Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20.382
Filtrar
1.
J Agric Food Chem ; 67(32): 9039-9049, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31353898

RESUMO

This study focused on the effects of oxidized tyrosine products (OTPs) and major component dityrosine (DT) on the brain and behavior of growing mice. Male and female mice were treated with daily intragastric administration of either tyrosine (Tyr; 420 µg/kg body weight), DT (420 µg/kg body weight), or OTPs (1909 µg/kg body weight) for 35 days. We found that pure DT and OTPs caused redox state imbalance, elevated levels of inflammatory factors, hippocampal oxidative damage, and neurotransmitter disorders while activating the mitochondrial apoptosis pathway in the hippocampus and downregulating the genes associated with learning and memory. These events eventually led to growing mice learning and memory impairment, lagging responses, and anxiety-like behaviors. Furthermore, the male mice exhibited slightly more oxidative damage than the females. These findings imply that contemporary diets and food-processing strategies of the modern world should be modified to reduce oxidized protein intake.


Assuntos
Transtornos da Memória/etiologia , Aprendizagem Espacial , Tirosina/análogos & derivados , Tirosina/efeitos adversos , Tirosina/química , Animais , Comportamento Animal , Feminino , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Humanos , Masculino , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Transtornos da Memória/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Estresse Oxidativo , Tirosina/metabolismo
2.
Gene ; 713: 143951, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31269464

RESUMO

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Rifampina/farmacologia , Tirosina/metabolismo , Antibióticos Antituberculose/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , RNA Bacteriano , Tirosina/genética
3.
J Agric Food Chem ; 67(31): 8500-8509, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298534

RESUMO

To map qualitative and quantitative metabolome alterations when Penicillium roqueforti is grown in an environment where l-tyrosine levels are perturbed, the recently established differential off-line LC-NMR (DOLC-NMR) approach was successfully applied in connection with an absolute metabolite quantitation using a quantitative 1H NMR protocol following the ERETIC 2 (Electronic REference To access In vivo Concentrations) methodology. Among the 23 influenced metabolites, amino acid degradation products like 2-(4-hydroxyphenyl)acetic acid and 2-(3,4-dihydroxyphenyl)acetic acid underwent a tremendous upregulation in the amino acid perturbed approach. Moreover, the output of secondary metabolites like andrastin A, eremofortin B, and the tetrapeptide d-Phe-l-Val-d-Val-l-Tyr was affected in the case of the presence or absence of the added aromatic amino acid. Furthermore, the isolated secondary metabolites of P. roqueforti have been quantified for the first time in five divergent Penicillium isolates by means of a validated LC-ECHO-MS/MS method. This technique is used to compensate the effect of co-extracted matrix compounds during the analysis and to utilize quasi-internal standards to quantify all metabolites of interest accurately. This screening outlined the great variety between the different fungi of the same species. The metabolite spectra of wild-type fungi included more toxic intermediates compared to a selected fungi used as a starter culture for blue-mold cheese production. In addition, these secondary metabolites were quantified in commercially available white- and blue-mold cheese samples. The main differences between the analyte profiles of white and blue cheeses were linked to the impact of the used starter culture. Specific metabolites detected from P. roqueforti like andrastin A and B or roquefortine C could not be detected in white cheese. Among the blue cheese samples, different metabolite pattern could be observed regarding various P. roqueforti starter cultures.


Assuntos
Queijo/microbiologia , Metaboloma , Penicillium/metabolismo , Metabolismo Secundário , Tirosina/metabolismo , Aminoácidos Aromáticos/análise , Aminoácidos Aromáticos/metabolismo , Androstadienos/análise , Androstadienos/metabolismo , Queijo/análise , Penicillium/química , Penicillium/crescimento & desenvolvimento , Peptídeos/análise , Peptídeos/metabolismo , Sesquiterpenos/análise , Sesquiterpenos/metabolismo , Espectrometria de Massas em Tandem
4.
Chem Commun (Camb) ; 55(67): 9927-9930, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31334708

RESUMO

Tyrosine phosphorylation regulates the upstream signaling pathway but accounts for less than 0.1% of total phosphorylation in human cells. Herein, molecularly imprinted mesoporous materials were first synthesized to recognize the phosphorylated tyrosine residue from other phosphorylated residues.


