MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation.

We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates

Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not.

Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism.The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling.Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation.

We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates

Prostanoid profiles for WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>Prostanoid production was measured by LC-MS/MS after 60 seconds incubations of membrane fractions with PGH₂ (10μM final concentration) and GSH (2.5mM final concentration) at room temperature. Denatured enzymes by boiling were incorporated in the activity assay as controls. All prostanoid measurements of membrane fractions are expressed as mean ± SD from two independent experiment performed in duplicates.</p

Suggested chemical mechanism of PGH₂ isomerization to PGE₂ by MPGES1 and its active site structure highlighting the amino acids altered.

<p>(A) The thiolate of glutathione (GSH) could be stabilized by Arg126 and attack the C9 oxygen of the PGH₂ endoperoxide forming a sulfenic acid ester. An unidentified proton donor protonates the developing C11 oxyanion. This is followed by proton abstraction at C9 via Asp49. A carbonyl forms and the oxygen sulfur bond is broken forming PGE₂. The leaving GSH thiolate could again be stabilized by Arg126. The unidentified proton donor could then take up the proton from Asp49. (B) The reaction starts by proton abstraction at C9 via Asp49. A carbonyl forms and the endoperoxide bridge is broken. The thiol of GSH functions as a proton donor to the developing C11 oxyanion. After that the proton taken up by Asp49 can reprotonate the GSH thiolate. (C) The interaction between MPGES1 and GSH highlighting the positions of Asp49, Arg126 as well as Ser127 that was proposed to stabilize the GSH thiolate.</p

Western Blot analysis of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>40μg of membrane fraction was loaded into each well. The exposure time was 5 minutes. As a positive control purified MPGES1 was loaded at different concentrations, yielding: 75ng, 100ng, 150ng, 200ng and 450ng.</p

Glutathione transferase (GST) activity assay of purified MPGES1, expressed in <i>Baculovirus</i> infected <i>SF9 cells</i>.

<p>The enzymatic activity of purified MPGES1 was measured by the conjugation of GSH to the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The spontaneous conjugation of CDNB to GSH was subtracted from each enzymatic reaction and the specific activity values were calculated based on the slope of the initial linear portion of the absorbance curve. The GST activity is expressed as mean ± SD from two independent experiment performed in triplicates.</p

Specific activity of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu and Ser127Ala, expressed and measured in <i>E</i>. <i>Coli</i> membrane fractions.

<p>Specific activity of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu and Ser127Ala, expressed and measured in <i>E</i>. <i>Coli</i> membrane fractions.</p