Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis.

Selenoproteins are rare proteins among all kingdoms of life containing the 21st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.

Homeostatic control of redox status and health.

Research on oxidants and electrophiles has shifted from focusing on damage to biomolecules to the more fine-grained physiological arena. Redox transitions as excursions from a steady-state redox set point are continually ongoing in maintenance of redox balance. Current excitement on these topics results from the fact that recent research provided mechanistic insight, which gives rise to more concrete and differentiated questions. This Commentary focuses on redox eustress and the feedback restoration of steady state as concepts in active maintenance of physiological health, with brief discussion of redox stress response to viral infection, exemplified by COVID-19.

Phytosome complex of curcumin as complementary therapy of advanced pancreatic cancer improves safety and efficacy of gemcitabine: Results of a prospective phase II trial.

A large body of biomedical evidence indicates that activation of Nrf2 by curcumin increases the nucleophilic tone and damps inflammation cumulatively supporting the malignant phenotype. Conversely, genetic analyses suggest a possible oncogenic nature of constitutive Nrf2 activation since an increased nucleophilic tone is alleged increasing chemoresistance of cancer cells. Aiming to contribute to solve this paradox, this study addressed the issue of safety and efficacy of curcumin as complementary therapy of gemcitabine on pancreatic cancer. This was a single centre, single arm prospective phase II trial. Patients received gemcitabine and Meriva®, a patented preparation of curcumin complexed with phospholipids. Primary endpoint was response rate, secondary endpoints were progression free survival, overall survival, tolerability and quality of life. Analysis of inflammatory biomarkers was also carried out. Fifty-two consecutive patients were enrolled. Forty-four (13 locally advanced and 31 metastatic) were suitable for primary endpoint evaluation. Median age was 66 years (range 42-87); 42 patients had Eastern Cooperative Oncology Group performance status 0-1. The median number of treatment cycle was 4.5 (range 2-14). We observed 27.3% of response rate and 34.1% of cases with stable disease, totalizing a disease control rate of 61.4%. The median progression free survival and overall survival were 8.4 and 10.2 months, respectively. Higher IL-6 and sCD40L levels before treatment were associated to a worse overall survival (p < 0.01). Increases in sCD40L levels after 1 cycle of chemotherapy were associated with a reduced response to the therapy. Grade 3/4 toxicity was observed (neutropenia, 38.6%; anemia, 6.8%). There were no significant changes in quality of life during therapy. In conclusion, the complementary therapy to gemcitabine with phytosome complex of curcumin is not only safe but also efficiently translate in a good response rate in first line therapy of advanced pancreatic cancer.

Protein cysteine oxidation in redox signaling: Caveats on sulfenic acid detection and quantification.

Oxidation of critical signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione. Previous studies have claimed that RSOH can be detected as an adduct (e.g., with 5,5-dimethylcyclohexane-1,3-dione; dimedone). Here, kinetic data are discussed which indicate that few proteins can form RSOH under physiological signaling conditions. We also present experimental evidence that indicates that (1) dimedone reacts rapidly with sulfenyl amides, and more rapidly than with sulfenic acids, and (2) that disulfides can react reversibly with amides to form sulfenyl amides. As some proteins are more stable as the sulfenyl amide than as a glutathionylated species, the former may account for some of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids.

Redox status in a model of cancer stem cells.

Reversible oxidation of Cys residues is a crucial element of redox homeostasis and signaling. According to a popular concept in oxidative stress signaling, the oxidation of targets of signals can only take place following an overwhelming of the cellular antioxidant capacity. This concept, however, ignores the activation of feedback mechanisms possibly leading to a paradoxical effect. In a model of cancer stem cells (CSC), stably overexpressing the TAZ oncogene, we observed that the increased formation of oxidants is associated with a globally more reduced state of proteins. Redox proteomics revealed that several proteins, capable of undergoing reversible redox transitions, are indeed more reduced while just few are more oxidized. Among the proteins more oxidized, G6PDH emerges as both more expressed and activated by oxidation. This accounts for the observed more reduced state of the NADPH/NADP+ couple. The dynamic redox flux generating this apparently paradoxical effect is rationalized in a computational system biology model highlighting the crucial role of G6PDH activity on the rate of redox transitions eventually leading to the reduction of reversible redox switches.

Expression of a Catalytically Inactive Mutant Form of Glutathione Peroxidase 4 (Gpx4) Confers a Dominant-negative Effect in Male Fertility.

The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4(-/-) embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility.

An overview of mechanisms of redox signaling.