Assuntos
Epitopos , Impressão Molecular , Nanopartículas/química , Compostos Organofosforados/química , Dióxido de Silício/química , Tirosina/metabolismo , Adsorção , Cinética , Fosfopeptídeos/química , Fosforilação , Porosidade , Titânio/química
5.
Clin Biochem ; 71: 24-30, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228435

RESUMO

OBJECTIVES: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1). MATERIALS AND METHODS: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter. RESULTS: No significant effect (p > 0.1) was observed up to 225 µmol/L of HPPA and HPLA, and up to 5000 µmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked. CONCLUSIONS: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Alcaptonúria/tratamento farmacológico , Alcaptonúria/metabolismo , Cicloexanonas/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Nitrobenzoatos/uso terapêutico , Fenilpropionatos/análise , Ácidos Fenilpirúvicos/análise , Tirosina/metabolismo , Alcaptonúria/sangue , Alcaptonúria/urina , Humanos , Fenilpropionatos/sangue , Fenilpropionatos/urina , Ácidos Fenilpirúvicos/sangue , Ácidos Fenilpirúvicos/urina
6.
Food Chem ; 297: 124924, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253284

RESUMO

Yeast nitrogen metabolism produces metabolites, whose origin in wines has scarcely been studied, with an important biological and organoleptic role. The present work focuses on comparing three intracellular extraction methods in order to elucidate efficiency of extraction while measuring the effect of temperature upon the integrity of the compounds related to the metabolism of tryptophan and tyrosine by yeast. Two UHPLC/HRMS methods to measure 16 metabolites were developed and validated. The validation provided optimum values of LOD (7.4·10-6 to 0.1 µg L-1), of LOQ (2·10-5 to 0.02 µg L-1) of precision (11-0.5% RSD) and repeatability (12-0.5% RSD). The removal of interfering molecules enabled matrix effects to be kept at low levels. The results pointed out that the low-temperature methods were more effective, providing better precision for 16 metabolites. The high-temperature extraction method may yield false enhanced compounds concentrations since they originate in cell wall macromolecules degradation.


Assuntos
Fracionamento Químico/métodos , Saccharomyces cerevisiae/metabolismo , Triptofano/análise , Tirosina/análise , Cromatografia Líquida de Alta Pressão , Temperatura Baixa , Congelamento , Glicerol/química , Espectrometria de Massas , Nitrogênio/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo , Vinho/análise
7.
Nat Commun ; 10(1): 2574, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189925

RESUMO

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Domínios Proteicos/genética , Adenosina Trifosfatases/genética , Ácido Glutâmico/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Relação Estrutura-Atividade , Tirosina/genética , Tirosina/metabolismo
8.
Anticancer Res ; 39(6): 2749-2756, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177110

RESUMO

BACKGROUND/AIM: The differentiation of the mouse breast epithelial cell line HC11 is known to require confluence as well as the addition of hydrocortisone, insulin and prolactin. MATERIALS AND METHODS: Since confluence, which triggers the engagement of the cell-to-cell adhesion molecule E-cadherin, induces a dramatic increase in the activity of signal transducer and activator of transcription-3 (Stat3), we examined the role of Stat3 in HC11 cell differentiation. RESULTS: Stat3 inhibition abolished differentiation, indicating that Stat3 activity is critically required. However, expression of the mutationally activated form of Stat3 (Stat3C), rather than promoting, it was found to block cell differentiation, even when expressed in low levels, and in the absence of full neoplastic conversion. CONCLUSION: The strength of the E-cadherin/Stat3 signal is key for the outcome of the differentiation process.


Assuntos
Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Animais , Caderinas/metabolismo , Diferenciação Celular , Feminino , Glândulas Mamárias Animais/metabolismo , Camundongos , Mutação , Fosforilação , Transdução de Sinais , Tirosina/metabolismo
9.
Biochemistry (Mosc) ; 84(6): 652-662, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238865

RESUMO

Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong oxidants that can damage not only foreign intruders but also host tissues, including blood plasma proteins. Here, we compared the MPO-induced oxidation of two plasma proteins with antioxidant properties - human serum albumin (HSA) and ceruloplasmin (CP). Incubation of both proteins with hypochlorite (NaOCl) or catalytically active MPO (MPO + H2O2), which synthesizes hypochlorous acid (HOCl) in the presence of chloride ions, resulted in the quenching of protein tryptophan fluorescence. Oxidation-induced changes in the structures of HSA and CP were different. HSA efficiently neutralized MPO-generated oxidants without protein aggregation, while CP oxidation resulted in the formation of large aggregates stabilized by strong covalent bonds between the aromatic amino acid residues. Tyrosine is present in the plasma as free amino acid and also as a component of the polypeptide chains of the proteins. The number of tyrosine residues in a protein does not determine its propensity for aggregate formation. In the case of CP, protein aggregation was primarily due to the high content of tryptophan residues in its polypeptide chain. MPO-dependent oxidation of free tyrosine results in the formation of tyrosyl radicals, that do not oxidize aromatic amino acid residues in proteins because of the high rate of recombination with dityrosine formation. At the same time, free tyrosine can influence MPO-induced protein oxidation due to its ability to modulate HOCl synthesis in the MPO active site.


Assuntos
Albuminas/metabolismo , Ceruloplasmina/metabolismo , Peroxidase/metabolismo , Tirosina/metabolismo , Antioxidantes/metabolismo , Humanos , Oxirredução
10.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083552

RESUMO

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5-7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.


Assuntos
Aminoacil-tRNA Sintetases/genética , Biologia Computacional/métodos , Biblioteca Gênica , Luz , Tirosina/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Temperatura Ambiente
11.
Eur J Med Chem ; 176: 326-342, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31112893

RESUMO

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis playing a central role in cardiovascular disease, obesity, and diabetes. These receptors show a high degree of stereoselectivity towards several classes of drugs. This review covers the most relevant findings that have been made in the last decade and takes into consideration only those compounds in which stereochemistry led to unexpected results or peculiar interactions with the receptors. These cases are reviewed and discussed with the aim to show how enantiomeric recognition originates at the molecular level. The structural characterization by crystallographic methods and docking experiments of complexes formed by PPARs with their ligands turns out to be an essential tool to explain receptor stereoselectivity.


Assuntos
Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Acetatos/química , Acetatos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Indóis/química , Indóis/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Oxazóis/química , Oxazóis/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Receptores Ativados por Proliferador de Peroxissomo/química , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina/metabolismo
12.
Nat Commun ; 10(1): 1676, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976006

RESUMO

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.


Assuntos
Divisão Celular/fisiologia , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Cristalografia por Raios X , Ciclina A/isolamento & purificação , Quinase 2 Dependente de Ciclina/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/isolamento & purificação , Proteínas de Fusão bcr-abl/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo , Tirosina/metabolismo , Quinases da Família src/isolamento & purificação , Quinases da Família src/metabolismo
13.
J Physiol Pharmacol ; 70(1)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-31019122

RESUMO

The influence of low-energy defibrillation on changes in the ET-1 levels in the myocardium and on disruptions in coronary blood flow and microcirculation being their consequence still remains unclear. Myocardial microcirculatory dysfunction is considered as a significant cause underlying myocardial dysfunction in post-cardiac arrest syndrome. This study is aimed at evaluating time-dependent changes in ET-1 levels in serum and the heart of a healthy rabbit following the application of a low-energy two-phase shock impulse. The research was conducted in 35 healthy rabbits at the age of 36 - 42 weeks and with body mass from 3200 to 4150 grams. The animals were divided in a randomized way into four groups depending on the dose of the electrical energy planned for the application during the experiment. The life parameters of the animals were monitored with the application of BeneView T5 patient monitor. The concentration of endothelin-1 in the groups was measured before, 15 and 360 minutes after the application of the low-energy double-phase straight-lined electrical impulse. A transthoracic low-energy defibrillation shock impulse causes a long-term increase in the endothelin-1 levels in the heart muscle and blood serum in a healthy rabbit. The increase in ET-1 levels results from the effect of electrical energy, independently of consequences of the ischemia/reperfusion injury. The increase in the endothelin-1 levels may lead to capillary blood flow abnormalities in the heart, contributing to the development of its dysfunction in the course of postresuscitation disease.


Assuntos
Estimulação Elétrica , Endotelina-1/metabolismo , Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Animais , Pressão Sanguínea , Proteínas de Ligação a Ácido Graxo/sangue , Feminino , Ventrículos do Coração/ultraestrutura , Masculino , Miocárdio/ultraestrutura , Coelhos , Tirosina/análogos & derivados , Tirosina/metabolismo , Função Ventricular
14.
Genes Cells ; 24(6): 422-435, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002205

RESUMO

Dictyostelium STATa is a homologue of metazoan signal transducers and activators of transcription (STATs) and is important for morphogenesis. STATa is activated by phosphorylation on Tyr702 when cells are exposed to extracellular cAMP. Although two tyrosine kinase-like (TKL) proteins, Pyk2 and Pyk3, have been definitively identified as STATc kinases, no kinase is known for STATa activation. Based on homology to the previously identified tyrosine-selective TKLs, we identified DrkA, a member of the TKL family and the Dictyostelium receptor-like kinase (DRK) subfamily, as a candidate STATa kinase. The drkA gene is almost exclusively expressed in prestalk A (pstA) cells, where STATa is activated. Transient over-expression of DrkA increased STATa phosphorylation, although over-expression of the protein causes a severe growth defect and cell death. Furthermore, recombinant DrkA protein is auto-phosphorylated on tyrosine and threonine residues, and an in vitro kinase assay shows that DrkA can phosphorylate STATa on Tyr702 in a STATa-SH2 (phosphotyrosine binding) domain-dependent manner. These observations strongly suggest that DrkA is one of the key regulators of STATa tyrosine phosphorylation and is consistent with it being the kinase that directly activates STATa.


Assuntos
Dictyostelium/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Transcrição STAT/metabolismo , Animais , Dictyostelium/citologia , Dictyostelium/genética , Morfogênese , Mutação , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/fisiologia , Tirosina/metabolismo
15.
Nat Commun ; 10(1): 1846, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015464

RESUMO

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca2+-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca2+-activated Cl- channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Anoctaminas/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/genética , Isoleucina/metabolismo , Lisina/genética , Lisina/metabolismo , Mutagênese , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Tirosina/metabolismo
16.
Microb Cell Fact ; 18(1): 74, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023316

RESUMO

BACKGROUND: Production of L-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is expected to increase due to increasing cases of Parkinson's disease a neurodegenerative disease. Attempts to overproduce L-tyrosine for conversion to L-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. RESULTS: An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in L-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of L-DOPA in shake flask and 5 L bioreactor, respectively. CONCLUSION: Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of L-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased L-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of L-tyrosine and enhancing enzyme activity ensures maximum L-DOPA productivity.


Assuntos
Escherichia coli/metabolismo , Glucose/metabolismo , Levodopa/biossíntese , Engenharia Metabólica , Tirosina/metabolismo , Reatores Biológicos , Escherichia coli/genética , Glicerol/metabolismo , Oxigenases de Função Mista/metabolismo , Ácido Chiquímico/metabolismo , Tirosina/genética
17.
Nat Commun ; 10(1): 991, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824700

RESUMO

6-Phosphogluconate dehydrogenase (6PGD) is a key enzyme that converts 6-phosphogluconate into ribulose-5-phosphate with NADP+ as cofactor in the pentose phosphate pathway (PPP). 6PGD is commonly upregulated and plays important roles in many human cancers, while the mechanism underlying such roles of 6PGD remains elusive. Here we show that upon EGFR activation, 6PGD is phosphorylated at tyrosine (Y) 481 by Src family kinase Fyn. This phosphorylation enhances 6PGD activity by increasing its binding affinity to NADP+ and therefore activates the PPP for NADPH and ribose-5-phosphate, which consequently detoxifies intracellular reactive oxygen species (ROS) and accelerates DNA synthesis. Abrogating 6PGD Y481 phosphorylation (pY481) dramatically attenuates EGF-promoted glioma cell proliferation, tumor growth and resistance to ionizing radiation. In addition, 6PGD pY481 is associated with Fyn expression, the malignancy and prognosis of human glioblastoma. These findings establish a critical role of Fyn-dependent 6PGD phosphorylation in EGF-promoted tumor growth and radiation resistance.


Assuntos
Neoplasias/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/efeitos da radiação , Proliferação de Células , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Células HEK293 , Humanos , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , NADP/metabolismo , Neoplasias/patologia , Via de Pentose Fosfato , Fosforilação , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Ribosemonofosfatos/metabolismo , Regulação para Cima
18.
Nutrients ; 11(3)2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30823411

RESUMO

Introduction: In phenylketonuria (PKU), evidence suggests that casein glycomacropeptide supplemented with rate-limiting amino acids (CGMP-AA) is associated with better protein utilisation and less blood phenylalanine (Phe) variability. Aim: To study the impact of CGMP-AA on blood Phe variability using 3 different dietary regimens in children with PKU. Methods: This was a 6-week randomised controlled cross-over study comparing CGMP-AA vs. Phe-free l-amino acids (l-AA) assessing blood Phe and tyrosine (Tyr) variability over 24 h in 19 children (7 boys) with PKU, with a median age of 10 years (6⁻16). Subjects were randomised to 3 dietary regimens: (1) R1, CGMP-AA and usual dietary Phe (CGMP + Phe); (2) R2, CGMP-AA - Phe content of CGMP-AA from usual diet (CGMP - Phe); and (3) R3, l-AA and usual dietary Phe. Each regimen was administered for 14 days. Over the last 48 h on days 13 and 14, blood spots were collected every 4 h at 08 h, 12 h, 16 h, 20 h, 24 h, and 04 h. Isocaloric intake and the same meal plan and protein substitute dosage at standardised times were maintained when blood spots were collected. Results: Eighteen children completed the study. Median Phe concentrations over 24 h for each group were (range) R1, 290 (30⁻580), R2, 220 (10⁻670), R3, 165 (10⁻640) µmol/L. R1 vs. R2 and R1 vs. R3 p < 0.0001; R2 vs. R3 p = 0.0009. There was a significant difference in median Phe at each time point between R1 vs. R2, p = 0.0027 and R1 vs. R3, p < 0.0001, but not between any time points for R2 vs. R3. Tyr was significantly higher in both R1 and R2 [70 (20⁻240 µmol/L] compared to R3 [60 (10⁻200) µmol/L]. In children < 12 years, blood Phe remained in the target range (120⁻360 µmol/L), over 24 h, for 75% of the time in R1, 72% in R2 and 64% in R3; for children aged ≥ 12 years, blood Phe was in target range (120⁻600 µmol/L) in R1 and R2 for 100% of the time, but 64% in R3. Conclusions: The residual Phe in CGMP-AA increased blood Phe concentration in children. CGMP-AA appears to give less blood Phe variability compared to l-AA, but this effect may be masked by the increased blood Phe concentrations associated with its Phe contribution. Reducing dietary Phe intake to compensate for CGMP-AA Phe content may help.


Assuntos
Caseínas/administração & dosagem , Caseínas/farmacologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacologia , Fenilalanina/sangue , Tirosina/sangue , Criança , Suplementos Nutricionais , Feminino , Humanos , Masculino , Fenilalanina/metabolismo , Fenilcetonúrias , Tirosina/metabolismo
19.
Chemistry ; 25(26): 6533-6541, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30820987

RESUMO

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.


Assuntos
Bacillus megaterium/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Hexosiltransferases/química , Hexosiltransferases/genética , Oligossacarídeos/metabolismo , Tirosina/química , Bacillus megaterium/química , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Reação de Cicloadição , Frutanos/química , Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA