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Regina Brigeliusflohe - One of the best experts on this subject based on the ideXlab platform.
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the putative Glutathione Peroxidase gene of plasmodium falciparum codes for a thioredoxin Peroxidase
Journal of Biological Chemistry, 2001Co-Authors: Helena Sztajer, Katja Becker, Regina Brigeliusflohe, Klausdieter Aumann, Benoit Gamain, Christian Slomianny, Leopold FloheAbstract:Abstract A putative Glutathione Peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide Glutathione Peroxidase of mammals, it proved to be monomeric. It was active with H2O2 and organic hydroperoxides but, unlike phospholipid hydroperoxide Glutathione Peroxidase, not with phosphatidylcholine hydroperoxide. With Glutathione Peroxidases it shares the ping-pong mechanism with infiniteV max and K m when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoPeroxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum(Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin Peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K mof 10 μm and a k 1′ of 0.55 s− 1. The apparent k 1′ for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 × 104 m − 1 s− 1, 3.3 × 103 m − 1s− 1, and 2.5 × 103 m − 1 s− 1, respectively. k 2′ for reduction by autologous thioredoxin is 5.4 × 104 m -1s− 1 (21.2m − 1 s− 1for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.
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tissue specific functions of individual Glutathione Peroxidases
Free Radical Biology and Medicine, 1999Co-Authors: Regina BrigeliusfloheAbstract:The family of Glutathione Peroxidases comprises four distinct mammalian selenoproteins. The classical enzyme (cGPx) is ubiquitously distributed. According to animal, cell culture and inverse genetic studies, its primary function is to counteract oxidative attack. It is dispensible in unstressed animals, and accordingly ranks low in the hierarchy of Glutathione Peroxidases. The gastrointestinal isoenzyme (GI-GPx) is most related to cGPx and is exclusively expressed in the gastrointestinal tract. It might provide a barrier against hydroperoxides derived from the diet or from metabolism of ingested xenobiotics. The extreme stability in selenium deficiency ranks this Glutathione Peroxidase highest in the hierarchy of selenoproteins and points to a more vital function than that of cGPx. Plasma GPx (pGPx) behaves similar to cGPx in selenium deficiency. It is directed to extracellular compartments and is expressed in various tissues in contact with body fluids, e.g., kidney, ciliary body, and maternal/fetal interfaces. It has to be rated as an efficient extracellular antioxidant device, though with low capacity because of the limited extracellular content of potential thiol substrates. Phospholipid hydroperoxide Glutathione Peroxidase (PHGPx), originally presumed to be a universal antioxidant enzyme protecting membrane lipids, appears to have adopted a variety of specific roles like silencing lipoxygenases and becoming an enzymatically inactive structural component of the mitochondrial capsule during sperm maturation. Thus, all individual isoenzymes are efficient Peroxidases in principle, but beyond their mere antioxidant potential may exert cell- and tissue-specific roles in metabolic regulation, as is evident for PHGPx and may be expected for others.
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mrna stability and selenocysteine insertion sequence efficiency rank gastrointestinal Glutathione Peroxidase high in the hierarchy of selenoproteins
FEBS Journal, 1999Co-Authors: Kirstin Wingler, Leopold Flohe, Michael Bocher, Heike Kollmus, Regina BrigeliusfloheAbstract:The recently described gastrointestinal Glutathione Peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes Glutathione Peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical Glutathione Peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.
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mrna stability and selenocysteine insertion sequence efficiency rank gastrointestinal Glutathione Peroxidase high in the hierarchy of selenoproteins
FEBS Journal, 1999Co-Authors: Kirstin Wingler, Leopold Flohe, Michael Bocher, Heike Kollmus, Regina BrigeliusfloheAbstract:The recently described gastrointestinal Glutathione Peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes Glutathione Peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical Glutathione Peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.
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interleukin 1 induced nuclear factor kappa b activation is inhibited by overexpression of phospholipid hydroperoxide Glutathione Peroxidase in a human endothelial cell line
Biochemical Journal, 1997Co-Authors: Regina Brigeliusflohe, Barbel Friedrichs, Stefanie Maurer, Manfred Schultz, Rudiger StreicherAbstract:Oxygen radicals are commonly accepted mediators in the tumour necrosis factor-mediated nuclear factor κB (NFκB) signalling cascade, but evidence for their role during interleukin-1 (IL-1) signalling is lacking. To test the involvement of hydroperoxides we investigated whether IL-1-induced NFκB activation could be influenced by Glutathione Peroxidases (GPx). These enzymes remove hydroperoxides with various specificities for the hydroperoxide substrate. By overexpressing phospholipid hydroperoxide Glutathione Peroxidase (PHGPx), which characteristically reacts with lipophilic hydroperoxides, the roles of H 2 O 2 and lipidhydroperoxides were assessed. A human umbilical endothelial cell line, ECV 304, was stably transfected with the genes for both PHGPx and selenophosphate synthetase ( selD ), which provides selenophosphate for selenoprotein biosynthesis. When grown in selenium-deficient culture medium, the double-transfected clone (ECV PHGPx+SelD+ ) expressed 5-fold higher ( P 2 O 2 removal was also significantly ( P 2 O 2 removal were enhanced substantially in control cells and transfected cells. Under these conditions, PHGPx activity was 1.7-fold ( P PHGPx+SelD+ , but H 2 O 2 removal was the same as in controls. IL-1-induced NFκB activation was inhibited by selenium supplementation in control cells. In ECV PHGPx+SelD+ under conditions of selenium restriction, IL-1 induced NFκB activation only to a similar extent as under conditions of selenium supplementation in controls, and activation was abolished with 50 nM sodium selenite. These results show that overexpressed PHGPx is sufficient to inhibit NFκB activation, and suggests that NFκB activation by IL-1 is mediated by a preferential substrate of PHGPx, such as a fatty acid hydroperoxide, rather than by H 2 O 2 , the preferred substrate of the more abundant cytosolic GPx.
Yasuhito Nakagawa - One of the best experts on this subject based on the ideXlab platform.
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Failure of the Expression of Phospholipid Hydroperoxide Glutathione Peroxidase in the Spermatozoa of Human Infertile Males1
2016Co-Authors: Kazuo Emoto, Masato Umeda, Yasuhito NakagawaAbstract:Phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of hu-man spermatozoa by immunostaining with anti-PHGPx mono-clonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of ex-pression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10 % of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26
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protection from inactivation of the adenine nucleotide translocator during hypoglycaemia induced apoptosis by mitochondrial phospholipid hydroperoxide Glutathione Peroxidase
Biochemical Journal, 2003Co-Authors: Hirotaka Imai, Tomoko Koumura, Ryo Nakajima, Kazuhiro Nomura, Yasuhito NakagawaAbstract:We demonstrated that mitochondrial phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) first suppressed the dissociation of cytochrome c (cyt c) from cardiolipin (CL) in mitochondrial inner membranes and then apoptosis caused by the hypoglycaemia by the prevention of peroxidation of CL [Nomura, Imai, Koumura, Arai and Nakagawa (1999) J. Biol. Chem. 274, 29294-29302; Nomura, Imai, Koumura, Kobayashi and Nakagawa (2000) Biochem. J. 351, 183-193]. The present study shows the involvement of peroxidation of CL in the inactivation of adenine nucleotide translocator (ANT) and the opening of permeability transition pores by using the system of ANT-reconstituted liposome and isolated mitochondria. ANT activity appeared in dioleoyl phosphatidylcholine proteoliposome containing 10% (mol/mol) CL or phosphatidylglycerol (PG), but not other classes of phospholipids. ANT activity was competitively inhibited by the addition of cardiolipin hydroperoxide (CLOOH) in reconstituted liposomes containing CL. However, phosphatidylcholine hydroperoxide failed to inactivate the activity of ANT. The activity of ANT in reconstituted liposomes, including CLOOH, recovered when CLOOH in reconstituted liposome was reduced to hydroxycardiolipin by incubation with PHGPx. The activity of ANT was determined in rat basophil leukaemia RBL2H3 cells after their exposure to 2-deoxyglucose. ANT activity decreased to 50% of the control level by 4 h in response to apoptosis. In parallel, cyt c and apoptosis-inducing factor (AIF) were released from mitochondria. Suppression of the accumulation of CLOOH by overexpression of PHGPx in mitochondria effectively prevented the inactivation of ANT, the opening of permeability transition pores and the release of cyt c and AIF from mitochondria in hypoglycaemia-induced apoptotic cells. These findings suggest that mitochondrial PHGPx might be involved in the modulation of the activity of ANT and the opening of pores for the release of cyt c via the modulation of levels of CLOOH in the mitochondria.
