RESUMO
Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic beta-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice. In beta-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low or almost undetectable levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and beta-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, beta-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the alpha- to beta-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas do Tecido Nervoso , Animais , Glicemia/análise , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Glucagon/metabolismo , Glucose/farmacologia , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 2 , Transportador de Glucose Tipo 3 , Homozigoto , Hiperglicemia/genética , Secreção de Insulina , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/metabolismoRESUMO
The high metabolic requirements of the mammalian central nervous system require specialized structures for the facilitated transport of nutrients across the blood-brain barrier. Stereospecific high-capacity carriers, including those that recognize glucose, are key components of this barrier, which also protects the brain against noxious substances. Facilitated glucose transport in vertebrates is catalyzed by a family of carriers consisting of at least five functional isoforms with distinct tissue distributions, subcellular localizations and transport kinetics. Several of these transporters are expressed in the mammalian brain. GLUT-1, whose sequence was originally deduced from cDNAs cloned from human hepatoma and rat brain, is present at high levels in primate erythrocytes and brain endothelial cells. GLUT1 has been cloned and positionally mapped to the short arm of chromosome 1 (1p35-p31.3; refs 6-8). Despite substantial metabolic requirements of the central nervous system, no genetic disease caused by dysfunctional blood-brain barrier transport has been identified. Several years ago, we described two patients with infantile seizures, delayed development and acquired microcephaly who have normal circulating blood glucose, low-to-normal cerebrospinal fluid (CSF) lactate, but persistent hypoglycorrachia (low CSF glucose) and diminished transport of hexose into isolated red blood cells (RBC). These symptoms suggested the existence of a defect in glucose transport across the blood brain barrier. We now report two distinct classes of mutations as the molecular basis for the functional defect of glucose transport: hemizygosity of GLUT1 and nonsense mutations resulting in truncation of the GLUT-1 protein.
Assuntos
Cromossomos Humanos Par 1 , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas de Transporte de Monossacarídeos/genética , Mutação Puntual , Polimorfismo Genético , Animais , Barreira Hematoencefálica , Encéfalo/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Deficiências do Desenvolvimento/genética , Feminino , Transportador de Glucose Tipo 1 , Haplótipos , Humanos , Hibridização in Situ Fluorescente , Neoplasias Hepáticas/metabolismo , Masculino , Microcefalia/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Linhagem , Reação em Cadeia da Polimerase , Ratos , Convulsões/genética , Pele/patologia , SíndromeRESUMO
In adipose and muscle cells, insulin stimulates a rapid and dramatic increase in glucose uptake, primarily by promoting the redistribution of the GLUT4 glucose transporter from its intracellular storage site to the plasma membrane. In contrast, the more ubiquitously expressed isoform GLUT1 is localized at the cell surface in the basal state, and shows a less dramatic translocation in response to insulin. To identify sequences involved in the differential subcellular localization and hormone-responsiveness of these isoforms, chimeric GLUT1/GLUT4 transporters were stably expressed in mouse 3T3-L1 adipocytes. The NH2 terminus of GLUT4 contains sequences capable of sequestering the transporter inside the cell, although not in an insulin-sensitive pool. In contrast, the COOH-terminal 30 amino acids of GLUT4 are sufficient for its correct localization to an intracellular storage pool which translocates to the cell surface in response to insulin. The dileucine motif within this domain, which is required for intracellular sequestration of chimeric transporters in fibroblasts, is not critical for targeting to the hormone-responsive compartment in adipocytes. Analysis of rates of internalization of chimeric transporter after the removal of insulin from cells, as well as the subcellular distribution of transporters in cells unexposed to or treated with insulin, leads to a three-pool model which can account for the data.
