RESUMEN
Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The â¼50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of transfer RNA (tRNA) wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human hepatocellular carcinoma cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to demonstrating an epitranscriptomic facet of arsenite toxicity and response, our results highlight the links between environmental exposures, stress tolerance, RNA modifications, and micronutrients.
Asunto(s)
Arsenitos , Epigénesis Genética , Guanina , ARN de Transferencia , Transcriptoma , Arsenitos/toxicidad , Línea Celular Tumoral , Codón/genética , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN de Transferencia/genéticaRESUMEN
Mitochondria serves a primary role in energy maintenance but also function to govern levels of mitochondria-derived reactive oxygen species (mROS). ROS have long been established to play a critical role in tumorigenesis and are now considered to be integral to the regulation of diverse signaling networks that drive proliferation, tumor cell survival and malignant progression. mROS can damage DNA, activate oncogenes, block the function of tumor suppressors and drive migratory signaling. The mitochondrion's oxidant scavenging systems including SOD2, Grx2, GPrx, Trx and TrxR are key of the cellular redox tone. These mitochondrial antioxidant systems serve to tightly control the levels of the primary ROS signaling species, H2O2. The coordinated control of mROS levels is also coupled to the activity of the primary H2O2 consuming enzymes of the mitochondria which are reliant on the epitranscriptomic control of selenocysteine incorporation. This review highlights the interplay between these many oncogenic signaling networks, mROS and the H2O2 emitting and consuming capacity of the mitochondria.
Asunto(s)
Mitocondrias/metabolismo , Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Metabolismo Energético , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Despite increasing numbers of patients on dialysis, the numbers of renal transplants performed yearly have remained relatively static. During the last 50 years, there have been many advances in the pharmacology of prevention of organ rejection. However, most patients will suffer from a slow but steady decline in renal function leading to graft loss. The most common cause of long-term graft loss is chronic allograft nephropathy (CAN). Therefore, elucidating and understanding the mechanisms involved in CAN is crucial for achieving better posttransplant outcomes. It is thought that the development of epithelial to mesenchymal transition (EMT) in proximal tubules is one of the first steps towards CAN, and has been shown to be a result of cellular senescence. Cells undergoing senescence acquire a senescence associated secretory phenotype (SASP) leading to the production of interleukin-1 alpha (IL-1α), which has been implicated in several degenerative and inflammatory processes including renal disease. A central mediator in SASP activation is the production of reactive oxygen species (ROS), which are produced in response to numerous physiological and pathological stimuli. This review explores the connection between SASP and the development of EMT/CAN in an effort to suggest future directions for research leading to improved long-term graft outcomes.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Rechazo de Injerto/etiología , Trasplante de Riñón , Trasplante Homólogo , Enfermedad Crónica , Transición Epitelial-Mesenquimal/inmunología , Humanos , Trasplante de Riñón/métodos , Tiempo , Trasplante Homólogo/efectos adversosRESUMEN
As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.
Asunto(s)
Proteínas Bacterianas/inmunología , Catalasa/inmunología , Francisella tularensis/inmunología , Inmunidad Innata , Macrófagos/inmunología , Canales Catiónicos TRPM/inmunología , Tularemia/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Calcio/inmunología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Catalasa/genética , Catalasa/metabolismo , Femenino , Francisella tularensis/enzimología , Francisella tularensis/genética , Eliminación de Gen , Peróxido de Hidrógeno/inmunología , Peróxido de Hidrógeno/metabolismo , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-6/metabolismo , Ionomicina/farmacología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Noqueados , Oxidación-Reducción/efectos de los fármacos , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Tularemia/genética , Tularemia/metabolismoRESUMEN
Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91(phox) subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.
Asunto(s)
Células de la Médula Ósea/metabolismo , Peróxido de Hidrógeno/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Receptores de IgG/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Células de la Médula Ósea/inmunología , Catalasa/genética , Catalasa/inmunología , Catalasa/metabolismo , Francisella tularensis/enzimología , Francisella tularensis/genética , Francisella tularensis/inmunología , Peróxido de Hidrógeno/inmunología , Inmunidad Innata/fisiología , Interleucina-6/genética , Interleucina-6/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de IgG/genética , Receptores de IgG/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H2O2 and intracellular Ca(2+) levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca(2+) promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca(2+) chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca(2+) chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca(2+) homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Senescencia Celular/fisiología , Interleucina-1alfa/biosíntesis , Proteolisis , Calpaína/genética , Calpaína/metabolismo , Línea Celular Tumoral , Activación Enzimática/fisiología , Transición Epitelial-Mesenquimal/fisiología , Humanos , Peróxido de Hidrógeno/farmacología , Interleucina-1alfa/genética , Interleucina-6/biosíntesis , Interleucina-6/genética , Interleucina-8/biosíntesis , Interleucina-8/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Oxidantes/farmacología , Oxidación-Reducción , Comunicación Paracrina/fisiología , Microambiente TumoralRESUMEN
Salivary gland hypofunction is highly prevalent in aged and diseased individuals leading to significant discomfort and morbidity. One factor that contributes to salivary gland hypofunction is cellular aging, or senescence. Senescent cells can impair gland function by secreting paracrine-acting growth factors and cytokines, known as senescence-associated secretory phenotype (SASP) factors. These SASP factors stimulate inflammation, propagate the senescent phenotype through the bystander effect, and stimulate fibrosis. As senotherapeutics that target senescent cells have shown effectiveness in limiting disease manifestations in other conditions, there is interest in the use of these drugs to treat salivary gland hypofunction. In this review, we highlight the contribution of senescence and fibrosis to salivary gland pathologies. We also discuss therapeutic approaches to eliminate or modulate the senescent SASP phenotype for treating age-related salivary gland diseases and extending health span.
RESUMEN
Selenium is a naturally found trace element, which provides multiple benefits including antioxidant, anticancer, and antiaging, as well as boosting immunity. One unique feature of selenium is its incorporation as selenocysteine, a rare 21st amino acid, into selenoproteins. Twenty-five human selenoproteins have been discovered, and a majority of these serve as crucial antioxidant enzymes for redox homeostasis. Unlike other amino acids, incorporation of selenocysteine requires a distinctive UGA stop codon recoding mechanism. Although many studies correlating selenium, selenoproteins, aging, and senescence have been performed, it has not yet been explored if the upstream events regulating selenoprotein synthesis play a role in senescence-associated pathologies. The epitranscriptomic writer alkylation repair homolog 8 (ALKBH8) is critical for selenoprotein production, and its deficiency can significantly decrease levels of selenoproteins that are essential for reactive oxygen species (ROS) detoxification, and increase oxidative stress, one of the major drivers of cellular senescence. Here, we review the potential role of epitranscriptomic marks that govern selenocysteine utilization in regulating the senescence program.
Asunto(s)
Selenio , Humanos , Selenio/metabolismo , Antioxidantes , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Codón de Terminación , Homólogo 8 de AlkB ARNt MetiltransferasaRESUMEN
Francisella tularensis is the etiologic agent of the highly infectious animal and human disease tularemia. Its extreme infectivity and virulence are associated with its ability to evade immune detection, which we now link to its robust reactive oxygen species-scavenging capacity. Infection of primary human monocyte-derived macrophages with virulent F. tularensis SchuS4 prevented proinflammatory cytokine production in the presence or absence of IFN-gamma compared with infection with the attenuated live vaccine strain. SchuS4 infection also blocked signals required for macrophage cytokine production, including Akt phosphorylation, IkappaB alpha degradation, and NF-kappaB nuclear localization and activation. Concomitant with SchuS4-mediated suppression of Akt phosphorylation was an increase in the levels of the Akt antagonist PTEN. Moreover, SchuS4 prevented the H(2)O(2)-dependent oxidative inactivation of PTEN compared with a virulent live vaccine strain. Mutation of catalase (katG) sensitized F. tularensis to H(2)O(2) and enhanced PTEN oxidation, Akt phosphorylation, NF-kappaB activation, and inflammatory cytokine production. Together, these findings suggest a novel role for bacterial antioxidants in restricting macrophage activation through their ability to preserve phosphatases that temper kinase signaling and proinflammatory cytokine production.
