RESUMO
Inflammatory bowel disease (IBD) is characterized by multiple alterations in cytokine expression and is a risk factor for colon cancer. The Omega class glutathione transferase GSTO1-1 regulates the release of the pro-inflammatory cytokines interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) by deglutathionylating NEK7 in the NLRP3 inflammasome. When treated with azoxymethane and dextran sodium sulphate (AOM/DSS) as a model of IBD, Gsto1-/- mice were highly sensitive to colitis and showed a significant increase in the size and number of colon tumours compared with wild-type (WT) mice. Gsto1-/- mice treated with AOM/DSS had significantly lower serum IL-1ß and IL-18 levels as well as significantly decreased interferon (IFN)-γ, decreased pSTAT1 and increased pSTAT3 levels in the distal colon compared with similarly treated WT mice. Histologically, AOM/DSS treated Gsto1-/- mice showed increased active chronic inflammation with macrophage infiltration, epithelial dysplasia and invasive adenocarcinoma compared with AOM/DSS treated WT mice. Thus, this study shows that GSTO1-1 regulates IL-1ß and IL-18 activation and protects against colorectal cancer formation in the AOM/DSS model of IBD. The data suggest that while GSTO1-1 is a new target for the regulation of the NLRP3 inflammasome-associated cytokines IL-1ß and IL-18 by small molecule inhibitors, there is a possibility that anti-inflammatory drugs targeting these cytokines may potentiate colon cancer in some situations.
Assuntos
Azoximetano/toxicidade , Proteínas de Transporte/fisiologia , Colite/complicações , Neoplasias Colorretais/prevenção & controle , Glutationa Transferase/fisiologia , Inflamação/prevenção & controle , Interleucina-18/sangue , Interleucina-1beta/sangue , Animais , Carcinógenos/toxicidade , Colite/induzido quimicamente , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Sulfato de Dextrana/toxicidade , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Drug metabolizing enzymes catalyze the biotransformation of many of drugs and chemicals. The drug metabolizing enzymes are distributed among several evolutionary families and catalyze a range of detoxication reactions, including oxidation/reduction, conjugative, and hydrolytic reactions that serve to detoxify potentially toxic compounds. This detoxication function requires that drug metabolizing enzymes exhibit substrate promiscuity. In addition to their catalytic functions, many drug metabolizing enzymes possess functions unrelated to or in addition to catalysis. Such proteins are termed 'moonlighting proteins' and are defined as proteins with multiple biochemical or biophysical functions that reside in a single protein. This review discusses the diverse moonlighting functions of drug metabolizing enzymes and the roles they play in physiological functions relating to reproduction, vision, cell signaling, cancer, and transport. Further research will likely reveal new examples of moonlighting functions of drug metabolizing enzymes.
Assuntos
Biotransformação , Humanos , OxirreduçãoRESUMO
BACKGROUND: Glutathione S-transferases omega class 1 (GSTO1-1) is a unique member of the GST family regulating cellular redox metabolism and innate immunity through the promotion of LPS/TLR4/NLRP3 signalling in macrophages. House dust mite (HDM) triggers asthma by promoting type 2 responses and allergic inflammation via the TLR4 pathway. Although linked to asthma, the role of GSTO1-1 in facilitating type 2 responses and/or HDM-driven allergic inflammation is unknown. OBJECTIVE: To determine the role of GSTO1-1 in regulating HDM-induced allergic inflammation in a preclinical model of asthma. METHODS: Wild-type and GSTO1-1-deficient mice were sensitized and aeroallergen challenged with HDM to induce allergic inflammation and subsequently hallmark pathophysiological features characterized. RESULTS: By contrast to HDM-challenged WT mice, exposed GSTO1-1-deficient mice had increased numbers of eosinophils and macrophages and elevated levels of eotaxin-1 and -2 in their lungs. M1 macrophage-associated factors, such as IL-1ß and IL-6, were decreased in GSTO1-1-deficient mice. Conversely, M2 macrophage factors such as Arg-1 and Ym1 were up-regulated. HIF-1α expression was found to be higher in the absence of GSTO1-1 and correlated with the up-regulation of M2 macrophage markers. Furthermore, HIF-1α was shown to bind and activate the eotaxin-2 promotor. Hypoxic conditions induced significant increases in the levels of eotaxin-1 and -2 in GSTO1-deficient BMDMs, providing a potential link between inflammation-induced hypoxia and the regulation of M2 responses in the lung. Collectively, our results suggest that GSTO1-1 deficiency promotes M2-type responses and increased levels of nuclear HIF-1α, which regulates eotaxin (s)-induced eosinophilia and increased disease severity. CONCLUSION & CLINICAL IMPLICATION: We propose that GSTO1-1 is a novel negative regulator of TLR4-regulated M2 responses acting as an anti-inflammatory pathway. The discovery of a novel HIF-1α-induced eotaxin pathway identifies an unknown connection between hypoxia and the regulation of the severity of allergic inflammation in asthma.
Assuntos
Asma/imunologia , Proteínas de Transporte/imunologia , Eosinófilos/imunologia , Glutationa Transferase/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/metabolismo , Animais , Asma/genética , Asma/patologia , Proteínas de Transporte/genética , Eosinófilos/patologia , Glutationa Transferase/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a ß-strand and repositioning of the functionally important "BB-loop" region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.
Assuntos
Glutationa/metabolismo , Glicoproteínas de Membrana , Processamento de Proteína Pós-Traducional , Receptores de Interleucina-1 , Transdução de Sinais , Substituição de Aminoácidos , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Glutationa/química , Glutationa/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Relação Estrutura-AtividadeRESUMO
Ryanodine receptor (RyR) Ca2+ channels are central to striated muscle function and influence signalling in neurons and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However, the correlation with submaximal opening is debated and quantitative evidence is lacking. Here, we have measured altered FKBP binding to RyRs and submaximal activity with addition of wild-type (WT) CLIC2, an inhibitory RyR ligand, or its H101Q mutant that hyperactivates RyRs, which probably causes cardiac and intellectual abnormalities. The proportion of sub-conductance opening increases with WT and H101Q CLIC2 and is correlated with reduced FKBP-RyR association. The sub-conductance opening reduces RyR currents in the presence of WT CLIC2. In contrast, sub-conductance openings contribute to excess RyR 'leak' with H101Q CLIC2. There are significant FKBP and RyR isoform-specific actions of CLIC2, rapamycin and FK506 on FKBP-RyR association. The results show that FKBPs do influence RyR gating and would contribute to excess Ca2+ release in this CLIC2 RyR channelopathy.
Assuntos
Canais de Cloreto/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Ativação do Canal Iônico , Potenciais da Membrana , Mutação de Sentido Incorreto , Ligação Proteica , Coelhos , Carneiro DomésticoRESUMO
BACKGROUND: Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)-infected gastric epithelial cells in culture. The CHAC1-induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa. MATERIALS AND METHODS: Both fresh-frozen and formalin-fixed paraffin-embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago-gastrectomy. Fresh samples were used for real-time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin-fixed samples were used for immunohistochemistry with anti-CHAC1 and anti-H. pylori monoclonal antibodies. Double-enzyme or fluorescence immunohistochemistry and immuno-electron microscopy were used for further analysis. RESULTS: Significant CHAC1 overexpression was detected in H. pylori-infected parietal cells that expressed the human proton pump/H,K-ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic-gland areas. Immuno-electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection. CONCLUSION: Cation transport regulator 1 overexpression in H. pylori-infected parietal cells may cause the H. pylori-induced somatic mutations that contribute to the development of gastric cancer.
Assuntos
Mucosa Gástrica/metabolismo , Infecções por Helicobacter/genética , Helicobacter pylori/fisiologia , gama-Glutamilciclotransferase/genética , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Humanos , Células Parietais Gástricas/metabolismo , Células Parietais Gástricas/microbiologia , Células Parietais Gástricas/patologia , gama-Glutamilciclotransferase/metabolismoRESUMO
Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and ß subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR ß subunit ß1a Removal of the intrinsically disordered N and C termini and the hook region of ß1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined ß isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of â¼2-3 °C between the ß1a and ß2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both ß isoforms by â¼6-8 °C. Similar to other ß isoforms, ß1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of ß1a while also providing a conduit for allosteric signaling events.
