RESUMEN
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
Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades de Proteína/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Escherichia coli/citología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Eliminación de Gen , Chaperonas Moleculares/genética , Estrés Oxidativo , Periplasma/metabolismo , Transporte de Proteínas , Canales de Translocación SECRESUMEN
Members of the CorA/Mrs2/Alr1 superfamily of transport proteins mediate magnesium uptake in all kingdoms of life. Family members have a low degree of sequence conservation but are characterized by a conserved extracellular loop. While the degree of sequence conservation in the loop deviates to some extent between family members, the GMN family signature motif is always present. Structural and functional data imply that the loop plays a central role in magnesium selectivity, and recent biochemical data suggest it is crucial for stabilizing the pentamer in the magnesium-free (putative open) conformation. In this study, we present a detailed structure-function analysis of the extracellular loop of CorA from Thermotoga maritima, which provides molecular insight into how the loop mediates these two functions. The data show that loop residues outside of the GMN motif can be substituted if they support the pentameric state, but the residues of the GMN motif are intolerant to substitution. We conclude that G(312) is absolutely required for magnesium uptake, M(313) is absolutely required for pentamer integrity in the putative open conformation, and N(314) plays a role in both of these functions. These observations suggest a molecular reason why the GMN motif is conserved throughout the CorA/Mrs2/Alr1 superfamily.
Asunto(s)
Secuencias de Aminoácidos , Proteínas de Transporte de Catión/química , Secuencia Conservada , Magnesio/metabolismo , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Crystal structures of the CorA Mg(2+) channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open "metal free" state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg(2+). We find that when Mg(2+) is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg(2+) is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.
Asunto(s)
Proteínas de Transporte de Catión/química , Magnesio/química , Thermotoga maritima/química , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Cristalografía por Rayos X , Transporte Iónico/fisiología , Magnesio/metabolismo , Periplasma/química , Periplasma/genética , Periplasma/metabolismo , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Thermotoga maritima/genética , Thermotoga maritima/inmunologíaRESUMEN
Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/prevención & control , Riñón/patología , Lípidos/toxicidad , Transducción de Señal , Factor B de Crecimiento Endotelial Vascular/metabolismo , Adulto , Anciano , Albuminuria/complicaciones , Albuminuria/metabolismo , Albuminuria/patología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/farmacología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Dislipidemias/complicaciones , Dislipidemias/metabolismo , Dislipidemias/patología , Proteínas de Transporte de Ácidos Grasos/metabolismo , Femenino , Eliminación de Gen , Humanos , Insulina/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/fisiopatología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/patología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Adulto JovenRESUMEN
Vascular endothelial growth factor (VEGF) B belongs to the VEGF family, but in contrast to VEGF-A, VEGF-B does not regulate blood vessel growth. Instead, VEGF-B controls endothelial fatty acid (FA) uptake and was identified as a target for the treatment of type 2 diabetes. The regulatory mechanisms controlling Vegfb expression have remained unidentified. We show that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) together with estrogen-related receptor α (ERR-α) regulates expression of Vegfb Mice overexpressing PGC-1α under the muscle creatine kinase promoter (MPGC-1αTG mice) displayed increased Vegfb expression, and this was accompanied by increased muscular lipid accumulation. Ablation of Vegfb in MPGC-1αTG mice fed a high-fat diet (HFD) normalized glucose intolerance, insulin resistance, and dyslipidemia. We suggest that VEGF-B is the missing link between PGC-1α overexpression and the development of the diabetes-like phenotype in HFD-fed MPGC-1αTG mice. The findings identify Vegfb as a novel gene regulated by the PGC-1α/ERR-α signaling pathway. Furthermore, the study highlights the role of PGC-1α as a master metabolic sensor that by regulating the expression levels of Vegfa and Vegfb coordinates blood vessel growth and FA uptake with mitochondrial FA oxidation.
Asunto(s)
Ácidos Grasos/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/fisiología , Factor B de Crecimiento Endotelial Vascular/genética , Animales , Células COS , Respiración de la Célula/genética , Células Cultivadas , Chlorocebus aethiops , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Respiratory complexes in both prokaryotes and eukaryotes contain multiple co-factors, which are coordinated in defined positions so that they can function as electron wires. Intriguingly, co-factors are usually buried deep within hetero-oligomeric protein complexes and it is not clear when or how they are incorporated. In this study we show that heme is incorporated into the cytochrome bo(3) complex of Escherichia coli at a late stage of assembly. Specifically the apo-form of subunit I (the catalytic subunit) interacts with subunits III and IV before accepting heme. Assembly of subunit II is stalled until heme is incorporated.
Asunto(s)
Citocromos/química , Citocromos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Hemo/metabolismo , Grupo Citocromo b , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structure-function relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.