Asunto(s)
Citocinas/biosíntesis , Francisella tularensis/metabolismo , Depuradores de Radicales Libres/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Catalasa/genética , Catalasa/metabolismo , Línea Celular Tumoral , Francisella tularensis/enzimología , Francisella tularensis/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Peróxido de Hidrógeno/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Interferón gamma/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Monocitos/citología , FN-kappa B/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/inmunología , Especificidad de la EspecieRESUMEN
The results from this study demonstrate the potential of an AlGaN/GaN high electron mobility transistor sensor for the detection of reactive and transient biological molecules such as hydrogen peroxide. A boronate-based fluorescent probe was used with this device to detect the presence of micromolar levels of hydrogen peroxide typically associated with intracellular processes. The real-time electrical response of the high electron mobility transistor sensor showed a gradual decrease in the two-dimensional electron gas current as the reaction proceeded over time. A corresponding increase in the emission intensity was measured from the fluorescent probe with the progression of the reaction. The fluorescence from the boronate probe was used as an indicator to confirm the detection of hydrogen peroxide. These results demonstrate the dynamic measurement capability of AlGaN/GaN high electron mobility transistor sensors in monitoring real-time reactions of reactive oxygen species such as hydrogen peroxide.
Asunto(s)
Compuestos de Aluminio/química , Técnicas Biosensibles , Ácidos Borónicos/química , Electrones , Galio/química , Peróxido de Hidrógeno/análisis , Sondas Moleculares/química , Transistores Electrónicos , Electricidad , Imagen Óptica , Espectrometría de FluorescenciaRESUMEN
The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state (H(2)O(2)) 3.5-fold (13.7-48.6 pM) relative to young cells. Restricting H(2)O(2) production through low O(2) exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H(2)O(2)-dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover.
Asunto(s)
Senescencia Celular/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Fibroblastos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Sistema de Señalización de MAP Quinasas/fisiología , Metaloproteinasa 1 de la Matriz/genética , Metaloporfirinas/metabolismo , Oxidantes/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Oxígeno/metabolismoRESUMEN
Critically important to the maintenance of the glutathione (GSH) redox cycle are the activities of many selenocysteine-containing GSH metabolizing enzymes whose translation is controlled by the epitranscriptomic writer alkylation repair homolog 8 (ALKBH8). ALKBH8 is a tRNA methyltransferase that methylates the wobble uridine of specific tRNAs to regulate the synthesis of selenoproteins. Here we demonstrate that a deficiency in the writer ALKBH8 (Alkbh8def), alters selenoprotein levels and engages senescence, regulates stress response genes and promotes mitochondrial reprogramming. Alkbh8def mouse embryonic fibroblasts (MEFs) increase many hallmarks of senescence, including senescence associated ß-galactosidase, heterocromatic foci, the cyclin dependent kinase inhibitor p16Ink4a, markers of mitochondrial dynamics as well as the senescence associated secretory phenotype (SASP). Alkbh8def cells also acquire a stress resistance phenotype that is accompanied by an increase in a number redox-modifying transcripts. In addition, Alkbh8def MEFs undergo a metabolic shift that is highlighted by a striking increase in the level of uncoupling protein 2 (UCP2) which enhances oxygen consumption and promotes a reliance on glycolytic metabolism. Finally, we have shown that the Alkbh8 deficiency can be exploited and corresponding MEFs are killed by glycolytic inhibition. Our work demonstrates that defects in an epitransciptomic writer promote senescence and mitochondrial reprogramming and unveils a novel adaptive mechanism for coping with defects in selenocysteine utilization.
Asunto(s)
Homólogo 8 de AlkB ARNt Metiltransferasa/genética , Perfilación de la Expresión Génica/métodos , Mitocondrias/metabolismo , Animales , Células Cultivadas , Senescencia Celular , Epigénesis Genética , Eliminación de Gen , Humanos , Ratones , Consumo de Oxígeno , Selenocisteína/metabolismo , Proteína Desacopladora 2/metabolismoRESUMEN
IMPACT STATEMENT: Through its ability to evoke responses from cells in a paracrine fashion, the senescence-associated secretory phenotype (SASP) has been linked to numerous age-associated disease pathologies including tumor invasion, cardiovascular dysfunction, neuroinflammation, osteoarthritis, and renal disease. Strategies which limit the amplitude and duration of SASP serve to delay age-related degenerative decline. Here we demonstrate that the SASP regulation is linked to shifts in intracellular Ca2+ homeostasis and strategies which rescue redox-dependent calcium entry including enzymatic H2O2 scavenging, TRP modulation, or mTOR inhibition block SASP and TRPC6 gene expression. As Ca2+ is indispensable for secretion from both secretory and non-secretory cells, it is exciting to speculate that the expression of plasma lamellar TRP channels critical for the maintenance of intracellular Ca2+ homeostasis may be coordinately regulated with the SASP.
Asunto(s)
Calcio/metabolismo , Senescencia Celular , Serina-Treonina Quinasas TOR/metabolismo , Señalización del Calcio/efectos de los fármacos , Catalasa/metabolismo , Línea Celular , Senescencia Celular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/toxicidad , Imidazoles/farmacología , Oxidación-Reducción/efectos de los fármacos , Canal Catiónico TRPC6/metabolismoRESUMEN
The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8def and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Homólogo 8 de AlkB ARNt Metiltransferasa/metabolismo , Epigénesis Genética , Pulmón/metabolismo , Naftalenos/toxicidad , Estrés Oxidativo , Transcriptoma , Homólogo 8 de AlkB ARNt Metiltransferasa/genética , Animales , Sistema Enzimático del Citocromo P-450/genética , Resistencia a Medicamentos , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Procesamiento Postranscripcional del ARN , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O(2)(-)). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (DeltasodC) and a F. tularensis DeltasodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB DeltasodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-gamma)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis DeltasodC and sodB DeltasodC mutants showed attenuated intramacrophage survival in IFN-gamma-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the DeltasodC mutant or inhibiting the IFN-gamma-dependent production of O(2)(-) or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The DeltasodC and sodB DeltasodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the DeltasodC mutant was restored in ifn-gamma(-/-), inos(-/-), and phox(-/-) mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.
Asunto(s)
Vacunas Bacterianas , Francisella tularensis/enzimología , Especies Reactivas de Oxígeno/farmacología , Superóxido Dismutasa/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Francisella tularensis/clasificación , Francisella tularensis/efectos de los fármacos , Francisella tularensis/patogenicidad , Regulación Bacteriana de la Expresión Génica/fisiología , Macrófagos Alveolares/microbiología , Ratones , Mutación , Especies de Nitrógeno Reactivo , Tularemia/microbiología , VirulenciaRESUMEN
Here we highlight the role of epitranscriptomic systems in post-transcriptional regulation, with a specific focus on RNA modifying writers required for the incorporation of the 21st amino acid selenocysteine during translation, and the pathologies linked to epitranscriptomic and selenoprotein defects. Epitranscriptomic marks in the form of enzyme-catalyzed modifications to RNA have been shown to be important signals regulating translation, with defects linked to altered development, intellectual impairment, and cancer. Modifications to rRNA, mRNA and tRNA can affect their structure and function, while the levels of these dynamic tRNA-specific epitranscriptomic marks are stress-regulated to control translation. The tRNA for selenocysteine contains five distinct epitranscriptomic marks and the ALKBH8 writer for the wobble uridine (U) has been shown to be vital for the translation of the glutathione peroxidase (GPX) and thioredoxin reductase (TRXR) family of selenoproteins. The reactive oxygen species (ROS) detoxifying selenocysteine containing proteins are a prime examples of how specialized translation can be regulated by specific tRNA modifications working in conjunction with distinct codon usage patterns, RNA binding proteins and specific 3' untranslated region (UTR) signals. We highlight the important role of selenoproteins in detoxifying ROS and provide details on how epitranscriptomic marks and selenoproteins can play key roles in and maintaining mitochondrial function and preventing disease.
Asunto(s)
Enfermedad/etiología , Epigénesis Genética , Procesamiento Postranscripcional del ARN , Especies Reactivas de Oxígeno/metabolismo , Selenio/metabolismo , Selenoproteínas/metabolismo , Transcriptoma , Animales , Humanos , Oxidación-Reducción , Biosíntesis de Proteínas , Transducción de SeñalRESUMEN
The transcoelomic metastasis pathway is an alternative to traditional lymphatic/hematogenic metastasis. It is most frequently observed in ovarian cancer, though it has been documented in colon and gastric cancers as well. In transcoelomic metastasis, primary tumor cells are released into the abdominal cavity and form cell aggregates known as spheroids. These spheroids travel through the peritoneal fluid and implant at secondary sites, leading to the formation of new tumor lesions in the peritoneal lining and the organs in the cavity. Models of this process that incorporate the fluid shear stress (FSS) experienced by these spheroids are few, and most have not been fully characterized. Proposed herein is the adaption of a known dynamic cell culture system, the orbital shaker, to create an environment with physiologically-relevant FSS for spheroid formation. Experimental conditions (rotation speed, well size and cell density) were optimized to achieve physiologically-relevant FSS while facilitating the formation of spheroids that are also of a physiologically-relevant size. The FSS improves the roundness and size consistency of spheroids versus equivalent static methods and are even comparable to established high-throughput arrays, while maintaining nearly equivalent viability. This effect was seen in both highly metastatic and modestly metastatic cell lines. The spheroids generated using this technique were fully amenable to functional assays and will allow for better characterization of FSS's effects on metastatic behavior and serve as a drug screening platform. This model can also be built upon in the future by adding more aspects of the peritoneal microenvironment, further enhancing its in vivo relevance.
