Functional Characterization of Transgenic Mice Overexpressing Human 15-Lipoxygenase-1 (ALOX15) under the Control of the aP2 Promoter.

Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory, hyperproliferative, neurodegenerative, and metabolic diseases, but the physiological function of ALOX15 still remains a matter of discussion. To contribute to this discussion, we created transgenic mice (aP2-ALOX15 mice) expressing human ALOX15 under the control of the aP2 (adipocyte fatty acid binding protein 2) promoter, which directs expression of the transgene to mesenchymal cells. Fluorescence in situ hybridization and whole-genome sequencing indicated transgene insertion into the E1-2 region of chromosome 2. The transgene was highly expressed in adipocytes, bone marrow cells, and peritoneal macrophages, and ex vivo activity assays proved the catalytic activity of the transgenic enzyme. LC-MS/MS-based plasma oxylipidome analyses of the aP2-ALOX15 mice suggested in vivo activity of the transgenic enzyme. The aP2-ALOX15 mice were viable, could reproduce normally, and did not show major phenotypic alterations when compared with wildtype control animals. However, they exhibited gender-specific differences with wildtype controls when their body-weight kinetics were evaluated during adolescence and early adulthood. The aP2-ALOX15 mice characterized here can now be used for gain-of-function studies evaluating the biological role of ALOX15 in adipose tissue and hematopoietic cells.

Male guanine-rich RNA sequence binding factor 1 knockout mice (Grsf1-/-) gain less body weight during adolescence and adulthood.

The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the heterogenous nuclear ribonucleoprotein H/F (hnRNP H/F) family that binds to guanine-rich RNA sequences forming G-quadruplex structures. In mice and humans there are single copy GRSF1 genes, but multiple transcripts have been reported. GRSF1 has been implicated in a number of physiological processes (e.g. embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of viral infections and hyperproliferative diseases. These postulated biological functions of GRSF1 originate from in vitro studies rather than complex in vivo systems. To assess the in vivo relevance of these findings, we created systemic Grsf1-/- knockout mice lacking exons 4 and 5 of the Grsf1 gene and compared the basic functional characteristics of these animals with those of wildtype controls. We found that Grsf1-deficient mice are viable, reproduce normally and have fully functional hematopoietic systems. Up to an age of 15 weeks they develop normally but when male individuals grow older, they gain significantly less body weight than wildtype controls in a gender-specific manner. Profiling Grsf1 mRNA expression in different mouse tissues we observed high concentrations in testis. Comparison of the testicular transcriptomes of Grsf1-/- mice and wildtype controls confirmed near complete knock-out of Grsf1 but otherwise subtle differences in transcript regulations. Comparative testicular proteome analyses suggested perturbed mitochondrial respiration in Grsf1-/- mice which may be related to compromised expression of complex I proteins. Here we present, for the first time, an in vivo complete Grsf1 knock-out mouse with comprehensive physiological, transcriptomic and proteomic characterization to improve our understanding of the GRSF1 beyond in vitro cell culture models.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Expression Regulation, Protein Chemistry and Functional Biology of the Guanine-Rich Sequence Binding Factor 1 (GRSF1).

In eukaryotic cells RNA-binding proteins have been implicated in virtually all post-transcriptional mechanisms of gene expression regulation. Based on the structural features of their RNA binding domains these proteins have been divided into several subfamilies. The presence of at least two RNA recognition motifs defines the group of heterogenous nuclear ribonucleoproteins H/F and one of its members is the guanine-rich sequence binding factor 1 (GRSF1). GRSF1 was first described 25 years ago and is widely distributed in eukaryotic cells. It is present in the nucleus, the cytoplasm and in mitochondria and has been implicated in a variety of physiological processes (embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of various diseases. This review summarizes our current understanding on GRSF1 biology, critically discusses the literature reports and gives an outlook of future developments in the field.

Identification of the COMM-domain containing protein 1 as specific binding partner for the guanine-rich RNA sequence binding factor 1.

