RESUMO
The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1â s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1â s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233â¢+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233â¢+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.
Assuntos
Heme/metabolismo , Parasitos/metabolismo , Parasitos/patogenicidade , Peroxidase/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Virulência/fisiologia , Animais , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Grupo dos Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Transporte de Elétrons/fisiologia , Feminino , Peróxido de Hidrogênio/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida/métodos , Oxirredução , Fenilalanina/metabolismo , Triptofano/metabolismoRESUMO
Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108 M-1â s-1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M-1â s-1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/fisiologia , Ácidos Graxos/química , Peróxido de Hidrogênio/química , Nitratos/química , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/metabolismo , Simulação de Acoplamento Molecular , Nitratos/metabolismoRESUMO
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g. high-fat diets) or overweight and insulin resistance (e.g. methionine-choline-deficient diets) or they are based on monogenetic defects (e.g. ob/ob mice). In the current study, a western-type diet containing soybean oil with high n 6-PUFA and 0.75% cholesterol (SOD+Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast a soybean oil-containing western-type diet without cholesterol (SOD) induced only mild steatosis but neither hepatic inflammation nor fibrosis, weight gain or insulin resistance. Another high-fat diet mainly consisting of lard and supplemented with fructose in drinking water (LAD+Fru) resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD+Cho but livers were devoid of inflammation and fibrosis. Although both LAD+Fru- and SOD+Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD+Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. Summarizing, dietary cholesterol in SOD+Cho diet may trigger hepatic inflammation and fibrosis. SOD+Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.
Assuntos
Colesterol na Dieta , Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica/etiologia , Óleo de Soja , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Dieta Hiperlipídica , Dieta Ocidental , Hepatócitos/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse OxidativoRESUMO
Tank experiments were performed at different water turbidities to examine relationships between the beam attenuation coefficient (c) and Weibull shape parameters derived from LiDAR waveforms measured with the Fine Structure Underwater LiDAR (FSUIL). Optical inversions were made at 532 nm, within a c range of 0.045-1.52 m-1, and based on a LiDAR system having two field-of-view (15 and 75.7 mrad) and two linear polarizations. Consistently, the Weibull scale parameter or P2 showed the strongest covariation with c and was a more accurate proxy with respect to the LiDAR attenuation coefficient.
RESUMO
Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts â¼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzyme's dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.
Assuntos
Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/enzimologia , Peroxirredoxinas/química , Multimerização Proteica , Especificidade por SubstratoRESUMO
Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Mycobacterium tuberculosis/metabolismo , Peroxirredoxinas/metabolismo , Biocatálise/efeitos dos fármacos , Cisteína/química , Dissulfetos/metabolismo , Glicopeptídeos/química , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Inositol/química , Cinética , Modelos Biológicos , NADP/metabolismo , Oxirredução/efeitos dos fármacosRESUMO
Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.
Assuntos
Eritrócitos/enzimologia , Proteínas de Homeodomínio/metabolismo , Nitrogênio/metabolismo , Estresse Oxidativo/fisiologia , Ácido Peroxinitroso/metabolismo , Animais , Domínio Catalítico , Echinococcus granulosus/enzimologia , Ativação Enzimática/fisiologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tirosina/metabolismoRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.
Assuntos
Proteínas de Protozoários/metabolismo , Superóxido Dismutase/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Sítios de Ligação/genética , Western Blotting , Domínio Catalítico , Doença de Chagas/parasitologia , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Ativação Enzimática/efeitos dos fármacos , Interações Hospedeiro-Parasita , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Nitratos/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologia , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMO
BACKGROUND: Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases - glutathione reductase and thioredoxin reductase - that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes. SCOPE OF REVIEW: This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms. MAJOR CONCLUSIONS: Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism. GENERAL SIGNIFICANCE: The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.
Assuntos
Glutationa/análogos & derivados , Glutationa/metabolismo , Espermidina/análogos & derivados , Trypanosomatina/metabolismo , Animais , Humanos , Oxidantes/metabolismo , Oxirredução , Espermidina/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M-1·s-1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.
Assuntos
Glutationa Peroxidase/metabolismo , Populus/enzimologia , Substituição de Aminoácidos , Domínio Catalítico , Cisteína/química , Dimerização , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Cinética , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Ácido Peroxinitroso/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/genética , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ácidos Sulfênicos/metabolismo , terc-Butil Hidroperóxido/metabolismoRESUMO
Poison frogs are chemically defended from predators by diverse alkaloids, almost all of which are sequestered unchanged from alkaloid-containing arthropods in the frog diet. Oribatid mites recently have been proposed as a major dietary source of poison frog alkaloids. Here, we report on alkaloids common to an oribatid mite and poison frogs. Gas chromatographic-mass spectrometric analysis of methanol extracts of adult Scheloribates laevigatus (Oribatida: Scheloribatidae) revealed nine alkaloids. Five of these have been detected previously in the skin glands of poison frogs: two isomers of the pumiliotoxin 291G, two isomers of the 5,6,8-trisubstituted indolizidine 209C, and the 5,6,8-trisubstituted indolizidine 195G. The other four alkaloids, a pumiliotoxin, a tricyclic (coccinelline-like), and two isomers of an izidine, were not previously known, but are similar in structure to alkaloids found in poison frogs. Alkaloids were not detected in immature S. laevigatus, suggesting that they are adult-specific and possibly the result of mite biosynthesis. Although most of the alkaloids detected in S. laevigatus are common to poison frogs, the geographic distributions of these organisms are not sympatric. The findings of this study indicate that oribatid mites, and in particular, members of the genus Scheloribates, represent a relatively unexplored arthropod repository for alkaloids and a significant dietary source of alkaloids in poison frogs.
Assuntos
Alcaloides/metabolismo , Anuros/metabolismo , Ácaros/metabolismo , Alcaloides/análise , Alcaloides/química , Ração Animal/análise , Animais , Anuros/fisiologia , Óxidos N-Cíclicos/química , Cromatografia Gasosa-Espectrometria de Massas , Compostos Heterocíclicos com 3 Anéis/química , Indolizidinas/metabolismo , Ácaros/química , Especificidade da EspécieRESUMO
PURPOSE: The purpose of this study is to provide a description of the learning environment at Folk High School for participants with high-functioning autism and to examine their learning experience at Folk High School. METHODS: A qualitative interview study was conducted with 21 participants who were enrolled at Folk High School which had been adapted to suit young adults with high-functioning autism. The interviews were analysed by means of a thematic content analysis which resulted in the identification of 6 themes related to learning experiences at Folk High School. RESULTS: The participants enjoyed themselves and felt secure at Folk High School. They felt that they and their academic endeavours were suitably recognised, acknowledged, and understood. They reported that the teaching was suitably adapted for them and they felt that they could succeed in their studies. A frequent report that they made concerned their experience of clear structures in the teaching process and its predictability. The participants stated that Folk High School has the ability to satisfy each participant's needs, which entailed lower levels of perceived stress than what they had experienced in their previous schooling. The participants experienced personal development during their time at Folk High School. CONCLUSIONS: Folk High School, and its special character, is able to successfully satisfy the needs of participants with high-functioning autism. Many of the participants, for the first time in their lives, experienced a sense of inclusion in an educational system and felt that they could succeed in their studies. However, there exists a risk that they become institutionalised, which entails that the participants function well primarily in Folk High School's safe and caring environment.Implications for rehabilitationA supportive environment including both formal and social learning is paramount for people with high-functioning autism.Individually adapted teaching that is structured and predictable improve the conditions under which they can focus on their studies and enjoy academic success.The teachers' relational competence and ability to show interest in each individual are crucial.Social- and special-pedagogic competencies need to co-exist so as to improve learning conditions.Internship/workplace training can provide an important social learning experience for participants, as they learn about themselves and others and as they develop their social competence.To practice living on one's own is a significant challenge, but it can create opportunities to learn about one's self and to develop a sense of responsibility and other social skills.
Assuntos
Sucesso Acadêmico , Transtorno Autístico , Humanos , Pesquisa Qualitativa , Instituições Acadêmicas , Estudantes , Suécia , Adulto JovemRESUMO
The proteasomal degradation system is one of the most important protein degradation systems in the cytosol and nucleus. This system is present in two major forms: the ATP-stimulated 26S/30 S proteasome or the ATP-independent 20S core proteasome. While the first recognize ubiquitin-tagged target proteins and degrade them, the 20S proteasome works also independent from ATP, but requires partially unfolded substrates. While the role of the proteasome in the selective removal of oxidized proteins is undoubted, the debate about a selective ubiquitination of oxidized proteins is still ongoing. Here we demonstrate, that under some conditions of oxidative stress an accumulation of oxidized and of K48-ubiquitinated proteins occurs. However, the removal of oxidized proteins seems not to be linked to ubiquitination. In further experiments, we could show that the accumulation of ubiquitinated proteins under certain oxidative stress conditions is rather a result of a different sensitivity of the 26S proteasome and the ubiquitination machinery towards oxidants.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteínas , Oxirredução , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , UbiquitinaçãoRESUMO
Urolithin A is a metabolite generated from ellagic acid and ellagitannins by the intestinal microbiota after consumption of fruits such as pomegranates or strawberries. The objective of this study was to determine the cytoprotective capacity of this polyphenol in Neuro-2a cells subjected to oxidative stress, as well as its direct radical scavenging activity and properties as an inhibitor of oxidases. Cells treated with this compound and H2O2 showed a greater response to oxidative stress than cells only treated with H2O2, as mitochondrial activity (MTT assay), redox state (ROS formation, lipid peroxidation), and the activity of antioxidant enzymes (CAT: catalase, SOD: superoxide dismutase, GR: glutathione reductase, GPx: glutathione peroxidase) were significantly ameliorated; additionally, urolithin A enhanced the expression of cytoprotective peroxiredoxins 1 and 3. Urolithin A also acted as a direct radical scavenger, showing values of 13.2 µM Trolox Equivalents for Oxygen Radical Absorbance Capacity (ORAC) and 5.01 µM and 152.66 µM IC50 values for superoxide and 2,2-diphenyss1-picrylhydrazyl (DPPH) radicals, respectively. Finally, inhibition of oxidizing enzymes, such as monoamine oxidase A and tyrosinase, was also detected in a dose-dependent manner. The cytoprotective effects of urolithin A could be attributed to the improvement of the cellular antioxidant battery, but also to its role as a direct radical scavenger and enzyme inhibitor of oxidases.
RESUMO
Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen's antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.
Assuntos
Cisteína/química , Mycobacterium tuberculosis/enzimologia , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mycobacterium tuberculosis/patogenicidade , Oxirredução , Peróxidos/antagonistas & inibidores , Peróxidos/metabolismo , Peróxidos/toxicidade , Ácido Peroxinitroso/metabolismo , Conformação Proteica , Especificidade por Substrato , Ácidos Sulfênicos/química , Compostos de Sulfidrila/química , TermodinâmicaRESUMO
Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K(M) of 2.4 microM for human thioredoxin and a very low K(M) for H2O2 (0.7 microM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H2O2 or peroxynitrite, were determined by competition kinetics, k(2) = 1.0 x 10(8) and 1.4 x 10(7) M(-1) s(-1) at 25 degrees C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K(d) < 10(-23) M(4) which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.
Assuntos
Eritrócitos/enzimologia , Peroxirredoxinas/sangue , Animais , Catálise , Cromatografia em Gel , Dimerização , Humanos , Peróxido de Hidrogênio/sangue , Peróxido de Hidrogênio/metabolismo , Cinética , Mamíferos , NADP/sangue , Oxidantes/sangue , Oxirredução , Oxirredutases/sangue , Peroxidases/sangue , Peroxirredoxinas/química , Peroxirredoxinas/isolamento & purificação , Tiorredoxinas/sangueRESUMO
Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions.
Assuntos
Cisteína/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Trifosfato de Adenosina/metabolismo , Cisteína/genética , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Mitocôndrias/genética , Oxirredução , Peroxirredoxina III/metabolismo , Peroxirredoxinas/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , UbiquitinaçãoRESUMO
Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2α pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging.
Assuntos
Envelhecimento/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Fibroblastos/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Aldeído Pirúvico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxidantes/metabolismo , Aldeído Pirúvico/toxicidade , PeleRESUMO
The production of reactive species is an inevitable by-product of metabolism and thus, life itself. Since reactive species are able to damage cellular structures, especially proteins, as the most abundant macromolecule of mammalian cells, systems are necessary which regulate and preserve a functional cellular protein pool, in a process termed "proteostasis". Not only the mammalian protein pool is subject of a constant turnover, organelles are also degraded and rebuild. The most important systems for these removal processes are the "ubiquitin-proteasomal system" (UPS), the central proteolytic machinery of mammalian cells, mainly responsible for proteostasis, as well as the "autophagy-lysosomal system", which mediates the turnover of organelles and large aggregates. Many age-related pathologies and the aging process itself are accompanied by a dysregulation of UPS, autophagy and the cross-talk between both systems. This review will describe the sources and effects of oxidative stress, preservation of cellular protein- and organelle-homeostasis and the effects of aging on proteostasis in mammalian cells.
Assuntos
Envelhecimento/metabolismo , Estresse Oxidativo , Proteostase , Animais , Autofagia , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Lipofuscina/metabolismoRESUMO
Mitochondria have been in the focus of oxidative stress and aging research for decades due to their permanent production of ROS during the oxidative phosphorylation. The hypothesis exists that mitochondria are involved in the formation of lipofuscin, an autofluorescent protein aggregate that accumulates progressively over time in lysosomes of post-mitotic and senescent cells. To investigate the influence and involvement of mitochondria in lipofuscinogenesis, we analyzed lipofuscin amounts as well as the mitochondrial function in young and senescent cells. In addition we used an aging model and Lon protease deficient HeLa cells to investigate the influence of mitochondrial degradation processes on lipofuscin formation. We were able to show that mitophagy is impaired in senescent cells resulting in an increased mitochondrial mass and superoxide formation. In addition, the inhibition of mitochondrial fission leads to increased lipofuscin formation. Moreover, we observed that Lon protease downregulation is linked to a higher lipofuscinogenesis whereas the application of the mitochondrial-targeted antioxidant mitoTEMPO is able to prevent the accumulation of this protein aggregate.