RESUMEN

Carotenoids produced by bacteria, yeasts, algae and plants inactivate Free Radicals (FR). However, FR may inactivate carotenoids and even turn them into free radicals. Oxidative metabolism is a source of the highly motile Reactive Oxygen Species (ROS). To evaluate carotenoid interactions with ROS, the yeast Rhodotorula mucilaginosa was grown in dextrose (YPD), a fermentative substrate where low rates of oxygen consumption and low carotenoid expression were observed, or in lactate (YPLac), a mitochondrial oxidative-phosphorylation (OxPhos) substrate, which supports high respiratory activity and carotenoid production. ROS were high in YPLac-grown cells and these were unmasked by the carotenoid production-inhibitor diphenylamine (DPA). In contrast, in YPD-grown cells ROS were almost absent. It is proposed that YPLac cells are under oxidative stress. In addition, YPLac-grown cells were more sensitive than YPD-grown cells to menadione (MD), a FR-releasing agent. To test whether carotenoids from cells grown in YPLac had been modified by ROS, carotenoids from each, YPD- and YPLac-grown cells were isolated and added back to cells, evaluating protection from MD. Remarkably, carotenoids extracted from cells grown in YPLac medium inhibited growth, while in contrast extracts from YPD-grown cells were innocuous or mildly protective. Results suggest that carotenoid-synthesis in YPLac-cells is a response to OxPhos-produced ROS. However, upon reacting with FR, carotenoids themselves may be inactivated or even become prooxidant themselves.

RESUMEN

The transmembrane nitrate reductase (Nar) is the first enzyme in the dissimilatory alternate anaerobic nitrate respiratory chain in denitrifying bacteria. To date, there has been no real-time method to determine its specific activity embedded in its native membrane; here, we describe such a new method, which is useful with the inside-out membranes of Paracoccus denitrificans and other denitrifying bacteria. This new method takes advantage of the native coupling of the endogenous NADH dehydrogenase or Complex I with the reduction of nitrate by Nar through the quinone pool of the inner membranes of P. denitrificans. This is achieved under previously reached anaerobic conditions. Inner controls confirming the specific Nar activity determined by this new method were made by the total inhibition of the Nar enzyme by sodium azide and cyanide, well-known Nar inhibitors. The estimation of the Michaelis-Menten affinity of Nar for NO3- using this so-called Nar-JJ assay gave a Km of 70.4 µM, similar to previously determined values. This new Nar-JJ assay is a suitable, low-cost, and reproducible method to determine in real-time the endogenous Nar activity not only in P. denitrificans, but in other denitrifying bacteria such as Brucella canis, and potentially in other entero-pathogenic bacteria.

Asunto(s)

Desnitrificación , Nitrato-Reductasa , Paracoccus denitrificans , Paracoccus denitrificans/enzimología , Paracoccus denitrificans/metabolismo , Nitrato-Reductasa/metabolismo , Nitratos/metabolismo , Cinética

RESUMEN

Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.

Asunto(s)

Calcio , Mitocondrias Cardíacas , Mitocondrias Hepáticas , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Ratas Wistar , Animales , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Masculino , Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Ratas , Consumo de Oxígeno , Hígado/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Ciclosporina/farmacología

RESUMEN

Rhodotorula mucilaginosa survives extreme conditions through several mechanisms, among them its carotenoid production and its branched mitochondrial respiratory chain (RC). Here, the branched RC composition was analyzed by biochemical and complexome profiling approaches. Expression of the different RC components varied depending on the growth phase and the carbon source present in the medium. R. mucilaginosa RC is constituted by all four orthodox respiratory complexes (CI to CIV) plus several alternative oxidoreductases, in particular two type-II NADH dehydrogenases (NDH2) and one alternative oxidase (AOX). Unlike others, in this yeast the activities of the orthodox and alternative respiratory complexes decreased in the stationary phase. We propose that the branched RC adaptability is an important factor for survival in extreme environmental conditions; thus, contributing to the exceptional resilience of R. mucilaginosa.

Asunto(s)

Extremófilos , Rhodotorula , Transporte de Electrón , Rhodotorula/química , Rhodotorula/metabolismo , Membranas Mitocondriales/metabolismo

RESUMEN

INTRODUCTION: Diarrheagenic Escherichia coli pathotypes are important aetiological agents of diarrhoeal illness among children from less developed areas, worldwide. Diarrheagenic E. coli pathotypes strains are increasingly becoming drug resistant, thus effective and accessible therapeutic alternatives are required for their treatment; herbal extracts may be a potential alternative. AIMS: to evaluate Echeveria craigiana, E. kimnachii, and E. subrigida methanol extracts antibacterial effect on six diarrheagenic E. coli reference strains and on human colorectal adenocarcinoma cells viability and cytokine production. METHODOLOGY: Diarrheagenic E. coli pathotypes reference strains: typical enteropathogenic E2348/69, enterotoxigenic H10407, enterohaemorrhagic O157:H7/EDL933, enteroinvasive E11, diffusely adherent C18451-A, and enteroaggregative 042 E. coli. E craigiana, E. kimnachii, and E. subrigida leaves, collected at Sinaloa, Mexico, were freeze-dried and macerated in methanol solvent. Antibacterial activity was determined by a novel method developed in our laboratory, bacterial oxygen consumption by polarographic oxygen electrode technique and membrane integrity by two methods (live/dead and protein leakage assays). Colorectal adenocarcinoma cells viability by MTT assay and cytokine production using a Cytometric Bead Array kit. RESULTS: Extracts concentrations of 100 µg/mL and 5-hour incubation, reduced more than 93% the growth of all diarrheagenic E. coli pathotypes tested strains and significantly decreased bacterial oxygen consumption, like bacteriostatic antibiotics. After 24-hour incubation methanol extracts had a differential antibacterial effect on each diarrheagenic E. coli pathotypes strain. Echeveria extracts did not have any effect on viability and cytokine production of colorectal adenocarcinoma cells. CONCLUSIONS: Echeveria methanol extracts have a bacteriostatic effect on all diarrheagenic E. coli pathotypes strains, thus potentially they could be used as antibacterial agents on diarrheagenic E. coli pathotypes-contaminated products and on patients with diarrheagenic E. coli pathotypes infections.

Asunto(s)

Escherichia coli Enteropatógena , Infecciones por Escherichia coli , Células CACO-2 , Niño , Diarrea/microbiología , Escherichia coli , Infecciones por Escherichia coli/microbiología , Humanos , Extractos Vegetales/farmacología

RESUMEN

The yeast Saccharomyces cerevisiae uses fermentation as the preferred pathway to obtain ATP and requires the respiratory chain to re-oxidize the NADH needed for activity of Glyceraldehyde-3-phosphate. This process is favored by uncoupling of oxidative phosphorylation (OxPhos), which is at least partially controlled by the mitochondrial unspecific pore (ScMUC). When mitochondrial ATP synthesis is needed as in the diauxic phase or during mating, a large rise in Ca2+ concentration ([Ca2+]) closes ScMUC, coupling OxPhos. In addition, ScMUC opening/closing is mediated by the ATP/ADP ratio, which indicates cellular energy needs. Here, opening and closing of ScMUC was evaluated in isolated mitochondria from S. cerevisiae at different incubation times and in the presence of different ATP/ADP ratios or varying [Ca2+]. Measurements of the rate of O2 consumption, mitochondrial swelling, transmembrane potential and ROS generation were conducted. It was observed that ScMUC opening was reversible, a high ATP/ADP ratio promoted opening and [Ca2+] closed ScMUC even after several minutes of incubation in the open state. In the absence of ATP synthesis, closure of ScMUC resulted in an increase in ROS.

RESUMEN

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus-mitochondria crosstalk in DENV infected mosquito cells.

