Ralf Morgenstern

Membrane Prostaglandin E Synthase-1: A Novel Therapeutic Target

Prostaglandin E2 (PGE2) is the most abundant prostaglandin in the human body. It has a large number of biological actions that it exerts via four types of receptors, EP1–4. PGE2 is formed from arachidonic acid by cyclooxygenase (COX-1 and COX-2)-catalyzed formation of prostaglandin H2 (PGH2) and further transformation by PGE synthases. The isomerization of the endoperoxide PGH2 to PGE2 is catalyzed by three different PGE synthases, viz. cytosolic PGE synthase (cPGES) and two membrane-bound PGE synthases, mPGES-1 and mPGES-2. Of these isomerases, cPGES and mPGES-2 are constitutive enzymes, whereas mPGES-1 is mainly an induced isomerase. cPGES uses PGH2 produced by COX-1 whereas mPGES-1 uses COX-2-derived endoperoxide. mPGES-2 can use both sources of PGH2. mPGES-1 is a member of the membrane associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily. It requires glutathione as an essential cofactor for its activity. mPGES-1 is up-regulated in response to various proinflammatory stimuli with a concomitant increased expression of COX-2. The coordinate increased expression of COX-2 and mPGES-1 is reversed by glucocorticoids. Differences in the kinetics of the expression of the two enzymes suggest distinct regulatory mechanisms for their expression. Studies, mainly from disruption of the mPGES-1 gene in mice, indicate key roles of mPGES-1-generated PGE2 in female reproduction and in pathological conditions such as inflammation, pain, fever, anorexia, atherosclerosis, stroke, and tumorigenesis. These findings indicate that mPGES-1 is a potential target for the development of therapeutic agents for treatment of several diseases.

Microsomal Glutathione S‐Transferase

Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.

Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Abstract Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).

The distribution of microsomal glutathione transferase among different organelles, different organs, and different organisms

In the present study we have used both enzyme assay with 1-chloro-2,4-dinitrobenzene as substrate and immunochemical quantitation to examine the distribution of microsomal glutathione transferase in different organelles, in different organs, and in different organisms. This enzyme was found to constitute 3% and 5%, respectively, of the total protein recovered in the microsomal and outer mitochondrial membrane fractions from rat liver. Microsomal glutathione transferase present in other subcellular fractions can be accounted for by contamination by the endoplasmic reticulum. In contrast to the situation with rat liver microsomes the glutathione transferase activities of microsomes from extrahepatic tissues of this same animal could not be activated by treatment with N-ethylmaleimide. Nonetheless, significant albeit low levels of a protein with the same molecular weight and immunochemical properties as the rat liver enzyme could be detected in microsomes from several extrahepatic tissues, notably the intestine, the adrenal, and the testis. Of those mammals for which fresh liver could be obtained, all demonstrated N-ethylmaleimide-activatable glutathione transferase activity in their liver microsomes. On the other hand, representatives for fish, birds, and amphibia did not demonstrate such activatable transferase activity in their liver microsomes. Toad was the only species that had a notable (twofold) sex difference in their level of hepatic microsomal glutathione transferase activity.

Structural basis for induced formation of the inflammatory mediator prostaglandin E2

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH2 to PGE2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.

Human glutathione dependent prostaglandin E synthase: gene structure and regulation

A P1 clone containing the gene for human glutathione dependent PGE synthase (PGES) was isolated and characterized. The gene is divided into three exons, spans 14.8 kb and was localized to chromosome 9q34.3. In A549 cells, the protein and activity levels of PGES were increased by interleukin‐1β. This increase was prevented by phenobarbital. Reporter constructs containing the 5′‐flanking region of exon 1, which exhibited strong promoter activity, responded accordingly, except that interleukin‐1β induced a transient increase followed by a decrease. As cyclooxygenase 2 expression has been reported to respond in a similar fashion, a transcriptional regulatory basis for the observed co‐regulation with PGES is implied. The strong down‐regulation by phenobarbital raises important issues concerning its mechanisms of action.

Bioinformatic and enzymatic characterization of the MAPEG superfamily

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5‐lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1‐chloro‐2,4‐dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N‐terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1‐chloro‐2,4‐dinitrobenzene of 11 µmol·min−1·mg−1. Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 µmol·min−1·mg−1, whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 µmol·min−1·mg−1. The purified pike enzyme is the most active MGST described so far with a specific activity of 285 µmol·min−1·mg−1. Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 µmol·min−1·mg−1, respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation

Abstract Evidence that rat liver musomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver musomes has been obtained. Activation of the musomal glutathione transferase in musomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated musomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the musomal glutathione transferase (Glutaryl- l -Cys-Gly and α- l -Glu- l -Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (α d -Glu- l -Cys-Gly, γ- d -Glu- l -Cys-Gly, β- l -Asp- l -Cys-Gly, α- l -Asp- l -Cys-Gly and α- d -Asp- l -Cys-Gly) shows the specificity of the protection and rules out any non-enzymic component. Inhibitors of selenium- dependent glutathione peroxidase (mercaptosuccinate at 50 μM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver musomes against lipid peroxidation. Purified musomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in musomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 μM NADPH and 10 μM Fe-EDTA (2: 1). This model system was used to investigate whether musomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional musomal glutathione transferase was included. This was not the case when the enzyme had been pre-inactivated with diethylpyrocarbonate. Furthermore, the protective effect of glutathione could be partly reversed by an inhibitor (100μM bromosulphophtalein) of the enzyme. Apparently, rat liver musomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.

Enzymology of Microsomal Glutathione S-Transferase

Publisher Summary The study of the microsomal glutathione transferase regarding molecular properties, substrate specificity, kinetic behavior, and activation mechanisms has advanced considerably over the past few years. Polyhalogenated hydrocarbons that form toxic and carcinogenic glutathione conjugates need to be characterized with the purified enzyme with respect to the molecular properties that determine catalysis. These and additional studies on the extrahepatic distribution of microsomal glutathione transferase may likely yield information on the sites of formation of conjugates and on the enzymes involved. The glutathione peroxidase activity of the microsoma1 glutathione transferase may protect the organism from reactive hydroperoxides formed during oxidative stress. The significance of the extrahepatic enzyme needs to be studied in this respect. Finally, studies on the interplay and functional significance of different activation mechanisms are interesting regarding enzyme regulation in general and protection against toxic insult in particular.

Activity of rat liver microsomal glutathione transferase toward products of lipid peroxidation and studies of the effect of inhibitors on glutathione-dependent protection against lipid peroxidation

Rat liver microsomal glutathione transferase displays glutathione peroxidase activity with linoleic acid hydroperoxide, linoleic acid ethyl ester hydroperoxide, and dilinoleoyl phosphatidylcholine hydroperoxide, with rates of 0.2, 0.3, and 0.3 mumol/min/mg, respectively. The activities are increased between three- and fourfold when the enzyme is activated with N-ethylmaleimide. Microsomal glutathione transferase can also conjugate 4-hydroxynon-2-enal with a specific activity of 0.5 mumol/min/mg. These findings show that the enzyme can remove harmful products of lipid peroxidation and thereby possibly protect intracellular membranes against oxidative stress. A set of glutathione transferase inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue, and bromosulfophtalein) which abolish the glutathione-dependent protection against lipid peroxidation in liver microsomes have been characterized. These inhibitors were found to be effective in the micromolar range and could prove valuable in studying the factor responsible for glutathione-dependent protection against lipid peroxidation.