PRDX6 contributes to selenocysteine metabolism and ferroptosis resistance.

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and has traditionally been thought to begin with the uptake of the Sec carrier selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP undergoes metabolisation via selenocysteine lyase (SCLY), producing selenide, a substrate used by selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor - selenophosphate - for the biosynthesis of the selenocysteine tRNA. Here, we report the discovery of an alternative pathway mediating Sec metabolisation that is independent of SCLY and mediated by peroxiredoxin 6 (PRDX6). Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the presence and functional significance of this alternative route in cancer cells where we reveal a notable association between elevated expression of PRDX6 with a highly aggressive neuroblastoma subtype. Altogether, our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering new avenues for therapeutic exploitation.

The role of ferroptosis in cell-to-cell propagation of cell death initiated from focal injury in cardiomyocytes.

AIMS: Ferroptosis has grown in importance as a key factor in ischemia-reperfusion (I/R) injury. This study explores the mechanism underlying fibrotic scarring extending along myofibers in cardiac ischemic injury and demonstrates the integral role of ferroptosis in causing a unique cell death pattern linked to I/R injury. MAIN METHODS: Cadaveric hearts from individuals who had ischemic injury were examined by histological assays. We created a novel model of inducing cell death in H9c2 cells, and used it to demonstrate ferroptotic cell death extending in a cell-to-cell manner. Ex vivo Langendorff-perfused hearts were used alongside the model to replicate cell death extension along myofibers while also demonstrating protective effects of a ferroptosis inhibitor, ferrostatin-1 (Fer-1). KEY FINDINGS: Human hearts from individuals who had I/R injury demonstrated scarring along myofibers that was consistent with mouse models, suggesting that cell death extended from cell-to-cell. Treatment with Ras-selective lethal 3 (RSL3), a ferroptosis inducer, and exposure to excess iron exacerbated cell death propagation in in vitro models, and inhibition of ferroptosis by Fer-1 blunted this effect in both settings. In ex vivo models, Fer-1 was sufficient to reduce cell death along the myofibers caused by external injury. SIGNIFICANCE: The unique I/R injury-induced pattern of cell death along myofibers requires novel injury models that mimic this phenomenon, thus we established new methods to replicate it. Ferroptosis is important in propagating injury between cells and better understanding this mechanism may lead to therapeutic responses that limit I/R injury.

Metabolism of Selenium, Selenocysteine, and Selenoproteins in Ferroptosis in Solid Tumor Cancers.

A potential target of precision nutrition in cancer therapeutics is the micronutrient selenium (Se). Se is metabolized and incorporated as the amino acid selenocysteine (Sec) into 25 human selenoproteins, including glutathione peroxidases (GPXs) and thioredoxin reductases (TXNRDs), among others. Both the processes of Se and Sec metabolism for the production of selenoproteins and the action of selenoproteins are utilized by cancer cells from solid tumors as a protective mechanism against oxidative damage and to resist ferroptosis, an iron-dependent cell death mechanism. Protection against ferroptosis in cancer cells requires sustained production of the selenoprotein GPX4, which involves increasing the uptake of Se, potentially activating Se metabolic pathways such as the trans-selenation pathway and the TXNRD1-dependent decomposition of inorganic selenocompounds to sustain GPX4 synthesis. Additionally, endoplasmic reticulum-resident selenoproteins also affect apoptotic responses in the presence of selenocompounds. Selenoproteins may also help cancer cells adapting against increased oxidative damage and the challenges of a modified nutrient metabolism that result from the Warburg switch. Finally, cancer cells may also rewire the selenoprotein hierarchy and use Se-related machinery to prioritize selenoproteins that are essential to the adaptations against ferroptosis and oxidative damage. In this review, we discuss both the evidence and the gaps in knowledge on how cancer cells from solid tumors use Se, Sec, selenoproteins, and the Se-related machinery to promote their survival particularly via resistance to ferroptosis.

Selenium and selenoproteins in thermogenic adipocytes.

Selenium (Se) is involved in energy metabolism in the liver, white adipose tissue, and skeletal muscle, and may also play a role in thermogenic adipocytes, i.e. brown and beige adipocytes. Thereby this micronutrient is a key nutritional target to aid in combating obesity and metabolic diseases. In thermogenic adipocytes, particularly in brown adipose tissue (BAT), the selenoprotein type 2 iodothyronine deiodinase (DIO2) is essential for the activation of adaptive thermogenesis. Recent evidence has suggested that additional selenoproteins may also be participating in this process, and a role for Se itself through its metabolic pathways is also envisioned. In this review, we discuss the recognized effects and the knowledge gaps in the involvement of Se metabolism and selenoproteins in the mechanisms of adaptive thermogenesis in thermogenic (brown and beige) adipocytes.

TLR7 Mediates Acute Respiratory Distress Syndrome in Sepsis by Sensing Extracellular miR-146a.

TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment in vitro. Both conditioned media of miR-146a-5p-treated macrophages (Mϕ) and septic sera of wild-type mice induce a marked EC barrier disruption in vitro, whereas Mϕ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the Mϕ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.

Pyruvate-Driven Oxidative Phosphorylation is Downregulated in Sepsis-Induced Cardiomyopathy: A Study of Mitochondrial Proteome.

BACKGROUND: Sepsis-induced cardiomyopathy (SIC) is a major contributing factor for morbidity and mortality in sepsis. Accumulative evidence has suggested that cardiac mitochondrial oxidative phosphorylation is attenuated in sepsis, but the underlying molecular mechanisms remain incompletely understood. METHODS: Adult male mice of 9 to 12 weeks old were subjected to sham or cecal ligation and puncture procedure. Echocardiography in vivo and Langendorff-perfused hearts were used to assess cardiac function 24 h after the procedures. Unbiased proteomics analysis was performed to profile mitochondrial proteins in the hearts of both sham and SIC mice. Seahorse respirator technology was used to evaluate oxygen consumption in purified mitochondria. RESULTS: Of the 665 mitochondrial proteins identified in the proteomics assay, 35 were altered in septic mice. The mitochondrial remodeling involved various energy metabolism pathways including subunits of the electron transport chain, fatty acid catabolism, and carbohydrate oxidative metabolism. We also identified a significant increase of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) and inhibition of PDH activity in septic hearts. Furthermore, compared to sham mice, mitochondrial oxygen consumption of septic mice was significantly reduced when pyruvate was provided as a substrate. However, it was unchanged when PDH was bypassed by directly supplying the Complex I substrate NADH, or by using the Complex II substrate succinate, or using Complex IV substrate, or by providing the beta-oxidation substrate palmitoylcarnitine, neither of which require PDH for mitochondrial oxygen consumption. CONCLUSIONS: These data demonstrate a broad mitochondrial protein remodeling, PDH inactivation and impaired pyruvate-fueled oxidative phosphorylation during SIC, and provide a molecular framework for further exploration.

The Impact of Selenium Deficiency on Cardiovascular Function.

Selenium (Se) is an essential trace element that is necessary for various metabolic processes, including protection against oxidative stress, and proper cardiovascular function. The role of Se in cardiovascular health is generally agreed upon to be essential yet not much has been defined in terms of specific functions. Se deficiency was first associated with Keshan's Disease, an endemic disease characterized by cardiomyopathy and heart failure. Since then, Se deficiency has been associated with multiple cardiovascular diseases, including myocardial infarction, heart failure, coronary heart disease, and atherosclerosis. Se, through its incorporation into selenoproteins, is vital to maintain optimal cardiovascular health, as selenoproteins are involved in numerous crucial processes, including oxidative stress, redox regulation, thyroid hormone metabolism, and calcium flux, and inadequate Se may disrupt these processes. The present review aims to highlight the importance of Se in cardiovascular health, provide updated information on specific selenoproteins that are prominent for proper cardiovascular function, including how these proteins interact with microRNAs, and discuss the possibility of Se as a potential complemental therapy for prevention or treatment of cardiovascular disease.

ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions.

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

mTOR-mediated calcium transients affect cardiac function in ex vivo ischemia-reperfusion injury.

The mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, cell growth, and survival. While previous studies using transgenic mice with cardiac-specific overexpression of mTOR (mTOR-Tg) demonstrated the protective effects of cardiac mTOR against ischemia-reperfusion (I/R) injury in both ex vivo and in vivo models, the mechanisms underlying the role of cardiac mTOR in cardiac function following I/R injury are not well-understood. Torin1, a pharmacological inhibitor of mTOR complex (mTORC) 1 and mTORC2, significantly decreased functional recovery of LV developed pressure in ex vivo I/R models (p < 0.05). To confirm the role of mTOR complexes in I/R injury, we generated cardiac-specific mTOR-knockout (CKO) mice. In contrast to the effects of Torin1, CKO hearts recovered better after I/R injury than control hearts (p < 0.05). Interestingly, the CKO hearts had exhibited irregular contractions during the reperfusion phase. Calcium is a major factor in Excitation-Contraction (EC) coupling via Sarcoplasmic Reticulum (SR) calcium release. Calcium is also key in opening the mitochondrial permeability transition pore (mPTP) and cell death following I/R injury. Caffeine-induced SR calcium release in isolated CMs showed that total SR calcium content was lower in CKO than in control CMs. Western blotting showed that a significant amount of mTOR localizes to the SR/mitochondria and that GSK3-ß phosphorylation, a key factor in SR calcium mobilization, was decreased. These findings suggest that cardiac mTOR located to the SR/mitochondria plays a vital role in EC coupling and cell survival in I/R injury.

Extracellular miR-146a-5p Induces Cardiac Innate Immune Response and Cardiomyocyte Dysfunction.

Previous studies have demonstrated that transient myocardial ischemia leads to release of cellular nucleic acids such as RNA. Extracellular RNA reportedly plays a pivotal role in myocardial inflammation and ischemic injury in animals. RNA profiling has identified that numerous microRNA (miRNAs), such as ss-miR-146a-5p, are upregulated in plasma following myocardial ischemia, and certain uridine-rich miRNAs exhibit strong proinflammatory effects in immune cells via ssRNA-sensing mechanism. However, the effect of extracellular miRNAs on myocardial inflammation and cardiac cell function remains unknown. In this study, we treated adult mouse cardiomyocytes with miR-146a-5p loaded in extracellular vesicles and observed a dose- and TLR7-dependent production of CXCL-2, IL-6, and TNF-α. In vivo, a single dose of myocardial injection of miR-146a-5p induced both cytokine expression (CXCL2, IL-6, and TNF-α) and innate immune cell activation (CD45+ leukocytes, Ly6Cmid+ monocytes, Ly6G+ neutrophils), which was significantly attenuated in the hearts of TLR7 KO mice. We discovered that conditioned media from miR-146a-treated macrophages stimulated proinflammatory cytokine production in adult cardiomyocytes and significantly inhibited their sarcomere shortening. Finally, using an electric cell impedance-sensing assay, we found that the conditioned media from miR-146a-treated cardiac fibroblasts or cardiomyocytes impaired the barrier function of coronary artery endothelial cells. Taken together, these data demonstrate that extracellular miR-146a-5p activates multiple cardiac cells and induces myocardial inflammation and cardiomyocyte dysfunction via intercellular interaction and innate immune TLR7 nucleic acid sensing.

The effects of Tel2 on cardiomyocyte survival.

AIMS: Overexpression of the mechanistic target of rapamycin (mTOR), a member of the PIKK (phosphoinositide kinase-related kinase) family, protects cardiomyocytes from cell death induced by pathological stimuli such as ischemia. We previously reported that posttranslational modification of mTOR plays an important role in regulating cardiac mTOR expression. The aim of this study was to see if Tel2 (telomere maintenance 2), a protein that regulates the abundance of PIKKs, confers similar cardioprotective effects as mTOR. Tel2 is not well-characterized in cardiomyocytes, therefore we examined the effects of Tel2 on cardiomyocyte viability under ischemic stress conditions. MATERIALS AND METHODS: We overexpressed Tel2 or silenced Tel2 with siRNA in the HL-1 cardiomyocyte cell line to survey the effects of Tel2 overexpression and downregulation on cell survival during hypoxia. Adult mouse cardiomyocytes transfected with Tel2 adenoviruses were used to test whether Tel2 sufficiently prevented cardiomyocyte cell death against hydrogen peroxide (H2O2). KEY FINDINGS: Overexpressing Tel2 increased mTOR expression with a concomitant increase in mTOR Complex 1 (mTORC1) and mTORC2 activity in HL-1 cells. Tel2 deletion decreased mTOR expression, and mTORC1 and mTORC2 activity accordingly. In both HL-1 cells and adult mouse cardiomyocytes, Tel2 overexpression protected cardiomyocytes under ischemic stress. These effects were mTOR-dependent, as mTOR inhibitors blunted the effects of Tel2. While gene silencing of Tel2 did not affect cell survival under normoxia, Tel2 silencing made cardiomyocytes more vulnerable to cell death under hypoxia. SIGNIFICANCE: Upregulating Tel2 expression increases mTOR-mediated cardiomyocyte survival and targeting Tel2 could be another therapeutic strategy against ischemic heart disease.

