RESUMEN
BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Arteriolas/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/farmacología , Rutina/farmacología , Antioxidantes/farmacología , Arteriolas/metabolismo , Células Cultivadas , Femenino , Glutamato-Cisteína Ligasa/metabolismo , Células HEK293 , Semivida , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Oxidación-Reducción/efectos de los fármacos , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)â µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)â µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.
Asunto(s)
Cistina/metabolismo , Estrés Oxidativo/fisiología , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxinas/metabolismo , Carcinoma de Células Escamosas , Cisteína/metabolismo , Activación Enzimática/fisiología , Glutatión/metabolismo , Células HEK293 , Células HT29 , Humanos , Peróxido de Hidrógeno/farmacología , Neoplasias Pulmonares , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , Óxido Nítrico/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Especificidad por Sustrato , Azufre/metabolismo , Tiorredoxina Reductasa 1/genética , Tiorredoxinas/genéticaRESUMEN
The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-ß receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-ß receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.
Asunto(s)
Activación Enzimática/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Tiorredoxinas/farmacología , Animales , Activación Enzimática/efectos de los fármacos , Fibroblastos , Técnicas de Inactivación de Genes , Violeta de Genciana , Ratones , Oxidación-Reducción , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxina Reductasa 1/deficienciaRESUMEN
Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 µg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-ß-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.
Asunto(s)
Glutatión/metabolismo , Sulfuros/metabolismo , Tiorredoxinas/metabolismo , Animales , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Glutatión Reductasa/metabolismo , Células HEK293 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratas , Tiorredoxina Reductasa 1/metabolismoRESUMEN
Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP(I)-ACP(II)-ACP(III)) within the CurA PKS module. We have determined the structure of the isolated holo-ACP(I) and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP(I), which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP(I) and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP(I)-ACP(II)-ACP(III) tridomain, our data provide insight into the specificity of the chlorination reaction.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Cianobacterias/química , Ciclopropanos/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Tiazoles/metabolismo , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de ProteínaRESUMEN
Palladium (Pd), platinum (Pt), and gold (Au) are noble metals, two of which have established medical use. Pt has anticancer efficacy, predominantly as cisplatin, whereas the gold compound auranofin is used against arthritis. Both compounds inhibit the selenoprotein thioredoxin reductase (TrxR), but Pd has not been studied in this regard. Using salts of Pd, Pt, and Au as well as cisplatin and auranofin we found that Pd and Au were strikingly more potent inhibitors of recombinant TrxR1 than Pt. The TrxR-related nonselenoprotein glutathione reductase in pure form (but less so in a cellular context), as well as cellular thioredoxin (Trx) activities, were inhibited by the gold salt KAuCl(4) but were little affected by auranofin or the other compounds. In an analysis of three cancer cell lines, the extent of inhibition of TrxR activity and decrease in cell viability depended upon the choice of both noble metal and ligand and also seemed independent of p53 status. During treatment of cells with cisplatin, covalent complexes of TrxR1 with either Trx1 or TRP14 (Trx-related protein of 14kDa) were formed, as verified by Western blot analyses and mass spectrometry. These results reveal that Au and Pd are strong inhibitors of TrxR, but Pt and cisplatin trigger highly specific cellular effects on the Trx system, including covalent cross-linking of TrxR1 with Trx1 and TRP14.