BACKGROUND: The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the hnRNP H/F family, which has been implicated in erythropoiesis, regulation of the redox homeostasis, embryonic brain development, mitochondrial function and cellular senescence. The molecular basis for GRSF1-RNA interaction has extensively been studied in the past but for the time being GRSF1 binding proteins have not been identified. METHODS: To search for GRSF1 binding proteins we first employed the yeast two-hybrid system and screened a cDNA library of human fetal brain for potential GRSF1 binding proteins. Subsequently, we explored the protein-protein-interaction of the recombiant proteins, carried out immunoprecipitation experiments to confirm the interaction of the native proteins in living cells and performed truncation studies to identify the protein-binding motif of GRSF1. RESULTS: Using the yeast two-hybrid system we identified the COMM-domain containing protein 1 (COMMD1) as specific GRSF1 binding protein and in vitro truncation studies suggested that COMMD1 interacts with the alanine-rich domain of GRSF1. Co-immunoprecipitation strategies indicated that COMMD1-GRSF1 interaction was RNA independent and also occurred in living cells expressing the two native proteins. CONCLUSION: In mammalian cells the COMM-domain containing protein 1 (COMMD1) specifically interacts with the Ala-rich domain of GRSF1 in an RNA-independent manner. GENERAL SIGNIFICANCE: This is the first report describing a specific GRSF1 binding protein.

Functional characterization of novel ALOX15 orthologs representing key steps in mammalian evolution supports the Evolutionary Hypothesis of reaction specificity.

Arachidonic acid lipoxygenases (ALOXs) are lipid-metabolizing enzymes that have been implicated in cell differentiation, but also in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Most mammalian genomes involve six or seven functional ALOX genes and among the corresponding ALOX-isoforms the ALOX15 orthologs are somewhat unique since they exhibit variable reaction specificity using arachidonic acid as substrate. The Evolutionary Hypothesis of mammalian ALOX15 reaction specificity (Prog. Lipid Res. 72, 55, 2018) suggests that ALOX15 orthologs of primates ranked higher in evolution than gibbons are 15-lipoxygenating enzymes. In contrast, mammals ranking lower than gibbons express dominantly 12-lipoxygenating lipoxygenases and gibbon ALOX15 constitutes a transition enzyme with pronounced dual reaction specificity. Here we predicted the reaction specificity of 95 different prototherian, metatherian and eutherian ALOX15 orthologs on the basis of their primary structures and characterized experimentally the reaction specificity of ten novel metatherian/eutherian enzymes representing different stages of mammalian evolution (gorilla, opossum, cape golden mole, dog, horseshoe bat, hedgehog, Sunda flying lemur, pika, chinchilla, kangaroo rat). We found that 97% of the currently sequenced mammalian ALOX15 including the enzymes of living and extinct hominids follow the Evolutionary Hypothesis. However, the ALOX15 orthologs of rabbits and of the Ord's kangaroo rat violate this mechanistic concept. Taken together, this data confirms the Evolutionary Hypothesis of ALOX15 reaction specificity and puts this concept on a more reliable experimental basis.

Monoamine oxidase-A promotes protective autophagy in human SH-SY5Y neuroblastoma cells through Bcl-2 phosphorylation.