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biological significance of phospholipid hydroperoxide Glutathione Peroxidase phgpx gpx4 in mammalian cells
Free Radical Biology and Medicine, 2003Co-Authors: Hirotaka Imai, Yasuhito NakagawaAbstract:Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide Glutathione Peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.
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overexpression of phospholipid hydroperoxide Glutathione Peroxidase modulates acetyl coa 1 o alkyl 2 lyso sn glycero 3 phosphocholine acetyltransferase activity
Journal of Biological Chemistry, 2002Co-Authors: Hikaru Sakamoto, Takaki Tosaki, Yasuhito NakagawaAbstract:Abstract The synthesis of platelet-activating factor (PAF) by A23187-stimulated RBL-2H3 cells was significantly suppressed by overexpression of phospholipid hydroperoxide Glutathione Peroxidase (PHGPx). When the cells overexpressing PHGPx (L9 cells) were pretreated with diethyl maleate, which reduces PHGPx activity, PAF synthesis uponA23187 stimulation rose to levels seen in mock-transfected cells (S1 cells). Hydroperoxide levels, which are reduced in L9 cells, are involved in regulating PAF synthesis, because the addition of hydroperoxyeicosatetraenoic acid increased PAF production inA23187-stimulated L9 cells to control cell levels. The activity of acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, which is involved in the last step of PAF synthesis, is also reduced in L9 cells. p38 kinase inhibitors block acetyltransferase activity in normal A23187-stimulated cells, suggesting that p38 kinase is involved in regulating acetyltransferase activity. Recombinant active p38 kinase activates acetyltransferase, whereas alkaline phosphatase reverses this, suggesting p38 kinase directly phosphorylates acetyltransferase. p38 kinase phosphorylation is blocked in L9 cells, indicating that high hydroperoxide levels are needed for the activation of p38 kinase. Thus, intracellular hydroperoxide levels participate in regulating p38 kinase phosphorylation, which in turn controls the activation of acetyltransferase and thus the synthesis of PAF. These observations suggest that PHGPx is an important component of the mechanisms regulating inflammation.
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involvement of phospholipid hydroperoxide Glutathione Peroxidase in the modulation of prostaglandin d2 synthesis
Journal of Biological Chemistry, 2000Co-Authors: Hikaru Sakamoto, Hirotaka Imai, Yasuhito NakagawaAbstract:Abstract Antigenic cross-linking of the high affinity IgE receptors on mast cells induced the synthesis of prostaglandin D2 (PGD2). The production of PGD2 in L9 cells, which overexpressed non-mitochondrial phospholipid Glutathione Peroxidase (PHGPx), was only one-third that in the control line of cells (S1 cells). The reduction in the formation of PGD2 in L9 cells was reversed upon inhibition of PHGPx activity by buthionine sulfoximine. Experiments with inhibitors demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme responsible for the production of PGD2 upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to prostaglandin H2 (PGH2) was strongly inhibited in L9 cells, whereas the rate of conversion of PGH2 to PGD2 was the same in L9 cells and S1 cells, indicating that PGHS was inactivated in L9 cells. The PGHS activity in L9 cells was about half that in S1 cells. However, PGHS activity in L9 cells increased to the level in S1 cells upon the addition of the hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of 3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2 in nucleus and endoplasmic reticulum via reductions in levels of the hydroperoxides that are required for full activation of PGHS. Therefore, it appears that PHGPx might function as a modulator of the production of prostanoids, in addition to its role as an antioxidant enzyme.
Leopold Flohe - One of the best experts on this subject based on the ideXlab platform.
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Selenium in mammalian spermiogenesis
Biological Chemistry, 2007Co-Authors: Leopold FloheAbstract:The role of selenium in male fertility is reviewed with special emphasis on selenoprotein P and phospholipid hydroperoxide Glutathione Peroxidase (GPx4) in spermiogenesis. Inverse genetics reveal that selenoprotein P is required for selenium supply to the testis. GPx4 is abundantly synthesized in spermatids. As a moonlighting protein it is transformed in the later stages of spermiogenesis from an active selenoPeroxidase into a structural protein that becomes a constituent of the mitochondrial sheath of spermatozoa. The transformation is paralleled by loss of Glutathione. Mechanistically, the process is an alternate substrate inactivation of GPx4 resulting from reactions of its selenenic form with thiols of GPx4 itself and other proteins. Circumstantial evidence and ongoing experimental genetics indicate that the mitochondrially expressed form of the GPx4 gene is the most relevant one in spermiogenesis, with the nuclear form being dispensable for fertility and the role of cytosolic GPx4 remaining unclear. Clinical data reveal a strong association of low sperm GPx4 with infertility. Thus, impaired GPx4 biosynthesis, due to selenium deficiency or to genetic defects in gpx4 itself or in proteins involved in Se distribution and selenoprotein biosynthesis, causes male infertility, but can also be an epiphenomenon due to any perturbation of testicular function.
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male fertility is linked to the selenoprotein phospholipid hydroperoxide Glutathione Peroxidase
Biology of Reproduction, 2002Co-Authors: Carlo Foresta, Leopold Flohe, Antonella Roveri, Fulvio Ursini, Andrea Garolla, Matilde MaiorinoAbstract:The selenoprotein phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) accounts for almost the entire selenium content of mammalian testis. PHGPx is abundantly expressed in spermatids as active Peroxidase but is transformed to an oxidatively inactivated protein in mature sperm, where it is a major constituent of the mitochondrial capsule in the midpiece. Male infertility in selenium-deficient animals, which is characterized by impaired sperm motility and morphological midpiece alterations, is considered to result from insufficient PHGPx content. We studied the relationship between sperm PHGPx, measured as rescued activity, and human fertility. Sperm specimens from 75 infertile men and 37 controls were analyzed for fertility-related parameters according to World Health Organization criteria. The PHGPx protein content was estimated after reductive solubilization of the spermatozoa by measuring the rescued PHGPx activity. Rescued PHGPx activity of infertile men ranged significantly below that of controls (93.2 6 60.1 units/mg sperm protein vs. 187.5 6 55.3 units/mg) and was particularly low in oligoasthenozoospermic specimens (61.93 6 45.42 units/mg; P , 0.001 compared with controls and asthenozoospermic samples). Rescued PHGPx activity was correlated positively with viability, morphological integrity, and most profoundly forward motility (r 5 0.35, 0.44, and 0.45, respectively). In isolated motile samples, motility decreased faster with decreasing PHGPx content. In humans, PHGPx appears to be indispensable for structural integrity of spermatozoa and to codetermine sperm motility and viability. Because the content of PHGPx, irrespective of the cause of alteration, is correlated with fertility-related parameters, PHGPx can be considered a predictive measure for fertilization capacity. fertilization, gamete biology, male sexual function, sperm, spermatogenesis
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the putative Glutathione Peroxidase gene of plasmodium falciparum codes for a thioredoxin Peroxidase
Journal of Biological Chemistry, 2001Co-Authors: Helena Sztajer, Katja Becker, Regina Brigeliusflohe, Klausdieter Aumann, Benoit Gamain, Christian Slomianny, Leopold FloheAbstract:Abstract A putative Glutathione Peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide Glutathione Peroxidase of mammals, it proved to be monomeric. It was active with H2O2 and organic hydroperoxides but, unlike phospholipid hydroperoxide Glutathione Peroxidase, not with phosphatidylcholine hydroperoxide. With Glutathione Peroxidases it shares the ping-pong mechanism with infiniteV max and K m when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoPeroxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum(Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin Peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K mof 10 μm and a k 1′ of 0.55 s− 1. The apparent k 1′ for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 × 104 m − 1 s− 1, 3.3 × 103 m − 1s− 1, and 2.5 × 103 m − 1 s− 1, respectively. k 2′ for reduction by autologous thioredoxin is 5.4 × 104 m -1s− 1 (21.2m − 1 s− 1for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.
