Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis.

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Systemic deficiency of mouse arachidonate 15-lipoxygenase induces defective erythropoiesis and transgenic expression of the human enzyme rescues this phenotype.

Arachidonic acid 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes, which has previously been implicated in the maturational breakdown of intracellular organelles and plasma membrane remodeling during reticulocyte-erythrocyte transition. Conventional Alox15-/- mice are viable, develop normally but do not exhibit a major defective erythropoietic phenotype. To characterize the putative in vivo relevance of Alox15 for red blood cell development, we explored the impact of systemic inactivation of the Alox15 gene on mouse erythropoiesis. We found that Alox15-/- mice exhibited reduced erythrocyte counts, elevated reticulocyte counts and red cell hyperchromia. The structure of the plasma membrane of Alox15-/- erythrocytes is altered and a significant share of the red cells was present as echinocytes and/or acanthocytes. An increased share of the Alox15-/- erythrocytes cells were annexin V positive, which indicates a loss of plasma membrane asymmetry. Erythrocytes of Alox15-/- mice were more susceptible to osmotic hemolysis and exhibited a reduced ex vivo life span. When we transgenically expressed human ALOX15 in Alox15-/- mice under the control of the aP2 promoter the defective erythropoietic system was rescued and the impaired osmotic resistance was normalized. Together these data suggest the involvement Alox15 in the maturational remodeling of the plasma membrane during red cell development.

Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction.

Glutathione peroxidases (GPX) are anti-oxidative enzymes that reduce organic and inorganic hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. The human genome involves eight GPX genes and five of them encode for selenocysteine-containing enzymes. Among the human GPX-isoforms, GPX4 is unique since it is capable of reducing complex hydroperoxy ester lipids such as hydroperoxy phospholipids and hydroperoxy cholesterolesters. Using a number of genetically modified mouse strains the biological role of GPX4 has comprehensively characterized but the molecular enzymology is less well explored. This lack of knowledge is partly related to the fact that mammalian selenoproteins are not high-level expressed in conventional overexpression systems. To explore the structural and functional properties of human GPX4 we expressed this selenoprotein in a cysteine-auxotrophic E. coli strain using a semi-chemical expression strategy. The recombinant enzyme was purified in mg amounts from the bacterial lysate to electrophoretic homogeneity and characterized with respect to its protein-chemical and enzymatic properties. Its crystal structure was solved at 1.3 Šresolution and the X-ray data indicated a monomeric protein, which contains the catalytic selenium at the redox level of the seleninic acid. These data suggest an alternative reaction mechanism involving three different redox states (selenol, selenenic acid, seleninic acid) of the catalytically active selenocysteine.

Male Subfertility Induced by Heterozygous Expression of Catalytically Inactive Glutathione Peroxidase 4 Is Rescued in Vivo by Systemic Inactivation of the Alox15 Gene.

Glutathione peroxidase 4 (GPX4) and arachidonic acid 15-lipoxygenase (ALOX15) are antagonizing enzymes in the metabolism of hydroperoxy lipids. In spermatoid cells and/or in the male reproductive system both enzymes are apparently expressed, and GPX4 serves as anti-oxidative enzyme but also as a structural protein. In this study we explored whether germ line inactivation of the Alox15 gene might rescue male subfertility induced by heterozygous expression of catalytically silent Gpx4. To address this question we employed Gpx4 knock-in mice expressing the Sec46Ala-Gpx4 mutant, in which the catalytic selenocysteine was replaced by a redox inactive alanine. Because homozygous Gpx4 knock-in mice (Sec46Ala-Gpx4+/+) are not viable we created heterozygous animals (Sec46Ala-Gpx4+/-) and crossed them with Alox15 knock-out mice (Alox15-/-). Male Sec46Ala-Gpx4+/- mice, but not their female littermates, were subfertile. Sperm extracted from the epididymal cauda showed strongly impaired motility characteristics and severe structural midpiece alterations (swollen mitochondria, intramitochondrial vacuoles, disordered mitochondrial capsule). Despite these structural alterations, they exhibited similar respiration characteristics than wild-type sperm. When Sec46Ala-Gpx4+/- mice were crossed with Alox15-deficient animals, the resulting males (Sec46Ala-Gpx4+/-+Alox15-/-) showed normalized fertility, and sperm motility was reimproved to wild-type levels. Taken together these data suggest that systemic inactivation of the Alox15 gene normalizes the reduced fertility of male Sec46Ala-Gpx4+/- mice by improving the motility of their sperm. If these data can be confirmed in humans, ALOX15 inhibitors might counteract male infertility related to GPX4 deficiency.