Protective effects of the mechanistic target of rapamycin against excess iron and ferroptosis in cardiomyocytes.

Clinical studies have suggested that myocardial iron is a risk factor for left ventricular remodeling in patients after myocardial infarction. Ferroptosis has recently been reported as a mechanism of iron-dependent nonapoptotic cell death. However, ferroptosis in the heart is not well understood. Mechanistic target of rapamycin (mTOR) protects the heart against pathological stimuli such as ischemia. To define the role of cardiac mTOR on cell survival in iron-mediated cell death, we examined cardiomyocyte (CM) cell viability under excess iron and ferroptosis conditions. Adult mouse CMs were isolated from cardiac-specific mTOR transgenic mice, cardiac-specific mTOR knockout mice, or control mice. CMs were treated with ferric iron [Fe(III)]-citrate, erastin, a class 1 ferroptosis inducer, or Ras-selective lethal 3 (RSL3), a class 2 ferroptosis inducer. Live/dead cell viability assays revealed that Fe(III)-citrate, erastin, and RSL3 induced cell death. Cotreatment with ferrostatin-1, a ferroptosis inhibitor, inhibited cell death in all conditions. mTOR overexpression suppressed Fe(III)-citrate, erastin, and RSL3-induced cell death, whereas mTOR deletion exaggerated cell death in these conditions. 2',7'-Dichlorodihydrofluorescein diacetate measurement of reactive oxygen species (ROS) production showed that erastin-induced ROS production was significantly lower in mTOR transgenic versus control CMs. These findings suggest that ferroptosis is a significant type of cell death in CMs and that mTOR plays an important role in protecting CMs against excess iron and ferroptosis, at least in part, by regulating ROS production. Understanding the effects of mTOR in preventing iron-mediated cell death will provide a new therapy for patients with myocardial infarction. NEW & NOTEWORTHY Ferroptosis has recently been reported as a new form of iron-dependent nonapoptotic cell death. However, ferroptosis in the heart is not well characterized. Using cultured adult mouse cardiomyocytes, we demonstrated that the mechanistic target of rapamycin plays an important role in protecting cardiomyocytes against excess iron and ferroptosis.

The mTOR Signaling Pathway in Myocardial Dysfunction in Type 2 Diabetes Mellitus.

PURPOSE OF REVIEW: T2DM (type 2 diabetes mellitus) is a risk factor for heart failure. The mTOR (mechanistic target of rapamycin) is a key mediator of the insulin signaling pathway. We will discuss the role of mTOR in myocardial dysfunction in T2DM. RECENT FINDINGS: In T2DM, chronically activated mTOR induces multiple pathological events, including a negative feedback loop that suppresses IRS (insulin receptor substrate)-1. While short-term treatment with rapamycin, an mTOR inhibitor, is a promising strategy for cardiac diseases such as acute myocardial infarction and cardiac hypertrophy in T2DM, there are many concerns about chronic usage of rapamycin. Two mTOR complexes, mTORC1 and mTORC2, affect many molecules and processes via distinct signaling pathways that regulate cardiomyocyte function and survival. Understanding mechanisms underlying mTOR-mediated pathophysiological features in the heart is essential for developing effective therapies for cardiac diseases in the context of T2DM.

Cardiac mTOR rescues the detrimental effects of diet-induced obesity in the heart after ischemia-reperfusion.

Diet-induced obesity deteriorates the recovery of cardiac function after ischemia-reperfusion (I/R) injury. While mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, the effects of cardiac mTOR in ischemic injury under metabolic syndrome remains undefined. Using cardiac-specific transgenic mice overexpressing mTOR (mTOR-Tg mice), we studied the effect of mTOR on cardiac function in both ex vivo and in vivo models of I/R injury in high-fat diet (HFD)-induced obese mice. mTOR-Tg and wild-type (WT) mice were fed a HFD (60% fat by calories) for 12 wk. Glucose intolerance and insulin resistance induced by the HFD were comparable between WT HFD-fed and mTOR-Tg HFD-fed mice. Functional recovery after I/R in the ex vivo Langendorff perfusion model was significantly lower in HFD-fed mice than normal chow diet-fed mice. mTOR-Tg mice demonstrated better cardiac function recovery and had less of the necrotic markers creatine kinase and lactate dehydrogenase in both feeding conditions. Additionally, mTOR overexpression suppressed expression of proinflammatory cytokines, including IL-6 and TNF-α, in both feeding conditions after I/R injury. In vivo I/R models showed that at 1 wk after I/R, HFD-fed mice exhibited worse cardiac function and larger myocardial scarring along myofibers compared with normal chow diet-fed mice. In both feeding conditions, mTOR overexpression preserved cardiac function and prevented myocardial scarring. These findings suggest that cardiac mTOR overexpression is sufficient to prevent the detrimental effects of diet-induced obesity on the heart after I/R, by reducing cardiac dysfunction and myocardial scarring.

Pseudomonas syringae coordinates production of a motility-enabling surfactant with flagellar assembly.

Using a sensitive assay, we observed low levels of an unknown surfactant produced by Pseudomonas syringae pv. syringae B728a that was not detected by traditional methods yet enabled swarming motility in a strain that exhibited deficient production of syringafactin, the main characterized surfactant produced by P. syringae. Random mutagenesis of the syringafactin-deficient strain revealed an acyltransferase with homology to rhlA from Pseudomonas aeruginosa that was required for production of this unidentified surfactant, subsequently characterized by mass spectrometry as 3-(3-hydroxyalkanoyloxy) alkanoic acid (HAA). Analysis of other mutants with altered surfactant production revealed that HAA is coordinately regulated with the late-stage flagellar gene encoding flagellin; mutations in genes involved in early flagellar assembly abolish or reduce HAA production, while mutations in flagellin or flagellin glycosylation genes increase its production. When colonizing a hydrated porous surface, the bacterium increases production of both flagellin and HAA. P. syringae was defective in porous-paper colonization without functional flagella and was slightly inhibited in this movement when it lacked surfactant production. Loss of HAA production in a syringafactin-deficient strain had no effect on swimming but abolished swarming motility. In contrast, a strain that lacked HAA but retained syringafactin production exhibited broad swarming tendrils, while a syringafactin-producing strain that overproduced HAA exhibited slender swarming tendrils. On the basis of further analysis of mutants altered in HAA production, we discuss its regulation in Pseudomonas syringae.

Novel high-throughput detection method to assess bacterial surfactant production.

A novel biosurfactant detection assay was developed for the observation of surfactants on agar plates. By using an airbrush to apply a fine mist of oil droplets, surfactants can be observed instantaneously as halos around biosurfactant-producing colonies. This atomized oil assay can detect a wide range of different synthetic and bacterially produced surfactants. This method could detect much lower concentrations of many surfactants than a commonly used water drop collapse method. It is semiquantitative and therefore has broad applicability for uses such as high-throughput mutagenesis screens of biosurfactant-producing bacterial strains. The atomized oil assay was used to screen for mutants of the plant pathogen Pseudomonas syringae pv. syringae B728a that were altered in the production of biosurfactants. Transposon mutants displaying significantly altered surfactant halos were identified and further analyzed. All mutants identified displayed altered swarming motility, as would be expected of surfactant mutants. Additionally, measurements of the transcription of the syringafactin biosynthetic cluster in the mutants, the principal biosurfactant known to be produced by B728a, revealed novel regulators of this pathway.