RESUMEN
AIM: Hypoxia is an important feature of multiple diseases like cancer and obesity and also an environmental stressor to high altitude travelers. Emerging research suggests the importance of redox signaling in physiological responses transforming the notion of oxidative stress into eustress and distress. However, the behavior of redox protein post-translational modifications (PTMs), and their correlation with stress acclimatization in humans remains sketchy. Scant information exists about modifications in redoxome during physiological exposure to environmental hypoxia. In this study, we investigated redox PTMs, nitrosylation and carbonylation, in context of extended environmental hypoxia exposure. METHODS: The volunteers were confirmed to be free of any medical conditions and matched for age and weight. The human global redoxome and the affected networks were investigated using TMT-labeled quantitative proteo-bioinformatics and biochemical assays. The percolator PSM algorithm was used for peptide-spectrum match (PSM) validation in database searches. The FDR for peptide matches was set to 0.01. 1-way ANOVA and Tukey's Multiple Comparison test were used for biochemical assays. p-value<0.05 was considered statistically significant. Three independent experiments (biological replicates) were performed. Results were presented as Mean ± standard error of mean (SEM). KEY FINDINGS: This investigation revealed direct and indirect interplay between nitrosylation and carbonylation especially within coagulation and inflammation networks; interlinked redox signaling (via nitrosylationcarbonylation); and novel nitrosylation and carbonylation sites in individual proteins. SIGNIFICANCE: This study elucidates the role of redox PTMs in hypoxia signaling favoring tolerance and survival. Also, we demonstrated direct and indirect interplay between nitrosylation and carbonylation is crucial to extended hypoxia tolerance.
Asunto(s)
Aclimatación/fisiología , Altitud , Proteínas Sanguíneas/metabolismo , Estrés Oxidativo/fisiología , Carbonilación Proteica , Adulto , Citocinas/sangre , Citocinas/metabolismo , Glutatión/sangre , Humanos , Hipoxia/fisiopatología , Inflamación/metabolismo , Masculino , Óxido Nítrico/sangre , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Factores de TiempoRESUMEN
Extended exposure to low pO2 has multiple effects on signaling cascades. Despite multiple exploratory studies, omics studies elucidating the signaling cascades essential for surviving extended low pO2 exposures are lacking. In this study, we simulated low pO2 (PB = 40 kPa; 7620 m) exposure in male Sprague-Dawley rats for 3, 7 and 14 days. Redox stress assays and proteomics based network biology were performed using lungs and plasma. We observed that redox homeostasis was achieved after day 3 of exposure. We investigated the causative events for this. Proteo-bioinformatics analysis revealed STAT3 to be upstream of lung cytoskeletal processes and systemic lipid metabolism (RXR) derived inflammatory processes, which were the key events. Thus, during prolonged low pO2 exposure, particularly those involving slowly decreasing pressures, redox homeostasis is achieved but energy metabolism is perturbed and this leads to an immune/inflammatory signaling impetus after third day of exposure. We found that an interplay of lung cytoskeletal elements, systemic energy metabolism and inflammatory proteins aid in achieving redox homeostasis and surviving extended low pO2 exposures. Qualitative perturbations to cytoskeletal stability and innate immunity/inflammation were also observed during extended low pO2 exposure in humans exposed to 14,000 ft for 7, 14 and 21 days.
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Apoptosis , Inflamación , Animales , Homeostasis , Inflamación/inducido químicamente , Inflamación/genética , Pulmón , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-DawleyRESUMEN
Apo-A1 is correlated with conditions like hyperlipidemia, cardiovascular diseases, high altitude pulmonary edema and etc. where hypoxia constitutes an important facet.Hypoxia causes oxidative stress, vaso-destructive and inflammatory outcomes.Apo-A1 is reported to have vasoprotective, anti-oxidative, anti-apoptotic, and anti-inflammatory effects. However, effects of Apo-A1 augmentation during hypoxia exposure are unknown.In this study, we investigated the effects of exogenously supplementing Apo-A1-mimetic peptide on SD rats during hypoxia exposure. For easing the processes of delivery, absorption and bio-availability, Apo-A1 mimetic peptide D4F was used. The rats were given 10 mg/kg BW dose (i.p.) of D4F for 7 days and then exposed to hypoxia. D4F was observed to attenuate both oxidative stress and inflammation during hypoxic exposure. D4F improved energy homeostasis during hypoxic exposure. D4F did not affect HIF-1a levels during hypoxia but increased MnSOD levels while decreasing CRP and Apo-B levels. D4F showed promise as a prophylactic against hypoxia exposure.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Apolipoproteína A-I/farmacología , Metabolismo Energético/efectos de los fármacos , Hipoxia/tratamiento farmacológico , Inflamación/prevención & control , Pulmón/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Apolipoproteínas B/sangre , Proteínas Portadoras/sangre , Modelos Animales de Enfermedad , Hipoxia/sangre , Hipoxia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/sangre , Inflamación/etiología , Pulmón/metabolismo , Masculino , Oxidación-Reducción , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismoRESUMEN
The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.
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Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Extensión de la Cadena Peptídica de Translación , Factor G de Elongación Peptídica/química , ARN Mitocondrial/química , ARN de Transferencia/química , Proteínas Ribosómicas/química , Ribosomas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Microscopía por Crioelectrón , Células HEK293 , Humanos , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Factor G de Elongación Peptídica/genética , Factor G de Elongación Peptídica/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/ultraestructura , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Intermittent hypoxia, initially associated with adverse effects of sleep apnea, has now metamorphosed into a module for improved sports performance. The regimen followed for improved sports performance is milder intermittent hypoxic training (IHT) as compared to chronic and severe intermittent hypoxia observed in sleep apnea. Although several studies have indicated the mechanism and enough data on physiological parameters altered by IH is available, proteome perturbations remain largely unknown. Altitude induced hypobaric hypoxia is known to require acclimatization as it causes systemic redox stress and inflammation in humans. In the present study, a short IHT regimen consisting of previously reported physiologically beneficial FIO2 levels of 13.5% and 12% was administered to human subjects. These subjects were then airlifted to altitude of 3500 m and their plasma proteome along with associated redox parameters were analyzed on days 4 and 7 of high altitude stay. We observed that redox stress and associated post-translational modifications, perturbed lipid metabolism and inflammatory signaling were induced by IHT exposure at Baseline. However, this caused activation of antioxidants, energy homeostasis mechanisms and anti-inflammatory responses during subsequent high-altitude exposure. Thus, we propose IHT as a beneficial non-pharmacological intervention that benefits individuals venturing to high altitude areas.
Asunto(s)
Homeostasis , Hipoxia/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Metabolismo de los Lípidos , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Adulto , Altitud , Biomarcadores , Biología Computacional/métodos , Citocinas/metabolismo , Humanos , Inmunohistoquímica , Inflamación/patología , Estrés Oxidativo , Proteoma , Proteómica/métodos , Especies Reactivas de Oxígeno , Transducción de Señal , Adulto JovenRESUMEN
Exposure to high altitude above 3000 m leads to two outcomes-acclimation or high-altitude maladies. To reach a particular outcome, the plasma proteome is modified differentially, either in context of an acclimation response or mal-acclimation response leading to disease. This ensures that hypoxia-responsive plasma protein trends reflect acclimation in acclimated individuals when compared with their levels prior to acclimation. Such protein trends could be used to assess acclimation in an individual and any significant deviation from this trend may indicate non-acclimation, thereby preventing high-altitude illnesses before they manifest. In this study, we investigate and statistically evaluate the trendlines of various hypoxia-responsive plasma protein levels, reported significantly perturbed in our previous studies, in individuals (male; n = 20) exposed to 3520 m at high-altitude day 1 (HAD1), HAD4, and HAD7L and to 4420 m at HAD7H, HAD30, and HAD120. We observe that thioredoxin (Trx), glutathione peroxidase 3 (GPx-3), and apolipoprotein AI (Apo-AI) are statistically robust markers to assess acclimation across the exposure duration while sulfotransferase 1A1 (ST1A1) is a capable negative control whose levels increase only in cases of HAPE. We also observe exposure day-specific and resident altitude-specific proteins capable of accurately assessing acclimation when compared with baseline levels or the lower altitude zone.
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Aclimatación , Altitud , Proteínas Sanguíneas/análisis , Hipoxia/sangre , Adulto , Apolipoproteína A-I/sangre , Arilsulfotransferasa/sangre , Biomarcadores/sangre , Ensayo de Inmunoadsorción Enzimática , Glutatión Peroxidasa/sangre , Humanos , Masculino , Personal Militar , Curva ROC , Tiorredoxinas/sangre , Factores de Tiempo , Adulto JovenRESUMEN
Altitude acclimatization describes the processes whereby lowland humans respond to decreased partial pressure of oxygen. It refers to the changes seen as beneficial and involves a series of physiological adjustments that compensate for reduced ambient PO2, as opposed to changes that are pathological. Although numerous reports document the physiological effects of exposure to hypobaric hypoxia of varying durations but an interesting aspect overlooked by many researchers is that of acclimatization related studies. As proteome, a dynamic entity responds immediately to external stimuli, protein markers and their trends can be studied to assess acclimatization status of an individual. Compared to blood, the use of saliva is advantageous because sample collection and processing are easy, minimally invasive, low cost and better tolerated by individuals. In this study, we employed iTRAQ based LC-MS/MS technique for comparing saliva samples from humans exposed to hypobaric hypoxia from 7 to 120 days with normoxic controls followed by analysis using Ingenuity Pathway Analysis software and validation by immunoassays. Nearly 67 proteins were found to be differentially expressed in the exposed groups as compared to normoxia indicating modulated canonical pathways as lipid metabolism; acute phase response signalling and proteins as carbonic anhydrase 6, alpha-enolase, albumin, and prolactin inducible protein. Collectively, this study provides the proof of concept for the non-invasive assessment of high altitude acclimatization.
Asunto(s)
Aclimatación , Altitud , Hipoxia/metabolismo , Proteómica , Proteínas y Péptidos Salivales/metabolismo , Adulto , Humanos , MasculinoRESUMEN
AIMS: HAPE remains the most common lethal high-altitude disease. Although its pathophysiology and other associated causal factors have been partially uncovered along with some potential biomarker proteins, it has not been completely elucidated. A major hindrance to improving the understanding of HAPE pathophysiology and associated molecular events has been the absence of a quick, reliable and definitive animal model of HAPE. This study is aimed at development of a rapid and reliable SD rat model of high altitude pulmonary edema (HAPE) that can be roentgenographically confirmed and be used to study protein markers of HAPE. MAIN METHODS: In this study, we detail the process of rapidly inducing HAPE in male SD rats within 18â¯h of simulated high-altitude exposure without causing high rates of mortality. Thereafter, we confirmed HAPE using roentgenography. We assessed Sulfotransferase 1A1 (SULT1A1), IL-1 beta, TNF- alpha and IFN-gamma using ELISA. Finally, H&E staining of lung tissues was also performed. KEY FINDINGS: A roentgenographically confirmed HAPE model was demonstrated. SULT 1A1 levels are found to be highest in rats suffering HAPE, as previously confirmed in human patients. Inflammation was also assessed based on levels of inflammatory proteins like IL-1b, TNF-a, and IFN-g in addition to H&E staining of lung tissues. Inflammation and HAPE were observed to be synergistic events and not cause and effect of each other. SIGNIFICANCE: This rat model of HAPE will help researchers and clinicians in evaluating performance of therapies, potential biomarker and also further elucidate underlying molecular processes causing HAPE.
Asunto(s)
Altitud , Arilsulfotransferasa/metabolismo , Biomarcadores/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/diagnóstico , Edema Pulmonar/diagnóstico , Animales , Humanos , Inflamación/diagnóstico por imagen , Inflamación/metabolismo , Masculino , Edema Pulmonar/diagnóstico por imagen , Edema Pulmonar/metabolismo , Radiografía , Ratas , Ratas Sprague-DawleyRESUMEN
Intermittent hypoxic training (IHT) is a discrete cost-effective method for improving athletic performance and high altitude acclimatization. Unfortunately, IHT protocols widely vary in terms of hypoxia severity, duration, and number of cycles affecting physiological outcomes. In the present study, we evaluated the efficacy of a moderate normobaric IHT protocol (12% FiO2 for 4 h, 4 days) on acclimatization to high altitude (3250 m). Global plasma proteomics studies revealed that IHT elicited acute-phase response proteins like C-reactive protein (CRP), serum amyloid A-1 protein (SAA), and alpha-1-acid glycoprotein 2 (AGP 2) as well as altered levels of several apolipoproteins. On subsequent exposure to high altitude, the IH trained volunteers exhibited significant higher arterial oxygen saturation with concomitant lower incidences of acute mountain sickness (AMS) as compared to controls. Interestingly, IH trained subjects exhibited lower levels of positive acute-phase proteins like C-reactive protein (CRP), serum amyloid A-1 protein (SAA), and fibrinogen (FGA, FGB, and FGG) both after days 4 and 7 of high altitude ascent. High altitude exposure also decreased the levels of HDL, LDL, and associated proteins as well as key enzymes for assembly and maturation of lipoprotein particles like lecithin-cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). In contrast, IHT curtailed hypoxia-induced alterations of HDL, LDL, Apo-AI, Apo-B, LCAT, CETP, and PLTP. Further validation of results also corroborated attenuation of hypoxia-induced inflammation and dyslipidemia by IHT. These results provide molecular evidences supporting the use of moderate IHT as a potential non-pharmacological strategy for high altitude acclimatization.
Asunto(s)
Aclimatación/fisiología , Dislipidemias/fisiopatología , Hipoxia/fisiopatología , Inflamación/fisiopatología , Adulto , Altitud , Mal de Altura/metabolismo , Mal de Altura/fisiopatología , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Dislipidemias/metabolismo , Femenino , Humanos , Hipoxia/metabolismo , Masculino , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Identification of molecular signatures having key roles in hypobaric hypoxia by analysing the salivary proteome. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being non-invasive. METHODOLOGY: We employed qualitative proteomics approach to develop discriminatory biomarker signatures from human saliva exposed to hypobaric hypoxia. Salivary proteins were analyzed and compared between age-matched healthy subjects exposed to high altitude (â¼13700 ft) for seven days (HAD7) with control subjects at sea level (Normoxia) by using 2-Dimensional gel electrophoresis/Mass Spectrometry approach. RESULTS: Several proteins with significant differential expression were found. The up-regulated proteins were apoptosis inducing factor-2, cystatin S, cystatin SN and carbonic anhydrase 6. The down regulated proteins were polymeric immunoglobulin receptor, alpha-enolase and prolactin-inducible protein. Further confirmation of the altered proteins such as alpha enolase, carbonic anhydrase 6, prolactin-inducible protein, apoptosis inducing factor 2, cystatin S and cystatin SN were performed using immunoblotting. The expression patterns of the selected proteins observed by immunoblot were in concurrence with 2-Dimesional gel electrophoresis results, therefore affirming the authenticity of the proteomic investigation. CONCLUSION: This study provides the proof of concept of salivary biomarkers for the non-invasive detection of hypobaric hypoxia induced effects. It is highly feasible to turn these biomarkers into an applicable clinical test after large scale validation.
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Altitud , Hipoxia , Proteómica/métodos , Proteínas y Péptidos Salivales/metabolismo , Adulto , Factor Inductor de la Apoptosis/metabolismo , Anhidrasas Carbónicas/metabolismo , Estudios de Casos y Controles , Electroforesis en Gel Bidimensional , Humanos , Masculino , Espectrometría de Masas , Estrés Oxidativo , Fosfopiruvato Hidratasa/metabolismo , Prolactina/metabolismo , Cistatinas Salivales/metabolismoRESUMEN
Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.
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Técnicas Biosensibles/tendencias , Técnicas Electroquímicas/tendencias , Inmunoensayo/tendencias , Superóxido Dismutasa/análisis , Animales , Humanos , Ratones , Óxido Nítrico , Estrés Oxidativo , Sistemas de Atención de Punto , Especies Reactivas de Oxígeno , Compuestos de SulfhidriloRESUMEN
The lower inhaled oxygen per volume at high altitude poses an intimidating challenge for humans to survive and reproduce. Indigenous populations of the Himalayas reportedly exhibit higher microcirculatory blood flow accompanied by higher orders of magnitude of nitric oxide (NO) products in lung, plasma and red blood cells as a vascular adaptation strategy for hypobaric hypoxia. The precise mechanism of such observed higher NO metabolites for hypoxia adaptation remains elusive. Studying high altitude native Ladakhi women, we observed significant higher eNOS mRNA and protein in blood/plasma as compared to lowland women. We also observed higher level of plasma l-citrulline and NOx (nitrates and nitrites) with concomitant lower levels of arginase mRNA and protein further suggesting higher eNOS activity and NO bioavailability. Interestingly, middle aged postmenopausal Ladakhi women exhibited significantly higher level of eNOS activity, NOx and cGMP as compared to age matched lowland women. Preferential phosphorylation of eNOS on stimulatory Ser1177 and Ser615 as well as dephosphorylation of inhibitory Thr495 site contributed to higher NO availability in Ladakhi women irrespective of age. We also observed higher levels of eNOS activating humoral factors like bradykinin and estrogen in both young and middle-aged Ladakhi women. These results suggest that an altered phosphorylation status, together with an enhanced expression of eNOS and potential humoral endothelial activators, are involved in enhanced activation of the eNOS-NO-cGMP pathway in Ladakhi women irrespective of age, reinforcing the hypothesis that NO metabolites play a major role in Himalayan pattern of hypoxia adaptation.
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Aclimatación/fisiología , Hipoxia/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Adulto , Altitud , Bradiquinina/metabolismo , Estrógenos/metabolismo , Femenino , Frecuencia Cardíaca/fisiología , Humanos , India , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/química , Fosforilación , Procesamiento Proteico-Postraduccional , Serina/química , Transducción de Señal/fisiología , Adulto JovenRESUMEN
Lack of zero side-effect, prescription-less prophylactics and diagnostic markers of acclimatization status lead to many suffering from high altitude illnesses. Although not fully translated to the clinical setting, many strategies and interventions are being developed that are aimed at providing an objective and tangible answer regarding the acclimatization status of an individual as well as zero side-effect prophylaxis that is cost-effective and does not require medical supervision. This short review brings together the twin problems associated with high-altitude acclimatization, i.e. acclimatization status and zero side-effect, easy-to-use prophylaxis, for the reader to comprehend as cogs of the same phenomenon. We describe current research aimed at preventing all the high-altitude illnesses by considering them an assault on redox and energy homeostasis at the molecular level. This review also entails some proteins capable of diagnosing either acclimatization or high-altitude illnesses. The future strategies based on bioinformatics and systems biology is also discussed.
Asunto(s)
Aclimatación , Mal de Altura/diagnóstico , Biomarcadores/sangre , Edema Encefálico/prevención & control , Mal de Altura/sangre , HumanosRESUMEN
Hydrogen Sulfide (H2S), recently identified as the third endogenously produced gaseous messenger, is a promising therapeutic prospect for multiple cardio-pathological states, including myocardial hypertrophy. The molecular niche of H2S in normal or diseased cardiac cells is, however, sparsely understood. Here, we show that ß-adrenergic receptor (ß-AR) overstimulation, known to produce hypertrophic effects in cardiomyocytes, rapidly decreased endogenous H2S levels. The preservation of intracellular H2S levels under these conditions strongly suppressed hypertrophic responses to adrenergic overstimulation, thus suggesting its intrinsic role in this process. Interestingly, unbiased global transcriptome sequencing analysis revealed an integrated metabolic circuitry, centrally linked by NADPH homeostasis, as the direct target of intracellular H2S augmentation. Within these gene networks, glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme (producing NADPH) in pentose phosphate pathway, emerged as the critical node regulating cellular effects of H2S. Utilizing both cellular and animal model systems, we show that H2S-induced elevated G6PD activity is critical for the suppression of cardiac hypertrophy in response to adrenergic overstimulation. We also describe experimental evidences suggesting multiple processes/pathways involved in regulation of G6PD activity, sustained over extended duration of time, in response to endogenous H2S augmentation. Our data, thus, revealed H2S as a critical endogenous regulator of cardiac metabolic circuitry, and also mechanistic basis for its anti-hypertrophic effects.
RESUMEN
Hypoxia, especially altitude associated hypoxia is known to cause severe physiological alterations and life-threatening conditions. Impaired redox balance along with oxidative stress, protein carbonylation and instigation of apoptotic events are common sub-cellular events that follow the hypoxic insult. The role of nitric oxide (NO) is very dynamic and versatile in preventing the ill effects of hypoxia vis-a-vis reacting with oxidative species and causing protein nitrosylation. Although several mechanisms of NO-mediated cytoprotection are known during hypoxic insult, limited pieces of evidence are available to support the relationship between two downstream events of oxidative stress, protein carbonylation (caused by carbonyl; CO radical) and protein nitrosylation/nitration (caused by NO/peroxynitrite; ONOO radical). In this study, we investigated an entirely new aspect of NO protection in hypoxia involving crosstalk between carbonylation and nitrosylation. Using standard NO inhibitor l-NAME and simulated hypoxic conditions in hypoxia-sensitive cell line H9c2, we evaluated the levels of radicals, cell death, mitochondrial membrane potential, levels of protein nitrosylation, protein nitration and carbonylation and glutathione content. The results were then carefully analyzed in light of NO bioavailability. Our study shows that reducing NO during hypoxia caused cell death via the increased degree of carbonylation in proteins. This provides a new aspect of NO benefits which furthers opens new possibilities to explore potential mechanisms and effects of cross-talk between nitrosylation, protein nitration and carbonylation, especially through some common antioxidant mediators such as glutathione and thioredoxin.
Asunto(s)
Óxido Nítrico/metabolismo , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular , Hipoxia de la Célula , Línea Celular , Citratos/metabolismo , Glutatión/metabolismo , Oxidación-Reducción , Carbonilación Proteica , Transducción de Señal , Compuestos de Sulfhidrilo/metabolismo , Tiorredoxinas/metabolismo , Transcripción GenéticaRESUMEN
Recent studies showed that silk and human hair fibers develop thermoelectric properties at optimal water, temperature and light conditions. The nature of charge carriers and the role of water in mediating charge conduction in these fibers is an unexplored issue. By studying four different classes of natural fibers, viz., silk cocoon, human hair, jute and corn silk, we uncover their common electrical transport properties and its dependence on water concentration and temperature. All these fibers uniformly exhibit nonlinear, hysteretic current - voltage characteristics, which scale with water concentration. The optimal electrical conductivity shows thermally activated hopping transport mechanism. Scanning tunneling microscope (STM) and dielectric measurements of silk cocoon fibers showed the electronic density of states and dielectric properties of the hydrated medium enhances with water concentration. Electron paramagnetic resonance (EPR) study reveals that the charge carriers in these membranes are electronic in nature. Our results are explained through the mechanism of hopping of a Polaron, which is an electron surrounded by positive charge fluctuations created by water molecules. The mechanism unravels the peculiar role water plays in mediating electrical activity in these membranes and also opens the possibility for exploring such charge transport mechanism in other biological membranes.
