Astrid Borchert

REGULATION OF ENZYMATIC LIPID PEROXIDATION: THE INTERPLAY OF PEROXIDIZING AND PEROXIDE REDUCING ENZYMES

For a long time lipid peroxidation has only been considered a deleterious process leading to disruption of biomembranes and thus, to cellular dysfunction. However, when restricted to a certain cellular compartment and tightly regulated, lipid peroxidation may have beneficial effects. Early on during evolution of living organisms special lipid peroxidizing enzymes, called lipoxygenases, appeared and they have been conserved during phylogenesis of plants and animals. In fact, a diverse family of lipoxygenase isoforms has evolved starting from a putative ancient precursor. As with other enzymes, lipoxygenases are regulated on various levels of gene expression and there are endogenous antagonists controlling their cellular activity. Among the currently known mammalian lipoxygenase isoforms only 12/15-lipoxygenases are capable of directly oxygenating ester lipids even when they are bound to membranes and lipoproteins. Thus, these enzymes represent the pro-oxidative part in the cellular metabolism of complex hydroperoxy ester lipids. Its metabolic counterplayer, representing the antioxidative part, appears to be the phospholipid hydroperoxide glutathione peroxidase. This enzyme is unique among glutathione peroxidases because of its capability of reducing ester lipid hydroperoxides. Thus, 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase constitute a pair of antagonizing enzymes in the metabolism of hydroperoxy ester lipids, and a balanced regulation of the two proteins appears to be of major cell physiological importance. This review is aimed at summarizing the recent developments in the enzymology and molecular biology of 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase, with emphasis on cytokine-dependent regulation and their regulatory interplay.

Redox Control in Mammalian Embryo Development

The development of an embryo constitutes a complex choreography of regulatory events that underlies precise temporal and spatial control. Throughout this process the embryo encounters ever changing environments, which challenge its metabolism. Oxygen is required for embryogenesis but it also poses a potential hazard via formation of reactive oxygen and reactive nitrogen species (ROS/RNS). These metabolites are capable of modifying macromolecules (lipids, proteins, nucleic acids) and altering their biological functions. On one hand, such modifications may have deleterious consequences and must be counteracted by antioxidant defense systems. On the other hand, ROS/RNS function as essential signal transducers regulating the cellular phenotype. In this context the combined maternal/embryonic redox homeostasis is of major importance and dysregulations in the equilibrium of pro- and antioxidative processes retard embryo development, leading to organ malformation and embryo lethality. Silencing the in vivo expression of pro- and antioxidative enzymes provided deeper insights into the role of the embryonic redox equilibrium. Moreover, novel mechanisms linking the cellular redox homeostasis to gene expression regulation have recently been discovered (oxygen sensing DNA demethylases and protein phosphatases, redox-sensitive microRNAs and transcription factors, moonlighting enzymes of the cellular redox homeostasis) and their contribution to embryo development is critically reviewed.

Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function.

Abstract Selenoproteins have been recognized as modulators of brain function and signaling. Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a unique member of the selenium-dependent glutathione peroxidases in mammals with a pivotal role in brain development and function. GPx4 exists as a cytosolic, mitochondrial, and nuclear isoform derived from a single gene. In mice, the GPx4 gene is located on chromosome 10 in close proximity to a functional retrotransposome that is expressed under the control of captured regulatory elements. Elucidation of crystallographic data uncovered structural peculiarities of GPx4 that provide the molecular basis for its unique enzymatic properties and substrate specificity. Monomeric GPx4 is multifunctional: it acts as a reducing enzyme of peroxidized phospholipids and thiols and as a structural protein. Transcriptional regulation of the different GPx4 isoforms requires several isoform-specific cis-regulatory sequences and trans-activating factors. Cytosolic and mitochondrial GPx4 are the major isoforms exclusively expressed by neurons in the developing brain. In stark contrast, following brain trauma, GPx4 is specifically upregulated in non-neuronal cells, i.e., reactive astrocytes. Molecular approaches to genetic modification in mice have revealed an essential and isoform-specific function for GPx4 in development and disease. Here we review recent findings on GPx4 with emphasis on its molecular structure and function and consider potential mechanisms that underlie neural development and neuropathological conditions.

The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.

Inverse regulation of lipid-peroxidizing and hydroperoxyl lipid-reducing enzymes by interleukins 4 and 13

12/15‐lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are opposite enzymes balancing the intracellular concentration of hydroperoxy lipids. We studied the regulation of both enzymes by interleukins 4 and 13 and found an inverse response. When human lung carcinoma cells A549 were cultured in vitro in the presence of these cytokines, an up‐regulation of the 12/15‐lipoxygenase and a down‐regulation of the phospholipid hydroperoxide glutathione peroxidase were observed. A similar inverse regulation was found in human peripheral monocytes. Interleukin 4‐treated A549 cells exhibited an impaired capability of reducing exogenous hydroperoxyl lipids and their levels of endogenous lipid hydroperoxides were markedly increased. To find out whether these regulatory processes also occur in vivo, arachidonic acid oxygenase and phospholipid hydroperoxide glutathione peroxidase activity was assayed in various tissues of transgenic mice that systemically overexpress interleukin 4. In lung, spleen, kidney, and heart, an increased arachidonic acid oxygenase activity was detected when transgenic mice were compared with inbred controls. The phospholipid hydroperoxide glutathione peroxidase activity was impaired in lung, liver, and spleen of the transgenic animals. These data indicate that lipid‐peroxidizing and lipid peroxide‐reducing enzymes are inversely regulated in various mammalian cells. Up‐regulation of the 12/15‐lipoxygenase and simultaneous down‐regulation of the phospholipid hydroperoxide glutathione peroxidase may lead to an increased oxidizing potential, which is reflected by an augmented intracellular peroxide tone.—Schnurr, K., Borchert, A., Kuhn, H. Inverse regulation of lipid‐peroxidizing and hydroperoxyl lipid‐reducing enzymes by interleukins 4 and 13. FASEB J. 13, 143–154 (1999)

Monoamine oxidases in development.

Monoamine oxidases (MAOs) are flavoproteins of the outer mitochondrial membrane that catalyze the oxidative deamination of biogenic and xenobiotic amines. In mammals there are two isoforms (MAO-A and MAO-B) that can be distinguished on the basis of their substrate specificity and their sensitivity towards specific inhibitors. Both isoforms are expressed in most tissues, but their expression in the central nervous system and their ability to metabolize monoaminergic neurotransmitters have focused MAO research on the functionality of the mature brain. MAO activities have been related to neurodegenerative diseases as well as to neurological and psychiatric disorders. More recently evidence has been accumulating indicating that MAO isoforms are expressed not only in adult mammals, but also before birth, and that defective MAO expression induces developmental abnormalities in particular of the brain. This review is aimed at summarizing and critically evaluating the new findings on the developmental functions of MAO isoforms during embryogenesis.

Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene

15‐Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH‐GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH‐GPx is less well investigated. In this study we cloned the PH‐GPx cDNA from a mouse fibroblast cDNA library and the PH‐GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5′‐flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies.

Monoamine Oxidase A Expression Is Vital for Embryonic Brain Development by Modulating Developmental Apoptosis

Monoamine oxidases (MAO-A, MAO-B) metabolize biogenic amines and have been implicated in neuronal apoptosis. Although apoptosis is an important process in embryo development, the role of MAO isoenzymes has not been investigated in detail. We found that expression of MAO-A and MAO-B can be detected early on during embryo development. Expression levels remained constant until around midgestation but then dropped to almost undetectable levels toward birth. Similar expression kinetics were observed in the brain. Isoform-specific expression silencing of MAO-A mediated by siRNA during in vitro embryogenesis induced developmental defects, as indicated by a reduction of the crown rump length and impaired cerebral development. These alterations were paralleled by elevated serotonin levels. Similar abnormalities were observed when embryos were cultured in the presence of the MAO-A inhibitor clorgyline or when the transcriptional inhibitor of MAO-A expression R1 was overexpressed. In contrast, no such alterations were detected when expression of MAO-B was knocked down. To explore the underlying mechanisms for the developmental abnormalities in MAO-A knockdown embryos, we quantified the degree of developmental apoptosis in the developing brain. MAO-A knockdown reduced the number of apoptotic cells in the neuroepithelium, which coincided with impaired activation of caspases 3 and 9. Moreover, we observed reduced cyclin D1 levels as an indicator of impaired cell proliferation in MAO-A knockdown embryos. This data highlights MAO-A as a vital regulator of embryonic brain development.

Synthesis of a New Seleninic Acid Anhydride and Mechanistic Studies into Its Glutathione Peroxidase Activity

Starting from low toxic salicyloylglycine, a new seleninic acid anhydride 7 that lacks SeN or SeO non-bonded interactions was synthesized. This compound exhibits a fourfold higher glutathione peroxidase-like (GPx-like) activity than ebselen and inhibits plant and mammalian 12/15-lipoxygenases at lower micromolar concentrations. Because of these pharmacological properties, 7 may constitute a new lead compound for the development of anti-inflammatory low-molecular-weight seleno-organic compounds. Analyzing the redox products of 7 with glutathione (GSH) and tBuOOH, we identified three potential catalytic cycles (A, B, C) of GPx-like activity that are interconnected by key metabolites. To study the relative contribution of these cycles to the catalytic activity, we prepared selected reaction intermediates and found that the activity of seleninic acid anhydride 7 and of the corresponding diselenide 11 and selenol 14 compounds were in the same range. In contrast, the GPx-like activity of monoselenide 9 was more than one order of magnitude lower. These data suggested that cycles A and B may constitute the major routes of GPx-like activity of 7, whereas cycle C may not significantly contribute to catalysis.

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.