Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.     

Cracks in the shell—zooming in on eggshell formation in the human parasite <Emphasis Type="Italic">Schistosoma mansoni</Emphasis>

Schistosomiasis, currently the second most common parasitic disease of humans in tropical regions is caused by the eggs of trematode worms of the genus Schistosoma. Understanding egg formation and specifically the synthesis of the eggshell comprises, consequently, a promising starting point to cure and prevent the disease. To shed light on the genetics of the latter process, we analysed the three known S. mansoni eggshell proteins P14, P19 and P48 against the background of the species inferred proteome and of eggshell proteins identified in other trematode species. Our results suggest that eggshell formation in Schistosoma involves a multitude of different proteins organised in currently three distinct protein families (P14, P48 and P34 eggshell protein family). The first two families are of simple structure. Their respective members share a substantial degree of sequence similarity and are, to date, observed only in the genus Schistosoma. In contrast, the P34 family of eggshell proteins is complex. Its in part highly diverged members share only a conserved motif of 67-aa length on average and are detected in various trematode species. The resulting widespread occurrence of this protein motif suggests an important role during eggshell formation in trematodes. Screening more than 7,000 putative proteins of S. mansoni, we could identify six new members of the P34 protein family that are likely to be involved in eggshell formation in this species.Electronic Supplementary Material Supplementary material is available for this article at .     

Cloning,characterization, and expression analysis of goat (Capra hircus) phospholipid hydroperoxide glutathione peroxidase (PHGPx)

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), as a ubiquitous antioxidant enzyme in the glutathione peroxidases (GPx) family, plays multiple roles in organisms. However, there is very little information on PHGPx in goats (Capra hircus). In this study, a full-length cDNA was cloned and characterized from Taihang black goat testes. The 844 bp cDNA contains an open reading frame (ORF) of 597 bp. The goat PHGPx nucleotide sequence contains a selenocysteine (sec) codon TGA^244-246, two potential start codons ATG^20-22 and ATG^108-110, a polyadenylation signal AATAAA^813-818 and selenocysteine insertion sequence (SECIS) motif AUGA^688-691, UGA^729-731 and AAA^703-705. As a selenoprotein, the active-site motifs and GPx family signature motifs LAFPCNQF^101-108 and WNFEK^165-170 were also found. The order of PHGPx mRNA expression levels was: testes >> heart > brain > epididymis > kidney > liver > lung > spleen > muscle. Real-time PCR and immunohistochemistry results revealed similar expression differences in different age testes, with high expression levels during adolescence. Immunofluorescence results suggested that PHGPx mainly expressed in Leydig cells and spermatids in mature goat testes.     

Analysis of <Emphasis Type="Italic">Acropora muricata</Emphasis> Calmodulin (<Emphasis Type="Italic">CaM</Emphasis>) Indicates That Scleractinian Corals Possess the Ancestral Exon/Intron Organization of the Eumetazoan <Emphasis Type="Italic">CaM</Emphasis> Gene

Calmodulin (CaM), belonging to the tropinin C (TnC) superfamily, is one of the calcium-binding proteins that are highly conserved in their protein and gene structure. Based on the structure comparison among published vertebrate and invertebrate CaM, it is proposed that the ancestral form of eumetazoan CaM genes should have five exons and four introns (four-intron hypothesis). In this study, we determined the gene structure of CaM in the coral Acropora muricata, an anthozoan cnidarian representing the basal position in animal evolution. A CaM clone was isolated from a cDNA library constructed from the spawned eggs of A. muricata. This clone was composed of 908 nucleotides, including 162 base pairs (bp) of 5′-untranslated region (UTR), 296 bp of 3′-UTR, and an open reading frame 450 bp in length. The deduced amino acid indicated that the Acropora CaM protein is identical to that of the actiniarian, Metridinium senile, and has four putative calcium-binding domains highly similar to those of other vertebrate or invertebrate CaMs. Southern blot analysis revealed that Acropora CaM is a putative single-copy gene in the nuclear genome. Genomic sequencing showed that Acropora CaM was composed of five exons and four introns, with intron II not corresponding to any region in the actiniarian CaM gene, which possesses only four exons and three introns. Our results highlight that the coral CaM gene isolated from A. muricata has four introns at the predicted positions of the early metazoan CaM gene organization, providing the first evidence from the basal eumetazoan phylum to support the four-intron hypothesis.     

Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals

Background  

Phospholipid hydroperoxide glutathione peroxidases (PHGPx), the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown.     

Composition and evolution of the vertebrate and mammalian selenoproteomes

Background

Selenium is an essential trace element in mammals due to its presence in proteins in the form of selenocysteine (Sec). Human genome codes for 25 Sec-containing protein genes, and mouse and rat genomes for 24.

