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Markus R Wenk - One of the best experts on this subject based on the ideXlab platform.

  • activity based lipid esterase profiling of m bovis bcg at different metabolic states using Tetrahydrolipstatin thl as bait
    Methods of Molecular Biology, 2017
    Co-Authors: Markus R Wenk, Madhu Sudhan Ravindran
    Abstract:

    This chapter provides a step-by-step protocol using activity-based protein profiling (ABPP) as a chemical-proteomic tool to survey the antibiotic properties of a small molecule. Here, we investigate the molecular mechanism behind the bactericidal activity of Tetrahydrolipstatin (THL). ABPP relies on small molecule probes that target the active site of specific enzymes in complex proteomes. These probes in turn are equipped with a reporter tag that allows capturing, visualization, enrichment, identification, and quantification of its targets either in vitro or in situ. THL possesses bactericidal activities, but its precise spectrum of molecular targets is poorly characterized. Here, we used THL analogs functionalized to enable Huisgen-base cycloaddition, commonly known as "click chemistry," to identify target proteins after enrichment from mycobacterial cell lysates obtained from different physiological conditions.

  • targeting lipid esterases in mycobacteria grown under different physiological conditions using activity based profiling with Tetrahydrolipstatin thl
    Molecular & Cellular Proteomics, 2014
    Co-Authors: Madhu Sudhan Ravindran, Xiamin Cheng, Ankit Shukla, Amaury Cazenavegassiot, Markus R Wenk
    Abstract:

    Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guerin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.

  • activity based proteome profiling of potential cellular targets of orlistat an fda approved drug with anti tumor activities
    Journal of the American Chemical Society, 2010
    Co-Authors: Pengyu Yang, Kai Liu, Mun Hong Ngai, Martin J Lear, Markus R Wenk, Shao Q Yao
    Abstract:

    Orlistat, or Tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

  • triacylglycerol utilization is required for regrowth of in vitro hypoxic nonreplicating mycobacterium bovis bacillus calmette guerin
    Journal of Bacteriology, 2009
    Co-Authors: Kai Leng Low, Srinivasa P S Rao, Guanghou Shui, Anne K Bendt, Kevin Pethe, Thomas Dick, Markus R Wenk
    Abstract:

    Mycobacteria store triacylglycerols (TGs) under various stress conditions, such as hypoxia, exposure to nitric oxide, and acidic environments. These stress conditions are known to induce nonreplicating persistence in mycobacteria. The importance of TG accumulation and utilization during regrowth is not clearly understood. Here we specifically determined the levels of accumulated TG and TG lipase activity in Mycobacterium bovis bacillus Calmette-Guerin (BCG) in various different physiological states (logarithmic growth, aerated stationary phase, hypoxia-induced dormancy, and regrowth from dormancy). We found extensive accumulation and degradation of TGs in the bacilli during entry into and exit from hypoxia-induced dormancy, respectively. These processes are accompanied by dynamic appearance and disappearance of intracellular TG lipid particles. The reduction in TG levels coincides with an increase in cellular TG lipase activity in the regrowing bacilli. Tetrahydrolipstatin, an inhibitor of TG lipases, reduces total lipase activity, prevents breakdown of TGs, and blocks the growth of mycobacteria upon resuscitation with air. Our results demonstrate that utilization of TGs is essential for the regrowth of mycobacteria during their exit from the hypoxic nonreplicating state.

Erica H Jaffé - One of the best experts on this subject based on the ideXlab platform.

  • Acute Immobilization Stress Modulate GABA Release from Rat Olfactory Bulb: Involvement of Endocannabinoids-Cannabinoids and Acute Stress Modulate GABA Release
    2020
    Co-Authors: Alejandra Delgado, Erica H Jaffé
    Abstract:

    We studied the effects of cannabinoids and acute immobilization stress on the regulation of GABA release in the olfactory bulb. Glutamate-stimulated 3H-GABA release was measured in superfused slices. We report that cannabinoids as WIN55, 212-2, methanandamide, and 2-arachidonoylglycerol were able to inhibit glutamate-and KCl-stimulated 3H-GABA release. This effect was blocked by the CB1 antagonist AM281. On the other hand, acute stress was able per se to increase endocannabinoid activity. This effect was evident since the inhibition of stimulated GABA release by acute stress was reversed with AM281 and Tetrahydrolipstatin. Inhibition of the endocannabinoid transport or its catabolism showed reduction of GABA release, antagonized by AM281 in control and stressed animals. These results point to endocannabinoids as inhibitory modulators of GABA release in the olfactory bulb acting through an autocrine mechanism. Apparently, stress increases the endocannabinoid system, modulating GABAergic synaptic function in a primary sensory organ

  • Acute Immobilization Stress Modulate GABA Release from Rat Olfactory Bulb: Involvement of Endocannabinoids—Cannabinoids and Acute Stress Modulate GABA Release
    Hindawi Limited, 2011
    Co-Authors: Alejandra Delgado, Erica H Jaffé
    Abstract:

    We studied the effects of cannabinoids and acute immobilization stress on the regulation of GABA release in the olfactory bulb. Glutamate-stimulated 3H-GABA release was measured in superfused slices. We report that cannabinoids as WIN55, 212-2, methanandamide, and 2-arachidonoylglycerol were able to inhibit glutamate- and KCl-stimulated 3H-GABA release. This effect was blocked by the CB1 antagonist AM281. On the other hand, acute stress was able per se to increase endocannabinoid activity. This effect was evident since the inhibition of stimulated GABA release by acute stress was reversed with AM281 and Tetrahydrolipstatin. Inhibition of the endocannabinoid transport or its catabolism showed reduction of GABA release, antagonized by AM281 in control and stressed animals. These results point to endocannabinoids as inhibitory modulators of GABA release in the olfactory bulb acting through an autocrine mechanism. Apparently, stress increases the endocannabinoid system, modulating GABAergic synaptic function in a primary sensory organ

  • Acute Immobilization Stress Modulate GABA Release from Rat Olfactory Bulb: Involvement of Endocannabinoids—Cannabinoids and Acute Stress Modulate GABA Release
    2011
    Co-Authors: Ra Delgado, Erica H Jaffé
    Abstract:

    License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We studied the effects of cannabinoids and acute immobilization stress on the regulation of GABA release in the olfactory bulb. Glutamate-stimulated 3H-GABA release was measured in superfused slices. We report that cannabinoids as WIN55, 212-2, methanandamide, and 2-arachidonoylglycerol were able to inhibit glutamate- and KCl-stimulated 3H-GABA release. This effect was blocked by the CB1 antagonist AM281. On the other hand, acute stress was able per se to increase endocannabinoid activity. This effect was evident since the inhibition of stimulated GABA release by acute stress was reversed with AM281 and Tetrahydrolipstatin. Inhibition of the endocannabinoid transport or its catabolism showed reduction of GABA release, antagonized by AM281 in control and stressed animals. These results point to endocannabinoids as inhibitory modulators of GABA release in the olfactory bulb acting through an autocrine mechanism. Apparently, stress increases the endocannabinoid system, modulating GABAergic synaptic function in a primary sensory organ. 1

Alain Eschalier - One of the best experts on this subject based on the ideXlab platform.

  • monoacylglycerols activate trpv1 a link between phospholipase c and trpv1
    PLOS ONE, 2013
    Co-Authors: Anna Ermund, Charlotte Simonsen, Bo A. G. Jönsson, Bryndis Birnir, Anders Blomgren, Pouya Movahed, David A Andersson, Stuart Bevan, Alain Eschalier
    Abstract:

    Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor Tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and Tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

  • Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1
    PLOS ONE, 2013
    Co-Authors: Anna Ermund, Charlotte Simonsen, Bo A. G. Jönsson, Bryndis Birnir, Anders Blomgren, Pouya Movahed, David A Andersson, Stuart Bevan, Alain Eschalier
    Abstract:

    Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor Tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and Tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

P Hadváry - One of the best experts on this subject based on the ideXlab platform.

  • surface behaviour of bile salts and Tetrahydrolipstatin at air water and oil water interfaces
    Chemistry and Physics of Lipids, 2001
    Co-Authors: Ali Tiss, P Hadváry, Stephane Ransac, Hans Lengsfeld, Alain Cagna, Robert Verger
    Abstract:

    The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of Tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.

  • identification of the active site serine in human pancreatic lipase by chemical modification with Tetrahydrolipstatin
    FEBS Letters, 1992
    Co-Authors: Qiaoqian Luthipeng, Hans Peter Marki, P Hadváry
    Abstract:

    A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C] Tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the delta-lactone Ro 40-4441 in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152 ... His-263 ... Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.

  • the lipase inhibitor Tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase
    Journal of Biological Chemistry, 1991
    Co-Authors: P Hadváry, W Sidler, W Meister, W Vetter, H Wolfer
    Abstract:

    Tetrahydrolipstatin (THL) is a selective inhibitor of fat absorption. In animal models, it has anti-obesity and anti-hypercholesterolemic activity and is presently in clinical trials for these indications. THL binds covalently to pancreatic lipase. Complete inhibition of lipolytic activity is obtained concomitant with the incorporation of 1 mol of THL/mol of enzyme. Pancreatic lipase is the best studied lipase, but published results concerning its catalytic mechanism are still controversial. In order to learn more about the inhibitory mechanism of THL, a selective lipase inhibitor interacting at or near the catalytic site, and therefore, to obtain more information on the catalytic mechanism of lipase, we have determined the amino acid residue to which THL is bound. After proteolytic degradation of porcine pancreatic lipase inhibited with radioactively labeled THL, the labeled peptides were isolated and analyzed by quantitative amino acid analysis, N-terminal sequencing, and by mass spectrometry with fast atom bombardment ionization. The data clearly show that THL is bound as an ester to the serine 152 of the lipase.

A. R. Prasad - One of the best experts on this subject based on the ideXlab platform.