RESUMEN
Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.
Asunto(s)
Glutatión Transferasa , Melaninas , Melanoma , Animales , Humanos , Ratones , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Melaninas/biosíntesis , Melanoma/genética , Melanoma/inmunología , Melanoma/fisiopatología , Pez Cebra/metabolismo , Oxidación-Reducción , Ratones Endogámicos C57BL , Línea Celular Tumoral , Proliferación Celular/genéticaRESUMEN
While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Melanoma , Humanos , Ratones , Animales , Glutatión Transferasa/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Estrés Oxidativo , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Glutatión/metabolismoRESUMEN
Prostaglandin E2 (PGE2) is a lipid mediator of inflammation and cancer progression. It is mainly formed via metabolism of arachidonic acid by cyclooxygenases (COX) and the terminal enzyme microsomal prostaglandin E synthase-1 (mPGES-1). Widely used non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity, resulting in decreased PGE2 production and symptomatic relief. However, NSAIDs block the production of many other lipid mediators that have important physiological and resolving actions, and these drugs cause gastrointestinal bleeding and/or increase the risk for severe cardiovascular events. Selective inhibition of downstream mPGES-1 for reduction in only PGE2 biosynthesis is suggested as a safer therapeutic strategy. This review covers the recent advances in characterization of new mPGES-1 inhibitors in preclinical models and their future clinical applications.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Prostaglandina-E Sintasas/antagonistas & inhibidores , Animales , Ensayos Clínicos como Asunto , Humanos , Prostaglandina-E Sintasas/metabolismoRESUMEN
Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.
Asunto(s)
Dipéptidos/química , Oxidorreductasas Intramoleculares/química , Microsomas/enzimología , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Glutatión/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Ligandos , Mutagénesis Sitio-Dirigida , Prostaglandina-E Sintasas , Conformación ProteicaRESUMEN
Microsomal glutathione transferase 1 (MGST1) has a unique ability to be activated, ≤30-fold, by modification with sulfhydryl reagents. MGST1 exhibits one-third-of-the-sites reactivity toward glutathione and hence heterogeneous binding to different active sites in the homotrimer. Limited turnover stopped-flow kinetic measurements of the activated enzyme allowed us to more accurately determine the KD for the "third" low-affinity GSH binding site (1.4 ± 0.3 mM). The rate of thiolate formation, k2 (0.77 ± 0.06 s-1), relevant to turnover, could also be determined. By deriving the steady-state rate equation for a random sequential mechanism for MGST1, we can predict KM, kcat, and kcat/KM values from these and previously determined pre-steady-state rate constants (all determined at 5 °C). To assess whether the pre-steady-state behavior can account for the steady-state kinetic behavior, we have determined experimental values for kinetic parameters at 5 °C. For reactive substrates and the activated enzyme, data for the microscopic steps account for the global mechanism of MGST1. For the unactivated enzyme and more reactive electrophilic substrates, pre-steady-state and steady-state data can be reconciled only if a more active subpopulation of MGST1 is assumed. We suggest that unactivated MGST1 can be partially activated in its unmodified form. The existence of an activated subpopulation (approximately 10%) could be demonstrated in limited turnover experiments. We therefore suggest that MSGT1 displays a preexisting dynamic equilibrium between high- and low-activity forms.
Asunto(s)
Glutatión Transferasa/metabolismo , Biocatálisis , Activación Enzimática , Glutatión Transferasa/química , Humanos , Cinética , Modelos Moleculares , Estructura MolecularRESUMEN
Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.
Asunto(s)
Glutatión Transferasa/metabolismo , Microsomas Hepáticos/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Estrés Oxidativo/fisiologíaRESUMEN
Previous studies that have investigated the association between B-vitamin supplement use and risk for cataract yield conflicting results. The aim of this study was to examine the association between use of high-dose B-vitamin supplements (approximately 10 times recommended daily intake) and risk for age-related cataract in a population-based prospective study of 13 757 women from the Swedish Mammography Cohort and 22 823 men from the Cohort of Swedish Men. Dietary supplement use and potential confounders were assessed using a questionnaire at baseline. Information on cataract diagnosis and extraction was obtained through linkage to registers. During the follow-up period between January 1998 and December 2011, we identified 8395 cataract cases (3851 for women and 4544 for men). The use of B vitamins plus other supplements and B vitamins only was associated with 9 % (95 % CI 2, 17) and 27 % (95 % CI 12, 43) increased risk for cataract, respectively. The hazard ratios for use of B vitamins only and risk for cataract stratified by different age groups were as follows: <60 years: 1·88 (95 % CI 1·47, 2·39); 60-69 years: 1·21 (95 % CI 0·96, 1·53); and ≥70 years: 1·09 (95 % CI 0·91, 1·31) (P interaction=0·002). Our results suggest that the use of high-dose B-vitamin supplements was associated with an increased risk for cataract. This association might be confined to younger participants.
Asunto(s)
Envejecimiento , Catarata/inducido químicamente , Catarata/epidemiología , Complejo Vitamínico B/administración & dosificación , Complejo Vitamínico B/efectos adversos , Anciano , Estudios de Cohortes , Dieta , Suplementos Dietéticos , Femenino , Humanos , Estilo de Vida , Masculino , Persona de Mediana Edad , Fenómenos Fisiológicos de la Nutrición , Estudios Prospectivos , Ingesta Diaria Recomendada , Factores de Riesgo , Encuestas y Cuestionarios , SueciaRESUMEN
Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA4 forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometry of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.
Asunto(s)
Dinitroclorobenceno/química , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Glutatión/química , Secuencia de Aminoácidos , Calorimetría , Dominio Catalítico , Dinitroclorobenceno/metabolismo , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Glutatión/metabolismo , Glutatión Transferasa/genética , Humanos , Cinética , Microsomas/enzimología , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Pichia/genética , Pichia/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.
Asunto(s)
Glutatión Transferasa/metabolismo , Profármacos/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Citostáticos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Etopósido/farmacología , Glutatión/metabolismo , Humanos , Células MCF-7 , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Glutathione transferases (GSTs) are the primary catalysts protecting from reactive electrophile attack. In this review, the quantitative levels and distribution of glutathione transferases in relation to physiological function are discussed. The catalytic properties (random sequential) tell us that these enzymes have evolved to intercept reactive intermediates. High concentrations of enzymes (up to several hundred micromolar) ensure efficient protection. Individual enzyme molecules, however, turn over only rarely (estimated as low as once daily). The protection of intracellular protein and DNA targets is linearly proportional to enzyme levels. Any lowering of enzyme concentration, or inhibition, would thus result in diminished protection. It is well established that GSTs also function as binding proteins, potentially resulting in enzyme inhibition. Here the relevance of ligand inhibition and catalytic mechanisms, such as negative co-operativity, is discussed. There is a lack of knowledge pertaining to relevant ligand levels in vivo, be they exogenous or endogenous (e.g., bile acids and bilirubin). The stoichiometry of active sites in GSTs is well established, cytosolic enzyme dimers have two sites. It is puzzling that a third of the site's reactivity is observed in trimeric microsomal glutathione transferases (MGSTs). From a physiological point of view, such sub-stoichiometric behavior would appear to be wasteful. Over the years, a substantial amount of detailed knowledge on the structure, distribution, and mechanism of purified GSTs has been gathered. We still lack knowledge on exact cell type distribution and levels in vivo however, especially in relation to ligand levels, which need to be determined. Such knowledge must be gathered in order to allow mathematical modeling to be employed in the future, to generate a holistic understanding of reactive intermediate protection.
Asunto(s)
Glutatión Transferasa , Glutatión Transferasa/metabolismo , Humanos , Cinética , AnimalesRESUMEN
Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 µM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of â¼0.2 µmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S exhibit distinct catalytic and mechanistic properties, reflecting adaptation to broad and specific physiological functions, respectively.
Asunto(s)
Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Catálisis , Dominio Catalítico , Glutatión/metabolismo , Glutatión Transferasa/genética , Humanos , Cinética , Leucotrieno C4/biosíntesis , Protones , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismoRESUMEN
We examined the associations of high-dose supplements of vitamins C and E and low-dose multivitamins with the risk of age-related cataract among 31,120 Swedish men, aged 45-79 years, in a population-based prospective cohort. Dietary supplement use was assessed from a questionnaire at baseline in 1998. During follow-up (January 1998-December 2006), 2,963 incident age-related cataract cases were identified. The multivariable-adjusted hazard ratio for men using vitamin C supplements only was 1.21 (95% confidence interval (CI): 1.04, 1.41) in a comparison with that of non-supplement users. The hazard ratio for long-term vitamin C users (≥10 years before baseline) was 1.36 (95% CI: 1.02, 1.81). The risk of cataract with vitamin C use was stronger among older men (>65 years) (hazard ratio = 1.92, 95% CI: 1.41, 2.60) and corticosteroid users (hazard ratio = 2.11, 95% CI: 1.48, 3.02). The hazard ratio for vitamin E use only was 1.59 (95% CI: 1.12, 2.26). Use of multivitamins only or multiple supplements in addition to vitamin C or E was not associated with cataract risk. These results suggest that the use of high-dose (but not low-dose) single vitamin C or E supplements may increase the risk of age-related cataract. The risk may be even higher among older men, corticosteroid users, and long-term users.
Asunto(s)
Ácido Ascórbico/administración & dosificación , Catarata/epidemiología , Corticoesteroides/administración & dosificación , Factores de Edad , Anciano , Consumo de Bebidas Alcohólicas/epidemiología , Catarata/inducido químicamente , Dieta , Suplementos Dietéticos , Escolaridad , Humanos , Incidencia , Estilo de Vida , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Riesgo , Fumar/epidemiología , Encuestas y Cuestionarios , Suecia/epidemiología , Vitamina E/administración & dosificación , Vitaminas/administración & dosificación , Circunferencia de la CinturaRESUMEN
We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the σ value in the same way as the non-enzymatic reaction, whereas the dependence of the σ value of the GST mu and pi was not as pronounced as that of GST alpha.
Asunto(s)
Colorantes Fluorescentes/química , Glutatión Transferasa/metabolismo , Nitrobencenos/química , Biocatálisis , HumanosRESUMEN
Glutathione transferases (GSTs) are a class of phase II detoxifying enzymes catalysing the conjugation of glutathione (GSH) to endogenous and exogenous electrophilic molecules, with microsomal glutathione transferase 1 (MGST1) being one of its key members. MGST1 forms a homotrimer displaying third-of-the-sites-reactivity and up to 30-fold activation through modification of its Cys-49 residue. It has been shown that the steady-state behaviour of the enzyme at 5 °C can be accounted for by its pre-steady-state behaviour if the presence of a natively activated subpopulation (~ 10%) is assumed. Low temperature was used as the ligand-free enzyme is unstable at higher temperatures. Here, we overcame enzyme lability through stop-flow limited turnover analysis, whereby kinetic parameters at 30 °C were obtained. The acquired data are more physiologically relevant and enable confirmation of the previously established enzyme mechanism (at 5 °C), yielding parameters relevant for in vivo modelling. Interestingly, the kinetic parameter defining toxicant metabolism, kcat /KM , is strongly dependent on substrate reactivity (Hammett value 4.2), underscoring that glutathione transferases function as efficient and responsive interception catalysts. The temperature behaviour of the enzyme was also analysed. Both the KM and KD values decreased with increasing temperature, while the chemical step k3 displayed modest temperature dependence (Q10 : 1.1-1.2), mirrored in that of the nonenzymatic reaction (Q10 : 1.1-1.7). Unusually high Q10 values for GSH thiolate anion formation (k2 : 3.9), kcat (2.7-5.6) and kcat /KM (3.4-5.9) support that large structural transitions govern GSH binding and deprotonation, which limits steady-state catalysis.
Asunto(s)
Glutatión Transferasa , Proteínas de la Membrana , Catálisis , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Cinética , Temperatura , Animales , RatasRESUMEN
Microsomal glutathione transferase 1 (MGST1) is a member of the MAPEG family (membrane associated proteins in eicosanoid and glutathione metabolism), defined according to enzymatic activities, sequence motifs, and structural properties. MGST1 is a homotrimer which can bind three molecules of glutathione (GSH), with one modified to a thiolate anion displaying one-third-of-sites-reactivity. MGST1 has both glutathione transferase and peroxidase activities. Each is based on stabilizing the GSH thiolate in the same active site. MGST1 is abundant in the liver and displays a broad subcellular distribution with high levels in endoplasmic reticulum and mitochondrial membranes, consistent with a physiological role in protection from reactive electrophilic intermediates and oxidative stress. In this review paper, we particularly focus on recent advances made in understanding MGST1 activation, induction, broad subcellular distribution, and the role of MGST1 in apoptosis, ferroptosis, cancer progression, and therapeutic responses.
Asunto(s)
Ferroptosis , Neoplasias , Humanos , Apoptosis , Glutatión , Glutatión TransferasaRESUMEN
Human leukotriene C4 synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C4 (LTC4). This molecule, together with its metabolites LTD4 and LTE4, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA4, presumably according to an S(N)2 reaction to form LTC4. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s⻹). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K(d((GS) = 14.3 µM). The release of the thiolate was assessed using a GSH release experiment (1.3 s⻹). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC4 production (k(cat) = 26 s⻹), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.
Asunto(s)
Glutatión Transferasa/metabolismo , Glutatión/análogos & derivados , Leucotrieno C4/metabolismo , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Arginina/química , Unión Competitiva , Biocatálisis , Dominio Catalítico/efectos de los fármacos , Cisteína/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glutatión/química , Glutatión/metabolismo , Glutatión/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Leucotrieno A4/metabolismo , Terapia Molecular Dirigida , Conformación Proteica/efectos de los fármacos , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E(2) (PGE(2)). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a naturally occurring degradation product of prostaglandin D(2), is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ(2) can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH(2)) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ(2) were determined. The question of whether the anti-inflammatory properties of 15d-PGJ(2) are due to inhibition of MPGES1 is discussed.
Asunto(s)
Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Microsomas/enzimología , Prostaglandina D2/análogos & derivados , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Sitios de Unión , Glutatión/análogos & derivados , Glutatión/química , Glutatión/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/metabolismo , Espectrometría de Masas , Microsomas/metabolismo , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacología , Prostaglandina-E SintasasRESUMEN
The inducible microsomal prostaglandin E(2) synthase 1 (MPGES1) is an integral membrane protein coexpressed with and functionally coupled to cyclooxygenase 2 (COX-2) generating the pro-inflammatory molecule PGE(2). The development of effective inhibitors of MPGES1 holds promise as a highly selective route for controlling inflammation. In this paper, we describe the use of backbone amide H/D exchange mass spectrometry to map the binding sites of different types of inhibitors of MPGES1. The results reveal the locations of specific inhibitor binding sites that include the GSH binding site and a hydrophobic cleft in the protein thought to accommodate the prostaglandin H(2) substrate. In the absence of three-dimensional crystal structures of the enzyme-bound inhibitors, the results provide clear physical evidence that three pharmacologically active inhibitors bind in a hydrophobic cleft composed of sections of transmembrane helices Ia, IIb, IIIb, and IVb at the interface of subunits in the trimer. In principle, the H/D exchange behavior of the protein can be used as a preliminary guide for optimization of inhibitor efficacy. Finally, a comparison of the structures and H/D exchange behavior of MPGES1 and the related enzyme MGST1 in the presence of glutathione and the inhibitor glutathione sulfonate confirms the unusual observation that two proteins from the same superfamily harbor GSH binding sites in different locations.
Asunto(s)
Inhibidores de la Ciclooxigenasa/metabolismo , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/química , Sitios de Unión , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Glutatión/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Oxidorreductasas Intramoleculares/metabolismo , Prostaglandina-E Sintasas , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E(2). Nonsteroidal anti-inflammatory drugs prevent prostaglandin E(2) production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH(2), or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Concentración 50 Inhibidora , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Prostaglandina-E Sintasas , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Human leukotriene C(4) synthase (hLTC(4)S) is an integral membrane enzyme that conjugates leukotriene (LT) A(4) with glutathione to form LTC(4), a precursor to the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC(4)S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3-99.9% of the specific activity against LTA(4). Steady-state kinetics of R104A and R104S revealed that the K(m) for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λ(max) at 239 nm, could be detected using R104A or R104S hLTC(4)S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pK(a) to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA(4). On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC(4)S by 88 and 70%, respectively, without significantly affecting the k(cat)/K(m) values for GSH, and a crystal structure of R31Q hLTC(4)S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC(4)S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.