Emerging roles for non-selenium containing ER-resident glutathione peroxidases in cell signaling and disease.

Maintenance of cellular redox control is pivotal for normal cellular functions and cell fate decisions including cell death. Among the key cellular redox systems in mammals, the glutathione peroxidase (GPX) family of proteins is the largest conferring multifaceted functions and affecting virtually all cellular processes. The endoplasmic reticulum (ER)-resident GPXs, designated as GPX7 and GPX8, are the most recently added members of this family of enzymes. Recent studies have provided exciting insights how both enzymes support critical processes of the ER including oxidative protein folding, maintenance of ER redox control by eliminating H2O2, and preventing palmitic acid-induced lipotoxicity. Consequently, numerous pathological conditions, such as neurodegeneration, cancer and metabolic diseases have been linked with altered GPX7 and GPX8 expression. Studies in mice have demonstrated that loss of GPX7 leads to increased differentiation of preadipocytes, increased tumorigenesis and shortened lifespan. By contrast, GPX8 deficiency in mice results in enhanced caspase-4/11 activation and increased endotoxic shock in colitis model. With the increasing recognition that both types of enzymes are dysregulated in various tumor entities in man, we deem a review of the emerging roles played by GPX7 and GPX8 in health and disease development timely and appropriate.

Loss of the cystine/glutamate antiporter in melanoma abrogates tumor metastasis and markedly increases survival rates of mice.

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

hIAPP forms toxic oligomers in plasma.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in ß-sheet rich oligomers with increased ß-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.

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.

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in ß-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

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.

ROS, thiols and thiol-regulating systems in male gametogenesis.

BACKGROUND: During maturation and storage, spermatozoa generate substantial amounts of reactive oxygen species (ROS) and are thus forced to cope with an increasingly oxidative environment that is both needed and detrimental to their biology. Such a janus-faceted intermediate needs to be tightly controlled and this is done by a wide array of redox enzymes. These enzymes not only have to prevent unspecific modifications of essential cellular biomolecules by quenching undesired ROS, but they are also required and often directly involved in critical protein modifications. SCOPE OF REVIEW: The present review is conceived to present an update on what is known about critical roles of redox enzymes, whereby special emphasis is put on the family of glutathione peroxidases, which for the time being presents the best characterized tasks during gametogenesis. MAJOR CONCLUSIONS: We therefore demonstrate that understanding the function of (seleno)thiol-based oxidases/reductases is not a trivial task and relevant knowledge will be mainly gained by using robust systems, as exemplified by several (conditional) knockout studies. We thus stress the importance of using such models for providing unequivocal evidence in the molecular understanding of redox regulatory mechanisms in sperm maturation. GENERAL SIGNIFICANCE: ROS are not merely detrimental by-products of metabolism and their proper generation and usage by specific enzymes is essential for vital functions as beautifully exemplified during male gametogenesis. As such, lessons learnt from thiol-based oxidases/reductases in male gametogenesis could be used as a general principle for other organs as it is most likely not only restricted to this developmental phase. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.