Selenoproteins are rare proteins among all kingdoms of life containing the 21 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 Gpx4 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.
Singlet oxygen ( 1 O 2 ) is a biologically relevant reactive oxygen species capable of efficiently reacting with cellular constituents. The resulting oxidatively generated damage to nucleic acids, membrane unsaturated lipids, and protein components has been shown to be implicated in several diseases, including arthritis, cataracts, and skin cancer. Singlet oxygen may be endogenously produced, among various possibilities, by myeloperoxidase, an enzyme implicated in inflammation processes, and also efficiently in skin by the UVA component of solar radiation through photosensitization reactions. Emphasis is placed in this Review on the description of the main oxidation reactions initiated by 1 O 2 and the resulting modifications within key cellular targets, including guanine for nucleic acids, unsaturated lipids, and targeted amino acids. Most of these reactions give rise to peroxides and dioxetanes, whose formation has been rationalized in terms of [4+2] cycloaddition and 1,2-cycloaddition with dienes + olefins, respectively. The use of [ 18 O]-labeled thermolabile endoperoxides as a source of [ 18 O]-labeled 1 O 2 has been applied to study mechanistic aspects and preferential targets of 1 O 2 in biological systems. A relevant major topic deals with the search for the molecular signature of the 1 O 2 formation in targeted biomolecules within cells. It may be anticipated that [ 18 O]-labeled 1 O 2 and labeled peroxides in association with sensitive mass spectrometric methods should constitute powerful tools for this purpose.
The decomposition of lipid hydroperoxides into peroxyl radicals is a potential source of singlet oxygen ((1)O(2)) in biological systems. We report herein on evidence of the generation of (1)O(2) from lipid hydroperoxides involving a cyclic mechanism from a linear tetraoxide intermediate proposed by Russell. Using (18)O-labeled linoleic acid hydroperoxide (LA(18)O(18)OH) in the presence of Ce(4+) or Fe(2+), we observed the formation of (18)O-labeled (1)O(2) ((18)[(1)O(2)]) by chemical trapping of (1)O(2) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) by high-performance liquid chromatography coupled to tandem mass spectrometry. Spectroscopic evidence for the generation of (1)O(2) was obtained by measuring (i) the dimol light emission in the red spectral region (lambda > 570 nm); (ii) the monomol light emission in the near-infrared (IR) region (lambda = 1270 nm); and (iii) the quenching effect of sodium azide. Moreover, the presence of (1)O(2) was unequivocally demonstrated by the direct spectral characterization of the near-IR light emission. For the sake of comparison, (1)O(2) deriving from the H(2)O(2)/OCl(-) and H(2)O(2)/MoO(4)(2)(-) systems or from the thermolysis of the endoperoxide of 1,4-dimethylnaphthalene was also monitored. These chemical trapping and photoemission properties clearly demonstrate that the decomposition of LA(18)O(18)OH generates (18)[(1)O(2)], consistent with the Russell mechanism and pointing to the involvement of (1)O(2) in lipid hydroperoxide mediated cytotoxicity.
Although the general mechanisms of lipid oxidation are known, the chemical steps through which photosensitizers and light permeabilize lipid membranes are still poorly understood. Herein we characterized the products of lipid photooxidation and their effects on lipid bilayers, also giving insight into their formation pathways. Our experimental system was designed to allow two phenothiazinium-based photosensitizers (methylene blue, MB, and DO15) to deliver the same amount of singlet oxygen molecules per second to 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine liposome membranes, but with a substantial difference in terms of the extent of direct physical contact with lipid double bonds; that is, DO15 has a 27-times higher colocalization with ω-9 lipid double bonds than MB. Under this condition, DO15 permeabilizes membranes at least 1 order of magnitude more efficiently than MB, a result that was also valid for liposomes made of polyunsaturated lipids. Quantification of reaction products uncovered a mixture of phospholipid hydroperoxides, alcohols, ketones, and aldehydes. Although both photosensitizers allowed the formation of hydroperoxides, the oxidized products that require direct reactions between photosensitizer and lipids were more prevalent in liposomes oxidized by DO15. Membrane permeabilization was always connected with the presence of lipid aldehydes, which cause a substantial decrease in the Gibbs free energy barrier for water permeation. Processes depending on direct contact between photosensitizers and lipids were revealed to be essential for the progress of lipid oxidation and consequently for aldehyde formation, providing a molecular-level explanation of why membrane binding correlates so well with the cell-killing efficiency of photosensitizers.
Soy isoflavonoids have attracted much attention because of their estrogenic activity. To study the intestinal absorption of the isoflavonoids, we investigated the cellular uptake and metabolism of genistein and daidzein and their glucosides, genistin and daidzin, by Caco-2 cell monolayers as a model of the human intestinal epithelium. When Caco-2 monolayers were incubated with genistein or daidzein at 10 micromol/L from the apical (mucosal) side, aglycone was lost from the apical solution for 2.0 h (P < 0.05) and the glucuronide/sulfates appeared at the level of 1-2 micromol/L. In the basolateral (serosal) solution, both intact aglycones and their glucuronide/sulfates increased (P < 0.05) with time and reached approximately 20 and 15% of the initial dose, respectively. The transport of their glucosides, genistin and daidzin, through Caco-2 monolayers was less than one tenth that of the aglycones. The cellular metabolism of genistein was compared with quercetin, kaempferol, luteolin and apigenin. Only genistein aglycone was transported intact to the basolateral solution. Transport was greater (P < 0.05) than that of flavonoid aglycones and was without an appreciable decrease of transepithelial resistance. Radical scavenging activity was not related to the susceptibility to conjugation of flavonoids/isoflavonoids. Affinity to the liposomal membrane was increased in the order of genistin = daidzin < daidzein < genistein << flavonoid aglycones. These results strongly suggest that isoflavone aglycones are taken up into enterocytes more efficiently than their glucosides because of their moderate lipophilicity. Furthermore, they are generally transported to the basolateral side in intact form in contrast to flavonoids, probably due to their unique isoflavonoid structure.
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