Methodology/Principal Findings

We characterized the selenoproteomes of 44 sequenced vertebrates by applying gene prediction and phylogenetic reconstruction methods, supplemented with the analyses of gene structures, alternative splicing isoforms, untranslated regions, SECIS elements, and pseudogenes. In total, we detected 45 selenoprotein subfamilies. 28 of them were found in mammals, and 41 in bony fishes. We define the ancestral vertebrate (28 proteins) and mammalian (25 proteins) selenoproteomes, and describe how they evolved along lineages through gene duplication (20 events), gene loss (10 events) and replacement of Sec with cysteine (12 events). We show that an intronless selenophosphate synthetase 2 gene evolved in early mammals and replaced functionally the original multiexon gene in placental mammals, whereas both genes remain in marsupials. Mammalian thioredoxin reductase 1 and thioredoxin-glutathione reductase evolved from an ancestral glutaredoxin-domain containing enzyme, still present in fish. Selenoprotein V and GPx6 evolved specifically in placental mammals from duplications of SelW and GPx3, respectively, and GPx6 lost Sec several times independently. Bony fishes were characterized by duplications of several selenoprotein families (GPx1, GPx3, GPx4, Dio3, MsrB1, SelJ, SelO, SelT, SelU1, and SelW2). Finally, we report identification of new isoforms for several selenoproteins and describe unusually conserved selenoprotein pseudogenes.

Conclusions/Significance

This analysis represents the first comprehensive survey of the vertebrate and mammal selenoproteomes, and depicts their evolution along lineages. It also provides a wealth of information on these selenoproteins and their forms.     

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H(2)O(2) challenge in Scylla paramamosain

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135?bp full-length cDNA contains a 9?bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562?bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec(40), or U(40)) residue which is encoded by an opal codon, (127)TGA(129), and forms an active site with residues Q(74) and W(142). Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 ((64)LAFPCNQF(71)), an active site motif ((152)WNFEKF(157)), a potential N-glycosylation site ((76)NTT(78)), and two residues (R(90) and R(168)) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p?相似文献   

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.     

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.     

3'UTRs of glutathione peroxidases differentially affect selenium-dependent mRNA stability and selenocysteine incorporation efficiency

Selenoprotein mRNAs are particular in several aspects. They contain a specific secondary structure in their 3'UTR, called Secis (selenocysteine inserting sequence), which is indispensable for selenocysteine incorporation, and they are degraded under selenium-limiting conditions according to their ranking in the hierarchy of selenoproteins. In the familiy of selenium-dependent glutathione peroxidases (GPx) the ranking is GI-GPx > or = PHGPx > cGPx = pGPx. This phenomenon was studied by mutually combining the coding regions of GI-GPx, PHGPx and cGPx with their 3'UTRs. HepG2 cells were stably transfected with the resulting constructs. Expression of glutathione peroxidases was estimated by activity measurement and Western blotting, the selenium-dependent mRNA stability by real-time PCR. Whereas 3'UTRs from stable PHGPx and GI-GPx could be exchanged without loss of stability, they were not able to stabilize cGPx mRNA. cGPx 3'UTR rendered GI-GPx and PHGPx mRNA unstable. Thus, cGPx mRNA contains selenium-responsive instability elements in both the translated and the untranslated region, which cannot be compensated by one of the stable homologs. Stabilizing efficiency of an individual GPx 3'UTR did not correlate with the efficiency of selenocysteine incorporation. PHGPx 3'UTR was equally effective as cGPx 3'UTR in enhancing GPx activity in all constructs, while GI-GPx 3'UTR showed a markedly lower efficacy. We conclude that different mRNA sequences and/or RNA-binding proteins might regulate mRNA stability and translation of selenoprotein mRNA.     

Characterization and expression analysis of phospholipid hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to cadmium

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is an antioxidant enzyme in the glutathione peroxidases (GPx) family that reduces hydroperoxides of phospholipids and maintains the integrity of biomembranes. Here, we report the identification and characterization of a full length cDNA of PHGPx from the ecotoxicologically important aquatic midge Chironomus riparius (CrPHGPx1) from the Expressed Sequence Tags (ESTs) database generated through pyrosequencing. The 837 base pair (bp) cDNA contained an open reading frame of 597 bp, and a 75 bp 5' and a 159 bp 3'untranslated region. The theoretical molecular mass of the deduced amino acid (aa) sequence (197 aa) was 22.40 kDa with an estimated pI of 8.77. The Cys-codon was present at residue 74 and also the active site residues Gln(91) and Trp(164). The active-site motifs and GPx family signature motifs LAFPCNQF(101-108) and WNFTK(163-168) were also found. Phylogenetic analysis showed that CrPHGPx1 is grouped with PHGPx1 from other species and is more closely related to insects belonging to the dipteran order. The mRNA of CrPHGPx1 was detected in larvae, pupae and adults. The expression of CrPHGPx1 is induced by cadmium exposure indicating that the mRNA expression of CrPHGPx1 is differently regulated in response to oxidative stress caused by environmental stressors.     

Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.     

Nonsense-mediated decay of mRNA for the selenoprotein phospholipid hydroperoxide glutathione peroxidase is detectable in cultured cells but masked or inhibited in rat tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.     

Nonsense-mediated decay of glutathione peroxidase 1 mRNA in the cytoplasm depends on intron position

mRNA for glutathione peroxidase 1 (GPx1) is subject to cytoplasmic nonsense-mediated decay (NMD) when the UGA selenocysteine (Sec) codon is recognized as nonsense. Here, we demonstrate by moving the sole intron of the GPx1 gene that either the Sec codon or a TAA codon in its place elicits NMD when located >/=59 bp but not 相似文献   

Glutathione peroxidases and redox-regulated transcription factors

Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides. cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-kappaB activation by interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis. GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells. GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways. In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs.