Dataset Information

Mapping the Binding Sites of UDP and Prostaglandin E2 Glyceryl Ester in the Nucleotide Receptor P2Y₆.

ABSTRACT: Cyclooxygenase-2 catalyzes the biosynthesis of prostaglandins from arachidonic acid and the biosynthesis of prostaglandin glycerol esters (PG-Gs) from 2-arachidonoylglycerol. PG-Gs are mediators of several biological actions such as macrophage activation, hyperalgesia, synaptic plasticity, and intraocular pressure. Recently, the human UDP receptor P2Y₆ was identified as a target for the prostaglandin E2 glycerol ester (PGE₂ -G). Here, we show that UDP and PGE₂ -G are evolutionary conserved endogenous agonists at vertebrate P2Y₆ orthologs. Using sequence comparison of P2Y₆ orthologs, homology modeling, and ligand docking studies, we proposed several receptor positions participating in agonist binding. Site-directed mutagenesis and functional analysis of these P2Y₆ mutants revealed that both UDP and PGE₂ -G share in parts one ligand-binding site. Thus, the convergent signaling of these two chemically very different agonists has already been manifested in the evolutionary design of the ligand-binding pocket.

SUBMITTER: Zimmermann A

PROVIDER: S-EPMC9305961 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Mapping the Binding Sites of UDP and Prostaglandin E2 Glyceryl Ester in the Nucleotide Receptor P2Y6.

Zimmermann Anne A Vu Oanh O Brüser Antje A Sliwoski Gregory G Marnett Lawrence J LJ Meiler Jens J Schöneberg Torsten T

ChemMedChem 20220128 7

Cyclooxygenase-2 catalyzes the biosynthesis of prostaglandins from arachidonic acid and the biosynthesis of prostaglandin glycerol esters (PG-Gs) from 2-arachidonoylglycerol. PG-Gs are mediators of several biological actions such as macrophage activation, hyperalgesia, synaptic plasticity, and intraocular pressure. Recently, the human UDP receptor P2Y6 was identified as a target for the prostaglandin E2 glycerol ester (PGE2 -G). Here, we show that UDP and PGE2 -G ...[more]

PMID: 35034430

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Project description:Human monocytic cells in blood have important roles in host defense and express the enzyme carboxylesterase 1 (CES1). This metabolic serine hydrolase plays a critical role in the metabolism of many molecules, including lipid mediators called prostaglandin glyceryl esters (PG-Gs), which are formed during cyclooxygenase-mediated oxygenation of the endocannabinoid 2-arachidonoylglycerol. Some PG-Gs have been shown to exhibit anti-inflammatory effects; however, they are unstable compounds, and their hydrolytic breakdown generates pro-inflammatory prostaglandins. We hypothesized that by blocking the ability of CES1 to hydrolyze PG-Gs in monocytes/macrophages, the beneficial effects of anti-inflammatory prostaglandin D2-glyceryl ester (PGD2-G) could be augmented. The goals of this study were to determine whether PGD2-G is catabolized by CES1, evaluate the degree to which this metabolism is blocked by small-molecule inhibitors, and assess the immunomodulatory effects of PGD2-G in macrophages. A human monocytic cell line (THP-1 cells) was pretreated with increasing concentrations of known small-molecule inhibitors that block CES1 activity [chlorpyrifos oxon (CPO), WWL229, or WWL113], followed by incubation with PGD2-G (10 μM). Organic solvent extracts of the treated cells were analyzed by liquid chromatography with tandem mass spectrometry to assess levels of the hydrolysis product PGD2. Further, THP-1 monocytes with normal CES1 expression (control cells) and "knocked-down" CES1 expression (CES1KD cells) were employed to confirm CES1's role in PGD2-G catabolism. We found that CES1 has a prominent role in PGD2-G hydrolysis in this cell line, accounting for about 50% of its hydrolytic metabolism, and that PGD2-G could be stabilized by the inclusion of CES1 inhibitors. The inhibitor potency followed the rank order: CPO > WWL113 > WWL229. THP-1 macrophages co-treated with WWL113 and PGD2-G prior to stimulation with lipopolysaccharide exhibited a more pronounced attenuation of pro-inflammatory cytokine levels (interleukin-6 and TNFα) than by PGD2-G treatment alone. In contrast, prostaglandin E2-glyceryl ester (PGE2-G) had opposite effects compared to those of PGD2-G, which appeared to be dependent on the hydrolysis of PGE2-G to PGE2. These results suggest that the anti-inflammatory effects induced by PGD2-G can be further augmented by inactivating CES1 activity with specific small-molecule inhibitors, while pro-inflammatory effects of PGE2-G are attenuated. Furthermore, PGD2-G (and/or its downstream metabolites) was shown to activate the lipid-sensing receptor PPARγ, resulting in altered "alternative macrophage activation" response to the Th2 cytokine interleukin-4. These findings suggest that inhibition of CES1 and other enzymes that regulate the levels of pro-resolving mediators such as PGD2-G in specific cellular niches might be a novel anti-inflammatory approach.