Monoamine oxidases (MAOs) are located on the outer mitochondrial membrane and are drug targets for the treatment of neurological disorders. MAOs control the levels of neurotransmitters in the brain via oxidative deamination and contribute to reactive oxygen species (ROS) generation through their catalytic by-product H2O2. Increased ROS levels may modulate mitochondrial function and mitochondrial dysfunction is implicated in a vast array of disorders. However, the downstream effects of MAO-A mediated ROS production in a neuronal model has not been previously investigated. In this study, using MAO-A overexpressing neuroblastoma cells, we demonstrate that higher levels of MAO-A protein/activity results in increased basal ROS levels with associated increase in protein oxidation. Increased MAO-A levels result in increased Lysine-63 linked ubiquitination of mitochondrial proteins and promotes autophagy through Bcl-2 phosphorylation. Furthermore, ROS generated locally on the mitochondrial outer membrane by MAO-A promotes phosphorylation of dynamin-1-like protein, leading to mitochondrial fragmentation and clearance without complete loss of mitochondrial membrane potential. Cellular ATP levels are maintained following MAO-A overexpression and complex IV activity/protein levels increased, revealing a close relationship between MAO-A levels and mitochondrial function. Finally, the downstream effects of increased MAO-A levels are dependent on the availability of amine substrates and in the presence of exogenous substrate, cell viability is dramatically reduced. This study shows for the first time that MAO-A generated ROS is involved in quality control signalling, and increase in MAO-A protein levels leads to a protective cellular response in order to mediate removal of damaged macromolecules/organelles, but substrate availability may ultimately determine cell fate. The latter is particularly important in conditions such as Parkinson's disease, where a dopamine precursor is used to treat disease symptoms and highlights that the fate of MAO-A containing dopaminergic neurons may depend on both MAO-A levels and catecholamine substrate availability.

Functional characterization of naturally occurring genetic variations of the human guanine-rich RNA sequence binding factor 1 (GRSF1).

The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D-models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.

Functional characterization of isolated RNA-binding domains of the GRSF1 protein.

The Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the heterogeneous nuclear ribonucleoprotein F/H family and has been implicated in RNA processing, RNA transport and translational regulation. Amino acid alignments and homology modeling suggested the existence of three distinct RNA-binding domains and two auxiliary domains. Unfortunately, little is known about the molecular details of GRSF1/RNA interactions. To explore the RNA-binding mechanisms we first expressed full-length human GRSF1 and several truncation mutants, which include the three separated qRRM domains in E. coli, purified the recombinant proteins and quantified their RNA-binding affinity by RNA electrophoretic mobility shift assays. The expression levels varied between 1 and 10mg purified protein per L bacterial liquid culture and for full-length human GRSF1 a binding constant (KD-value) of 0.5µM was determined. In addition, our mechanistic experiments with different truncation mutants allowed the following conclusions: i) Deletion of either of the three RNA-binding domains impaired the RNA-binding affinity suggesting that the simultaneous presence of the three domains is essential for high-affinity RNA-binding. ii) Deletion of the Ala-rich auxiliary domain did hardly affect RNA-binding. Thus, this structural subunit may not be involved in RNA interaction. iii) Deletion of the acidic auxiliary domain improved the RNA-binding suggesting a regulatory role for this structural motif. iv) The isolated RNA-binding domains did not exhibit sizeable RNA-binding affinities. Taken together these data suggest that a cooperative interaction of the three qRRMs is required for high affinity RNA-binding.

The lipoxygenase pathway in zebrafish. Expression and characterization of zebrafish ALOX5 and comparison with its human ortholog.

The zebrafish (Danio rerio) is frequently employed as model organism to explore vertebrate embryogenesis but little is known about the lipoxygenase pathway in this lower vertebrate. When we searched the zebrafish genome for lipoxygenase genes we detected seven different genes localized on four different chromosomes. Four of these genes (ALOX2, ALOX3a, ALOX3b, ALOX3c) share a high degree of sequence conservation with the human ALOX5 gene, which encodes for the key enzyme of mammalian leukotriene biosynthesis. Expression profiles of the corresponding transcripts indicated that the ALOX2 gene is high level expressed during zebrafish embryogenesis whereas transcripts originating from the other three paralog genes could not be detected. To functionally compare human ALOX5 with the putative zebrafish ortholog (zebrafish ALOX2) we cloned and expressed the zebrafish enzyme in pro- and eukaryotic expression systems and characterized it as arachidonic acid 5S-lipoxygenating enzyme, which also exhibits leukotriene synthase activity. Mutagenesis studies of the triad determinants (Phe359Trp, Ala424Ile, Asn425Met) altered the reaction specificity from 5S- to dominant 15S-lipoxygenation. The finding that zebrafish expresses a functional ALOX5 together with the observation that most other human leukotriene relevant genes have an ortholog in the zebrafish genome suggests the biological relevance of leukotriene signaling in lower vertebrates.

Mutagenesis of triad determinants of rat Alox15 alters the specificity of fatty acid and phospholipid oxygenation.

Among lipoxygenases ALOX15 orthologs are somewhat peculiar because of their capability of oxygenating polyenoic fatty acids even if they are incorporated in complex lipid-protein assemblies. ALOX15 orthologs of different species have been characterized before, but little is known about the corresponding rat enzyme. Since rats are frequently employed as models in biomedical research we expressed rat Alox15 as recombinant protein in pro- and eukaryotic expression systems and characterized the enzyme with respect to its enzymatic properties. The enzyme oxygenated free arachidonic acid mainly to 12S-HpETE with 15S-HpETE only contributing 10% to the product mixture. Multiple directed mutagenesis studies indicated applicability of the triad concept with particular importance of Leu353 and Ile593 as specificity determinants. Ala404Gly exchange induced subtle alterations in enantioselectivity suggesting partial applicability of the Coffa/Brash concept. Wildtype rat Alox15 and its 15-lipoxygenating Leu353Phe mutant are capable of oxygenating ester lipids of biomembranes and high-density lipoproteins. For the wildtype enzyme 13S-HODE and 12S-HETE were identified as major oxygenation products but for the Leu353Phe mutant 13S-HODE and 15S-HETE prevailed. These data indicate for the first time that mutagenesis of triad determinants modifies the reaction specificity of ALOX15 orthologs with free fatty acids and complex ester lipids in a similar way.

Grsf1-induced translation of the SNARE protein Use1 is required for expansion of the erythroid compartment.

Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5'UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5'UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5'UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.

Differential expression of secretoglobins in normal ovary and in ovarian carcinoma--overexpression of mammaglobin-1 is linked to tumor progression.

Secretoglobins (SCGB), such as mammaglobin 1 (MGB1, SCGB2A2), mammaglobin 2 (MGB2, SCGB2A1) and lipophilin B (LIPB, SCGB1D2), have been related to carcinogenesis. We profiled expression of MGB1, MGB2 and LIPB in human tissues and ovarian carcinoma and explored the impact of SCGB overexpression on cell proliferation. MGB1, MGB2 and LIPB mRNA are expressed at variable levels in most human tissues and we observed significant bilateral correlations between the different secretoglobins. Concerted overexpression of MGB1 and LIPB resulted in significant increase in cell proliferation. In clinical specimens of ovarian carcinoma we measured elevated concentrations of secretoglobin mRNA and for MGB1 this up-regulation was confirmed on the protein level. Overexpression of MGB1 positively correlated with the FIGO stage, the tumor grade and the mitotic index suggesting a patho-physiological role of the protein. Our data indicate that MGB1, MGB2 and LIPB mRNAs are expressed at low levels in human tissues but basal expression is upregulated in ovarian cancer. The in vivo correlation between nuclear MGB1 localization and the mitotic rate in ovarian cancer as well as the increased cell proliferation induced by secretoglobin overexpression in ovarian cancer cell lines suggest a pathophysiological role of these proteins in ovarian cancer.

Serotonin receptor 6 mediates defective brain development in monoamine oxidase A-deficient mouse embryos.

