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1.
Enzyme Microb Technol ; 150: 109884, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489037

RESUMO

Tyrosinase plays an essential role in melanin biosynthesis and inherently exhibits both monophenolase and diphenolase activity. A first derivative synchronous fluorometric assay was established for directly monitoring monophenolase activity. The zero-crossing point at 322 nm for the first-derivative under synchronous fluorescence with Δλ = 67 nm was utilized to selectively quantify tyrosine in the presence of the reaction product dihydroxyphenylalanine (DOPA). The limit of detection (LOD) for tyrosine was 0.54 µM. The fluorescence intensity of tyrosine was monitored at intervals of 30 s to establish the time course of tyrosine consumption. The LOD for the monophenolase activity was 0.0706 U⋅ mL-1. The Michaelis-Menten e constant and maximum speed were 21.83 µM and 1.12 µM min-1, respectively. Zinc ions competitively inhibited the monophenolase activity, with an IC50 value of 14.36 µM. This assay is easily and rapidly executed and is of great significance for analyzing the kinetics of enzymatic reactions and in fundamental research on monophenolase. This approach has potential applications in the discovery of tyrosinase inhibitors for medicine and cosmetics, as well as in the industrial synthesis of substituted o-diphenol intermediates.


Assuntos
Monofenol Mono-Oxigenase , Oxirredutases , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Oxirredutases/metabolismo , Tirosina/metabolismo
2.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360651

RESUMO

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.


Assuntos
Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Gases em Plasma/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo , Apoptose , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Triptofano/química , Tirosina/química
3.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
4.
Molecules ; 26(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208577

RESUMO

Metal-organic frameworks (MOFs) have been rapidly developed for their broad applications in many different chemistry and materials fields. In this work, a multi-dentate building block 5-(4-(tetrazol-5-yl)phenyl)-isophthalic acid (H3L) containing tetrazole and carbolxylate moieties was employed for the synthesis of a two-dimensional (2D) lanthanide MOF [La(HL)(DMF)2(NO3)] (DMF = N,N-dimethylformamide) (1) under solvothermal condition. The fluorescent sensing application of 1 was investigated. 1 exhibits high sensitivity recognition for antibiotic nitrofurantoin (Ksv: 3.0 × 103 M-1 and detection limit: 17.0 µM) and amino acid l-tyrosine (Ksv: 1.4 × 104 M-1 and detection limit: 3.6 µM). This work provides a feasible detection platform of 2D MOFs for highly sensitive discrimination of antibiotics and amino acids.


Assuntos
Elementos da Série dos Lantanídeos/química , Nitrofurantoína/química , Tirosina/química , Antibacterianos/química , Cristalografia por Raios X/métodos , Corantes Fluorescentes/química , Estruturas Metalorgânicas/química , Nitrofurantoína/metabolismo , Tirosina/metabolismo
5.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208040

RESUMO

(1) Background: The pro-resolving lipid mediator Resolvin D1 (RvD1) has already shown protective effects in animal models of diabetic retinopathy. This study aimed to investigate the retinal levels of RvD1 in aged (24 months) and younger (3 months) Balb/c mice, along with the activation of macro- and microglia, apoptosis, and neuroinflammation. (2) Methods: Retinas from male and female mice were used for immunohistochemistry, immunofluorescence, transmission electron microscopy, Western blotting, and enzyme-linked immunosorbent assays. (3) Results: Endogenous retinal levels of RvD1 were reduced in aged mice. While RvD1 levels were similar in younger males and females, they were markedly decreased in aged males but less reduced in aged females. Both aged males and females showed a significant increase in retinal microglia activation compared to younger mice, with a more marked reactivity in aged males than in aged females. The same trend was shown by astrocyte activation, neuroinflammation, apoptosis, and nitrosative stress, in line with the microglia and Müller cell hypertrophy evidenced in aged retinas by electron microscopy. (4) Conclusions: Aged mice had sex-related differences in neuroinflammation and apoptosis and low retinal levels of endogenous RvD1.


Assuntos
Envelhecimento/patologia , Ácidos Docosa-Hexaenoicos/farmacologia , Inflamação/patologia , Retina/patologia , Caracteres Sexuais , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Caspase 3/metabolismo , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Células Ependimogliais/ultraestrutura , Feminino , Masculino , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Microglia/ultraestrutura , NF-kappa B/metabolismo , Retina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
6.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208786

RESUMO

The accumulation of lipid intermediates may interfere with energy metabolism pathways and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B). Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium (IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmitoylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to insulin stimulation.


