RESUMEN
2-Cys peroxiredoxins (Prxs) rapidly reduce H2O2, thereby acting as antioxidants and also as sensors and transmitters of H2O2 signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H2O2 levels. Under these conditions, H2O2 oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (CPSOH) to the sulfinate (CPSO2-) and sulfonated (CPSO3-) forms, redirecting the CPSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the CPSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H2O2 and was selected here to investigate the effect of a physiological concentration of HCO3-/CO2 (25 mm) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO3-/CO2 increases Prx1 hyperoxidation and inactivation both in the presence of excess H2O2 and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO3-/CO2 was due to HCO4-, a peroxide present in equilibrated solutions of H2O2 and HCO3-/CO2 Indeed, additional experiments and calculations uncovered that HCO4- oxidizes CPSOH to CPSO2- with a second-order rate constant 2 orders of magnitude higher than that of H2O2 ((1.5 ± 0.1) × 105 and (2.9 ± 0.2) × 103 m-1·s-1, respectively) and that HCO4- is 250 times more efficient than H2O2 at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO3-/CO2 pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity.
Asunto(s)
Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/química , Antioxidantes/metabolismo , Bicarbonatos/química , Bicarbonatos/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Catálisis , Cisteína/química , Cisteína/metabolismo , Humanos , Cinética , NADP/química , NADP/metabolismo , Oxidación-Reducción , Peróxidos/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by degeneration of upper and lower motor neurons. While the fundamental causes of the disease are still unclear, the accumulation of Cu,Zn-superoxide dismutase (SOD1) immunoreactive aggregates is associated with familial ALS cases. Cholesterol 5,6-secosterol aldehydes (Seco A and Seco B) are reported to contribute to neurodegenerative disease pathology by inducing protein modification and aggregation. Here we have investigated the presence of secosterol aldehydes in ALS SOD1-G93A rats and their capacity to induce SOD1 aggregation. Secosterol aldehydes were analyzed in blood plasma, spinal cord and motor cortex of ALS rats at the pre-symptomatic and symptomatic stages. Seco B was significantly increased in plasma of symptomatic ALS rats compared to pre-symptomatic animals, suggesting an association with disease progression. In vitro experiments showed that both Seco A and Seco B induce the formation of high molecular weight (HMW) SOD1 aggregates with amorphous morphology. SOD1 adduction to ω-alkynyl-secosterols analyzed by click assay showed that modified proteins are only detected in the HMW region, indicating that secosterol adduction generates species highly prone to aggregate. Of note, SOD1-secosterol adducts containing up to five secosterol molecules were confirmed by MALDI-TOF analysis. Interestingly, mass spectrometry sequencing of SOD1 aggregates revealed preferential secosterol adduction to Lys residues located at the electrostatic loop (Lys 122, 128 and 136) and nearby the dimer interface (Lys 3 and 9). Altogether, our results show that secosterol aldehydes are increased in plasma of symptomatic ALS rats and represent a class of aldehydes that can potentially modify SOD1 enhancing its propensity to aggregate.
Asunto(s)
Aldehídos/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Colesterol/análogos & derivados , Agregación Patológica de Proteínas/metabolismo , Superóxido Dismutasa-1/metabolismo , Aldehídos/sangre , Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/genética , Animales , Colesterol/sangre , Colesterol/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Mutación Puntual , Agregado de Proteínas , Agregación Patológica de Proteínas/sangre , Agregación Patológica de Proteínas/genética , Ratas Sprague-Dawley , Ratas Transgénicas , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/ultraestructuraRESUMEN
The development of resistance to antibiotics by microorganisms is a major problem for the treatment of bacterial infections worldwide, and therefore, it is imperative to study new scaffolds that are potentially useful in the development of new antibiotics. In this regard, we propose the design, synthesis and biological evaluation of hybrid sulfonylhydrazone bioisosters/furoxans with potential antibacterial (Escherichia coli) activity. The most active compound of the series, (E)-3-methyl-4-((2-tosylhydrazono)methyl)-1,2,5-oxadiazole 2-oxide, with a MIC=0.36µM, was not cytotoxic when tested on Vero cells (IC50>100µM). To complement the in vitro screening, we also studied the interaction of the test compounds with ß-ketoacyl acyl carrier protein synthase (FabH), the target for the parent compounds, and we observed three important hydrogen-bonding interactions with two important active site residues in the catalytic site of the enzyme, providing complementary evidence to support the target of the new hybrid molecules.
Asunto(s)
Acetiltransferasas/antagonistas & inhibidores , Antibacterianos/química , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Acetiltransferasas/metabolismo , Animales , Antibacterianos/síntesis química , Antibacterianos/toxicidad , Sitios de Unión , Candida albicans/efectos de los fármacos , Dominio Catalítico , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/toxicidad , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/metabolismo , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Oxadiazoles/toxicidad , Electricidad Estática , Relación Estructura-Actividad , Células VeroRESUMEN
The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.
Asunto(s)
Superóxido Dismutasa/química , Triptófano/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bicarbonatos/química , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Agregación Patológica de Proteínas , Carbonilación Proteica , Multimerización de Proteína , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo, we studied the effects of tempol on hSOD1 oxidation in vitro. The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 µM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5-75 µM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32â¢. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences.