Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities

Abstract

Reactive oxygen species, such as superoxide and hydrogen peroxide, are generated in all cells by mitochondrial and enzymatic sources. Left unchecked, these reactive species can cause oxidative damage to DNA, proteins, and membrane lipids. Glutathione peroxidase-1 (GPx-1) is an intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. Certain reactive oxygen species, such as hydrogen peroxide, are also essential for growth factor-mediated signal transduction, mitochondrial function, and maintenance of normal thiol redox-balance. Thus, by limiting hydrogen peroxide accumulation, GPx-1 also modulates these processes. This review explores the molecular mechanisms involved in regulating the expression and function of GPx-1, with an emphasis on the role of GPx-1 in modulating cellular oxidant stress and redox-mediated responses. As a selenocysteine-containing enzyme, GPx-1 expression is subject to unique forms of regulation involving the trace mineral selenium and selenocysteine incorporation during translation. In addition, GPx-1 has been implicated in the development and prevention of many common and complex diseases, including cancer and cardiovascular disease. This review discusses the role of GPx-1 in these diseases and speculates on potential future therapies to harness the beneficial effects of this ubiquitous antioxidant enzyme.

FIG. 1.

Modulation of cellular reactive oxygen species (ROS). Superoxide originates from normal mitochondrial respiration or from enzymatic sources, such as NADPH oxidases (NOX), uncoupled endothelial nitric oxide synthase (eNOS), or p-450 (CYP) isoforms. Superoxide is dismutated spontaneously or enzymatically to hydrogen peroxide. Extracellular superoxide dismutase (ECSOD), intracellular copper, zinc SOD (Cu, ZnSOD), or mitochondrially localized manganese SOD (MnSOD) are enzymatic sources of this conversion. Hydrogen peroxide can also be produced directly by xanthine oxidase and NADPH oxidase subtype 4. Under certain oxidative stress conditions such as ischemia-reperfusion, hydrogen peroxide can react with free iron to promote the formation of hydroxyl radical. Under normal cellular conditions the amount of free iron in the cell is low limiting the flux through this pathway (illustrated by light gray). Hydrogen peroxide is subsequently enzymatically reduced by glutathione peroxidases (GPxs), including GPx-1, as well as catalase and peroxiredoxins (Prxs). Catalase is primarily in the peroxisomes, whereas various Prxs localize to the mitochondria (e.g., Prx 3,5) or cytosol (such as Prx 1,2). Most Prxs utilize thioredoxin as a source of reducing equivalents, although Prx 6 appears to function as a reduced glutathione (GSH)-dependent peroxidase. Importantly, GPx-1 can be found in the cytosol, in mitochondria, and also in peroxisomes. GPx-1 utilizes GSH as a cofactor to reduce hydrogen peroxide, resulting in the formation of oxidized glutathione (GSSG). For simplicity, peroxisomes, thioredoxin, and mitochondrial GSH, are not represented in this figure.

FIG. 2.

Modulation of oxidative and reductive stress by GPx-1. GPx-1 is one of several cellular enzymes that may modulate overall redox stress. Decreased GPx-1 activity can promote susceptibility to oxidative stress by allowing for the accumulation of harmful oxidants, whereas excess GPx-1 may promote reductive stress, characterized by a lack of essential ROS needed for cellular signaling processes. Excess oxidants or loss of essential ROS can each lead to diminished cell growth and promote apoptotic pathways.

FIG. 3.

Reduction of hydrogen peroxide by GPx-1. The enzymatic inactivation of peroxides by GPx-1 involves the formation of several stable intermediary modifications to the active-site selenocysteine (Sec) (127, 204, 248). Thus, the selenol of GPx-SeH (with -SeH representing the Sec active site) forms a selenenic acid (Se-OH) after reacting with peroxides (no. 1 in the figure). One molecule of GSH reduces selenenic acid leading to the Se-SG intermediate (no. 2 in the figure), which is reduced by the second GSH, resulting in the formation of GSSG (no. 3 in the figure). The net reaction is shown in the lower part of the figure.

FIG. 4.

Redox pathways involved in maintaining the cofactors necessary for the activity of GPx-1. GPx-1 reductively inactivates hydrogen peroxide and lipid hydroperoxides at the expense of GSH, which is oxidized to form GSSG. The enzyme glutathione reductase (GR) recycles GSSG to GSH using NADPH as a source of reducing equivalents, and glucose-6-phosphate dehydrogenase (G6PD) maintains cellular stores of NADPH.

FIG. 5.

Alignment of predicted human GPx proteins. Protein sequences for the Sec-containing GPxs-1–4 were aligned using the MacVector analysis program (version 8.1.2 from Accelyrs). Shown are single-letter amino acid codes for the precursor proteins derived from GenBank reference sequences NM_002083, NM_002084, and NM_002085 for GPx-2, 3, and 4, respectively, that were based on published cDNA sequences (5, 112, 346). The GPx-1 sequence was derived from a human cDNA (384) based on published GPx-1 sequences (263, 340). Conserved residues are boxed in gray, and conserved bases involved in the formation of the enzymatic active site are boxed in white and indicated with asterisks, including the Sec (represented with a U), Trp, and Gln residues (107). A conserved Asn residue that is part of the active site is marked with an “#” (359, 361). Arg residues involved in stabilizing the GSH and GPx-1 interactions are circled in white. Note that these Arg residues are conserved in the highly similar GPx-2 but not in GPx-3 and GPx-4.

FIG. 6.

Regulation of GPx-1 expression and function. GPx-1 can be regulated by transcriptional, post-transcriptional, translational, or post-translational means. Shown is an overview of the factors that regulate the expression and activity of GPx-1. In addition to factors that regulate its transcription, GPx-1 can also be regulated post-transcriptionally by the presence or absence of selenium and cofactors involved in Sec biosynthesis and insertion. Represented in the figure is the stem loop structure or SECIS element formed in the 3′ untranslated region (UTR) of the GPx-1 transcript. Absence of selenium promotes RNA degradation due to nonsense-mediated decay (NMD) and/or the presence of SECIS binding factors that interfere with normal translation. Translation involves special SECIS binding factors, a Sec-specific tRNA (black cloverleaf structure), elongation factor (white oval), and SECIS binding protein 2 (SBP2) (gray oval). Post-translationally, Sec in GPx-1 (reduced form SeH in the figure) may be oxidatively inactivated by excess ROS or by excess NO (SeNO in the figure). In addition Hg and Pb may inactivate GPx-1 and specific protein–protein interactions (black oval represents regulatory protein that binds GPx-1) may also inhibit GPx-1 activity. Kinases, such as c-abl, may activate GPx-1 by phosphorylation (indicated by a P in the figure).

FIG. 7.

Diagram of GPx-1 gene organization and nuclear factor binding sites. The GPx-1 gene consists of two exons (rectangular boxes). The 5′ and 3′ UTRs are shown as white boxes, whereas the protein coding region is shown as a gray box. The TGA Sec codon is in the first exon. Notably, the stop codon is a TAG nucleotide sequence (amber stop codon). The promoter region is expanded on the lower line to illustrate the position of nuclear factor binding sites thought to be important for GPx-1 transcription. The GATA and NFκB nuclear factor binding sites have primarily been studied in mice. All other sites are identified from studies with the human GPx-1 gene.

FIG. 8.

Comparison of cysteine (Cys) and Sec. Sec is the 21st amino acid. Its structure is similar to that of Cys, and, functionally, like Cys, Sec is also redox active.

FIG. 9.

Incorporation of Sec at UGA codons. The recognition of the UGA (opal) codon as a site for incorporation of Sec rather than a stop codon involves many specialized factors, such as SBP2, which binds to the stem-loop or Sec incorporation sequence (SECIS) element in the 3′ UTR of the GPx-1 transcript. SBP2 recruits the specific Sec tRNA and the Sec elongation factor (eEF^sec) that are necessary for insertion of Sec at the ribosomes. Other factors, such as ribosomal L30, NSEP1, and nucleolin, may also bind to the SECIS element to modulate incorporation (28, 99). In the absence of selenium, eIF4a3 competes for SBP2 binding, essentially inhibiting GPx-1 transcription (not shown in figure). UAG (amber) stop codon specifies translational termination of GPx-1.

FIG. 10.

Role of GPx-1 in modulating apoptosis. The extrinsic pathway of apoptosis involves activation of caspase pathways that promote cell death. ROS activates both survival and apoptotic pathways. GPx-1 by modulating cellular hydrogen peroxide can inhibit both pro-survival and pro-apoptotic pathways. The end result (cell death or survival) may depend on the extent and levels of ROS generated. Interestingly, excess ROS as found in GPx-1 deficiency may alter nuclear factor κB (NFκB) signaling to promote pro-apoptotic responses. Normally, NFκB activation results in the upregulation of IAPs and other antiapoptotic genes. Similarly, extracellular signal-related kinase (ERK) and Akt activation can promote IAP or FLICE-inhibitory protein (FLIP) expression to inhibit caspase cascades. c-Jun-amino terminal (stress-activated) kinase (JNK) activation, which can be attenuated by NFκB, augments apoptotic pathways by further stimulating ROS production. GPx-1 overexpression has been shown to specifically suppress the activation of Akt and NFκB pathways. GPx-1 specifically blocks NFκB activation by preventing the degradation of the NFκB inhibitor inhibitor of κB. The intrinsic pathway of apoptosis involves the release of apoptogens like apoptosis-inducing factor (AIF) or cytochrome c from mitochondria. These pathways may be activated by ROS (including hydrogen and lipid hydroperoxides) and caspase cascades that promote Bid (Bcl-2 interacting domain) cleavage. GPx-1 has been shown to attenuate AIF release and enhance the expression of Bcl-2, an antiapoptotic factor.

FIG. 11.

Role of ROS in cell signaling. Cellular ROS is necessary for the cellular responses to growth factor stimulation, the protective responses to excess ROS, and cell proliferation and growth. One of the ways ROS can mediate these pathways is by the oxidative inactivation of phosphatases, such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which antagonizes the action of phosphatidylinositol-3-kinase (PI3K). Inactivation of PTEN promotes Akt signaling. In addition, ROS can modulate growth-factor-mediated trans-activation. ROS from mitochondria are essential in these signaling pathways.

FIG. 12.

GPx-1 gene polymorphisms. There are two common sets of polymorphisms in the GPx-1 protein coding region. One is at the N-terminal region and involves the presence of five, six, or seven in-frame GCG repeats that result in 5–7 Ala residues. The presence of seven Ala repeats may reduce the expression of the protein in response to selenium. Similarly, the single-nucleotide polymorphism at amino acid 198 involves a T for C change that results in a Leu-containing polypeptide (Leu encoded by CTC) with reduced expression in response to selenium compared to the Pro-containing form. The noncoding polymorphisms at −602 upstream of the start site and +2 from the start site in linkage disequilibrium: these pairs of linked changes apparently affect promoter activity in a reporter gene assay.

FIG. 13.

Protective and harmful effects of GPx. This diagram illustrates some of the protective effects (top half) and harmful actions (bottom half) of GPx-1 that are mediated by decreasing cellular ROS. Thus, elimination of excess ROS can protect against apoptotic cell loss or injury during ischemia-reperfusion (including coronary and neuronal); protect against cell toxicity to drugs (preserving neurons, cardiomyocytes, and other cells); and protect cells and susceptible cells against high ROS, including endothelial progenitor cells (EPCs), and β-cells in pancreatic islets. In addition, removal of excess ROS can preserve bioavailable NO, preserving vascular function, which also decreases thrombosis and is antiatherogenic. GPx-1 has also been shown to protect against inflammatory stimuli that may promote a proatherogenic state and foster carcinogenesis. In addition, GPx-1 prevents DNA mutagenesis, which also decreases carcinogenic potential. Harmful effects of GPx-1. Note that many of the harmful effects of GPx-1 occur under conditions of excess GPx-1, which may be induced by physiological conditions such as mechanical stress in the vasculature (375) or under pathological conditions such as in certain cardiomyopathies (297, 405). Overall, by reducing excess oxidants, GPx-1 may allow for tumor survival and growth. Further, removal of essential oxidants can create a reductive stress characterized by loss of normal physiological responses, including reduced vascular responses to hydrogen peroxide and arachidonic acid; decreased mitochondrial function and growth factor (GF) signaling that can cause insulin insensitivity and decreased cell proliferation; and development of cardiomyopathies by mechanisms not completely understood but presumed to involve a reduction in essential levels of hydrogen peroxide.