RESUMEN

BACKGROUND: Long-non-coding RNAs, a class of transcripts with lengths > 200 nt, play key roles in tumour progression. Previous reports revealed that LINC00052 (long intergenic non-coding RNA 00052) was strongly downregulated during breast cancer multicellular spheroids formation and suggested a role in cell migration and oxidative metabolism. OBJECTIVE: To examine the function of LINC00052 in MCF-7 breast cancer cells. METHODS: Loss-of-function studies were performed to evaluate LINC00052 role on MCF-7 breast cancer cells. Microarray expression assays were performed to determine genes and cellular functions modified after LINC00052 knockdown. Next, the impact of LINC00052 depletion on MCF-7 cell respiration and migration was evaluated. RESULTS: 1,081 genes were differentially expressed upon LINC00052 inhibition. Gene set enrichment analysis, Gene Ontology and Key Pathway Advisor analysis showed that signalling networks related to cell migration and oxidative phosphorylation were enriched. However, whereas LINC00052 knockdown in MCF-7 cells revealed marginal difference in oxygen consumption rates when compared with control cells, LINC00052 inhibition enhanced cell migration in vitro and in vivo, as observed using a Zebrafish embryo xenotransplant model. CONCLUSION: Our data show that LINC00052 modulates MCF-7 cell migration. Genome-wide microarray experiments suggest that cancer cell migration is affected by LINC00052 through cytoskeleton modulation and Notch/ß-catenin/NF-κB signalling pathways.

Asunto(s)

Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/genética , ARN Largo no Codificante/genética , Animales , Neoplasias de la Mama/patología , Movimiento Celular , Femenino , Humanos , Pez Cebra

RESUMEN

Synaptic aging has been associated with neuronal circuit dysfunction and cognitive decline. Reduced mitochondrial function may be an early event that compromises synaptic integrity and neurotransmission in vulnerable brain regions during physiological and pathological aging. Thus, we aimed to measure mitochondrial function in synapses from three brain regions at two different ages in the 3xTg-AD mouse model and in wild mice. We found that aging is the main factor associated with the decline in synaptic mitochondrial function, particularly in synapses isolated from the cerebellum. Accumulation of toxic compounds, such as tau and Aß, that occurred in the 3xTg-AD mouse model seemed to participate in the worsening of this decline in the hippocampus. The changes in synaptic bioenergetics were also associated with increased activation of the mitochondrial fission protein Drp1. These results suggest the presence of altered mechanisms of synaptic mitochondrial dynamics and their quality control during aging and in the 3xTg-AD mouse model; they also point to bioenergetic restoration as a useful therapeutic strategy to preserve synaptic function during aging and at the early stages of Alzheimer's disease (AD).

Asunto(s)

Envejecimiento/genética , Disfunción Cognitiva/genética , Dinaminas/genética , Mitocondrias/metabolismo , Dinámicas Mitocondriales/genética , Envejecimiento/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Cerebelo/metabolismo , Cerebelo/fisiopatología , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Modelos Animales de Enfermedad , Dinaminas/metabolismo , Femenino , Regulación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Transgénicos , Mitocondrias/patología , Neuronas/metabolismo , Neuronas/patología , Especificidad de Órganos , Sinapsis/metabolismo , Sinapsis/patología , Sinaptosomas/metabolismo , Sinaptosomas/patología , Proteínas tau/genética , Proteínas tau/metabolismo

RESUMEN

Mycobacterium tuberculosis (MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 µM), while increasing the dose (50 µM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP in vitro and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion.

RESUMEN

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.

RESUMEN

Mitochondrial uncoupling proteins (UCP) transport protons from the intermembrane space to the mitochondrial matrix uncoupling oxidative phosphorylation. In mammals, these proteins have been implicated in several cellular functions ranging from thermoregulation to antioxidant defense. In contrast, their invertebrate homologs have been much less studied despite the great diversity of species. In this study, two transcripts encoding mitochondrial uncoupling proteins were, for the first time, characterized in crustaceans. The white shrimp Litopenaeus vannamei transcript LvUCP4 is expressed in all tested shrimp tissues/organs, and its cDNA includes a coding region of 954 bp long which encodes a deduced protein 318 residues long and a predicted molecular weight of 35.3 kDa. The coding region of LvUCP5 transcript is 906 bp long, encodes a protein of 302 residues with a calculated molecular weight of 33.17 kDa. Both proteins share homology with insect UCPs, their predicted structures show the conserved motifs of the mitochondrial carrier proteins and were confirmed to be located in the mitochondria through a Western blot analysis. The genic expression of LvUCP4 and LvUCP5 was evaluated in shrimp at oxidative stress conditions and results were compared to some antioxidant enzymes to infer about their antioxidant role. LvUCP4 and LvUCP5 genes expression did not change during hypoxia/re-oxygenation, and no coordinated responses were detected with antioxidant enzymes at the transcriptional level. Results confirmed UCPs as the first uncoupling mechanism reported in this species, but their role in the oxidative stress response remains to be confirmed.