Assuntos
Canais de Cálcio Tipo L/química , Guanilato Quinases/química , Regulação Alostérica , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Cristalografia por Raios X , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Camundongos , Estrutura Secundária de Proteína , Transdução de Sinais , Domínios de Homologia de srcRESUMO
Macrophages mediate innate immune responses that recognise foreign pathogens, and bacterial lipopolysaccharide (LPS) recruits a signalling pathway through Toll-like receptor 4 (TLR4) to induce pro-inflammatory cytokines and reactive oxygen species (ROS). LPS activation also skews the metabolism of macrophages towards a glycolytic phenotype. Here, we demonstrate that the LPS-triggered glycolytic switch is significantly attenuated in macrophages deficient for glutathione transferase omega-1 (GSTO1, note that GSTO1-1 refers to the dimeric molecule with identical type 1 subunits). In response to LPS, GSTO1-1-deficient macrophages do not produce excess lactate, or dephosphorylate AMPK, a key metabolic stress regulator. In addition, GSTO1-1-deficient cells do not induce HIF1α, which plays a key role in maintaining the pro-inflammatory state of activated macrophages. The accumulation of the TCA cycle intermediates succinate and fumarate that occurs in LPS-treated macrophages was also blocked in GSTO1-1-deficient cells. These data indicate that GSTO1-1 is required for LPS-mediated signalling in macrophages and that it acts early in the LPS-TLR4 pro-inflammatory pathway.
Assuntos
Proteínas de Transporte/metabolismo , Glutationa Transferase/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Proteínas de Transporte/genética , Glutationa Transferase/deficiência , Glutationa Transferase/genética , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genéticaRESUMO
Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin.
Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Ácido Dicloroacético/uso terapêutico , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Animais , Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The Omega-class cytosolic glutathione transferases (GSTs) have distinct structural and functional attributes that allow them to perform novel roles unrelated to the functions of other GSTs. Mammalian GSTO1-1 has been found to play a previously unappreciated role in the glutathionylation cycle that is emerging as significant mechanism regulating protein function. GSTO1-1-catalyzed glutathionylation or deglutathionylation of a key signaling protein may explain the requirement for catalytically active GSTO1-1 in LPS-stimulated pro-inflammatory signaling through the TLR4 receptor. The observation that ML175 a specific GSTO1-1 inhibitor can block LPS-stimulated inflammatory signaling has opened a new avenue for the development of novel anti-inflammatory drugs that could be useful in the treatment of toxic shock and other inflammatory disorders. The role of GSTO2-2 remains unclear. As a dehydroascorbate reductase, it could contribute to the maintenance of cellular redox balance and it is interesting to note that the GSTO2 N142D polymorphism has been associated with multiple diseases including Alzheimer's disease, Parkinson's disease, familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, age-related cataract and breast cancer.
Assuntos
Glutationa Transferase/metabolismo , Glutationa/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Predisposição Genética para Doença , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , Inativação Metabólica , Oxirredução , Polimorfismo Genético , Conformação Proteica , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified ß-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.
Assuntos
Glutationa Transferase/metabolismo , Glutationa/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Actinas/genética , Actinas/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Linhagem Celular Tumoral , Glutationa/genética , Glutationa Transferase/genética , Humanos , Mutação de Sentido Incorreto , Polimorfismo GenéticoRESUMO
Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.
Assuntos
Evolução Molecular , Glutationa Transferase/química , Óxido Nítrico/química , Animais , Bactérias/enzimologia , Bactérias/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Óxido Nítrico/genética , Óxido Nítrico/metabolismoRESUMO
BACKGROUND: The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions. SCOPE OF REVIEW: The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism. MAJOR CONCLUSIONS: All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca(2+) channels in skeletal and cardiac muscle. GENERAL SIGNIFICANCE: In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione.
Assuntos
Glutationa Transferase/metabolismo , Glutationa/metabolismo , Animais , Biotransformação , Catálise , Citosol/metabolismo , Glutationa/genética , Glutationa Transferase/genética , Humanos , Transdução de Sinais , Xenobióticos/metabolismo , Xenobióticos/farmacocinéticaRESUMO
Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca(2+) release channels. These RyR proteins play a major role in the regulation of Ca(2+) signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca(2+) signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via 'leaky mutations' can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca(2+) release through RyRs in neuronal cells and cardiac muscle cells.
Assuntos
Cardiomegalia/genética , Canalopatias/genética , Canais de Cloreto/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Sequência de Aminoácidos , Cálcio/metabolismo , Cardiomegalia/complicações , Canalopatias/complicações , Canais de Cloreto/metabolismo , Família , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Humanos , Masculino , Dados de Sequência Molecular , Miócitos Cardíacos/metabolismo , Linhagem , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Accumulating evidence shows that glutathione peroxidase (GPX, EC.1.11.1.9), one of the most important antioxidant selenoenzymes, plays an essential role in protecting cells and tissues against oxidative damage by catalyzing the reduction of hydrogen peroxide by glutathione. Unfortunately, because of the limited availability and poor stability of GPX, it has not been used clinically to protect against oxidative stress. To overcome these problems, it is necessary to generate mimics of GPX. In this study, we have used directed mutagenesis and the inclusion of a selenocysteine (Sec) insertion sequence to engineer the expression in eukaryotic cells of human glutathione transferase zeta1-1 (hGSTZ1-1) with Sec in the active site (seleno-hGSTZ1-1). This modification converted hGSTZ1-1 into an active GPX and is the first time this has been achieved in eukaryotic cells. The GPX activity of seleno-hGSTZ1-1 is higher than that of GPX from bovine liver, indicating Sec at the active site plays an important role in the determination of catalytic specificity and performance. Kinetic studies revealed that the ping-pong catalytic mechanism of Se-hGSTZ1-1 is similar to that of the natural GPX.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/genética , Selenocisteína/química , Selenocisteína/genética , Catálise , Domínio Catalítico , Glutationa Peroxidase/química , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Selênio/química , Selênio/metabolismo , Selenocisteína/metabolismo , TemperaturaRESUMO
This study reports the development of a new assay for the rapid determination of protein glutathionylation in tissues and cell lines using commercially available reagents and standard instrumentation. In this method cells are homogenized in the presence of N-ethylmaleimide to eliminate free thiols and the proteins are precipitated with acetone. Subsequently, the disulfide-bound glutathione is eluted from the protein by the addition of tris(2-carboxyethyl)phosphine and reacted with 2,3-napthalenedicarboxaldehyde to generate a highly fluorescent product. Lymphoblastoid cell lines were found to have glutathionylation levels in the range of 0.3-3 nmol/mg protein, which were significantly elevated after treatment of the cells with S-nitrosoglutathione. Mouse tissues including liver, kidney, lung, heart, brain, spleen, and testes were found to have glutathionylation levels between 1 and 2.5 nmol/mg protein and the levels tended to increase after treatment of mice with doxorubicin. In contrast, mouse skeletal muscle glutathionylation was significantly higher (4.2 ± 0.33 nmol/mg, p < 0.001) than in other tissues in untreated mice and decreased to 1.9 ± 0.15 nmol/mg after doxorubicin treatment. This new method allows rapid measurement of cellular glutathionylation in a high-throughput 96-well plate format.
Assuntos
Fluorometria/métodos , Glutationa/metabolismo , Naftalenos/química , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular Tumoral , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de ÓrgãosRESUMO
Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the ß(1a) subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide ß(1a)V490-M524, corresponding to the C-terminal 35 residues of ß(1a), reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in ß(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type ß(1a)A474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue ß(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Canais de Cálcio Tipo L/genética , Acoplamento Excitação-Contração , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Homologia de Sequência de Aminoácidos , Propriedades de SuperfícieRESUMO
S-Glutathionyl-hydroquinone reductases (GS-HQRs) are a new class of glutathione transferases, widely present in bacteria, halobacteria, fungi, and plants. They catalyze glutathione (GSH)-dependent reduction of GS-trichloro-p-hydroquinone to trichloro-p-hydroquinone. Since GS-trichloro-p-hydroquinone is uncommon in nature, the extensive presence of GS-HQRs suggests they use common GS-hydroquinones. Here we demonstrate that several benzoquinones spontaneously reacted with GSH to form GS-hydroquinones via Michael addition, and four GS-HQRs from yeast and bacteria reduced the GS-hydroquinones to the corresponding hydroquinones. The spontaneous and enzymatic reactions led to the reduction of benzoquinones to hydroquinones with the concomitant oxidation of GSH to oxidized glutathione (GS-SG). The enzymes did not use GS-benzoquinones or other thiol-hydroquinones, for example, S-cysteinyl-hydroquinone, as substrates. Apparent kinetic parameters showed the enzymes preferred hydrophobic, bulky substrates, such as GS-menadiol. The broad substrate range and their wide distribution suggest two potential physiological roles: channeling GS-hydroquinones back to hydroquinones and reducing benzoquinones via spontaneous formation of GS-hydroquinones and then enzymatic reduction to hydroquinones. The functions are likely important in metabolic pathways with quinone intermediates.
Assuntos
Benzoquinonas/química , Glutationa Transferase/química , Glutationa/química , Hidroquinonas/química , Benzoquinonas/metabolismo , Cupriavidus necator/enzimologia , Proteínas Fúngicas/química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Hidroquinonas/metabolismo , Oxirredução , Phanerochaete/enzimologia , Proteínas Recombinantes de Fusão/química , Saccharomyces cerevisiae/enzimologiaRESUMO
A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and ß-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.