RESUMEN
The signaling networks that drive the aging process, associated functional deterioration, and pathologies has captured the scientific community's attention for decades. While many theories exist to explain the aging process, the production of reactive oxygen species (ROS) provides a signaling link between engagement of cellular senescence and several age-associated pathologies. Cellular senescence has evolved to restrict tumor progression but the accompanying senescence-associated secretory phenotype (SASP) promotes pathogenic pathways. Here, we review known biological theories of aging and how ROS mechanistically control senescence and the aging process. We also describe the redox-regulated signaling networks controlling the SASP and its important role in driving age-related diseases. Finally, we discuss progress in designing therapeutic strategies that manipulate the cellular redox environment to restrict age-associated pathology.
Asunto(s)
Envejecimiento/genética , Senescencia Celular/genética , Oxidación-Reducción , Envejecimiento/patología , Humanos , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Manganese superoxide dismutase (MnSOD, SOD2) is an essential primary antioxidant enzyme which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix. MnSOD plays a prominent role in protection against many apoptotic stimuli. Its absence may therefore impair the cellular redox balance and enhance apoptosis. Our data show that in Jurkat T cells, following oligomerization of the Fas receptor, MnSOD is selectively degraded during apoptosis. In the presence of cycloheximide, an inhibitor of protein synthesis, the rates of cell death and MnSOD degradation were accelerated. Fas-induced MnSOD cleavage was partially inhibited in the presence of the pan-caspase inhibitor, z-VAD-fmk. MnSOD in the mitochondrial fractions was cleaved in vitro by treatment with the cytosolic fraction of Fas-activated cells. Moreover, two possible cleavage sites of recombinant hMnSOD by direct interaction with recombinant caspase-3 were noted. Cellular and mitochondrial factors were found to be necessary for the interaction. These factors include intracellular mobilization of calcium. Our data indicate that inactivation of MnSOD in receptor-mediated apoptosis by caspase-specific degradation would render the mitochondria sensitive to the steady-state production of superoxide, decrease the steady-state flux of H(2)O(2), expedite the loss of mitochondrial function, and potentiate apoptosis.
Asunto(s)
Apoptosis , Superóxido Dismutasa/metabolismo , Receptor fas/inmunología , Secuencia de Aminoácidos , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Calcio/fisiología , Caspasas/fisiología , Humanos , Células Jurkat , Modelos Biológicos , Datos de Secuencia Molecular , Desnaturalización Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptor fas/químicaRESUMEN
Manganese superoxide dismutase (SOD2) has been well established to be essential for protection from a variety of apoptotic stimuli. Here we demonstrate that the antiapoptotic effects of SOD2 are attributed to its ability to generate H(2)O(2) and that its efficient removal resensitizes cells to tumor necrosis factor (TNF)-alpha-induced apoptosis. SOD2 overexpression in HT-1080 cells leads to a decrease in the fluorescence of the superoxidesensitive fluorophore, dihydroethidium, and a concomitant increase in oxidation of the H2O2-sensitive dye, dichlorodihydrofluorescein diacetate (DCFDA). The rate of aminotriazole-inhibited catalase activity also was increased when SOD2 is overexpressed and reflects a 1.6-fold increase in the steady-state production of H(2)O(2). The increase in H(2)O(2) was associated with decreased sensitivity to TNF-alpha-mediated apoptosis, as measured by monitoring the loss of mitochondrial membrane potential (MMP), caspase activation, poly-ADP ribose polymerase (PARP) cleavage, and accumulation of hypodiploid DNA content. Both the increase in H2O2 and resistance to TNF-mediated apoptosis were reversed by coexpression of catalase. The lipid hydroperoxide scavengers, beta-hydroxytoluene and trolox, and the iron chelator, desferroxamine, showed partial recovery of TNF-induced apoptosis. These findings indicate that increases in the intracellular steady-state production of H(2)O(2) by SOD2 can block the activation of key processes fundamental to the process of programmed cell death.