RESUMEN
Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Acilcoenzima A/metabolismo , Hexanoles/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/genética , Dominio Catalítico , Redes y Vías Metabólicas , Modelos Moleculares , Ingeniería de Proteínas/métodos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Hydrogen sulfide (H(2)S), a bacterial metabolite present in the lumen of the large intestine, is able to exert deleterious effects on the colonic epithelium. The mechanisms involved are still poorly understood, the reported effect of sulfide being its capacity to reduce n-butyrate beta-oxidation in colonocytes. In this work, we studied both the acute effect of the sodium salt of H(2)S on human colonic epithelial cell metabolism and the adaptative response of these cells to the pre-treatment with this agent. Using the human colon carcinoma epithelial HT-29 Glc(-/+) cell model, we found that the acute effect of millimolar concentrations of NaHS was to inhibit l-glutamine, n-butyrate and acetate oxidation in a dose-dependent manner. Using micromolar concentrations of NaHS, a comparable effect but largely reversible was observed for O(2) consumption and cytochrome c oxidase activity. Pre-treatment with 1 mM NaHS induced several adaptative responses. Firstly, increased lactate release and decreased cellular oxygen consumption evidenced a Pasteur-like effect which only partly compensated for the altered mitochondrial ATP production. Thus, a decrease in the proliferation rate with a constant adenylate charge was observed. Secondly, in these pre-treated cells, NaHS induced a hypoxia-like effect on cytochrome c oxidase subunits I and II which were decreased. Thirdly, a mild uncoupling of mitochondrial respiration possibly resulting from an increase of UCP 2 protein was observed. The NaHS antimitotic activity was not due to cellular apoptosis and/or necrosis but to a proportional slowdown in all cell cycle phases. These results are compatible with a metabolic adaptative response of the HT-29 colonic epithelial cells to sulfide-induced O(2) consumption reduction which, through the maintenance of a constant energetic load and an increased mitochondrial proton leak, would participate in the preservation of cellular viability.
Asunto(s)
Metabolismo Energético/fisiología , Sulfuro de Hidrógeno/administración & dosificación , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Consumo de Oxígeno/fisiología , Oxígeno/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/fisiología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Medicamentos , Metabolismo Energético/efectos de los fármacos , Células HT29 , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Consumo de Oxígeno/efectos de los fármacosRESUMEN
The longevity of birds is surprising since they exhibit high metabolic rates and elevated blood sugar levels compared with mammals of the same body size, which presumably expose them to higher rates of free oxygen radical production, which is implicated in accelerated senescence. Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane and their physiological activity is still a subject of debate. Avian UCP was found in birds but data on its activity are scarce. Avian UCP (Gallus gallus) was overexpressed in yeast and we assessed its ability to prevent mitochondrial reactive oxygen species (ROS) production by measuring ROS damage (aconitase activity) and antioxidant defences (MnSOD activity). We show that avian UCP protects yeast mitochondria against the deleterious impact of ROS, but without stimulation of superoxide dismutase activity. Avian UCP protein was specifically immunodetected and retinoic acid, which belongs to the carotenoid family, was found to trigger its activity. These data show that avian UCP basal activity protects against ROS damage. However, when activated by retinoic acid, avian UCP can also operate as the mammalian thermogenic UCP1. The hypothesis that avian UCP activities are state- and species-dependent is further discussed.
Asunto(s)
Proteínas Aviares/metabolismo , Pollos/genética , Radicales Libres/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Aconitato Hidratasa/metabolismo , Análisis de Varianza , Animales , Proteínas Aviares/genética , Fumarato Hidratasa/metabolismo , Proteínas Mitocondriales/genética , Proteínas Desacopladoras Mitocondriales , Superóxido Dismutasa/metabolismo , Tretinoina/metabolismoRESUMEN
The mitochondrial carrier family transports a variety of metabolites across the inner mitochondrial membrane. We identified and cloned a new member of this family, KMCP1 (kidney mitochondrial carrier protein-1), that is highly homologous to the previously identified protein BMCP1 (brain mitochondrial carrier protein-1). Western blotting and in situ experiments showed that this carrier is expressed predominantly within the kidney cortex in the proximal and distal tubules. KMCP1 was increased during fasting and during the regenerative phase of glycerol-induced renal failure. We show that both situations are associated with transiently increased expression of superoxide-generating enzymes, followed by increased mitochondrial metabolism and antioxidant defenses. Given that KMCP1 expression occurs simultaneously with these latter events, we propose that KMCP1 is involved in situations in which mitochondrial metabolism is increased, in particular when the cellular redox balance tends toward a pro-oxidant status.
Asunto(s)
Antioxidantes/farmacología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Túbulos Renales/fisiología , Riñón/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/fisiología , Regeneración , Regulación hacia Arriba , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Células COS , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Glutamina/química , Glicerol/química , Glicerol/metabolismo , Inmunoprecipitación , Canales Iónicos , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidantes/farmacología , Oxidación-Reducción , Estrés Oxidativo , Oxígeno/metabolismo , Consumo de Oxígeno , Filogenia , ARN/química , ARN/metabolismo , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Distribución Tisular , Proteína Desacopladora 1RESUMEN
Reactive oxygen species (ROS)-induced damage on host cells and molecules has been considered the most likely proximal mechanism responsible for the age-related decline in organismal performance. Organisms have two possible ways to reduce the negative effect of ROS: disposing of effective antioxidant defenses and minimizing ROS production. The unbalance between the amount of ROS produced and the availability of antioxidant defenses determines the intensity of so-called oxidative stress. Interestingly, most studies that deal with the effect of oxidative stress on organismal performance have focused on the antioxidant defense compartment and, surprisingly, have neglected the mechanisms that control ROS production within mitochondria. Uncoupling proteins (UCPs), mitochondrial transporters of the inner membrane, are involved in the control of redox state of cells and in the production of mitochondrial ROS. Given their function, UCPs might therefore represent a major mechanistic link between metabolic activity and fitness. We suggest that by exploring the role of expression and function of UCPs both in experimental as well as in comparative studies, evolutionary biologists may gain better insight into this link.
Asunto(s)
Envejecimiento/fisiología , Evolución Biológica , Canales Iónicos/genética , Canales Iónicos/fisiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Aminoácidos/análisis , Animales , Aves , Ecosistema , Membranas Mitocondriales/fisiología , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Termodinámica , Proteína Desacopladora 1RESUMEN
BACKGROUND: Mitochondrial respiration is the main source of energy in aerobic animal cells and is adapted to the energy demand by respiratory coupling. Uncoupling proteins (UCPs) perturb respiratory coupling by inducing a proton leak through the mitochondrial inner membrane. Although this could lead to deleterious energy waste, it may prevent the production of oxygen radicals when the rate of phosphorylation of ADP into ATP is low, whereas oxygen and substrate availability to mitochondria is high. The latter conditions are encountered during cardiac reperfusion after ischemia and are highly relevant to heart infarction. METHODS AND RESULTS: Heart function of 6 transgenic mice expressing high amounts of UCP1 and of 6 littermate controls was compared in isolated perfused hearts in normoxia, after 40-minute global ischemia, and on reperfusion. In normoxia, oxygen consumption, contractility (quantified as the rate-pressure product), and their relationship (energetic yield) were similar in controls and transgenic mice. Although UCP1 expression did not alter the sensitivity to ischemia, it significantly improved functional recovery on reperfusion. After 60 minutes of reperfusion, contractility was 2-fold higher in transgenic mice than in controls. Oxygen consumption remained significantly depressed in controls (53+/-27% of control), whereas it recovered strikingly to preischemic values in transgenic mice, showing uncoupling of respiration by UCP1 activity. Glutathione and aconitase, markers of oxidative damage, indicated lower oxidative stress in transgenic mice. CONCLUSIONS: UCP1 activity is low under normoxia but is induced during ischemia-reperfusion. The presence of UCP1 mitigates reperfusion-induced damage, probably because it lowers mitochondrial hyperpolarization at reperfusion.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Isquemia Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Aconitato Hidratasa/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Hipoxia de la Célula , Regulación de la Expresión Génica , Glutatión/metabolismo , Canales Iónicos , Masculino , Potenciales de la Membrana , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Ratones , Ratones Transgénicos , Mitocondrias Cardíacas/fisiología , Proteínas Mitocondriales/fisiología , Isquemia Miocárdica/genética , Daño por Reperfusión Miocárdica/genética , Estrés Oxidativo , Consumo de Oxígeno , Ratas , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
The mitochondrial uncoupling protein of brown adipose tissue (UCP1) was expressed in skeletal muscle and heart of transgenic mice at levels comparable with the amount found in brown adipose tissue mitochondria. These transgenic mice have a lower body weight, and when related to body weight, food intake and energy expenditure are increased. A specific reduction of muscle mass was observed but varied according to the contractile activity of muscles. Heart and soleus muscle are unaffected, indicating that muscles undergoing regular contractions, and therefore with a continuous mitochondrial ATP production, are protected. In contrast, the gastrocnemius and plantaris muscles showed a severely reduced mass and a fast to slow shift in fiber types promoting mainly IIa and IIx fibers at the expense of fastest and glycolytic type IIb fibers. These observations are interpreted as a consequence of the strong potential dependence of the UCP1 protonophoric activity, which ensures a negligible proton leak at the membrane potential observed when mitochondrial ATP production is intense. Therefore UCP1 is not deleterious for an intense mitochondrial ATP production and this explains the tolerance of the heart to a high expression level of UCP1. In muscles at rest, where ATP production is low, the rise in membrane potential enhances UCP1 activity. The proton return through UCP1 mimics the effect of a sustained ATP production, permanently lowering mitochondrial membrane potential. This very likely constitutes the origin of the signal leading to the transition in fiber types at rest.