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Bombyx/fisiología , Conductividad Eléctrica , Membranas/química , Seda/química , Estrés Mecánico , Agua/química , Animales , Transporte de Electrón , Resistencia al CorteRESUMEN
Hypobaric hypoxia elicits several patho-physiological manifestations, some of which are known to be lethal. Among various molecular mechanisms proposed so far, perturbation in redox state due to imbalance between radical generation and antioxidant defence is promising. These molecular events are also related to hypoxic status of cancer cells and therefore its understanding has extended clinical advantage beyond high altitude hypoxia. In present study, however, the focus was to understand and propose a model for rapid acclimatization of high altitude visitors to enhance their performance based on molecular changes. We considered using simulated hypobaric hypoxia at some established thresholds of high altitude stratification based on known physiological effects. Previous studies have focused on the temporal aspect while overlooking the effects of varying pO2 levels during exposure to hypobaric hypoxia. The pO2 levels, indicative of altitude, are crucial to redox homeostasis and can be the limiting factor during acclimatization to hypobaric hypoxia. In this study we present the effects of acute (24h) exposure to high (3049m; pO2: 71kPa), very high (4573m; pO2: 59kPa) and extreme altitude (7620m; pO2: 40kPa) zones on lung and plasma using semi-quantitative redox specific transcripts and quantitative proteo-bioinformatics workflow in conjunction with redox stress assays. It was observed that direct exposure to extreme altitude caused 100% mortality, which turned into high survival rate after pre-exposure to 59kPa, for which molecular explanation were also found. The pO2 of 59kPa (very high altitude zone) elicits systemic energy and redox homeostatic processes by modulating the STAT3-RXR-Nrf2 trio. Finally we posit the various processes downstream of STAT3-RXR-Nrf2 and the plasma proteins that can be used to ascertain the redox status of an individual.
Asunto(s)
Mal de Altura/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxígeno/metabolismo , Receptores X Retinoide/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Aclimatación , Altitud , Animales , Metabolismo Energético , Homeostasis , Humanos , Masculino , Oxidación-Reducción , Estrés Oxidativo , Ratas Sprague-Dawley , Biología de SistemasRESUMEN
GNE myopathy is a rare neuromuscular genetic disorder characterized by early adult onset and muscle weakness due to mutation in sialic acid biosynthetic enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). More than 180 different GNE mutations are known all over the world with unclear pathomechanism. Although hyposialylation of glycoproteins is speculated to be the major cause, but cellular mechanism leading to loss of muscle mass has not yet been deciphered. Besides sialic acid biosynthesis, GNE affects other cellular functions such as cell adhesion and apoptosis. In order to understand the effect of mutant GNE protein on cellular functions, differential proteome profile of HEK293 cells overexpressing pathologically relevant recombinant mutant GNE protein (D207V and V603L) was analyzed. These cells, along with vector control and wild-type GNE-overexpressing cells, were subjected to two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF MS/MS). In the study, 10 differentially expressed proteins were identified. Progenesis same spots software revealed downregulation of peroxiredoxin IV (PrdxIV), an ER-resident H2O2 sensor that regulates neurogenesis. Significant reduction in mRNA and protein levels of PrdxIV was observed in GNE mutant cell lines compared with vector control. However, neither total reactive oxygen species was altered nor H2O2 accumulation was observed in GNE mutant cell lines. Interestingly, ER redox state was significantly affected due to reduced normal GNE enzyme activity. Our study indicates that downregulation of PrdxIV affects ER redox state that may contribute to misfolding and aggregation of proteins in GNE myopathy.