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dual function of the selenoprotein phgpx during sperm maturation
Science, 1999Co-Authors: Fulvio Ursini, Matilde Maiorino, Antonella Roveri, Sabina Heim, Michael Kiess, Josef Wissing, Leopold FloheAbstract:The selenoprotein phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble Peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.
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mrna stability and selenocysteine insertion sequence efficiency rank gastrointestinal Glutathione Peroxidase high in the hierarchy of selenoproteins
FEBS Journal, 1999Co-Authors: Kirstin Wingler, Leopold Flohe, Michael Bocher, Heike Kollmus, Regina BrigeliusfloheAbstract:The recently described gastrointestinal Glutathione Peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes Glutathione Peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical Glutathione Peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.
Hirotaka Imai - One of the best experts on this subject based on the ideXlab platform.
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lipid peroxidation dependent cell death regulated by gpx4 and ferroptosis
Current Topics in Microbiology and Immunology, 2016Co-Authors: Hirotaka Imai, Masaki Matsuoka, Takeshi Kumagai, Taro Sakamoto, Tomoko KoumuraAbstract:Glutathione Peroxidase 4 (Phospholipid hydroperoxide Glutathione Peroxidase, PHGPx) can directly reduce phospholipid hydroperoxide. Depletion of GPx4 induces lipid peroxidation-dependent cell death in embryo, testis, brain, liver, heart, and photoreceptor cells of mice. Administration of vitamin E in tissue specific GPx4 KO mice restored tissue damage in testis, liver, and heart. These results indicate that suppression of phospholipid peroxidation is essential for cell survival in normal tissues in mice. Ferroptosis is an iron-dependent non-apoptotic cell death that can elicited by pharmacological inhibiting the cystine/glutamate antiporter, system Xc− (type I) or directly binding and loss of activity of GPx4 (Type II) in cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression, but not in normal cells. Ferroptosis by Erastin (Type I) and RSL3 (RAS-selective lethal 3, Type II) treatment was suppressed by an iron chelator, vitamin E and Ferrostatin-1, antioxidant compound. GPx4 can regulate ferroptosis by suppression of phospholipid peroxidation in erastin and RSL3-induced ferroptosis. Recent works have identified several regulatory factors of erastin and RSL3-induced ferroptosis. In our established GPx4-deficient MEF cells, depletion of GPx4 induce iron and 15LOX-independent lipid peroxidation at 26 h and caspase-independent cell death at 72 h, whereas erastin and RSL3 treatment resulted in iron-dependent ferroptosis by 12 h. These results indicated the possibility that the mechanism of GPx4-depleted cell death might be different from that of ferroptosis induced by erastin and RSL3.
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depletion of selenoprotein gpx4 in spermatocytes causes male infertility in mice
Journal of Biological Chemistry, 2009Co-Authors: Hirotaka Imai, Nao Hakkaku, Ryo Iwamoto, Jyunko Suzuki, Toshiyuki Suzuki, Yoko Tajima, Kumiko Konishi, Shintaro Minami, Shizuko Ichinose, Kazuhiro IshizakaAbstract:Abstract Phospholipid hydroperoxide Glutathione Peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674–683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.
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biological significance of lipid hydroperoxide and its reducing enzyme phospholipid hydroperoxide Glutathione Peroxidase in mammalian cells
Yakugaku Zasshi-journal of The Pharmaceutical Society of Japan, 2004Co-Authors: Hirotaka ImaiAbstract:Excessive production of reactive oxygen species (ROS) may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Recently, ROS have gained attention as important second messengers. ROS lifetimes can be very short, and many types of ROS cannot penetrate organelle membranes. It is therefore thought that only ROS signal molecules that are generated locally in an organelle are transduced when cells are stimulated. Lipid hydroperoxides are one type of ROS of which the biological function has not yet been clarified. The phospholipid hydroperoxide Glutathione Peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme and separately distributed to the mitochondria, nucleus, nucleoli, and cytosol, where it regulates phospholipid hydroperoxide and fatty acid hydroperoxide as signal molecules. This review focuses on the structure and biological functions of PHGPx in mammalian cells. Overexpression of different types of PHGPx in the RBL2H3 cell line provides a useful model system with which to clarify the ability of different types of PHGPx to modulate cellular function and the importance of lipid hydroperoxides as signal molecules. Transformant studies show that lipid hydroperoxide is an activator of lipoxygenase and cyclooxygenase and participates in inflammation, cardiolipin hydroperoxide is the signal molecule for the release of cytochrome c during apoptotic cell death, and PHGPx is a signal regulator in the IgE receptor-mediated signaling pathway. It is becoming clear that PHGPx has an important role in spermatogenesis, sperm function, and embryonic development, and its deficiency is implicated in human infertility and in embryonic lethality of PHGPx knockout mice.
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protection from inactivation of the adenine nucleotide translocator during hypoglycaemia induced apoptosis by mitochondrial phospholipid hydroperoxide Glutathione Peroxidase
Biochemical Journal, 2003Co-Authors: Hirotaka Imai, Tomoko Koumura, Ryo Nakajima, Kazuhiro Nomura, Yasuhito NakagawaAbstract:We demonstrated that mitochondrial phospholipid hydroperoxide Glutathione Peroxidase (PHGPx) first suppressed the dissociation of cytochrome c (cyt c) from cardiolipin (CL) in mitochondrial inner membranes and then apoptosis caused by the hypoglycaemia by the prevention of peroxidation of CL [Nomura, Imai, Koumura, Arai and Nakagawa (1999) J. Biol. Chem. 274, 29294-29302; Nomura, Imai, Koumura, Kobayashi and Nakagawa (2000) Biochem. J. 351, 183-193]. The present study shows the involvement of peroxidation of CL in the inactivation of adenine nucleotide translocator (ANT) and the opening of permeability transition pores by using the system of ANT-reconstituted liposome and isolated mitochondria. ANT activity appeared in dioleoyl phosphatidylcholine proteoliposome containing 10% (mol/mol) CL or phosphatidylglycerol (PG), but not other classes of phospholipids. ANT activity was competitively inhibited by the addition of cardiolipin hydroperoxide (CLOOH) in reconstituted liposomes containing CL. However, phosphatidylcholine hydroperoxide failed to inactivate the activity of ANT. The activity of ANT in reconstituted liposomes, including CLOOH, recovered when CLOOH in reconstituted liposome was reduced to hydroxycardiolipin by incubation with PHGPx. The activity of ANT was determined in rat basophil leukaemia RBL2H3 cells after their exposure to 2-deoxyglucose. ANT activity decreased to 50% of the control level by 4 h in response to apoptosis. In parallel, cyt c and apoptosis-inducing factor (AIF) were released from mitochondria. Suppression of the accumulation of CLOOH by overexpression of PHGPx in mitochondria effectively prevented the inactivation of ANT, the opening of permeability transition pores and the release of cyt c and AIF from mitochondria in hypoglycaemia-induced apoptotic cells. These findings suggest that mitochondrial PHGPx might be involved in the modulation of the activity of ANT and the opening of pores for the release of cyt c via the modulation of levels of CLOOH in the mitochondria.
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biological significance of phospholipid hydroperoxide Glutathione Peroxidase phgpx gpx4 in mammalian cells
Free Radical Biology and Medicine, 2003Co-Authors: Hirotaka Imai, Yasuhito NakagawaAbstract:Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide Glutathione Peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.