Project description:AimsUDP-sugars can act as extracellular signalling molecules, but relatively little is known about their cardiovascular actions. The P2Y14 receptor is a Gi/o-coupled receptor which is activated by UDP-glucose and related sugar nucleotides. In this study we sought to investigate whether P2Y14 receptors are functionally expressed in the porcine coronary artery using a selective P2Y14 receptor agonist, MRS2690, and a novel selective P2Y14 receptor antagonist, PPTN (4,7-disubstituted naphthoic acid derivative).Methods and resultsIsometric tension recordings were used to evaluate the effects of UDP-sugars in porcine isolated coronary artery segments. The effects of the P2 receptor antagonists suramin and PPADS, the P2Y14 receptor antagonist PPTN, and the P2Y6 receptor antagonist MRS2578, were investigated. Measurement of vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry was used to assess changes in cAMP levels. UDP-glucose, UDP-glucuronic acid UDP-N-acetylglucosamine (P2Y14 receptor agonists), elicited concentration-dependent contractions of the porcine coronary artery. MRS2690 was a more potent vasoconstrictor than the UDP-sugars. Concentration dependent contractile responses to MRS2690 and UDP-sugars were enhanced in the presence of forskolin (activator of cAMP), where the level of basal tone was maintained by addition of U46619, a thromboxane A2 mimetic. Contractile responses to MRS2690 were blocked by PPTN, but not by MRS2578. Contractile responses to UDP-glucose were also attenuated by PPTN and suramin, but not by MRS2578. Forskolin-induced VASP-phosphorylation was reduced in porcine coronary arteries exposed to UDP-glucose and MRS2690, consistent with P2Y14 receptor coupling to Gi/o proteins and inhibition of adenylyl cyclase activity.ConclusionsOur data support a role of UDP-sugars as extracellular signalling molecules and show for the first time that they mediate contraction of porcine coronary arteries via P2Y14 receptors.

Project description:RationaleAutologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF.ObjectiveTo improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress.Methods and resultsc-Kit+ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway.ConclusionsProliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF.

Project description:BackgroundInteractions between adipocytes and macrophages are associated with metabolic disorders. Production of pro-inflammatory mediators and the release of free fatty acids (FFAs) increase when these cells are co-cultured; butyrate significantly diminishes these effects by suppressing both the macrophage inflammatory and adipocyte lipolysis pathways. Butyrate is known to up-regulate the expression of prostaglandin E2 (PGE2). Therefore, we hypothesized that PGE2 is associated with the suppression of lipolysis by butyrate in co-culture.MethodsUsing contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the release of PGE2 into the medium and on lipolysis in adipocytes. To elucidate the underlying mechanism, we examined the effects of butyrate on cyclooxygenase-2 (COX2) and phospholipase A2 (PLA2) in co-cultured cells, and cyclic adenine monophosphate (cAMP) and protein kinase A type 1-α regulatory subunit (PRKAR1A) in co-cultured adipocytes. Silent interfering (si)RNA targeting of G-protein-coupled receptor (GPR)41 and 109A was employed to examine the effect on lipolysis in TNF-α-stimulated adipocytes.ResultsCo-culture increased PGE2 release into the medium, compared with cells cultured separately. Butyrate significantly increased PGE2 production. Co-culture elevated COX2 expression in macrophages and adipocytes, and butyrate further enhanced this effect. Co-culture enhanced cytosolic PLA2 activity in macrophages, which was further enhanced by butyrate. As for lipolysis, co-culture increased the release of FFAs and free glycerol into the medium, whereas butyrate (and to a lesser extent, PGE2) suppressed FFAs and free glycerol release. An inhibition study using a prostaglandin E receptor 3-selective antagonist suggested that approximately 40% of the suppressive effect of butyrate depends on the PGE2-mediated pathway, whereas 60% depends on a non-PGE2-mediated pathway. Co-culture increased cAMP and PRKAR1A levels in adipocytes, whereas butyrate restored the levels to those of the control. Similarly, in TNF-α-stimulated adipocytes, butyrate reduced FFAs and free glycerol release. siRNA inhibition of GPR41 and GPR109A suggested that the GPR109A-mediated pathway predominates, but the GPR41-mediated pathway also regulates the effect of butyrate on lipolysis in TNF-α-stimulated 3T3-L1 cells.ConclusionsButyrate attenuates lipolysis in adipocytes co-cultured with macrophages via non-PGE2-mediated and PGE2-mediated pathways.

Dataset Information

Mapping the Binding Sites of UDP and Prostaglandin E2 Glyceryl Ester in the Nucleotide Receptor P2Y₆.

Publications

Mapping the Binding Sites of UDP and Prostaglandin E2 Glyceryl Ester in the Nucleotide Receptor P2Y<sub>6</sub>.

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