Assuntos
Compartimento Celular/fisiologia , Glucose/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Células 3T3/citologia , Células 3T3/metabolismo , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/fisiologia , Simulação por Computador , Expressão Gênica/fisiologia , Transportador de Glucose Tipo 4 , Leucina/fisiologia , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A strategy of metallizing peptides to serve as conduits of electronic signals that bridge between a redox enzyme and a carbon-nanotube electrode has been developed with enhanced results. In conjunction, a protocol to link the biological elements to the tips of carbon nanotubes has been developed to optimize contact and geometry between the redox enzyme and the carbon nanotube electrode array. A peptide nanowire of 33 amino acids, comprised of a leucine zipper motif, was mutated to bind divalent metals, conferring conductivity into the peptide. Reaction between a thiolate of the peptide with the sulfenic acid of the NADH peroxidase enzyme formed a peptide-enzyme assembly that are fully primed to transduce electrons out of the enzyme active site to an electrode. Scanning electron microscopy shows immobilization and linking of the assembly specifically to the tips of carbon nanotube electrodes, as designed. Isothermal titration calorimetry and mass spectrometry indicate a binding stoichiometry of at least three metals bound per peptide strand. Overall, these results highlight the gain that can be achieved when the signal tranducing units of a biosensor are aligned through directed peptide chemistry.
Assuntos
Técnicas Biossensoriais/métodos , Nanotubos de Carbono/química , Nanofios/química , Peptídeos/análise , Peroxidases/análise , Técnicas Biossensoriais/instrumentação , Eletrodos , Microscopia Eletrônica de Varredura , Estrutura Molecular , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Nanotubos de Carbono/ultraestrutura , Peptídeos/química , Peroxidases/química , Peroxidases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We report on a highly ordered array of carbon nanotubes (CNTs) that serves as a universally direct nanoelectrode interface for redox proteins and provides an efficient conduit for electron transfer. The site-selective, covalent docking of the enzyme glucose oxidase (GO(x)) on the CNT tips is found to have a marked effect on enhancing electron transfer properties, as measured by cyclic voltammetry. A unimolecular electron transfer rate of 1500 s(-1) has been measured for this system, a value exceeding the rate of oxygen reduction by glucose oxidase. Furthermore, the redox enzyme-CNT array conjugate can be utilized as a quantitative, substrate-specific biosensor.
Assuntos
Técnicas Biossensoriais/instrumentação , Glucose Oxidase/química , Glucose/análise , Glucose/química , Microeletrodos , Nanotubos de Carbono/química , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Desenho de Equipamento , Análise de Falha de Equipamento , Nanotubos de Carbono/ultraestrutura , Oxirredução , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transdução de SinaisRESUMO
A primary objective of this review is to facilitate the application of the chemical and structural approaches that are currently being employed in the identification of Cys-SOH, as both transient intermediates and stable redox forms, in biochemical systems where these derivatives are suspected of playing key roles in redox catalysis or regulation. These range from high-resolution crystallographic analyses benefiting from recent technological advances in rapid data collection at cryogenic temperatures to 13C NMR investigations of [3-(13)C]Cys-labeled proteins and chemical modification protocols that can be integrated with both UV-visible and fluorescence spectroscopic as well as mass spectrometric (especially ESI, MALDI-TOF, and even FT ion-cyclotron-resonance) analyses. In summarizing the diversity of biological functions currently identified with Cys-SH reversible Cys-SOH redox cycles (Fig. 17), it should also be [figure: see text] emphasized that in at least one protein (nitrile hydratase) stable Cys-SOH and Cys-SO2H derivatives play important structural roles while also modulating the electronic properties of the iron center; in neither case is the Cys-SOH residue itself involved in reduction and oxidation. The somewhat incomplete structural descriptions of the oxidized Cys forms involved in redox regulation of some transcription factors (e.g., BPV-1 E2 protein and activator protein-1) indicate that there is ample room for the application of the types of investigations employed, for example, with NADH peroxidase and the AhpC peroxiredoxin, with a view toward defining the potential roles of Cys-SOH in these very important contexts of intracellular redox signaling. These advances will also build on the recent progress in defining sulfenic acid stabilization and properties in small molecule model systems, as evidenced in the work of Okazaki, Goto, and others. When viewed in the perspective of Allison's 1976 review on the subject of sulfenic acids in proteins, the reader will hopefully come to appreciate the conclusion that the concept of protein-sulfenic acids has now become a very well-defined and established principle of biochemistry, with current efforts in this and other laboratories being directed to bring about still more detailed understanding of Cys-SOH function in both redox and nonredox modes of enzyme catalysis and regulation of protein function.