Asunto(s)

Proteínas de Artrópodos/biosíntesis , Regulación de la Expresión Génica/fisiología , Mitocondrias/metabolismo , Proteínas Desacopladoras Mitocondriales/biosíntesis , Penaeidae/metabolismo , Animales , Proteínas de Artrópodos/genética , Mitocondrias/genética , Proteínas Desacopladoras Mitocondriales/genética , Especificidad de Órganos/fisiología , Penaeidae/genética

RESUMEN

Diabetes affects a variety of tissues including the central nervous system; moreover, some evidence indicates that memory and learning processes are disrupted. Also, oxidative stress triggers alterations in different tissues including the brain. Recent studies indicate mitochondria dysfunction is a pivotal factor for neuron damage. Therefore, we studied mitochondrial activity in three brain regions at early type I-diabetes induction. Isolated mitochondria from normal hippocampus, cortex and cerebellum revealed different rates of oxygen consumption, but similar respiratory controls. Oxygen consumption in basal state 4 significantly increased in the mitochondria from all three brain regions from diabetic rats. No relevant differences were observed in the activity of respiratory complexes, but hippocampal mitochondrial membrane potential was reduced. However, ATP content, mitochondrial cytochrome c, and protein levels of ß-tubulin III, synaptophysin, and glutamine synthase were similar in brain regions from normal and diabetic rats. In addition, no differences in total glutathione levels were observed between normal and diabetic rat brain regions. Our results indicated that different regions of the brain have specific metabolic responses. The changes in mitochondrial activity we observed at early diabetes induction did not appear to cause metabolic alterations, but they might appear at later stages. Longer-term streptozotocin treatment studies must be done to elucidate the impact of hyperglycemia in brain metabolism and the function of specific brain regions.

Asunto(s)

Encéfalo/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Tipo 1/inducido químicamente , Mitocondrias/metabolismo , Oxígeno/análisis , Animales , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hipocampo/metabolismo , Masculino , Estrés Oxidativo , Ratas , Estreptozocina

RESUMEN

Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species' mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.

Asunto(s)

Artemia/química , Transporte de Electrón , Mitocondrias/metabolismo , Consumo de Oxígeno , Penaeidae/química , Adaptación Fisiológica , Animales , Glicerolfosfato Deshidrogenasa , Mitocondrias/química , Proteínas Mitocondriales , Oxidación-Reducción , Oxidorreductasas , Proteínas de Plantas , Especificidad por Sustrato

RESUMEN

Chitosan is a stressing molecule that affects the cells walls and plasma membrane of fungi. For chitosan derivatives, the action mode is not clear. In this work, we used the yeast Ustilago maydis to study the effects of these molecules on the plasma membrane, focusing on physiologic and stress responses to chitosan (CH), oligochitosan (OCH), and glycol-chitosan (GCH). Yeasts were cultured with each of these molecules at 1 mg·mL-1 in minimal medium. To compare plasma membrane damage, cells were cultivated in isosmolar medium. Membrane potential (Δψ) as well as oxidative stress were measured. Changes in the total plasma membrane phospholipid and protein profiles were analyzed using standard methods, and fluorescence-stained mitochondria were observed. High osmolarity did not protect against CH inhibition and neither affected membrane potential. The OCH did produce higher oxidative stress. The effects of these molecules were evidenced by modifications in the plasma membrane protein profile. Also, mitochondrial damage was evident for CH and OCH, while GCH resulted in thicker cells with fewer mitochondria and higher glycogen accumulation.

Asunto(s)

Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Quitina/análogos & derivados , Quitosano/farmacología , Ustilago/efectos de los fármacos , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular , Pared Celular/ultraestructura , Quitina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Oligosacáridos , Concentración Osmolar , Fosfolípidos/metabolismo , Poliaminas/farmacología , Polielectrolitos , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismo , Ustilago/metabolismo , Ustilago/ultraestructura

RESUMEN

Lovastatin is a commercially important secondary metabolite produced by Aspergillus terreus, either by solid-state fermentation or by submerged fermentation. In a previous work, we showed that reactive oxygen species (ROS) accumulation in idiophase positively regulates lovastatin biosynthetic genes. In addition, it has been found that lovastatin-specific production decreases with aeration in solid-state fermentation (SSF). To study this phenomenon, we determined ROS accumulation during lovastatin SSF, under high and low aeration conditions. Paradoxically, high aeration caused lower ROS accumulation, and this was the underlying reason of the aeration effect on lovastatin production. Looking for a mechanism that is lowering ROS production under those conditions, we studied alternative respiration. The alternative oxidase provides an alternative route for electrons passing through the electron transport chain to reduce oxygen. Here, we showed that an alternative oxidase (AOX) is expressed in SSF, and only during idiophase. It was shown that higher aeration induces higher alternative respiration (AOX activity), and this is a mechanism that limits ROS generation and keeps them within healthy limits and adequate signaling limits for lovastatin production. Indeed, the aox gene was induced in idiophase, i.e., at the time of ROS accumulation. Moreover, exogenous ROS (H2O2), added to lovastatin solid-state fermentation, induced higher AOX activity. This suggests that high O2 availability in SSF generates dangerously high ROS, so alternative respiration is induced in SSF, indirectly favoring lovastatin production. Conversely, alternative respiration was not detected in lovastatin-submerged fermentation (SmF), although exogenous ROS also induced relatively low AOX activity in SmF.

Asunto(s)

Fermentación , Proteínas Fúngicas/metabolismo , Lovastatina/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Aspergillus/enzimología , Aspergillus/genética , Medios de Cultivo/química , Proteínas Fúngicas/genética , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecular-weight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1ΔC15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1ΔC15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcO assembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcO biogenesis and that it is important for supercomplex formation/stability.

Asunto(s)

Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sustitución de Aminoácidos , Complejo IV de Transporte de Electrones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación Missense , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The oleaginous yeast Debaryomyces hansenii is a good model to understand molecular mechanisms involved in halotolerance because of its impressive ability to survive under a wide range of salt concentrations. Several cellular adaptations are implicated in this response, including the presence of a cyanide-insensitive ubiquinol oxidase (Aox). This protein, which is present in several taxonomical orders, has been related to different stress responses. However, little is known about its role in mitochondria during transitions from low to high saline environments. In this report, we analyze the effects of Aox in shifts from low to high salt concentrations in the culture media. At early stages of a salt insult, we observed that this protein prevents the overflow of electrons on the mitochondrial respiratory chain, thus, decreasing the production of reactive oxygen species. Interestingly, in the presence of high osmolite concentrations, Aox activity is able to sustain a stable membrane potential when coupled to complex I, despite a compromised cytochrome pathway. Taken together, our results suggest that under high osmolarity conditions Aox plays a critical role regulating mitochondrial physiology.

Asunto(s)

Ascomicetos/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales , Concentración Osmolar , Oxidorreductasas , Proteínas de Plantas , Respiración de la Célula , Supervivencia Celular , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Potencial de la Membrana Mitocondrial , Oxidación-Reducción , Estrés Fisiológico

RESUMEN

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Asunto(s)

Adenilil Ciclasas/metabolismo , AMP Cíclico/biosíntesis , Hepatocitos/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Transducción de Señal , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/genética , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hepatocitos/citología , Masculino , Cultivo Primario de Células , Ratas , Ratas Wistar , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/genética

RESUMEN

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca2+ buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn2+ superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction.

Asunto(s)

Mitocondrias Cardíacas/metabolismo , Ovariectomía , Fosforilación Oxidativa , Consumo de Oxígeno/fisiología , Especies Reactivas de Oxígeno/metabolismo , Aconitato Hidratasa/metabolismo , Animales , Femenino , Malondialdehído/metabolismo , Ratas , Superóxido Dismutasa/metabolismo