RESUMO
CONTEXT: The impact of sugar-sweetened beverages (SSB) on lipid metabolism when consumed in moderate amounts by normal weight subjects is debated. OBJECTIVE: The objective of the study was to investigate the effect of different types of sugars in SSB on fatty acid metabolism (ie, fatty acid synthesis and oxidation) in healthy young men. DESIGN: Thirty-four normal-weight men were studied in a randomized crossover study. Four isocaloric 3-week interventions with SSB were performed in random order: medium fructose (MF; 40 g/d); high fructose (HF; 80 g/d), high sucrose (HS; 80 g/d), and high glucose (HG; 80g/d). Fasting total plasma fatty acid composition was measured after each intervention. Acylcarnitines were measured in the fasting state and after a euglycemic hyperinsulinemic clamp in nine subjects. RESULTS: The relative abundance of palmitate (16:0) and the molar fatty acid ratio of palmitate to linoleic acid (16:0 to18:2) as markers of fatty acid synthesis were increased after HF [relative abundance of palmitate: 22.97% ± 5.51% (percentage of total fatty acids by weight ±SD)] and MF (26.1% ± 1.7%) compared with HS (19.40% ± 2.91%, P < .001), HG (19.43% ±3.12 %, P < .001), or baseline (19.40% ± 2.79%, P < .001). After HS and HG, the relative abundance of palmitate was equal to baseline. Fasting palmitoylcarnitine was significantly increased after HF and HS (HF and HS vs. HG: P = .005), decreasing after inhibition of lipolysis by insulin in the clamp. CONCLUSIONS: When consumed in moderate amounts, fructose but not sucrose or glucose in SSB increases fatty acid synthesis (palmitate), whereas fasting long-chain acylcarnitines are increased after both fructose and sucrose, indicating an impaired ß-oxidation flux.
Assuntos
Bebidas , Carboidratos da Dieta/farmacologia , Ácidos Graxos/metabolismo , Frutose/administração & dosagem , Metabolismo dos Lipídeos/efeitos dos fármacos , Sacarose/administração & dosagem , Edulcorantes/administração & dosagem , Adulto , Estudos Cross-Over , Ácidos Graxos/sangue , Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: The fatty acid mixture of human milk is ideal for the newborn but little is known about its composition in the first few weeks of lactation. Of special interest are the levels of long-chain PUFAs (LCPUFAs), since these are essential for the newborn's development. Additionally, the LCPUFAs arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are precursors for lipid mediators which regulate inflammation. METHODS: We determined the composition of 94 human milk samples from 30 mothers over the first month of lactation for fatty acids using GC-MS and quantified lipid mediators using HPLC-MS/MS. RESULTS: Over the four weeks period, DHA levels decreased, while levels of γC18:3 and αC18:3 steadily increased. Intriguingly, we found high concentrations of lipid mediators and their hydroxy fatty acid precursors in human milk, including pro-inflammatory leukotriene B4 (LTB4) and anti-inflammatory and pro-resolving lipoxin A4 (LXA4), resolvin D1 (RvD1) and resolvin E1 (RvE1). Lipid mediator levels were stable with the exception of two direct precursors. CONCLUSIONS: Elevated levels of DHA right after birth might represent higher requirements of the newborn and the high content of anti-inflammatory and pro-resolving lipid mediators and their precursors may indicate their role in neonatal immunity and may be one of the reasons for the advantage of human milk over infant formula.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Lactação/metabolismo , Metabolismo dos Lipídeos , Lipoxinas/biossíntese , Leite Humano/metabolismo , Eicosanoides/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Inflamação/metabolismo , Inflamação/patologia , Leite Humano/química , Estados UnidosRESUMO
Hemoglobin disorders consist of two different groups, the structural hemoglobin variants and the thalassemias. The structural hemoglobin variants typically are based on the point mutations in the alpha- or beta-globin chain that results in a single-amino acid substitution in the corresponding globin chain, whereas thalassemias are caused by quantitative reduction in globin chain synthesis. Various techniques are applied for the laboratory investigation of these diseases, among them mass spectrometry (MS) for the detection and identification of structural hemoglobin variants and array techniques for the thalassemias. In this review, we present in the first part the most important mass spectrometric techniques applied in hemoglobin variant detection and identification and discuss several important aspects of this analysis technique in hematology. In the second part, the DNA analysis techniques used in hemoglobin analysis, such as reverse hybridization or microarray-based comparative genomic hybridization (CGH) techniques, are briefly discussed.
