0.086 microM and 0.050+/-0.0074 microM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K' values ( identical with[I](2)(0.5) extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20+/-0.096 microM, 0.0018+/-0.0015 microM, 0.92+/-0.23 microM and 0.23+/-0.08 microM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.
ESTHER : Yucel_2008_Arch.Biochem.Biophys_478_201
PubMedSearch : Yucel_2008_Arch.Biochem.Biophys_478_201
PubMedID: 18656440