Assuntos
Carnitina/análogos & derivados , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Animais , Células CHO , Carnitina/farmacologia , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Insulina/metabolismo , Resistência à Insulina , Modelos Biológicos , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/química
7.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2334-2341, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327899

RESUMO

Tyrosine phosphorylation is one of the important protein phosphorylations in eukaryotes responsible for a variety of biological processes including cell signaling transduction, cell migration, and apoptosis. In the study of phosphoproteomics, due to the low stoichiometry of tyrosine phosphorylation (pTyr) proteins and sometimes limited initial sample, traditional phosphoproteomics enrichment technology is inefficient for the enrichment of pTyr peptides. Here, we review the substantial progress in tyrosine phosphoproteomics by preparation of limited amount sample and the newly introduced SH2 superbinder.


Assuntos
Peptídeos , Tirosina , Movimento Celular , Fosforilação , Tecnologia , Tirosina/metabolismo
8.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066283

RESUMO

Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: -17.65 kcal mol-1 (D6), -18.07 kcal mol-1 (D2), -18.13 (D5) kcal mol-1, and -10.31 kcal mol-1 (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (-12.84 kcal mol-1) and L-tyrosine (-9.04 kcal mol-1) in melanogenesis.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Pironas/química , Pironas/farmacologia , Domínio Catalítico , Humanos , Levodopa/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Melanoma/metabolismo , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Neoplasias Cutâneas/metabolismo , Relação Estrutura-Atividade , Tirosina/metabolismo
9.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073081

RESUMO

We studied the effects of the addition of large neutral amino acids, such as tyrosine (Tyr) and tryptophan (Trp), in mice DBA/2J and tetrahybrid mice DBCB receiving a high-fat, high-carbohydrate diet (HFCD) for 65 days. The locomotor activity, anxiety, muscle tone, mass of internal organs, liver morphology, adipokines, cytokines, and biochemical indices of animals were assessed. The Tyr supplementation potentiated increased anxiety in EPM and contributed to a muscle tone increase, a decrease in the AST/ALT ratio, and an increase in protein anabolism in both mice strains. Tyr contributed to a decrease in liver fatty degeneration and ALT reduction only in DBCB that were sensitive to the development of obesity. The addition of Trp caused an increase in muscle tone and potentiated an increase in anxiety with age in animals of both genotypes. Trp had toxic effects on the livers of mice, which was manifested in increased fatty degeneration in DBCB, edema, and the appearance of micronuclei in DBA/2J. The main identified effects of Tyr on mice are considered in the light of its modulating effect on the dopamine neurotransmitter metabolism, while for the Trp supplement, effects were presumably associated with the synthesis of its toxic metabolites by representatives of the intestinal microflora.


Assuntos
Suplementos Nutricionais , Obesidade/metabolismo , Triptofano , Tirosina , Animais , Dieta Hiperlipídica/efeitos adversos , Dopamina/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Triptofano/administração & dosagem , Triptofano/metabolismo , Tirosina/administração & dosagem , Tirosina/metabolismo
10.
Methods Mol Biol ; 2276: 383-396, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060056

RESUMO

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Immunoblotting/métodos , Espectrometria de Massas/métodos , Mitocôndrias Cardíacas/química , Proteínas Mitocondriais/análise , Ácido Peroxinitroso/química , Tirosina/análogos & derivados , Animais , Bovinos , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Tirosina/análise , Tirosina/metabolismo
11.
Ecotoxicol Environ Saf ; 221: 112418, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34146982

RESUMO

BACKGROUND: Bisphenol A (BPA), a widely used plastic monomer and plasticizer, is detectable in blood, urine and semen of a healthy people, with concentrations ranging from 0.1 nM to 10 nM. It has been shown that in vitro exposure of BPA as low as 0.001 nM could significantly inhibited mouse sperm motility and acrosome reaction. However, it is still unclear whether BPA at those physiologically detectable concentration affects human sperm. METHODS: The effects of different concentrations of BPA (0, 10-3, 10-2, 10-1, 10, 103 nM) on sperm functions were examined, including human sperm viability, kinematic parameters, hyperactivation and capacitation. RESULTS: BPA caused a remarkable decline in human sperm viability, motility and progressive motility, hyperactivation, capacitation and progesterone-induced acrosome reaction. Mechanism studies showed that BPA could suppress the protein tyrosine phosphorylation level of human sperm, but had no effect on sperm calcium signaling. CONCLUSIONS: Physiologically detectable concentrations of BPA may impair human sperm functions via suppressing protein tyrosine phosphorylation of human sperm, implying that environmental pollution of BPA might be a factor contributing to male infertility.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Plastificantes/toxicidade , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Progesterona/metabolismo , Proteínas/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/fisiologia , Tirosina/metabolismo
12.
Appl Microbiol Biotechnol ; 105(13): 5433-5447, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34181032

RESUMO

We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via L-DOPA, which is synthesized by hydroxylation of L-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for L-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 µM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes. Graphical abstract The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli KEY POINTS: • There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase. • Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli. • New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.


Assuntos
Benzilisoquinolinas , Escherichia coli , Animais , Drosophila melanogaster , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxilação , Tirosina/metabolismo
13.
FASEB J ; 35(7): e21708, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34169549

RESUMO

Metabolic reprogramming occurs in cancer cells and is regulated partly by the opposing actions of tyrosine kinases and tyrosine phosphatases. Several members of the protein tyrosine phosphatase (PTP) superfamily have been linked to cancer as either pro-oncogenic or tumor-suppressive enzymes. In order to investigate which PTPs can modulate the metabolic state of cancer cells, we performed an shRNA screen of PTPs in HCT116 human colorectal cancer cells. Among the 72 PTPs efficiently targeted, 24 were found to regulate mitochondrial respiration, 8 as negative and 16 as positive regulators. Of the latter, we selected TC-PTP (PTPN2) for further characterization since inhibition of this PTP resulted in major functional defects in oxidative metabolism without affecting glycolytic flux. Transmission electron microscopy revealed an increase in the number of damaged mitochondria in TC-PTP-null cells, demonstrating the potential role of this PTP in regulating mitochondrial homeostasis. Downregulation of STAT3 by siRNA-mediated silencing partially rescued the mitochondrial respiration defect observed in TC-PTP-deficient cells, supporting the role of this signaling axis in regulating mitochondrial activity. In addition, mitochondrial stress prevented an increased expression of electron transport chain-related genes in cells with TC-PTP silencing, correlating with decreased ATP production, cellular proliferation, and migration. Our shRNA-based metabolic screen revealed that PTPs can serve as either positive or negative regulators of cancer cell metabolism. Taken together, our findings uncover a new role for TC-PTP as an activator of mitochondrial metabolism, validating this PTP as a key target for cancer therapeutics.


Assuntos
Metabolismo Energético/fisiologia , Dinâmica Mitocondrial/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Tirosina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células HCT116 , Células HEK293 , Humanos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia
14.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
15.
Int J Biol Macromol ; 182: 1278-1291, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33991558

RESUMO

The aggregation of α-synuclein is linked to neurological disorders, and of these, Parkinson's disease (PD) is among the most widely studied. In this background, we have investigated here the effects of three α, ß-unsaturated carbonyl based plant metabolites, daidzein, fisetin and scopoletin on α-Syn aggregation. The ThT and light scattering kinetics studies establish that these compounds have ability to inhibit α-Syn fibrillation to different extents; this is confirmed by TEM studies. It is pertinent to note here that daidzein and scopoletin have been predicted to be able to cross the blood brain barrier. ANS binding assays demonstrate that the compounds interfere in the hydrophobic interactions. The tyrosine quenching, molecular docking and MD simulation studies showed that the compounds bind with α-Syn and provide structural rigidity which delays onset of structural transitions, which is confirmed by CD spectroscopy. The results obtained here throw light on the mechanisms underlying inhibition of α-Syn fibrillation by these compounds. Thus, the current work has significant therapeutic implications for identifying plant based potent therapeutic molecules for PD and other synucleinopathies, an area which needs extensive exploration.


Assuntos
Flavonóis/farmacologia , Isoflavonas/farmacologia , Metaboloma , Agregados Proteicos/efeitos dos fármacos , Escopoletina/farmacologia , alfa-Sinucleína/metabolismo , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Linhagem Celular , Flavonóis/química , Fluorescência , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoflavonas/química , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Escopoletina/química , Tirosina/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/ultraestrutura
16.
Cell Mol Life Sci ; 78(13): 5381-5395, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34028590

RESUMO

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.


Assuntos
Acetilcolina/metabolismo , Neurônios/metabolismo , Tirosina/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Quinases da Família src/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Fosforilação , Transdução de Sinais , Receptor Nicotínico de Acetilcolina alfa7/genética , Quinases da Família src/genética
17.
Mol Carcinog ; 60(7): 481-496, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34018249

RESUMO

c-Hepatocyte growth factor receptor (Met) inhibitors have demonstrated clinical benefits in some types of solid tumors. However, the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we discovered that c-Met inhibitors induced "Signal Transducer and Activator of Transcription (STAT3)-addiction" in ESCC cells, and the feedback activation of STAT3 in ESCC cells limits the tumor response to c-Met inhibition. Mechanistically, c-Met inhibition increased the autocrine of several cytokines, including CCL2, interleukin 8, or leukemia inhibitory factor, and facilitated the interactions between the receptors of these cytokines and Janus Kinase1/2 (JAK1/2) to resultantly activate JAKs/STAT3 signaling. Pharmacological inhibition of c-Met together with cytokines/JAKs/STAT3 axis enhanced cancer cells regression in vitro. Importantly, combined c-Met and STAT3 inhibitors synergistically suppressed tumor growth and promoted the apoptosis of tumor cells without producing systematic toxicity. These findings suggest that inhibition of the STAT3 feedback loop may augment the response to c-Met inhibitors via the STAT3-mediated oncogene addiction in ESCC cells.


Assuntos
Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Ácidos Aminossalicílicos/administração & dosagem , Ácidos Aminossalicílicos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/mortalidade , Retroalimentação Fisiológica/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Plant Cell ; 33(3): 671-696, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33955484

RESUMO

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.


Assuntos
Plântula/metabolismo , Triptofano/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Arabidopsis/metabolismo , Cicloexenos/metabolismo , Fenilalanina/metabolismo , Ácido Chiquímico/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
19.
Inorg Chem ; 60(11): 7844-7856, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34008401

RESUMO

Cysteine dioxygenase (CDO) is a nonheme mononuclear iron enzyme, which catalyzes the oxidation of cysteine to cysteine sulfinic acid. Crystal structure studies of mammalian CDO showed that there is a cross-linked cysteine-tyrosine (Cys-Tyr) cofactor in its active site. Moreover, the formation of the Cys-Tyr cofactor requires the metal cofactor (Fe2+) and O2, and it was previously considered to substantially enhance the catalytic efficiency and half-life of CDO. Recently, a pure human CDO (F2-CDO) without including the Cys-Tyr cofactor was crystalized by the site-directed mutagenesis approach in the anaerobic condition. In this work, to gain insights into the formation mechanism of the Cys-Tyr cofactor and whether it can really promote the catalytic reactivity of CDO, a series of computational models have been constructed, and quantum mechanical/molecular mechanical (QM/MM) calculations have been performed. Our calculation results reveal that WT-CDO and F2-CDO follow different mechanisms for the formation of the Cys-Tyr cofactor. In F2-CDO, the cofactor formation contains the H-abstraction, C-S bond formation, intramolecular F migration, and aromatization of the residue F2Y157, in which the Fe-coordinate dioxygen can be recovered after the formation cofactor; however, in the WT-CDO, the cofactor formation shows some differences. During the reaction, hydrogen peroxide is generated, and the final aromatization requires the assistance of one water molecule. Furthermore, the overall barriers of cofactor formation are always higher than l-cysteine oxidation for both WT-CDO and F2-CDO irrespective of the absence or presence of the cofactor. Thus, we can theoretically confirm that the Cys-Tyr cofactor is not essential for the oxidation activity of CDO, and cofactor formation is just an accompanying reaction but not a prerequisite for the oxidation reaction. These results may provide useful information for understanding the catalysis of CDO.


Assuntos
Cisteína Dioxigenase/metabolismo , Cisteína/metabolismo , Teoria da Densidade Funcional , Tirosina/metabolismo , Biocatálise , Cisteína/química , Cisteína Dioxigenase/química , Simulação de Dinâmica Molecular , Conformação Proteica , Tirosina/química
20.
J Biol Chem ; 296: 100720, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33932405

RESUMO

Platelets are key mediators of physiological hemostasis and pathological thrombosis, whose function must be carefully balanced by signaling downstream of receptors such as protease-activated receptor (PAR)4. Protein kinase C (PKC) is known to regulate various aspects of platelet function. For instance, PKCδ is known to regulate dense granule secretion, which is important for platelet activation. However, the mechanism by which PKCδ regulates this process as well as other facets of platelet activity is unknown. We speculated that the way PKCδ regulates platelet function may be because of the phosphorylation of tyrosine residues on PKCδ. We investigated phosphorylation of PKCδ following glycoprotein VI-mediated and PAR4-mediated platelet activation and found that Y311 is selectively phosphorylated when PAR4 is activated in human platelets. Therefore, we generated PKCδ Y311F knock-in mice, which are viable and have no gross abnormalities. However, PKCδY311F mice have significantly enhanced tail-bleeding times compared with WT littermate controls, which means hemostasis is interrupted. Furthermore, PKCδY311F mice exhibit longer time to carotid artery occlusion compared with WT control using a ferric chloride in vivo thrombosis model, indicating that the phosphorylation of PKCδ Y311 is prothrombotic. Washed platelets from PKCδY311F mice have reduced reactivity after stimulation with a PAR-4 agonist indicating its importance in platelet signaling. The phenotype observed in Y311F mouse platelets is because of reduced thromboxane generation, as an inhibitor of thromboxane generation equalizes the PKCδY311F platelet response to that of WT. Therefore, phosphorylation of PKCδ on Y311 is important for regulation of platelet function and specifically thromboxane generation, which reinforces platelet activation.


Assuntos
Plaquetas/metabolismo , Proteína Quinase C-delta/química , Proteína Quinase C-delta/metabolismo , Tromboxanos/biossíntese , Tirosina/metabolismo , Animais , Humanos , Camundongos , Modelos Moleculares , Fosforilação , Conformação Proteica
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