Glutathione peroxidase 4

Discovery

GPX4 was first discovered in biochemistry laboratories of the University of Padua, where it was described as an enzyme capable of protecting against peroxidation. Its role as an inhibitor of cellular death was only discovered in 2012 by a research group Columbia University.

Function

The antioxidant enzyme glutathione peroxidase 4 (GPX4) belongs to the family of glutathione peroxidases, which consists of 8 known mammalian isoenzymes (GPX1–8). GPX4 catalyzes the reduction of hydrogen peroxide, organic hydroperoxides, and lipid peroxides at the expense of reduced glutathione and functions in the protection of cells against oxidative stress. The oxidized form of glutathione (glutathione disulfide), which is generated during the reduction of hydroperoxides by GPX4, is recycled by glutathione reductase and NADPH/H+. GPX4 differs from the other GPX family members in terms of its monomeric structure, a less restricted dependence on glutathione as reducing substrate, and the ability to reduce lipid-hydroperoxides inside biological membranes.

Inactivation of GPX4 leads to an accumulation of lipid peroxides, resulting in ferroptotic cell death. Mutations in GPX4 cause spondylometaphyseal dysplasia. In vitro studies suggest that GPX4 protects cells against cold-induced cell death.

Therapy-resistant cancer cells in a high-mesenchymal state depend on a lipid peroxidase pathway to suppress ferroptosis, indicating a critical survival mechanism in this cellular context. Drug-tolerant persister cells exhibit a specific dependency on the lipid hydroperoxidase GPX4 for survival; inhibition of GPX4 induces ferroptotic cell death in these cells.

Structure

Mammalian GPX1, GPX2, GPX3, and GPX4 (this protein) have been shown to be selenium-containing enzymes, whereas GPX6 is a selenoprotein in humans with cysteine-containing homologues in rodents. In selenoproteins, the amino acid selenocysteine is inserted in the nascent polypeptide chain during the process of translational recoding of the UGA stop codon. GPX4 shares the amino acid motif of selenocysteine, glutamine, and tryptophan (catalytic triad) with other glutathione peroxidases.

Reaction mechanism

GPX4 catalyzes the following reaction:

• 2 glutathione + lipid–hydroperoxide → glutathione disulfide + lipid–alcohol + H2O

This reaction occurs at the selenocysteine within the catalytic center of GPX4. During the catalytic cycle of GPX4, the active selenol (-SeH) is oxidized by peroxides to selenenic acid (-SeOH), which is then reduced with glutathione (GSH) to an intermediate selenodisulfide (-Se-SG). GPX4 is eventually reactivated by a second glutathione molecule, releasing glutathione disulfide (GS-SG).

Subcellular distribution of isoforms

In mouse and rat, three distinct GPX4 isoforms with different subcellular localization are produced through alternative splicing and transcription initiation; cytosolic GPX4, mitochondrial GPX4 (mGPX4), and nuclear GPX4 (nGPX4). Cytosolic GPX4 has been identified as the only GPX4 isoform being essential for embryonic development and cell survival. The GPX4 isoforms mGPX4 and nGPX4 have been implicated in spermatogenesis and male fertility. In humans, experimental evidence for alternative splicing exists; alternative transcription initiation and the cleavage sites of the mitochondrial and nuclear transit peptides need to be experimentally verified.

Animal models

Knockout mice of GPX4 die at embryonic day 8 and conditional inducible deletion in adult mice (neurons) results in degeneration and death in less than a month. Targeted disruption of the mitochondrial GPX4 isoform (mGPX4) caused infertility in male mice and disruption of the nuclear GPX4 isoform (nGPX4) reduced the structural stability of sperm chromatin, yet both knockout mouse models (for mGPX4 and nGPX4) were fully viable. Surprisingly, knockout of GPX4 heterozygously in mice (GPX4+/−) increases their median life span. Knockout studies with GPX1, GPX2, or GPX3 deficient mice showed that cytosolic GPX4 is so far the only glutathione peroxidase that is indispensable for embryonic development and cell survival. As mechanisms to dispose of both hydrogen peroxide and lipid hydroperoxides are essential to life, this indicates that in contrast to the multiple metabolic pathways that can be utilized to dispose of hydrogen peroxide, pathways for the disposal of lipid hydroperoxides are limited.

While mammals have only one copy of the GPX4 gene, fish have two copies, GPX4a and GPX4b. The GPX4's appear to play a greater role in the fish GPX system than in mammals. For example, in fish GPX4 activity contributes to a greater extent to total GPX activity, GPX4a is the most highly expressed selenoprotein mRNA (in contrast to mammals where it is GPX1 mRNA) and GPX4a appears to be highly inducible to changes within the cellular environment, such as changes in methylmercury and selenium status.

Pathology

The interaction of GPX4 with the autophagic degradation pathway further modulates cell's response to oxidative stress. Impaired GPX4 function plays a role in tumorigenesis, neurodegeneration, infertility, inflammation, immune disorders, and ischemia-reperfusion injury. Additionally, the R152H mutation in GPX4 is involved in the development of Sedaghatian-type spinal metaphyseal dysplasia, a rare and fatal disease in newborn babies.

References

References

1. (July 1994). "Cloning and sequencing of the cDNA encoding a human testis phospholipid hydroperoxide glutathione peroxidase". Gene.
2. (January 2014). "Regulation of ferroptotic cancer cell death by GPX4". Cell.
3. (December 2014). "Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice". Nature Cell Biology.
4. (July 2014). "Mutations in the enzyme glutathione peroxidase 4 cause Sedaghatian-type spondylometaphyseal dysplasia". Journal of Medical Genetics.
5. (2024). "Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death". eLife.
6. (July 2017). "Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway". Nature.
7. (November 2017). "Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition". Nature.
8. (September 2009). "Mitochondrial glutathione peroxidase 4 disruption causes male infertility". FASEB Journal.
9. "Entrez Gene: GPX4 glutathione peroxidase 4 (phospholipid hydroperoxidase)".
10. (February 2003). "The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults". Free Radical Biology & Medicine.
11. (August 2007). "Trends in oxidative aging theories". Free Radical Biology & Medicine.
12. (September 2008). "Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death". Cell Metabolism.
13. (September 2007). "Reduction in glutathione peroxidase 4 increases life span through increased sensitivity to apoptosis". The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences.
14. (2012). "Composition and evolution of the vertebrate and mammalian selenoproteomes". PLOS ONE.
15. (November 2011). "Relationship between oxidizable fatty acid content and level of antioxidant glutathione peroxidases in marine fish". The Journal of Experimental Biology.
16. (2013). "Transcriptomic analyses of sexual dimorphism of the zebrafish liver and the effect of sex hormones". PLOS ONE.
17. (October 2014). "Selenium prevents downregulation of antioxidant selenoprotein genes by methylmercury". Free Radical Biology & Medicine.
18. (October 2023). "GPX4 in cell death, autophagy, and disease". Autophagy.