Assuntos
Cisteína/análogos & derivados , Cisteína/metabolismo , Ácidos Sulfênicos/metabolismo , Animais , Catálise , Cisteína/química , Humanos , Estrutura Molecular , Oxirredução , Ácidos Sulfênicos/químicaRESUMO
The high-resolution structures of the wild-type periplasmic domain of the bacterial aspartate receptor have been determined in the absence and presence of bound aspartate to 1.85 and 2.2 A resolution, respectively. As we reported earlier, in the refined structure of the complexed form of the crosslinked cysteine mutant receptor, the binding of the aspartate at the first site was mediated through four bridging water molecules while the second site showed an occupant electron density that best fit a sulfate group, which was present in the crystallization solution at high concentration. In the wild-type periplasmic domain structure two aspartate residues are bound per dimer, but with different occupancies. There exists a "strong" aspartate-binding site whose binding is again mediated by four water molecules while the second site contains aspartate whose B-factor is about 10% higher, signifying weaker binding. The interaction between the second, "weaker" aspartate with the three ligand-binding arginine side-chains is slightly different from the first site. The major difference is that there are three water molecules mediating the binding of aspartate at the second site, whereas in the first site there are four bridging water molecules. The fact that aspartate-complexed crystals of the wild-type were grown with a large excess aspartate while the cross-linked crystals were grown with equal molar aspartate may explain the difference in the stoichiometry observed. The conservation of the four bridging water molecules in the strong aspartate site of both the cross-linked and wild-type periplasmic domain may reflect an important binding motif. The periplasmic domain in the apo form is a symmetrical dimer, in which each of the subunits is equivalent, and the two aspartate binding sites are identical. Upon the binding of aspartate, the subunits are no longer symmetrical. The main difference between the aspartate-bound and unbound forms is in a small, rigid-body rotation between the subunits within a dimer. The rotation is similar in both direction and magnitude in the crosslinked and wild-type periplasmic domains. The presence of the second aspartate in the wild-type structure does not make any additional rotation compared to the single-site binding. The conservation of the small angular change in vitro suggests that the inter-subunit rotation may have relevance to the understanding of the mechanism of transmembrane signal transduction in vivo.
Assuntos
Ácido Aspártico , Receptores de Aminoácido/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Salmonella typhimurium/enzimologia , Especificidade por Substrato , Água/metabolismoRESUMO
OBJECTIVE: To study the role of the Gly971Arg variant of the insulin receptor substrate 1 (IRS-1) gene in the development of NIDDM in the Chinese population living in Taiwan. RESEARCH DESIGN AND METHODS: A total of 82 unrelated normal control subjects, 89 subjects with NIDDM, and 23 multiplex families were recruited in Taiwan. All of them were Han Chinese. Pedigree members without a history of diabetes were studies by the standard 75-g oral glucose tolerance test. Detection of the Gly971Arg variant of the IRS-1 gene was performed by polymerase chain reaction and restriction fragment-length polymorphism analysis. RESULTS: The frequency of Gly971Arg variant of the IRS-1 gene in the normal population was 1.2% which was lower than frequencies reported in white populations. The prevalence of the Gly971Arg variant was not significantly increased in both the nonselected NIDDM population (1.1%) and the probands of the multiplex families (4.3%). More importantly, the Gly971Arg variant of the IRS-1 gene did not cosegregate with BMI and NIDDM in these families, CONCLUSIONS: The Gly971Arg variant of the IRS-1 gene is an infrequent normal allele among Taiwanese. This variant is neither associated nor cosegregated with NIDDM in the Taiwanese population and families. Gly971Arg of IRS-1 gene does not play an important role in the development of NIDDM in this population.
Assuntos
Arginina , Diabetes Mellitus Tipo 2/genética , Variação Genética , Glicina , Fosfoproteínas/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Criança , Primers do DNA , Feminino , Intolerância à Glucose/genética , Humanos , Proteínas Substratos do Receptor de Insulina , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Valores de Referência , TaiwanRESUMO
OBJECTIVE: Catechol O-methyltransferase (COMT) is involved in the degradation of catecholamine neurotransmitters. Recent linkage studies of schizophrenia and molecular studies of velocardiofacial syndrome suggest that the COMT gene might be a candidate gene for schizophrenia. METHOD: The authors systematically searched for mutations and microdeletion of the COMT gene in 177 Chinese schizophrenic patients from Taiwan; 99 comparison subjects were also studied. RESULTS: Five molecular variants were identified: c.186C > T at exon 3, c.408C > G at exon 4, c.472G > A at exon 4, c.597G > A at exon 5, and c.821-827insC at the 3' untranslated region. However, no differences in the genotype and haplotype frequencies of these molecular variants between the schizophrenic and comparison subjects were detected. Furthermore, no microdeletion was identified among the patients. CONCLUSIONS: These data suggest that the COMT gene does not play a major role in the pathogenesis of schizophrenia, and the genotypic overlap between schizophrenia and velocardiofacial syndrome was rare in this cohort.
Assuntos
Catecol O-Metiltransferase/genética , Cromossomos Humanos Par 22/genética , Esquizofrenia/genética , Aberrações Cromossômicas/genética , Estudos de Coortes , Análise Mutacional de DNA , Éxons/genética , Feminino , Deleção de Genes , Marcadores Genéticos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , TaiwanRESUMO
AIMS: To compare intraocular pressure (IOP) readings between Tono-Pen tonometry and GAT, between noncontact tonometry (NCT) and GAT, and between dynamic contour tonometry (DCT) and Goldmann applanation tonometry (GAT). The correlation between IOP reading and possible confounder was identified. METHODS: This observational cross-sectional study enrolled sixty-two healthy subjects. All IOP and ocular pulse amplitude (OPA) measurements were taken by a single ophthalmologist; mean keratometric power (MK), central corneal thickness (CCT), and lens thickness (LT) were measured by a single experienced technician. RESULTS: Stepwise multiple regression analysis indicated that GAT (P=0.017) and DCT (P=0.002) readings correlated positively with MK; GAT, NCT, and Tono-Pen readings correlated positively with CCT (P<0.05); NCT (P=0.035), and DCT (P=0.016) readings correlated negatively with LT; GAT (P=0.006) and Tono-Pen (P=0.009) readings correlated positively with OPA. CONCLUSIONS: The K, CCT, LT, and OPA are confounders in tonometry readings.