Assuntos
Hemoglobinopatias/diagnóstico , Animais , DNA/química , DNA/genética , Variação Genética , Hemoglobinopatias/genética , Humanos , Espectrometria de Massas , Análise Serial de Proteínas , Talassemia/diagnóstico , Talassemia/genéticaRESUMO
A baby girl, born at term, presented with severe cyanosis and received oxygen supplementation. Consecutive arterial blood gas analysis showed a pronounced right shift of the saturation curve, suggesting the presence of a hemoglobin (Hb) variant. A new (G)γ-globin variant was detected, namely HBG2:c.308G, which we have named Hb F-Sarajevo, the city from where the baby's parents originate. This A to C transversion exists in cis to the common (A)γ(T) and the resulting mutant Hb molecule exhibits very low oxygen affinity and cooperativity. Its analogue in the ß-globin gene is Hb Kansas [ß102(G4)AsnâThr, AAC>ACC].
Assuntos
Cianose/genética , Hemoglobina Fetal/genética , Oxigênio/metabolismo , Mutação Puntual , gama-Globinas/genética , Sequência de Bases , Cianose/diagnóstico , Cianose/metabolismo , Análise Mutacional de DNA , Feminino , Hemoglobinas Anormais/genética , Humanos , Recém-Nascido , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH(4)), the cofactor of PAH. METHODS: A robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice. RESULTS: The PAH assay was linear for phenylalanine and tyrosine (r(2)≥0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 µmol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions.
Assuntos
Cromatografia Líquida , Mutação/genética , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Encéfalo/enzimologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Marcação por Isótopo , Rim/enzimologia , Fígado/enzimologia , Camundongos , Fenótipo , Fenilalanina/metabolismo , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/genética , Tirosina/metabolismoRESUMO
The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) enhances oxidative metabolism in skeletal muscle. Excessive lipid oxidation and electron transport chain activity can, however, lead to the accumulation of harmful metabolites and impair glucose homeostasis. Here, we investigated the effect of over-expression of PGC-1α on metabolic control and generation of insulin desensitizing agents in extensor digitorum longus (EDL), a muscle that exhibits low levels of PGC-1α in the untrained state and minimally relies on oxidative metabolism. We demonstrate that PGC-1α induces a strictly balanced substrate oxidation in EDL by concomitantly promoting the transcription of activators and inhibitors of lipid oxidation. Moreover, we show that PGC-1α enhances the potential to uncouple oxidative phosphorylation. Thereby, PGC-1α boosts elevated, yet tightly regulated oxidative metabolism devoid of side products that are detrimental for glucose homeostasis. Accordingly, PI3K activity, an early phase marker for insulin resistance, is preserved in EDL muscle. Our findings suggest that PGC-1α coordinately coactivates the simultaneous transcription of gene clusters implicated in the positive and negative regulation of oxidative metabolism and thereby increases metabolic flexibility. Thus, in mice fed a normal chow diet, over-expression of PGC-1α does not alter insulin sensitivity and the metabolic adaptations elicited by PGC-1α mimic the beneficial effects of endurance training on muscle metabolism in this context.
Assuntos
Regulação da Expressão Gênica , Genes Mitocondriais , Resistência à Insulina , Insulina/fisiologia , Mitocôndrias Musculares/genética , Músculo Esquelético/metabolismo , Transativadores/metabolismo , Animais , Ciclo do Ácido Cítrico/genética , DNA Mitocondrial/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Espécies Reativas de Oxigênio/metabolismo , Transativadores/genética , Fatores de Transcrição , Transcrição GênicaRESUMO
The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Defeitos Congênitos da Glicosilação/metabolismo , Dolicóis/metabolismo , Inibidores Enzimáticos/farmacologia , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Oligossacarídeos/biossíntese , Ácidos Tricarboxílicos/farmacologia , Antígenos CD59/biossíntese , Antígenos CD59/genética , Células Cultivadas , Colesterol/biossíntese , Colesterol/genética , Defeitos Congênitos da Glicosilação/genética , Dolicóis/genética , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Oligossacarídeos/genéticaRESUMO
BACKGROUND: Myocardial energy metabolism is a strong predictor of postoperative cardiac function. This study profiled the metabolites and metabolic changes in the myocardium exposed to sevoflurane, propofol, and Intralipid and investigated the underlying molecular mechanisms. METHODS: Sevoflurane (2 vol%) and propofol (10 and 100 microM) in the formulation of 1% Diprivan (AstraZeneca Inc., Mississauga, ON, Canada) were compared for their effects on oxidative energy metabolism and contractility in the isolated working rat heart model. Intralipid served as a control. Substrate flux through the major pathways for adenosine triphosphate generation in the heart, that is, fatty acid and glucose oxidation, was measured using [H]palmitate and [C]glucose. Biochemical analyses of nucleotides, acyl-CoAs, ceramides, and 32 acylcarnitine species were used to profile individual metabolites. Lipid rafts were isolated and used for Western blotting of the plasma membrane transporters CD36 and glucose transporter 4. RESULTS: Metabolic profiling of the hearts exposed to sevoflurane and propofol revealed distinct regulation of fatty acid and glucose oxidation. Sevoflurane selectively decreased fatty acid oxidation, which was closely related to a marked reduction in left ventricular work. In contrast, propofol at 100 microM but not 10 microM increased glucose oxidation without affecting cardiac work. Sevoflurane decreased fatty acid transporter CD36 in lipid rafts/caveolae, whereas high propofol increased pyruvate dehydrogenase activity without affecting glucose transporter 4, providing mechanisms for the fuel shifts in energy metabolism. Propofol increased ceramide formation, and Intralipid increased hydroxy acylcarnitine species. CONCLUSIONS: Anesthetics and their solvents elicit distinct metabolic profiles in the myocardium, which may have clinical implications for the already jeopardized diseased heart.
Assuntos
Antígenos CD36/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Coração/efeitos dos fármacos , Éteres Metílicos/farmacologia , Miocárdio/metabolismo , Propofol/farmacologia , Anestésicos Intravenosos/farmacologia , Animais , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Coração/fisiologia , Técnicas In Vitro , Masculino , Metaboloma/efeitos dos fármacos , Metaboloma/fisiologia , Miocárdio/enzimologia , Oxirredução/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , SevofluranoRESUMO
We report the characterization of five novel delta-globin gene mutations detected during routine screening for thalassemia. Three missense mutations were identified, resulting in the following delta chain hemoglobin (Hb) variants: Hb A(2)-Acacias [delta4 (ACT>AGT), Thr-->Ser, HBD c.14C>G], Hb A(2)-Toronto [delta74 (GGC>GAC), Gly-->Asp, HBD c.224G>A], and Hb A(2)-Calgary [delta99 (GAT>GGT), Asp-->Gly, HBD c.299A>G]. Two other mutations most likely result in delta(0)-thalassemia (delta(0)-thal). One mutation altered the translation initiation codon from ATG to ATA (HBD c.3G>A), and another changed the canonical splice donor sequence of IVS-II from GT to AT (HBD C.315+1G>A).
Assuntos
Mutação , Talassemia beta/genética , Globinas delta/genética , Adulto , Alelos , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação/genética , Análise Mutacional de DNA , Feminino , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Sítios de Splice de RNA/genética , Talassemia beta/diagnósticoRESUMO
A 14-year-old male child presented with microcytosis, a known alpha(+)-thalassemia (alpha-thal) heterozygote and a hemizygous glucose-6-phosphate dehydrogenase (G6PD) deficiency. Furthermore, cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A. The peak area of the variant was equal to that of Hb A, suggesting a beta-globin variant. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis confirmed the mutation at the protein level. The variant was detectable by isoelectric focusing (IEF) or by reversed phase HPLC. DNA sequencing revealed a heterozygous mutation at codon 135 of the beta gene, already described as Hb Alperton. Hb Alperton showed decreased oxygen affinity. Neither biochemical nor clinical characteristics for Hb Alperton have been reported so far.
Assuntos
Hemoglobinas Anormais/metabolismo , Oxigênio/metabolismo , Globinas beta/genética , Adolescente , Glucose-6-Fosfato/deficiência , Hemoglobinas Anormais/genética , Heterozigoto , Humanos , Masculino , Análise de Sequência de DNA , Talassemia alfa/genética , Talassemia alfa/metabolismoRESUMO
The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patient's fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairments.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Erros Inatos do Metabolismo/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Adolescente , Células Cultivadas , Criança , Feminino , Fibroblastos/metabolismo , Glicosilação , Humanos , Erros Inatos do Metabolismo/genética , Mutação , Splicing de RNARESUMO
A new alpha-globin mutation [alpha 138(H21)Ser-->Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an alpha-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the alpha2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser-->Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.
Assuntos
Hemoglobinas Anormais/genética , Oxigênio/metabolismo , alfa-Globinas/genética , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Saúde da Família , Hemoglobinas Anormais/metabolismo , Humanos , Focalização Isoelétrica , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mutação Puntual , Policitemia , SuíçaRESUMO
We describe a Hb S/beta-thalassemia (beta-thal) mutation involving an AT transition at codon 132 of the beta-globin gene. The mutation, in the heterozygous state, unlike several other mutations in exon 3, shows no signs of dominant thalassemia but those of a typical beta(0) carrier. Compound heterozygosity with Hb S [beta6(A3)GluVal, GAGGTG] showed a severe clinical picture.
Assuntos
Códon/genética , Éxons/genética , Hemoglobina Falciforme/genética , Heterozigoto , Mutação/genética , Talassemia beta/genética , África Ocidental , Família , Feminino , Humanos , MasculinoRESUMO
Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Estaurosporina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Proteína Forkhead Box O1 , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fator de Transcrição PAX3 , Fosforilação , Processamento Pós-Transcricional do RNA , Estaurosporina/farmacologiaRESUMO
BACKGROUND: More than 900 hemoglobin (Hb) variants are currently known. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. In our laboratory, we routinely apply chromatographic methods. To ascertain whether Hb variants are missed with our procedures, we additionally analyzed all samples with mass spectrometry (MS). METHODS: Database evaluation was performed using all entries made in the Hb variant database HbVar, and possible Hb variants were calculated based on DNA variations. During a 5-year period, we analyzed 2105 lysates with cation-exchange HPLC (PolyCAT A column) and reversed-phase HPLC and additionally with electrospray ionization or MALDI-TOF MS. Globin chains were identified by their molecular masses. RESULTS: Database evaluation revealed that 43.2% of all possible Hbalpha- and beta-chain variants were found to date (considering only single-point mutations). Currently, 68.2% of the possible charge difference variants and only 28.7% of the neutral variants are found. Among 2105 Hb samples we identified 4 samples with Hb variants that were detected only with the MS method; 2 were new Hb variants (Hb Zurich-Hottingen and Hb Zurich-Langstrasse). With cation-exchange HPLC, 1 sample was found to be a beta-thalassemia and was identified by MS to be a beta-variant (Hb Malay). More common variants, such as Hb C, Hb D, and Hb E, and thalassemias could not be detected with the MS method. CONCLUSIONS: Application of MS improves the sensitivity of Hb analysis. The combination of MS with electrophoretic and chromatographic methods is optimal for the detection of Hb variants.
Assuntos
Hemoglobinas/química , Hemoglobinas/genética , Cromatografia Líquida de Alta Pressão/métodos , Bases de Dados Factuais , Feminino , Variação Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation.
Assuntos
Citrulina/metabolismo , Cabelo/metabolismo , Hidrolases/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas S100/metabolismo , Substituição de Aminoácidos , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Citrulina/genética , Cabelo/citologia , Humanos , Hidrolases/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Estrutura Quaternária de Proteína/fisiologia , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Proteínas S100/genética , Zinco/metabolismoRESUMO
Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Tubulina (Proteína)/metabolismo , Antígenos Virais de Tumores/fisiologia , Axônios/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Viral , Clonagem Molecular , Escherichia coli/genética , Glutationa Transferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Confocal , Neuroblastoma/patologia , Neurônios/metabolismo , Testes de Precipitina , Estrutura Terciária de Proteína , Proteína 1 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Proteínas Recombinantes de Fusão/metabolismo , Vírus 40 dos Símios/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tubulina (Proteína)/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha1-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. On the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Proteoma/metabolismo , Erros Inatos do Metabolismo dos Carboidratos/sangue , Eletroforese em Gel Bidimensional/métodos , Glicosilação , Humanos , Orosomucoide/análise , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transferrina/análiseRESUMO
3-Nitrotyrosine (3-NT) is considered as a marker of oxidative stress, which occurs during inflammation. Since 3-NT levels in exhaled breath condensate (EBC) are very low, we applied a specific and sensitive gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) method and high performance liquid chromatography (HPLC) with electrochemical detection for the analysis of free 3-NT in EBC. A total of 42 children (aged 5-17 years) were enrolled in this study, including children with asthma (n=12), cystic fibrosis (n=12), and healthy controls (n=18). Additionally, 14 healthy non-smoking adults (aged 18-59 years) were included. An EcoScreen system was used for the collection of EBC samples. Free 3-NT levels in EBC ranged from 0.54-6.8 nM. Median (interquartile range) concentrations (nM) were similar in all groups: 1.46 (0.97-2.49) in healthy adults, 2.51 (1.22-3.51) in healthy children, 1.46 (0.88-2.02) in children with asthma, and 1.97 (1.37-2.35) in CF children, respectively (p=0.24, Kruskall-Walis test). No difference was found between the children with airway disease and age-matched healthy controls. In healthy subjects, there was no effect of age on 3-NT concentrations. HPLC analyses provided similar concentration ranges for EBC 3-NT when compared with GC-NICI-MS. Our study has clearly demonstrated that free 3-NT in EBC fails as a marker for oxidative stress in children with stable CF and asthma.
Assuntos
Asma/metabolismo , Biomarcadores/metabolismo , Testes Respiratórios , Fibrose Cística/metabolismo , Expiração , Estresse Oxidativo , Tirosina/análogos & derivados , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tirosina/metabolismoRESUMO
A new electrophoretically and clinically silent beta-globin variant has been detected by DNA analysis. The mutation was demonstrated at the protein level by reversed phase high performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS).