Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Ouellet, Marc; Falgueyret, Jean‐Pierre; Ear, Po Hien; Pen, Ally; Mancini, Joseph A.; Riendeau, Denis; Percival, M. David

doi:10.1016/s1046-5928(02)00566-1

Cited by 51 publications

(65 citation statements)

References 16 publications

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“…1 depicts a substratelimited assay that measured absolute product formation (nanograms) as described in Materials and Methods. Incubations with native enzyme in the presence of GSH resulted in near total conversion of PGH 2 to PGE 2 and are concurrent with the specific activity previously reported in the literature (∼4 μmol·min −1 ·mg −1 at 0°C) (28). A negative control involved microsomal preparations of WT mPGES-1 resuspended in buffer containing the GSH analog, GOH, that differs from the native cofactor only by the replacement of the thiol moiety by a hydroxyl group and did not produce product above background levels.…”

Section: Significancesupporting

confidence: 89%

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

Brock

Hámberg

Balagunaseelan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Microsomal prostaglandin E 2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E 2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.is an abundant lipid mediator that signals via four receptors (EP1-4) to induce an array of important biological actions in physiology as well as pathophysiology (1). Under proinflammatory conditions, biosynthesis of PGE 2 proceeds from arachidonic acid, which is converted to the unstable endoperoxide PGH 2 by cyclooxygenase type 2 (COX-2). PGH 2 is further isomerized into PGE 2 by microsomal PGE synthase type 1 (mPGES-1) (2, 3). mPGES-1 is encoded by PTGES and is up-regulated by mitogens and cytokines in a pathway that is functionally coupled to COX-2 (2, 4). Because of its key role in PGE 2 synthesis, mPGES-1 has attracted attention as a potential drug target in the areas of inflammation, pain, fever, and cancer (5).mPGES-1 is a member of the MAPEG (Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily of enzymes (6), which also includes two key proteins in the leukotriene (LT) cascade, viz. 5-lipoxygenase activating protein and LT C 4 synthase (LTC4S). All MAPEG members are integral, homotrimeric membrane proteins, and structural information on this family has been scarce. However, significant progress has recently been made in this area with several high-resolution structures being solved by X-ray crystallography (7-9). In particular, the crystal structures of human LTC4S provided detailed structural information, including an arginine residue that was later shown to activate the glutathione (GSH) thiolate (10, 11). We have previously proposed ...

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Section: Significancesupporting

confidence: 89%

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

Brock

Hámberg

Balagunaseelan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Possible explanations can be the difference in lipid composition between eukaryotic and prokaryotic cells, alternatively different post-transcriptional processing. In addition, we report significant but low GST and peroxidase activities in contrast to Ouellet et al (52), findings that are principally very important for the further understanding of the mPGES-1 reaction.…”

Section: Discussioncontrasting

confidence: 49%

Human Microsomal Prostaglandin E Synthase-1

Thorén

Weinander

Saha

et al. 2003

Journal of Biological Chemistry

153

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Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathionedependent conversion of PGH 2 to PGE 2 (V max ; 170 mol min ؊1 mg ؊1 ) and high k cat /K m (310 mM ؊1 s ؊1 ). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG 2 to 15-hydroperoxy-PGE 2 (V max ; 250 mol min ؊1 mg ؊1 ). The formation of 15-hydroperoxy-PGE 2 represents an alternative pathway for the synthesis of PGE 2 , which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 mol min ؊1 mg ؊1 ), 5-hydroperoxyeicosatetraenoic acid (0.043 mol min ؊1 mg ؊1 ), and 15-hydroperoxy-PGE 2 (0.04 mol min ؊1 mg ؊1 ). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 mol min ؊1 mg ؊1 ). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm 3 /g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.Prostaglandin (PG) 1 E 2 is a prostanoid with potent biological functions; among those functions, its role as a mediator of pain and fever in inflammatory reactions is considered of major importance (1-4). The biosynthesis of PGE 2 from arachidonic acid is catalyzed in a sequential action by PGH synthase (PGHS) forming first the endoperoxide PGG 2 and then PGH 2 by reduction. Subsequently, PGE synthase (PGES) (EC 5.3.99.3) converts PGH 2 into PGE 2 (5). Two forms of PGHS exist, PGHS-1 and PGHS-2, with similar enzymatic properties but distinctly different biological functions. PGHS-1 is constitutively expressed in many cells and organs and takes part in housekeeping functions such as the regulation of vascular homeostasis. PGHS-2, in contrast, is strongly induced in response to proinflammatory stimuli and takes part in various pathophysiological events (6, 7). PGES activity, in most cases glutathione (GSH)-dependent, has been detected both in microsomal and cytosolic fractions of various cells, and apparently, more than one form of PGES exist (8 -12). Microsomal, inducible PGES-1 (mPGES-1) is a member of the membrane-associated proteins in eicosanoid and glutathione metabo...

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“…PGD 3 was the predominant EPAderived metabolite produced in EPA-supplemented cells and was about threefold higher than in control cells; however, EPA COX metabolites were 100-fold lower than AA prostanoids. H-PGDS and mPGES-1 have about 17% and 30% activity with PGH 3 vs. PGH 2 (8,(19)(20)(21)(22), respectively.…”

Section: Effects Of Pufa Supplementation On Tlr4-stimulated Eicosanoidmentioning

confidence: 97%

Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling

Norris

Dennis²

2012

Proc. Natl. Acad. Sci. U.S.A.

166

129

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Dietary fish oil omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit cardioprotective and anti‐inflammatory effects through unresolved mechanisms. EPA and DHA may reduce arachidonic acid (AA) metabolism and pro‐inflammatory effects by competition and inhibition at multiple levels. Here, we report the effects of AA, EPA, and DHA supplementation on membrane incorporation, phospholipase A2 release, and eicosanoid production in RAW264.7 macrophages. Each PUFA supplemented increased by similar amounts in membrane phospholipids, while half of the increased AA and EPA were elongated to adrenic acid (AdA) and docosapentaenoic acid (DPA), respectively; and were subsequently released by PLA2 at levels comparable to AA. TLR‐4 stimulated COX‐2 AA production did not increase with AA supplementation and was inhibited by 20% and 50% after EPA and DHA supplementation, respectively. ATP stimulated COX‐1 AA production was inhibited to a greater extent by EPA and DHA; 5‐LOX AA metabolites increased 2‐fold with EPA and DHA supplementation, and 5‐LOX metabolized EPA and DHA to a greater extent than COX‐1/COX‐2. Altogether, AA, EPA and DHA supplementation decreased TLR‐4 stimulated AA COX‐2 prostanoid metabolism; increased the purinergic stimulated AA 5‐LOX/COX‐1 ratio; and significantly increased elongated PUFA levels that may be converted to novel mediators.

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Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Cited by 51 publications

References 16 publications

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

Human Microsomal Prostaglandin E Synthase-1

Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling

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Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Cited by 51 publications

References 16 publications

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

Human Microsomal Prostaglandin E Synthase-1

Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling

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Product

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A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E ₂ synthase