Assuntos
Pressão Intraocular/fisiologia , Tonometria Ocular/métodos , Adulto , Idoso , Córnea/fisiologia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Tonometria Ocular/instrumentação , Adulto JovemRESUMO
AIMS/HYPOTHESIS: The gene encoding solute carrier family 2, facilitated glucose transporter, member 10 (SLC2A10, previously known as glucose transporter 10 [GLUT10]) is a promising candidate gene for type 2 diabetes since it is highly expressed in liver and pancreas and is located on human chromosome region 20q12-q13.1, a region previously shown to harbour type 2 diabetes susceptibility genes. We investigated whether the SLC2A10 gene could be a type 2 diabetes susceptibility gene in the Taiwanese population. SUBJECTS AND METHODS: Sequencing of SLC2A10 gene from 48 diabetic subjects detected short tandem repeat polymorphisms in the promoter region, but did not detect any other sequence variants or new single-nucleotide polymorphisms (SNPs) other than those already in the SNPper database ( http://snpper.chip.org ) (30 June 2005). RESULTS: Using these genetic polymorphisms, we divided the SLC2A10 gene into four distinct linkage disequilibrium blocks and performed a case-control association study in a group of type 2 diabetes subjects (n = 375) and normoglycaemic individuals (n=377). The HapD (A-G-T-C) haplotype in block 3, a rare haplotype, which consisted of four SNPs (rs3092412, rs2235491, rs2425904 and rs1059217), was modestly associated with type 2 diabetes with a haplotype score of -2.95567 (p = 0.012 with the haplotype-specific test). CONCLUSIONS/INTERPRETATION: Our results suggest that SLC2A10 genetic variations do not appear to be major determinants for type 2 diabetes susceptibility in the Taiwanese population.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Idoso , Sequência de Bases , Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Éxons , Feminino , Frequência do Gene , Predisposição Genética para Doença , Variação Genética , Hemoglobinas Glicadas/análise , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Taiwan , Triglicerídeos/sangueRESUMO
Heme degradation plays important biological roles, ranging from generating light-absorbing compounds in plants to facilitating iron homeostasis in mammals. The X-ray crystal structure of human heme oxygenase-1, which instigates the degradation process, reveals insights into the enzymatic mechanism of this important process.
Assuntos
Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Animais , Sítios de Ligação , Carbono/química , Carbono/metabolismo , Cristalização , Cristalografia por Raios X , Heme Oxigenase-1 , Humanos , Ligantes , Proteínas de Membrana , Conformação Proteica , Dobramento de Proteína , Solubilidade , Especificidade por SubstratoRESUMO
A flash-annealing method has been developed that increases the diffraction limits, and simultaneously decreases the mosaicity of glycerol kinase crystals. This technique utilizes brief thawing and rapid freezing cycles of the crystal in the cold nitrogen stream. The effective resolution limits increased almost by 0.8 A, from 3.6 to 2.8 A, and mosaicity values halved.
Assuntos
Glicerol Quinase/química , Cristalização , Congelamento , Temperatura Alta , Difração de Raios XRESUMO
Insulin regulates hexose uptake by the redistribution of glucose transport proteins from intracellular compartments to the cell surface. We have submitted the trafficking of GLUT1, GLUT4, and GLUT1/GLUT4 chimeras to a mathematical analysis in the context of different models. Our data suggest that a model with one intracellular and one cell surface compartment can describe the glucose transporter-trafficking kinetics in fibroblasts. Moreover, the difference in cellular distribution between GLUT1 and GLUT4 overexpressed in fibroblasts is best explained by a slower rate of movement of GLUT4 to the plasma membrane. In 3T3-L1 adipocytes, glucose transporter-trafficking kinetics is adequately described by a three-pool model which includes flow of transporters from the endosomal compartment to cell surface. The kinetic roles of previously identified motifs in GLUT4 trafficking were defined in proposed fibroblast and adipocyte glucose transporter-trafficking models. The C-terminus is important in reducing the exocytosis rate from the endosomal compartment to the cell surface in both fibroblasts and adipocytes, and the N-terminus behaves similarly in adipocytes. The C-terminus has an additional signal(s) that allows GLUT4 to be sequestered more efficiently into the insulin responsive vesicle compartment. Mutation of the dileucine motif in the C-terminus significantly reduces the endocytosis of GLUT4 in both fibroblasts and adipocytes, but these amino acids appear not to be primarily responsible for the different kinetics of wild-type GLUT1 and GLUT4.
Assuntos
Adipócitos/metabolismo , Fibroblastos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Animais , Transporte Biológico , Glucose/metabolismo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Humanos , Cinética , Modelos Teóricos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at -160 degrees C was applied. The structure of the native enzyme to 2.8 A resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid. The Cys42-sulfenic acid atoms C alpha-C beta-S gamma-O roughly define a planar arrangement which is stacked parallel to the si face of the FAD isoalloxazine and positions the sulfenyl oxygen atom only 3.3 A from FAD-C4A. His10-N epsilon 2 contributes a hydrogen bond to the sulfenic acid oxygen, at a distance of 3.2 A. Although one oxygen atom (OX1) of the non-native Cys42-sulfonic acid derivative identified in the earlier wild-type peroxidase structure was taken to represent the native Cys42-sulfenic acid oxygen [Stehle, T., Ahmed, S. A., Claiborne, A., & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344], this structure shows that the sulfenic acid oxygen does not occupy this position, nor is it hydrogen-bonded to Cys42-N as was OX1. Comparison of the native Cys42-sulfenic acid structure with that of two-electron reduced glutathione reductase provides an insight into the sulfenic acid FAD charge-transfer interaction observed with both wild-type and His10 mutant peroxidases. A model of the E.NADH intermediate recently observed in stopped-flow analyses of the enzyme [Crane, E. J., III, Parsonage, D., Poole, L. B., & Claiborne, A. (1995) Biochemistry 34, 14114-14124] has also been generated to assist in analyzing the chemical mechanism of sulfenic acid reduction.
Assuntos
Cisteína/análogos & derivados , Enterococcus faecalis/enzimologia , Peroxidases/química , Estrutura Secundária de Proteína , Ácidos Sulfênicos , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X/métodos , Escherichia coli , Flavina-Adenina Dinucleotídeo , Substâncias Macromoleculares , Modelos Moleculares , Oxirredução , Peroxidases/metabolismo , Proteínas Recombinantes/químicaRESUMO
The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of =4.5. NADH titration of the mutant peroxidase yields the same EH(2) intermediate, but in contrast to the behavior of wild-type enzyme, this species can be reduced directly to an EH(4).NAD(+) complex. Kinetic analyses demonstrate that the R303M mutant is severely compromised, although active, with k(cat) = 3 s(-)(1) at pH 7.0, 5 degrees C; enzyme-monitored turnover results indicate that the steady-state consists predominantly of an E-FADH(2).NAD(+) species. When the oxidized mutant is reacted anaerobically with 0.9 equiv of NADH/FAD, a clearly biphasic pattern is observed at 450 nm; relatively rapid flavin reduction is followed by reoxidation at 2.6-2.7 s(-)(1) ( approximately k(cat)). Thus replacement of Arg303 with Met leads to an altered peroxidase form in which the rate-limiting step in turnover is the intramolecular transfer of electrons from FADH(2) --> Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.
Assuntos
Peroxidases/genética , Peroxidases/metabolismo , Substituição de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Peroxidases/química , Conformação Proteica , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
The three-dimensional structures of the ligand-binding domain of the wild-type Salmonella typhimurium aspartate receptor have been determined in the absence (apo) and presence of bound aspartate (complex) and compared to a cross-linked mutant containing a cysteine at position 36 which does not change signaling behavior of the intact receptor. The structures of the wild-type forms were determined in order to assess the effects of cross-linking on the structure and its influence on conformational changes upon ligand binding. As in the case of the cross-linked mutant receptor, the non-cross-linked ligand-binding domain is dimeric and is composed of 4-alpha-helical bundle monomer subunits related by a crystallographic 2-fold axis in the unbound form and by a non-crystallographic axis in the aspartate-bound form. A comparative study between the non-cross-linked and cross-linked structures has led to the following observations: 1) The long N-terminal helices of the individual subunits in the cross-linked structures are bent toward each other to accommodate the disulfide bond. 2) The rest of the subunit conformation is very similar to that of the wild-type. 3) The intersubunit angle of the cross-linked apo structure is larger by about 13 degrees when compared to the wild-type apo structure. 4) The nature and magnitude of the aspartate-induced conformational changes in the non-cross-linked wild-type structures are very similar to those of the cross-linked structures.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Estrutura Secundária de Proteína , Receptores de Aminoácido/química , Receptores de Aminoácido/metabolismo , Receptores de Superfície Celular , Salmonella typhimurium/metabolismo , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Células Quimiorreceptoras , Reagentes de Ligações Cruzadas , Ligantes , Substâncias Macromoleculares , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Receptores de Aminoácido/genéticaRESUMO
Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3'-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.
Assuntos
Androstadienos/farmacologia , Hexoses/metabolismo , Músculos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Hipóxia/metabolismo , Insulina/farmacologia , Masculino , Contração Muscular , Fosfatidilinositol 3-Quinases , Fosfatidilinositóis/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , WortmaninaRESUMO
While it has been known for more than 20 years that unusually stable cysteine-sulfenic acid (Cys-SOH) derivatives can be introduced in selected proteins by mild oxidation, only recently have chemical and crystallographic evidence for functional Cys-SOH been presented with native proteins such as NADH peroxidase and NADH oxidase, nitrile hydratase, and the hORF6 and AhpC peroxiredoxins. In addition, Cys-SOH forms of protein tyrosine phosphatases and glutathione reductase have been suggested to play key roles in the reversible inhibition of these enzymes during tyrosine phosphorylation-dependent signal transduction events and nitrosative stress, respectively. Substantial chemical data have also been presented which implicate Cys-SOH in redox regulation of transcription factors such as Fos and Jun (activator protein-1) and bovine papillomavirus-1 E2 protein. Functionally, the Cys-SOHs in NADH peroxidase, NADH oxidase, and the peroxiredoxins serve as either catalytically essential redox centers or transient intermediates during peroxide reduction. In nitrile hydratase, the active-site Cys-SOH functions in both iron coordination and NO binding but does not play any catalytic redox role. In Fos and Jun and the E2 protein, on the other hand, a key Cys-SH serves as a sensor for intracellular redox status; reversible oxidation to Cys-SOH as proposed inhibits the corresponding DNA binding activity. These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-SOH stabilization and function.
Assuntos
Cisteína/análogos & derivados , Enzimas/química , Enzimas/metabolismo , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismo , Animais , Catálise , Cisteína/química , Cisteína/metabolismo , Humanos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Peroxidases/química , Peroxidases/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismoRESUMO
OBJECTIVE: Vitamin D and its receptor have been suggested to play a role in the pathogenesis of type 1 diabetes mellitus. We have therefore studied the influence of vitamin D receptor (VDR) gene polymorphisms on susceptibility to type 1 diabetes, and rates of glutamic acid decarboxylase (GAD65) autoantibody and islet cell autoantibody (ICA512) positivity. SUBJECTS: AND MEASUREMENTS One hundred and fifty-seven type 1 diabetic patients and 248 unrelated normal controls were recruited for this study. Genomic DNA was extracted from peripheral blood leucocytes. All type 1 diabetic patients and controls were genotyped using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), for three restriction sites in the VDR gene, BsmI, ApaI and TaqI. The chi2 test was used to compare the frequency of the VDR gene polymorphisms in patients and normal controls. The association of VDR gene polymorphisms in type 1 diabetes with the presence of GAD65 and ICA512 autoantibodies were also examined using the chi2 test. RESULTS: The allele frequency of the BsmI and ApaI polymorphisms, but not TaqI polymorphism, differed between patients and controls (BsmI P = 0.015; ApaI P = 0.018; TaqI P = 0.266). However, after correction for the three different polymorphisms tested, only the BsmI was significant (pc = 0.045). CONCLUSIONS: Vitamin D receptor gene polymorphisms were associated with type 1 diabetes in a Taiwanese population. However, functional studies are needed to establish the role of the vitamin D receptor in the pathogenesis of type 1 diabetes mellitus.