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interleukin 1 induced nuclear factor kappa b activation is inhibited by overexpression of phospholipid hydroperoxide Glutathione Peroxidase in a human endothelial cell line
Biochemical Journal, 1997Co-Authors: Regina Brigeliusflohe, Barbel Friedrichs, Stefanie Maurer, Manfred Schultz, Rudiger StreicherAbstract:Oxygen radicals are commonly accepted mediators in the tumour necrosis factor-mediated nuclear factor κB (NFκB) signalling cascade, but evidence for their role during interleukin-1 (IL-1) signalling is lacking. To test the involvement of hydroperoxides we investigated whether IL-1-induced NFκB activation could be influenced by Glutathione Peroxidases (GPx). These enzymes remove hydroperoxides with various specificities for the hydroperoxide substrate. By overexpressing phospholipid hydroperoxide Glutathione Peroxidase (PHGPx), which characteristically reacts with lipophilic hydroperoxides, the roles of H 2 O 2 and lipidhydroperoxides were assessed. A human umbilical endothelial cell line, ECV 304, was stably transfected with the genes for both PHGPx and selenophosphate synthetase ( selD ), which provides selenophosphate for selenoprotein biosynthesis. When grown in selenium-deficient culture medium, the double-transfected clone (ECV PHGPx+SelD+ ) expressed 5-fold higher ( P 2 O 2 removal was also significantly ( P 2 O 2 removal were enhanced substantially in control cells and transfected cells. Under these conditions, PHGPx activity was 1.7-fold ( P PHGPx+SelD+ , but H 2 O 2 removal was the same as in controls. IL-1-induced NFκB activation was inhibited by selenium supplementation in control cells. In ECV PHGPx+SelD+ under conditions of selenium restriction, IL-1 induced NFκB activation only to a similar extent as under conditions of selenium supplementation in controls, and activation was abolished with 50 nM sodium selenite. These results show that overexpressed PHGPx is sufficient to inhibit NFκB activation, and suggests that NFκB activation by IL-1 is mediated by a preferential substrate of PHGPx, such as a fatty acid hydroperoxide, rather than by H 2 O 2 , the preferred substrate of the more abundant cytosolic GPx.
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interleukin 1 induced nuclear factor kappa b activation is inhibited by overexpression of phospholipid hydroperoxide Glutathione Peroxidase in a human endothelial cell line
Biochemical Journal, 1997Co-Authors: Regina Brigeliusflohe, Barbel Friedrichs, Stefanie Maurer, Manfred Schultz, Rudiger StreicherAbstract:Oxygen radicals are commonly accepted mediators in the tumour necrosis factor-mediated nuclear factor kappa B (NF kappa B) signalling cascade, but evidence for their role during interleukin-1 (IL-1) signalling is lacking. To test the involvement of hydroperoxides we investigated whether IL-1-induced NF kappa B activation could be influenced by Glutathione Peroxidases (GPx). These enzymes remove hydroperoxides with various specificities for the hydroperoxide substrate. By overexpressing phospholipid hydroperoxide Glutathione Peroxidase (PHGPx), which characteristically reacts with lipophilic hydroperoxides, the roles of H2O2 and lipid hydroperoxides were assessed. A human umbilical endothelial cell line, ECV 304, was stably transfected with the genes for both PHGPx and selenophosphate synthetase (selD), which provides selenophosphate for selenoprotein biosynthesis. When grown in selenium-deficient culture medium, the double-transfected clone (ECVPHGPx+SelD+) expressed 5-fold higher (P<0.005) PHGPx activity (measured by phosphatidylcholine hydroperoxide removal) than controls. The rate of H2O2 removal was also significantly (P<0.01) higher in this clone. When grown with high levels of extracellular selenium (up to 100 nM selenite), PHGPx activity and H2O2 removal were enhanced substantially in control cells and transfected cells. Under these conditions, PHGPx activity was 1.7-fold (P<0.005) higher in ECVPHGPx+SelD+, but H2O2 removal was the same as in controls. IL-1-induced NF kappa B activation was inhibited by selenium supplementation in control cells. In ECVPHGPx+SelD+ under conditions of selenium restriction, IL-1 induced NF kappa B activation only to a similar extent as under conditions of selenium supplementation in controls, and activation was abolished with 50 nM sodium selenite. These results show that overexpressed PHGPx is sufficient to inhibit NF kappa B activation, and suggests that NF kappa B activation by IL-1 is mediated by a preferential substrate of PHGPx, such as a fatty acid hydroperoxide, rather than by H2O2, the preferred substrate of the more abundant cytosolic GPx.