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Oxidative stress-mediated formation of protein hydroperoxides can induce irreversible fragmentation of the peptide backbone and accumulation of cross-linked protein aggregates, leading to cellular toxicity, dysfunction, and death. However, how bacteria protect themselves from damages caused by protein hydroperoxidation is unknown. Here, we show that YjbI, a group II truncated haemoglobin from Bacillus subtilis , prevents oxidative aggregation of cell-surface proteins by its protein hydroperoxide peroxidase-like activity, which removes hydroperoxide groups from oxidised proteins. Disruption of the yjbI gene in B. subtilis lowered biofilm water repellence, which associated with the cross-linked aggregation of the biofilm matrix protein TasA. YjbI was localised to the cell surface or the biofilm matrix, and the sensitivity of planktonically grown cells to generators of reactive oxygen species was significantly increased upon yjbI disruption, suggesting that YjbI pleiotropically protects labile cell-surface proteins from oxidative damage. YjbI removed hydroperoxide residues from the model oxidised protein substrate bovine serum albumin and biofilm component TasA, preventing oxidative aggregation in vitro. Furthermore, the replacement of Tyr 63 near the haem of YjbI with phenylalanine resulted in the loss of its protein peroxidase-like activity, and the mutant gene failed to rescue biofilm water repellency and resistance to oxidative stress induced by hypochlorous acid in the yjbI -deficient strain. These findings provide new insights into the role of truncated haemoglobin and the importance of hydroperoxide removal from proteins in the survival of aerobic bacteria.

The incidence of atherosclerosis increases with age. Oxidative changes in proteins and lipids are considered to be among the molecular mechanisms leading to endothelial dysfunction. Paraoxonase (PON1) is exclusively associated with high-density lipoprotein (HDL) and protects both HDL and low-density lipoprotein (LDL) from oxidation. PON1 has two cysteine residues for its antioxidant function. We investigated the relation between PON1 activity and protein oxidation parameters such as protein hydroperoxides (P-OOH), protein carbonyl (PCO), total thiol (T-SH) and advanced oxidation protein products (AOPP). Our study also covered other oxidative stress parameters such as oxidised LDL (oxLDL) and superoxide dismutase activity in the plasma of young, middle-aged and elderly individuals. PON1 activity of elderly and middle-aged individuals was decreased significantly compared with that in the young group. oxLDL levels of elderly individuals were increased significantly compared with those of both the young and middle-aged individuals. P-OOH, PCO and AOPP levels of the elderly and middle aged individuals were higher compared with those of the young. On the other hand, T-SH levels of the elderly and middle-aged individuals were lower compared with those of the young. Side by side with the decrease in the T-SH levels in the middle-aged and elderly groups as compared to the young, the increase we have observed in other protein oxidation parameters in the groups leading to decreasing PON1 activity might, we think, create a predisposition to atherosclerosis.

The reasons for the difference in life expectancy between males and females are still unknown. Previous studies have provided compelling evidence for the presence of oxidized proteins, and lipids in advanced human atherosclerotic lesions. The gender factor responsible for such protein oxidation is unknown and controversial. Our aim was to reveal the difference between myocardial protein and lipid oxidation parameters of male and female aged rats. We investigated the relation between myocardial protein carbonyl (PCO) and other protein oxidation parameters such as advanced oxidation protein products (AOPP), nitrotyrosine (NT), protein hydroperoxide (P-OOH), and protein thiol (P-SH). Our study also covered other oxidative stress parameters, such as total thiol (T-SH), non-protein thiol (Np-SH), 4-hydroxyalkenal (4-HAE), malondialdehyde (MDA), reduced glutathione (GSH), and the glutathione disulfide (GSSG). Among the studied parameters, myocardial PCO, AOPP, NT, Np-SH, GSH, Fe(2+) levels and the redox index (RI) of male rats were significantly higher than in the female group. On the other hand, P-OOH, P-SH, T-SH, 4-HAE, and MDA levels were all found to be not different. These data support the hypothesis that elevated levels of PCO, AOPP, and NT contribute to the extent of protein, but not lipid, oxidation in aged male rats. We are of the conviction that the increased myocardial Np-SH, GSH and RI levels that we have determined in aged male rats may be a protective factor in propagation of protein oxidation. Our findings support our conviction that protein and lipid oxidation, in the myocardial tissue of aged rats, have a controlling role in differing regulating mechanisms through gender differences.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids 1 , 2 . The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols 3 , 4 . Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions 2 , and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death 5 . However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines 6 , which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR–Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q 10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents. A synthetic lethal CRISPR–Cas9 screen identifies ferroptosis suppressor protein 1 as a key ferroptosis-resistance factor, the expression of which correlates with ferroptosis resistance in hundreds of cancer cell lines.

System x c − contributes to glutathione (GSH) synthesis and protects cells against ferroptosis by importing cystine and exchanging it with glutamate. Transforming growth factor β1 (TGF-β1) induces redox imbalance; however, its role in system x c − regulation remains poorly understood. The present study was the first to show that TGF-β1 repressed the protein and mRNA levels of xCT, a catalytic subunit of system x c − , in PLC/PRF/5, Huh7, Huh6, and HepG2 cells with an early TGF-β1 gene signature but not in SNU387, SNU449, SNU475, and SK-Hep1 cells with a late TGF-β1 gene signature. TGF-β1 treatment for 24 h reduced xCT expression in a dose-dependent manner but this TGF-β1-induced repression was blunted by pretreatment with a TGF-β1 receptor inhibitor. TGF-β1-mediated xCT repression was prevented by Smad3, but not Smad2 or Smad4, knockdown, whereas it was enhanced by Smad3 overexpression. TGF-β1 decreased GSH levels in control cells but not xCT-overexpressed cells. Furthermore, TGF-β1 increased reactive oxygen species (ROS) levels in PLC/PRF/5 cells and enhanced tert-butyl hydroperoxide-induced ROS levels in Huh7 cells; these changes were reversed by xCT overexpression. TGF-β1 treatment ultimately induced the ferrostatin-1- and deferoxamine-dependent lipid peroxidation after 2 days and 8 days in PLC/PRF/5 and Huh7 cells but not in SNU475 and SK-Hep1 cells. Pre-treatment of TGF-β1 for 2 days enhanced the reduction of cell viability induced by RSL3, a GSH peroxidase 4 (GPX4) inhibitor, in PLC/PRF/5 and Huh7 cells. In conclusion, TGF-β1 represses xCT expression via Smad3 activation and enhances lipid peroxidation in hepatocellular carcinoma cells with an early TGF-β1 signature, which would benefit from the targeting of GPX4.

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 ( Fsp1 ) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 ( Gpx4 cys/- ) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis. Necroptosis, a form of cell death, occurs in acute renal injury. Here, the authors show that ferroptosis—a form of cell death dependent on iron - also occurs during acute kidney injury, and show that an inhibitor of ferroptosis can improve survival in a mouse model of acute kidney damage.

This study aimed to elucidate the role of glutathione peroxidase 4 (GPX4) on the sterol regulatory element binding proteins (SREBPs)-proliferation pathway in oral cancer cells, and determine its protein expression in oral cancer tissues. Quantitative RT-PCR and immunoblot analysis were carried out. Cell viability assay, apoptosis detection assay, immunohistochemistry and GPX4 knockdown were performed. The levels of both GPX4 mRNA and protein were highest in SAS cells. GPX4 knockdown in SAS cells, a human oral squamous cell carcinoma cell line, using GPX4 siRNA resulted in a reduction in cell number, which appeared to be due to non-apoptotic cell death such as ferroptosis. Furthermore, SREBP was clearly down-regulated by GPX4 knockdown in SAS cells. Immunopositivity for GPX4 was revealed on the membrane of human oral squamous cell carcinoma cells, and this was correlated with p53 immunoreactivity. GPX4 appears to play an important role in oral cancer proliferation.

Ferroptosis is a more recently recognized form of cell death that relies on iron-mediated oxidative damage. Here, we evaluate the impact of high-iron diets or depletion of Gpx4 , an antioxidant enzyme reported as an important ferroptosis suppressor, in the pancreas of mice with cerulean- or L-arginine-induced pancreatitis, and in an oncogenic Kras murine model of spontaneous pancreatic ductal adenocarcinoma (PDAC). We find that either high-iron diets or Gpx4 depletion promotes 8-OHG release and thus activates the TMEM173/STING-dependent DNA sensor pathway, which results in macrophage infiltration and activation during Kras -driven PDAC in mice. Consequently, the administration of liproxstatin-1 (a ferroptosis inhibitor), clophosome-mediated macrophage depletion, or pharmacological and genetic inhibition of the 8-OHG-TMEM173 pathway suppresses Kras -driven pancreatic tumorigenesis in mice. GPX4 is also a prognostic marker in patients with PDAC. These findings provide pathological and mechanistic insights into ferroptotic damage in PDAC tumorigenesis in mice. Ferroptosis is an iron-dependent mechanism of cell death. In this mouse study, the authors show that diets high in iron or depletion of the antioxidant Gpx4 potentiates pancreatic damage and tumour formation by activating the DNA damage pathway and recruiting macrophages to the pancreas.

Ferroptosis, a form of regulated cell death that is induced by excessive lipid peroxidation, is a key tumour suppression mechanism 1 – 4 . Glutathione peroxidase 4 (GPX4) 5 , 6 and ferroptosis suppressor protein 1 (FSP1) 7 , 8 constitute two major ferroptosis defence systems. Here we show that treatment of cancer cells with GPX4 inhibitors results in acute depletion of N -carbamoyl- l -aspartate, a pyrimidine biosynthesis intermediate, with concomitant accumulation of uridine. Supplementation with dihydroorotate or orotate—the substrate and product of dihydroorotate dehydrogenase (DHODH)—attenuates or potentiates ferroptosis induced by inhibition of GPX4, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4 low ). Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4 low cancer cells, and synergizes with ferroptosis inducers to induce these effects in GPX4 high cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity). The DHODH inhibitor brequinar selectively suppresses GPX4 low tumour growth by inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis-inducing activity, synergistically induces ferroptosis and suppresses GPX4 high tumour growth. Our results identify a DHODH-mediated ferroptosis defence mechanism in mitochondria and suggest a therapeutic strategy of targeting ferroptosis in cancer treatment. DHO dehydrogenase regulates ferroptosis by preventing mitochondrial lipid peroxidation and its inhibition suppresses growth in tumours with low levels of GPX4.

Ferroptosis is a non-apoptotic form of cell death that can be triggered by small molecules or conditions that inhibit glutathione biosynthesis or the glutathione-dependent antioxidant enzyme glutathione peroxidase 4 (GPX4). This lethal process is defined by the iron-dependent accumulation of lipid reactive oxygen species and depletion of plasma membrane polyunsaturated fatty acids. Cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression may be sensitized to this process. Conversely, a number of small molecule inhibitors of ferroptosis have been identified, including ferrostatin-1 and liproxstatin-1, which can block pathological cell death events in brain, kidney and other tissues. Recent work has identified a number of genes required for ferroptosis, including those involved in lipid and amino acid metabolism. Outstanding questions include the relationship between ferroptosis and other forms of cell death, and whether activation or inhibition of ferroptosis can be exploited to achieve desirable therapeutic ends.