Nebraska Redox Biology Center Educational Portal

Glutathione Peroxidases

Glutathione peroxidases (GPXs) are ubiquitous class of thioredoxin fold thiol peroxidases. [ 1, 2, 3]. GPXs play a wide range of physiological functions in organisms and are involved in hydrogen peroxide signaling, detoxification of hydroperoxides, and maintaining cellular redox homeostasis. [ 1, 3, 4, 5]. There are 24 solved Glutathione Peroxidase structures in Protein Data Bank at this time (June 2015).

3D structure of Human Glutathione Peroxidase 4,

Reduction of hydroperoxides by GPXs consumes glutathione (GSH). [ 1, 3]. Not all GPXs utilize GSH and some of them have specificity for thioredoxin or other thiol oxidoreductases. The reduction of hydroperoxides by GPXs involves the conversion of the active-site Sec or Cys residue to the selenenic or sulfenic acid intermediate (-Se-OH or -S-OH), reduction of such intermediate by GSH, leading to (-Se-SG) or thiol (-S-SG) intermediate. As next step, this intermediate reacts with a second GSH molecule restoring active site Sec or Cys and producing oxidized glutathione (GSSG). GSSG is subsequently reduced by glutathione reductase (GR), which consumes NADPH as a source of reducing equivalents. [ 3, 6, 7, 8].

Glutathione peroxidase reaction cycle.

Yeast Saccharomyces Cerevisiae cytosolic Glutathione Peroxidases:

>gi|6322826|ref|NP_012899.1| Gpx1 [Saccharomyces cerevisiae]

>gi|6319721|ref|NP_009803.1| Gpx2 [Saccharomyces cerevisiae]

>gi|6322228|ref|NP_012303.1| Gpx3 [Saccharomyces cerevisiae]

There are eight glutathione peroxidase paralogs in mammals. GPX1, GPX2, GPX3, GPX4, and GPX6 are sepenoproteins and contain a Sec residue in their active site. GPX5, GPX7, and GPX8 are Cys-containing glutathione peroxidases. [ 1, 9 ]. In some mammals GPX6 homologs are not selenoproteins and have a Cys in their active site. The Cys-containing GPXs are most abundant in bacteria, protozoa, fungi, and terrestrial plants [ 9 ].

GPX1 is known as most abundant selenoprotein and major hydrogen peroxide scavenging enzyme in mammals. [ 9, 10 ]. Reduced activity of GPX1 leads to elevated intracellular H2O2 level and GPX1 overexpression of GPX1 may lead to reductive stress and may disrupt H2O2 signaling. Mammalian GPX1 is localized in cytosol and forms a homotetramers. GPX1 is expressed in all cell types, with the highest expression levels observed in the liver and kidney. [9]. GPX1 belongs to a group of stress-related selenoproteins and is highly regulated by Se availability. [ 9, 12, 13 ].

Mammalian GPX2 is primarily found in the epithelium of the gastrointestinal tract and has been shown to play a role in the development of cancer. [ 14, 15 ]. GPX3 is secreted primarily from kidney and is the major GPX form in plasma. [ 16 ]. GPX4 is expressed in a wide range of cell types and tissues, and GPx6 is only found in olfactory epithelium and during embryonic development. [ 9 ]. Similarly to GPX1, GPX2 and GPX3 are homotetrameric forms of GPXs.

CPX4 is involved in sperm maturation and serves a structural function in mature sperm cells. [ 17 ]. There are three different alternatively spliced GPx4 mRNA isoforms that code for cytosolic (cGPX4), mitochondrial (mGPX4), and nuclear (nGPX4) proteins. The cytosolic (short) form of GPx4 is ubiquitously expressed both during embryonic development and in adult organs and tissues. However, nuclear and mitochondrial isoforms, which differ from the cytosolic form by their NH2-terminal sequences, are expressed only in testes. The isoform-specific cGPX4 knockout in mice leads to embryonic lethality considering the role of GPX4 in inhibiting lipid peroxidation during early embryo development. [ 17, 18 19, 20 ]. GPX4 has also been implicated in regulating protein tyrosine phosphatases (PTPs) by reversible oxidation of the Cys residue in the active site of PTPs. [ 21 ].

GPX7 and GPX8 are localized in ER and are involved in ER hyrdroperoxides detoxification. [ 9 ].

Mammalian GPX1, GPX2, GPX3, GPX7 and GPX8 are specific for soluble low molecular weight hydroperoxides. GPX4 is involved in the reduction of complex phospholipid hydroperoxides such as phospatidylcholine hydroperoxide and cholesterol hydroperoxide that are associated with membranes [ 17, 22 23 ].

Human Glutathione Peroxidases:

>gi|41406084|ref|NP_000572.2| glutathione peroxidase 1 [Homo sapiens]

>gi|54038169|gb|AAH05277.1| Gastrointestinal glutathione peroxidase 2 [Homo sapiens]

>gi|6006001|ref|NP_002075.2| Plasma glutathione peroxidase 3 [Homo sapiens]

>gi|90903238|ref|NP_001034936.1| Glutathione peroxidase 4 [Homo sapiens]

>gi|3288455|emb|CAA06463.1| Glutathione peroxidase type 5 [Homo sapiens]

>gi|33186887|ref|NP_874360.1| Glutathione peroxidase 6 [Homo sapiens]

>gi|119627182|gb|EAX06777.1| Glutathione peroxidase 7 [Homo sapiens]

>gi|192455698|ref|NP_001008398.2| Glutathione peroxidase 8 [Homo sapiens]

Glutathione peroxidases and signaling.

Recent stadies shown that peroxiredoxins and glutathione peroxidases (thiol peroxidases) are involved in H2O2 mediated signaling and suggested that the role of thiol peroxidases in oxidative stress defense may be overestimated. [ 4, 5, 21, 24 ]. They oxidize regulatory and signaling proteins by transferring of oxidative equvalent from peroxide to signaling proteins, resulting in transcriptional responses and signaling programs. The examples, the response to H2O2 was inhibited in Saccharomyces cerevisiae cells lacking multiple thiol peroxidases. Saccharomyces cerevisiae Gpx3 can serve as an H2O2 sensor and activate the transcription factor Yap1 by forming a disulfide in this protein, and a Schizosaccharomyces pombe thiol peroxidase Tsa1 was found to stimulate signaling through a MAP kinase pathway. Schizosaccharomyces pombe thiol peroxidase Tpx1 similarly regulates transcription factor Pap1. In addition, the ability to transfer oxidative equivalents was demonstrated for mammalian GPX4, PRX4 and signaling kinase 1 (ASK1).

A model of redox regulation of gene expression in yeast. Schematic presentation of the current and proposed models of H2O2 mediated signaling. To activate hydroperoxide-dependent gene expression programs, H2O2 initially oxidizes glutathione peroxidases (glutathione system example), which in turn oxidize transcription factors, kinases and other target proteins in yeast cells. Oxidation of these targets then elicits transcriptional response, redox regulation, signaling pathways and other programs. The model proposes that thiol peroxidases mediate gene expression, whereas a direct interaction between H2O2 and target proteins (dashed arrow) plays a secondary role. The involvement of multiple thiol peroxidases and their regulated interactions with target proteins could explain specificity of the system. Red arrows indicate the direction of electron flow, which is opposite to the direction of thiol peroxidase-mediated oxidative signals.

The major assay for Glutathione Peroxidase activity measurement.

Spectrophotometric assay of NADPH oxidation: Final oxidation of NADPH is followed as the decrease of absorbance at 340 nm. The decrease of NADPH is proportional to GPX activity. [ 25, 26, 27 ].

1) R-OOH + 2GSH + GPX → R-OH + GSSH + H2O ;

2) GSSG + NADPH + H+ + GR → 2GSH + NADP+ ;

GPX is glutathione peroxidase, GR is glutathione reductase, and R-OOH is inorganic or organic hydroperoxide.

Tert-butyl hydroperoxide (t-Bu-OOH) is not metabolized by catalase and can be used for measurement of the selenium-containing glutathione peroxidase. Cumene hydroperoxide can be used as the substrate at a concentration of 0.25-1.0 mM. This will measure the total GPX (Se and non-Se enzymes) activity. The difference between the activity observed with cumene hydroperoxide and the tert-butyl hydroperoxide activity is the non-Se glutathione peroxidase activity.

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