Incorporation of Tellurocysteine into Glutathione Transferase Generates High Glutathione Peroxidase Efficiency
Xiaoman LiuLouis A. SilksCuiping LiuMorgane Ollivault‐ShiflettXin HuangJing LiGuimin LuoYa–Ming HouJunqiu LiuJiacong Shen
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Abstract:
A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.Keywords:
GPX3
GPX6
GPX1
GPX4
Glutathione reductase
Glutathione transferase
Glutathione S-transferase
Transferase
Residue (chemistry)
GPX1
Selenoprotein
GPX4
GPX3
DIO2
GPX6
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cDNA probes of human glutathione peroxidase (GSHPx) genes, including the classic GPX1 (GSHPx-1), the newly characterized GPX2 (GSHPx-GI), the plasma enzyme GPX3 (GSHPx-P), and the phospholipid hydroperoxide glutathione peroxidase GPX4 (PHGPX), were hybridized to Southern blots containing genomic DNA from human × hamster somatic cell hybrids. GPX2 was mapped to chromosome 14, GPX3 to chromosome 5 and GPX4 to chromosome 19. Additionally, human chromosomes 3 and 21 and the X chromosome were shown to contain sequences homologous to GPX1, as reported previously.
GPX3
GPX1
Phospholipid-hydroperoxide glutathione peroxidase
GPX6
GPX4
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A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.
GPX3
GPX6
GPX1
GPX4
Glutathione reductase
Glutathione transferase
Glutathione S-transferase
Transferase
Residue (chemistry)
Cite
Citations (71)
A set of human tumor cell lines was characterized in terms of the GPx isoenzymes GPx1, -2, -3 and -4. Semiquantitative PCR was used to investigate the GPx mRNA transcripts and the GPx activity was determined photometrically. As a result of culturing under standard conditions, diverse distribution of GPx mRNA and basic GPx activity was found in the investigated cell lines. PCR results showed nearly ubiquitous existence of the isoenzymes GPx1 and GPx4. GPx2 mRNA transcript was only detected in the colonic cell line CaCo-2. After detection of the GPx3 mRNA transcripts in most of the tested cell lines, an ELISA was performed to investigate if the GPx3 protein is present as well. However, the GPx3 protein could not be detected. Glutathione peroxidases contain the amino acid selenocysteine in their active centre. Selenocysteine contains selenium instead of sulfur in cysteine. Therefore, the influence of selenium on GPx activity and GPx isoenzyme distribution was investigated. Cell culturing with additional selenium showed a clear elevation of GPx activity in Mono Mac 6 cells but no gain of mRNA transcripts or any change in the isoenzyme's distribution.
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Selenocysteine
GPX3
Selenoprotein
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GPX6
Phospholipid-hydroperoxide glutathione peroxidase
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Five out of eight human glutathione peroxidases (GPXs) are selenoproteins, representing proteins that contain selenium as part of the amino acid selenocysteine. The GPXs are important for reducing hydroperoxides in a glutathione-consuming manner and thus regulate cellular redox homeostasis. GPX1, GPX2, and GPX4 represent the three main cytosolic GPXs, but they differ in their expression patterns with GPX1 and GPX4 being expressed ubiquitously, whereas GPX2 is mainly expressed in epithelial cells. GPX1 and GPX2 have been described to reduce soluble hydroperoxides, while GPX4 reduces complex lipid hydroperoxides, thus protecting cells from lipid peroxidation and ferroptosis. But most of these data are derived from cells that are devoid of one of the isoforms and thus, compensation or other cellular effects might affect the conclusions. So far, the use of isolated recombinant human selenoprotein glutathione peroxidases in pure enzyme assays has not been employed to study their substrate specificities side by side. Using recombinant GPX1, GPX2, and GPX4 produced in E. coli we here assessed their GPX activities by a NADPH-consuming glutathione reductase-coupled assay with 17 different peroxides (all at 50 μM) as substrates. GPX4 was clearly the only isoform able to reduce phosphatidylcholine hydroperoxide. In contrast, small soluble hydroperoxides such as H2O2, cumene hydroperoxide, and tert-butyl hydroperoxide were reduced by all three isoforms, but with approximately 10-fold higher efficiency for GPX1 in comparison to GPX2 and GPX4. Also, several fatty acid-derived hydroperoxides were reduced by all three isoforms and again GPX1 had the highest activity. Interestingly, the stereoisomerism of the fatty acid-derived hydroperoxides clearly affected the activity of the GPX enzymes. Overall, distinct substrate specificity is obvious for GPX4, but not so when comparing GPX1 and GPX2. Clearly GPX1 was the most potent isoform of the three GPXs in terms of turnover in reduction of soluble and fatty-acid derived hydroperoxides.
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GPX1
Selenoprotein
Selenocysteine
Phospholipid-hydroperoxide glutathione peroxidase
GPX6
GPX3
Glutaredoxin
Cumene hydroperoxide
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Glutathione reductase
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GPX4
GPX6
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AbstractPhospholipid hydroperoxide glutathione peroxidase (PHGPX or GPX4, E.C. 1.11.1.12) is one of the four identified selenium-dependent glutathione peroxidases (GPX) in mammals (1). Both of the pig (2.8 kb) and the mouse (4.0 kb) GPX4 genes contain seven exons and six introns, with putative regulatory elements or binding sites for transcriptional factors (2,3). There is 95% homology among amino acid sequences deduced from the GPX4 cDNA of rat (4), mouse (5) and human (6). In contrast, the homology between GPX1 and GPX4 is less than 40%. There are two forms of GPX4: the long form (23 kDa) with a leader sequence for transportation to mitochondria, and the short form (20 kDa) or the non-mitochondrial form (7). Although GPX4 was initially considered mainly an antioxidant enzyme by reducing phospholipid hydroperoxides (8), it may be involved in sperm maturation (9) as there are abundant GPX4 activity and mRNA in testis (10,11) and rat epididymal spermatozoa (12). It exists as a soluble peroxidase in spermatids, but loses its activity in mature spermatozoa and persists as an oxidatively cross-linked insoluble protein (13). Besides, GPX4 is expressed in all major tissues studied so far. Comparatively, GPX4 mRNA and activity are less affected by changes in tissue selenium status than those of GPX1 or GPX3 (14). Because there is a selenium-independent enzyme that reduces phospholipid hydroperoxides (15), GPX4 mRNA analysis becomes a specific tool to distinguish these two enzymes for various biochemical and physiological studies.KeywordsPhospholipid HydroperoxidePutative Regulatory ElementSaran WrapPhospholipid Hydroperoxide Glutathione PeroxidaseGPX4 mRNAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Phospholipid-hydroperoxide glutathione peroxidase
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In the blood fluke Schistosoma mansoni a functionally active, monomeric, phospholipid hydroperoxide glutathione peroxidase (PHGPx) has been purified and characterized. This enzyme contains a catalytically active selenocysteine. The protein has been shown to be the product of a cloned gene, previously referred to as a glutathione peroxidase gene. S. mansoni PHGPx has been found 5 times more abundant in female than in male worm extract. As in vertebrate PHGPx, homology alignment indicates that the residues involved in the glutathione binding by the tetrameric cellular glutathione peroxidase are mutated in the S. mansoni enzyme. Thus, this aspect appears a landmark of the PHGPx-type of glutathione peroxidases,which might be of functional relevance
Phospholipid-hydroperoxide glutathione peroxidase
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Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 (hGPx4) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1-leader-GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a "ping–pong" mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo. © 2013 IUBMB Life, 65(11):951–956, 2013
Phospholipid-hydroperoxide glutathione peroxidase
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GPX1
GPX6
GPX3
HEK 293 cells
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