A principal characteristic of redox signaling is that it involves an oxidation-reduction reaction or covalent adduct formation between the sensor signaling protein and second messenger. Non-redox signaling may involve alteration of the second messenger as in hydrolysis of GTP by G proteins, modification of the signaling protein as in farnesylation, or simple non-covalent binding of an agonist or second messenger. The chemistry of redox signaling is reviewed here. Specifically we have described how among the so-called reactive oxygen species, only hydroperoxides clearly fit the role of a second messenger. Consideration of reaction kinetics and cellular location strongly suggests that for hydroperoxides, particular protein cysteines are the targets and that the requirements for redox signaling is that these cysteines are in microenvironments in which the cysteine is ionized to the thiolate, and a proton can be donated to form a leaving group. The chemistry described here is the same as occurs in the cysteine and selenocysteine peroxidases that are generally considered the primary defense against oxidative stress. But, these same enzymes can also act as the sensors and transducer for signaling. Conditions that would allow specific signaling by peroxynitrite and superoxide are also defined. Signaling by other electrophiles, which includes lipid peroxidation products, quinones formed from polyphenols and other metabolites also involves reaction with specific protein thiolates. Again, kinetics and location are the primary determinants that provide specificity required for physiological signaling although enzymatic catalysis is not likely involved. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.

BACKGROUND: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. METHODS: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. RESULTS: Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2µM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. CONCLUSIONS: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. GENERAL SIGNIFICANCE: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Cardiolipin drives the catalytic activity of GPX4 on membranes: Insights from the R152H mutant.

The aim of this study was to examine, in biochemical detail, the functional role of the Arg152 residue in the selenoprotein Glutathione Peroxidase 4 (GPX4), whose mutation to His is involved in Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD). Wild-type and mutated recombinant enzymes with selenopcysteine (Sec) at the active site, were purified and structurally characterized to investigate the impact of the R152H mutation on enzymatic function. The mutation did not affect the peroxidase reaction's catalytic mechanism, and the kinetic parameters were qualitatively similar between the wild-type enzyme and the mutant when mixed micelles and monolamellar liposomes containing phosphatidylcholine and its hydroperoxide derivatives were used as substrate. However, in monolamellar liposomes also containing cardiolipin, which binds to a cationic area near the active site of GPX4, including residue R152, the wild-type enzyme showed a non-canonical dependency of the reaction rate on the concentration of both enzyme and membrane cardiolipin. To explain this oddity, a minimal model was developed encompassing the kinetics of both the enzyme interaction with the membrane and the catalytic peroxidase reaction. Computational fitting of experimental activity recordings showed that the wild-type enzyme was surface-sensing and prone to "positive feedback" in the presence of cardiolipin, indicating a positive cooperativity. This feature was minimal, if any, in the mutant. These findings suggest that GPX4 physiology in cardiolipin containing mitochondria is unique, and emerges as a likely target of the pathological dysfunction in SSMD.

Studying interactions by molecular dynamics simulations at high concentration.

Molecular dynamics simulations have been used to study molecular encounters and recognition. In recent works, simulations using high concentration of interacting molecules have been performed. In this paper, we consider the practical problems for setting up the simulation and to analyse the results of the simulation. The simulation of beta 2-microglobulin association and the simulation of the binding of hydrogen peroxide by glutathione peroxidase are provided as examples.

A white paper on Phospholipid Hydroperoxide Glutathione Peroxidase (GPx4) forty years later.

The purification of a protein inhibiting lipid peroxidation led to the discovery of the selenoperoxidase GPx4 forty years ago. Thus, the evidence of the enzymatic activity was reached after identifying the biological effect and unambiguously defined the relationship between the biological function and the enzymatic activity. In the syllogism where GPx4 inhibits lipid peroxidation and its inhibition is lethal, cell death is operated by lipid peroxidation. Based on this rationale, this form of cell death emerged as regulated iron-enforced oxygen toxicity and was named ferroptosis in 2012. In the last decades, we learned that reduction of lipid hydroperoxides is indispensable and, in cooperation with prooxidant systems, controls the critical steady state of lipid peroxidation. This concept defined the GPx4 reaction as both the target for possible anti-cancer therapy and if insufficient, as cause of degenerative diseases. We know the reaction mechanism, but the details of the interaction at the membrane cytosol interface are still poorly defined. We know the gene structure, but the knowledge about expression control is still limited. The same holds true for post-transcriptional modifications. Reverse genetics indicate that GPx4 has a role in inflammation, immunity, and differentiation, but the observations emerging from these studies need a more specifically addressed biochemical evidence. Finally, the role of GPx4 in spermatogenesis disclosed an area unconnected to lipid peroxidation. In its mitochondrial and nuclear form, the peroxidase catalyzes the oxidation of protein thiols in two specific aspects of sperm maturation: stabilization of the mid-piece and chromatin compaction. Thus, although available evidence converges to the notion that GPx4 activity is vital due to the inhibition of lipid peroxidation, it is reasonable to foresee other unknown aspects of the GPx4 reaction to be disclosed.

Grape Seeds Proanthocyanidins: Advanced Technological Preparation and Analytical Characterization.

A "green" solvent-free industrial process (patent pending) is here described for a grape seed extract (GSE) preparation (Ecovitis™) obtained from selected seeds of Veneto region wineries, in the northeast of Italy, by water and selective tangential flow filtration at different porosity. Since a comprehensive, non-ambiguous characterization of GSE is still a difficult task, we resorted to using an integrated combination of gel permeation chromatography (GPC) and electrospray ionization high resolution mass spectrometry (ESI-HRMS). By calibration of retention time and spectroscopic quantification of catechin as chromophore, we succeeded in quantifying GPC polymers up to traces at n = 30. The MS analysis carried out by the ESI-HRMS method by direct-infusion allows the detection of more than 70 species, at different polymerization and galloylation, up to n = 13. This sensitivity took advantage of the nanoscale shotgun approach, although paying the limit of missed separation of stereoisomers. GPC and MS approaches were remarkably well cross-validated by overlapping results. This simple integrated analytical approach has been used for quality control of the production of Ecovitis™. The emerging feature of Ecovitis™ vs. a popular benchmark in the market, produced by a different technology, is the much lower content of species at low n and the corresponding increase of species at high n.

Production and purification of homogenous recombinant human selenoproteins reveals a unique codon skipping event in E. coli and GPX4-specific affinity to bromosulfophthalein.

Selenoproteins are translated via animal domain-specific elongation machineries that redefine dedicated UGA opal codons from termination of translation to selenocysteine (Sec) insertion, utilizing specific tRNA species and Sec-specific elongation factors. This has made recombinant production of mammalian selenoproteins in E. coli technically challenging but recently we developed a methodology that enables such production, using recoding of UAG for Sec in an RF1-deficient host strain. Here we used that approach for production of the human glutathione peroxidases 1, 2 and 4 (GPX1, GPX2 and GPX4), with all these three enzymes being important antioxidant selenoproteins. Among these, GPX4 is the sole embryonically essential enzyme, and is also known to be essential for spermatogenesis as well as protection from cell death through ferroptosis. Enzyme kinetics, ICP-MS and mass spectrometry analyses of the purified recombinant proteins were used to characterize selenoprotein characteristics and their Sec contents. This revealed a unique phenomenon of one-codon skipping, resulting in a lack of a single amino acid at the position corresponding to the selenocysteine (Sec) residue, in about 30% of the recombinant GPX isoenzyme products. We furthermore confirmed the previously described UAG suppression with Lys or Gln as well as a minor suppression with Tyr, together resulting in about 20% Sec contents in the full-length proteins. No additional frameshifts or translational errors were detected. We subsequently found that Sec-containing GPX4 could be further purified over a bromosulfophthalein-column, yielding purified recombinant GPX4 with close to complete Sec contents. This production method for homogenously purified GPX4 should help to further advance the studies of this important selenoprotein.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis.

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.

Aerobic pyruvate metabolism sensitizes cells to ferroptosis primed by GSH depletion.

Ferroptosis is a non-accidental, regulated form of cell death operated by lipid peroxidation under strict control of GPx4 activity. This is consistent with the notion that lipid peroxidation is initiated by radicals produced from decomposition of traces of pre-existing lipid hydroperoxides. The question, therefore, emerges about the formation of these traces of lipid hydroperoxides interacting with Fe2+. In the most realistic option, they are produced by oxygen activated species generated during aerobic metabolism. Screening for metabolic sources of superoxide supporting ferroptosis induced by GSH depletion, we failed to detect, in our cell model, a role of respiratory chain. We observed instead that the pyruvate dehydrogenase complex -as other α keto acid dehydrogenases already known as a major source of superoxide in mitochondria- supports ferroptosis. The opposite effect on ferroptosis by silencing either the E1 or the E3 subunit of the pyruvate dehydrogenase complex pointed out the autoxidation of dihydrolipoamide as the source of superoxide. We finally observed that GSH depletion activates superoxide production, seemingly through the inhibition of the specific kinase that inhibits pyruvate dehydrogenase. In summary, this set of data is compatible with a scenario where the more electrophilic status produced by GSH depletion not only activates ferroptosis by preventing GPx4 activity, but also favors the formation of lipid hydroperoxides. In an attractive perspective of tissue homeostasis, it is the activation of energetic metabolism associated to a decreased nucleophilic tone that, besides supporting energy demanding proliferation, also sensitizes cells to a regulated form of death.

Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation.

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (H2O2) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate H2O2, have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner. Compared with its epimer trans-WOOH and H2O2, cis-WOOH reacts slower with the major arterial forms of glutathione peroxidases and peroxiredoxins while it reacts more readily with its target, protein kinase G 1α. Our results indicate a paradigm of redox signaling by H2O2 via its enzymatic conversion to an amino acid-derived hydroperoxide that 'escapes' effective reductive inactivation to engage in selective oxidative activation of key target proteins.

Signaling functions of reactive oxygen species.

We review signaling by reactive oxygen species, which is emerging as a major physiological process. However, among the reactive oxygen species, H(2)O(2) best fulfills the requirements of being a second messenger. Its enzymatic production and degradation, along with the requirements for the oxidation of thiols by H(2)O(2), provide the specificity for time and place that are required in signaling. Both thermodynamic and kinetic considerations suggest that among possible oxidation states of cysteine, formation of sulfenic acid derivatives or disulfides can be relevant as thiol redox switches in signaling. In this work, the general constraints that are required for protein thiol oxidation by H(2)O(2) to be fast enough to be relevant for signaling are discussed in light of the mechanism of oxidation of the catalytic cysteine or selenocysteine in thiol peroxidases. While the nonenzymatic reaction between thiol and H(2)O(2) is, in most cases, too slow to be relevant in signaling, the enzymatic catalysis of thiol oxidation by these peroxidases provides a potential mechanism for redox signaling.

Estradiol binding prevents ApoB-100 misfolding in electronegative LDL(-).

Seeking for a modified lipoprotein present in plasma that could account for the atherogenic effect of high cholesterol, several years ago electronegative LDL(-) was identified. The peculiar feature of LDL(-) is an apoprotein misfolding that triggers the formation of aggregates, perfectly fitting in size the subendothelial droplets observed in early phases of atherogenesis. Apoprotein misfolding was therefore proposed as a possible atherogenic modification. LDL(-) can be spontaneously produced in vitro by plasma incubation through phospholipid hydrolysis catalyzed by the activity of endogenous phospholipases. As a consequence, apoprotein is misfolded. 17beta-Estradiol (E2), a specific ligand to apoB-100, was used to unravel the relationship between negative charge of the lipoprotein and apoprotein structural/conformational shift. Although E2 addition to plasma does not prevent LDL(-) generation nor phospholipase activity, it deeply stabilizes apoB-100 structure, thus preventing its structural and conformational shift. Apoprotein stabilization extends to lipids. Indeed, while a loosening of lipid packing is observed together with apoprotein misfolding, conversely, when E2 stabilizes apoprotein, lipid structure is preserved. Finally, even in the presence of LDL(-), the E2-stabilized LDL is resistant to aggregation, unambiguously demonstrating that misfolding, but not negative charge, primes aggregation. In conclusion, electronegative charge and misfolding are independent and distinct features of LDL(-), and apoprotein misfolding rather than the increase in the negative charge emerges both as a valid biomarker and as an appealing pharmacological and nutritional target.

Catalytic mechanisms and specificities of glutathione peroxidases: variations of a basic scheme.

Kinetics and molecular mechanisms of GPx-type enzymes are reviewed with emphasis on structural features relevant to efficiency and specificity. In Sec-GPxs the reaction takes place at a single redox centre with selenocysteine as redox-active residue (peroxidatic Sec, U(P)). In contrast, most of the non-vertebrate GPx have the U(P) replaced by a cysteine (peroxidatic Cys, C(P)) and work with a second redox centre that contains a resolving cysteine (C(R)). While the former type of enzymes is more or less specific for GSH, the latter are reduced by "redoxins". The common denominator of the GPx family is the first redox centre comprising the (seleno)cysteine, tryptophan, asparagine and glutamine. In this architectural context the rate of hydroperoxide reduction by U(P) or C(P), respectively, is enhanced by several orders of magnitude compared to that of free selenolate or thiolate. Mammalian GPx-1 dominates H(2)O(2) metabolism, whereas the domain of GPx-4 is the reduction of lipid hydroperoxides with important consequences such as counteracting 12/15-lipoxygenase-induced apoptosis and regulation of inflammatory responses. Beyond, the degenerate GSH specificity of GPx-4 allows selenylation and oxidation to disulfides of protein thiols. Heterodimer formation of yeast GPx with a transcription factor is discussed as paradigm of a redox sensing that might also be valid in vertebrates.

Mitochondrial glutathione peroxidase 4 disruption causes male infertility.

Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.