Monoamine oxidases A and B (MAO-A and MAO-B) are enzymes of the outer mitochondrial membrane that metabolize biogenic amines. In the adult central nervous system, MAOs have important functions for neurotransmitter homeostasis. Expression of MAO isoforms has been detected in the developing embryo. However, suppression of MAO-B does not induce developmental alterations. In contrast, targeted inhibition and knockdown of MAO-A expression (E7.5-E10.5) caused structural abnormalities in the brain. Here we explored the molecular mechanisms underlying defective brain development induced by MAO-A knockdown during in vitro embryogenesis. The developmental alterations were paralleled by diminished apoptotic activity in the affected neuronal structures. Moreover, dysfunctional MAO-A expression led to elevated levels of embryonic serotonin (5-hydroxytryptamine (5-HT)), and we found that knockdown of serotonin receptor-6 (5-Htr6) expression or pharmacologic inhibition of 5-Htr6 activity rescued the MAO-A knockdown phenotype and restored apoptotic activity in the developing brain. Our data suggest that excessive 5-Htr6 activation reduces activation of caspase-3 and -9 of the intrinsic apoptotic pathway and enhances expression of antiapoptotic proteins Bcl-2 and Bcl-XL. Moreover, we found that elevated 5-HT levels in MAO-A knockdown embryos coincided with an enhanced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and a reduction of proliferating cell numbers. In summary, our findings suggest that excessive 5-HT in MAO-A-deficient mouse embryos triggers cellular signaling cascades via 5-Htr6, which suppresses developmental apoptosis in the brain and thus induces developmental retardations.

Monoamine oxidase-A knockdown in human neuroblastoma cells reveals protection against mitochondrial toxins.

The study examined how the mitochondrial enzyme monoamine oxidase-A (MAO-A), which produces hydrogen peroxide as a catalytic by-product, influences death and survival mechanisms. Targeted microRNA (miRNA) was used to stably knock down MAO-A mRNA, protein, and catalytic activity by 60-70% in SH-SY5Y human neuroblastoma cells. The effects of MAO-A knockdown (KD) on ATP, oxidative stress, electron transport chain, and survival following exposure to mitochondrial toxins were assessed. In control cells, complex I inhibition resulted in caspase-mediated cell death linked with ROS production and reduced ATP, followed by up-regulation of MAO-A mRNA, protein, and enzyme activity levels. Inhibition of complex III and IV resulted in a similar increase in MAO-A expression, while up-regulation of MAO-A was lower following complex II inhibition. MAO-A KD decreased basal reactive oxygen species levels by 50% and increased levels of ATP and reduced glutathione and Bcl-2. MAO-A KD specifically increased the activity of complex I but had no effect on complex II-IV activities. Furthermore, MAO-A KD protected against inhibitors of complex I, III, and IV. In summary, endogenous MAO-A levels influence mitochondrial function, notably complex I activity, and MAO-A may be a target for protection against neurodegenerative conditions that involve oxidative stress and mitochondrial dysfunction as underlying pathogenic factors.

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.

The biology of the RNA binding protein guanine-rich sequence binding factor 1.

The mechanisms that drive the expression of a gene into its final protein product can be sub-divided into three levels: transcriptional, post-transcriptional and post-translational events. To facilitate the development and maintenance of a multi-cellular organism precise regulatory circuits are needed to ensure the survival of the organism and its ability to respond to changes in its environment. The key element of post-transcriptional regulation is RNA. Within the cell RNA exists in the form of ribonucleoproteins (RNPs), which are characterised by the underlying RNA and the proteins that are associated to it. The eukaryotic cell contains a vast plethora of RNA-binding proteins (RBPs) that control the complex fate of cellular RNAs. One of such RBPs is Guanine-rich sequence binding factor 1 (Grsf1). Grsf1 belongs to a group of heterogeneous nuclear RNPs that are characterised by the presence of an RNA binding domain designated RNA recognition motif (RRM). Grsf1 is present in most eukaryotic cells and is located in the nucleus as well as in the cytoplasm. Thus, its activity has been related to nuclear processes (RNA splicing) as well as cytoplasmic events (translation initiation). However, its full functional significance is not yet understood. Grsf1 has been implicated in the influenza viral life cycle, embryonic brain development and the regulation of apoptosis. Moreover, Grsf1 is a functional component of several cellular signalling pathways as well as of the regulation of the cellular redox homeostasis. This review summarises the present knowledge of Grsf1 biology to bring the scattered reports of Grsf1 function into a proper context.

The Roles of Glutathione Peroxidases during Embryo Development.

Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on GPx4.

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.