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1.
Phytochemistry ; 178: 112467, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32771675

RESUMO

Mucuna pruriens L., commonly known as velvetbean or cow-itch, is a self-pollinated tropical legume of the family Fabaceae, known for its medicinal properties. The active principle L-DOPA extracted from the plant is a potent drug used in the treatment of Parkinson's disease. Although, it is hypothesized that a single step reaction can produce L-DOPA, the presence of optional routes makes the pathway more intricate. For instance, the catecholamine biosynthetic pathway, which leads to L-DOPA production, could occur by hydroxylation of tyrosine to L-DOPA either by polyphenol oxidase (PPO) or tyrosine hydroxylase (TH). Furthermore, Cytochrome P450 (CYP) enzymes can also cause hydroxylation of tyrosine, resulting in L-DOPA synthesis. Therefore, the present investigation was focused on validating the step, which catalyzes the synthesis of L-DOPA, at the biochemical and molecular levels. Enzyme inhibitor studies showed significant inhibition of PPO enzyme with corresponding decrease in L-DOPA synthesis while TH and CYP inhibition had no effect on L-DOPA synthesis. Activity staining of non-denaturing PAGE gel for PPO and TH showed activity only to PPO enzyme. Following in-gel assay and tryptic digestion of the excised stained gel portion, peptide recovery and LC-MS/MS analysis were performed. Degenerate primers based on peptide sequence resulted in an 800bp amplicon. The subsequent sub-cloning, RACE analysis and BLAST search resulted in the isolation of full-length PPO coding sequence of 1800 bp. Structure prediction and phylogenetic analysis of the obtained sequence revealed strong similarity to other plant PPO's like Glycine max, Vigna radiata and Vicia faba of the same family.


Assuntos
Mucuna , Animais , Catecol Oxidase , Bovinos , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450 , Feminino , Levodopa , Redes e Vias Metabólicas , Filogenia , Espectrometria de Massas em Tandem , Tirosina , Tirosina 3-Mono-Oxigenase
2.
Nature ; 584(7819): 148-153, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32699417

RESUMO

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.


Assuntos
Vias Biossintéticas , Colchicina/biossíntese , Engenharia Metabólica , Vias Biossintéticas/genética , Colchicaceae/enzimologia , Colchicaceae/genética , Colchicaceae/metabolismo , Colchicina/química , Colchicina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Fenilalanina/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transcriptoma , Tirosina/metabolismo
3.
Bioresour Technol ; 315: 123845, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32707504

RESUMO

The main aim of this work was to study the allethrin binding interactions with esterase and its bioremediation potential using an isolated bacterial strain CW7, identified as Pseudomonas nitroreducens. The degradation conditions with strain CW7 were optimized using response surface methodology at pH 7.0, a temperature of 32 °C, and an inocula concentration of 150 mg·L-1, with 96% allethrin degradation observed over 7 days. The kinetic parameters qmax, Ks, and Ki were calculated to be 0.512 day-1, 4.97 mg·L-1, and 317.13 mg·L-1, respectively. Nine intermediate metabolites were identified after analysing the degradation products by gas chromatography-mass spectrometry. Strain CW7 effectively degraded a wide variety of pyrethroids as a carbon source. Molecular modeling, docking, and enzyme kinetics were used to investigate the binding pocket of the esterase containing amino acids such as alanine, arginine, valine, proline, cysteine, glycine, isoleucine, phenylalanine, serine, asparagine, and threonine, which play active roles in allethrin degradation.


Assuntos
Aletrinas , Histidina , Alanina , Arginina , Biodegradação Ambiental , Esterases , Glutamatos , Leucina , Lisina , Metionina , Pseudomonas , Serina , Tirosina
4.
Ecotoxicol Environ Saf ; 201: 110801, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32502906

RESUMO

Mercury (Hg) pollution poses global human health and environmental risks. However, still knowledge gaps exist on both exposures and health effects. Here, we combined transcriptome sequencing technique to further investigate the specific mechanisms of inorganic Hg toxicity in the kidney. Strikingly, transcriptomic analysis revealed that 4174 unigenes (including 2646 upregulated and 1528 downregulated unigenes) were differentially expressed under acute HgCl2 (5 mg/kg) exposure in the kidney. Additionally, we observed that HgCl2 selectively induced tumor necrosis factor superfamily (TNFSF) to participate in renal damage, which was consistent with the high-throughput sequencing data. The phenomenon is accompanied by NLRP3 inflammasome and NF-κB signal activation in the kidney. Simultaneously, ELISA results shown that TNF-α, IL-1ß and IL-6 concentrations in the kidney were significant increased. KEGG enrichment analysis showed that peroxisome proliferators-activated receptors (PPAR) signaling pathway might be vital toxic mechanism of Hg in the kidney. Then, our data showed that PPARγ agonist (GW 1929) attenuated HgCl2 (15 µg/ml)-induced apoptosis and NLRP3 inflammasome activation via decreasing translocation of NF-κB and increasing Bcl2 levels in vitro. Along with this, we demonstrated that PPARγ antagonists (GW9662) effectively aggravated HgCl2-induced nephrotoxicity. Overall, our results suggested that PPARγ signaling pathway is considered to be a protective mechanism to combat against HgCl2-triggered NLRP3 inflammasome activation and apoptosis.


Assuntos
Poluentes Ambientais/toxicidade , Inflamassomos/metabolismo , Rim/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , NF-kappa B/metabolismo , PPAR gama/agonistas , Transcriptoma/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Benzofenonas/farmacologia , Regulação para Baixo , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transporte Proteico , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/farmacologia , Regulação para Cima
5.
PLoS One ; 15(6): e0234991, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584853

RESUMO

The breast cancer (BC) biomarker HER2 (Human Epidermal Receptor 2) is overexpressed in 25% of BC. Only patients with HER2-positive tumors receive HER2-targeting therapies, like trastuzumab (Herceptin). However, some women with a HER2-negative BC could benefit from trastuzumab. This could be explained by the activation/phosphorylation of HER2 that can be recognized by trastuzumab. The aim of this study is to examine trastuzumab effects on HER2 phosphorylation at tyrosine Y877 (pHER2Y877). HER2 and pHER2Y877 status were evaluated in a cohort of BC patients representative of molecular subtypes distribution (n = 497) and in a series of BC cell lines (n = 7). Immunohistochemistry against pHER2Y877 was performed on tissue micro arrays. Cellular proliferation assays were performed on BC cell lines presenting different combinations of HER2 and pHER2Y877 status and treated with increasing doses of trastuzumab (0-150 µg/ml). The prevalence of pHER2Y877 in this cohort was 6%. Nearly 5% of patients with HER2-negative tumors (n = 406, 82%) overexpressed pHER2Y877. Among triple negative BC patients (n = 39, 8%), 7.7% expressed pHER2Y877. Trastuzumab treatment decreased cell proliferation in HER2-/pHER2Y877+ BC cell lines, to an extent comparable to what occurs in HER2+ cell lines, but did not affect HER2-/pHER2Y877- cell lines. Trastuzumab sensitivity in HER2-/pHER2Y877+ cell line is specific to HER2 tyrosine 877 phosphorylation. Hence, with further confirmation in a bigger cohort, trastuzumab treatment could be envisaged as a treatment option to women presenting with HER2-/pHER2+ tumors, representing more than 1000 BC women in Canada in 2019.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Mama/patologia , Neoplasias da Mama/patologia , Canadá , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Análise Serial de Tecidos , Trastuzumab/uso terapêutico , Tirosina/metabolismo
6.
Chin J Physiol ; 63(3): 128-136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32594066

RESUMO

Glucose ingestion attenuates the water ingestion-induced increase in the total peripheral vascular resistance and orthostatic tolerance. We investigated the gastrointestinal physiology of glucose by examining the effect of glucose ingestion on the functional expression of focal adhesion kinase (FAK) in red blood cell (RBC) membrane. This study was performed in 24 young, healthy subjects. Blood samples were collected at 5 min before and 25 min and 50 min after an ingestion of 10% glucose water 500 mL, water 500 mL, or normal saline 500 mL. We determined glucose and osmolality in plasma, and phosphorylation of aquaporin 1 (AQP1), glucose transporter 1 (Glut1), and FAK in RBC membrane. Our results showed that glucose ingestion reduced the rise of peripheral vascular resistance after water ingestion and upregulated the serine phosphorylation of Glut1. It also lowered both the serine phosphorylation of FAK and tyrosine phosphorylation of AQP1, compared with the ingestion of either water or saline. In an ex vivo experiment, glucose activated the Glut1 receptor and subsequently reduced the expression of FAK compared with 0.8% saline alone. We concluded that glucose activates Glut1 and subsequently lowers the functional expression of FAK, a cytoskeleton protein of RBCs. The functional change in the RBC membrane proteins in connection with the attenuation of osmopressor response may elucidate the pathophysiology of glucose in postprandial hypotension.


Assuntos
Eritrócitos , Proteína-Tirosina Quinases de Adesão Focal , Glucose , Humanos , Fosforilação , Tirosina
7.
PLoS Genet ; 16(6): e1008715, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559233

RESUMO

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.


Assuntos
Proteínas de Drosophila/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas ras/metabolismo , Animais , Células Cultivadas , Sequência Conservada , Drosophila , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Janus Quinase 2/metabolismo , Fosforilação , Tirosina/química , Tirosina/genética , Ubiquitinação , Proteínas ras/química , Proteínas ras/genética , Quinases da Família src/metabolismo
8.
Nat Commun ; 11(1): 3154, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572025

RESUMO

An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a crucial prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. Besides, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine, and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification.


Assuntos
Aminoácidos/biossíntese , Código Genético , RNA de Transferência , Biologia Sintética/métodos , Alanina/análogos & derivados , Quimera/genética , Quimera/metabolismo , Escherichia coli , Células HEK293 , Histidina/análogos & derivados , Humanos , Lisina/análogos & derivados , Fenilalanina/análogos & derivados , RNA de Transferência/genética , RNA de Transferência/metabolismo , Triptofano/análogos & derivados , Tirosina/análogos & derivados
9.
PLoS One ; 15(6): e0229276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32542016

RESUMO

Tyrosine is mainly degraded in the liver by a series of enzymatic reactions. Abnormal expression of the tyrosine catabolic enzyme tyrosine aminotransferase (TAT) has been reported in patients with hepatocellular carcinoma (HCC). Despite this, aberration in tyrosine metabolism has not been investigated in cancer development. In this work, we conduct comprehensive cross-platform study to obtain foundation for discoveries of potential therapeutics and preventative biomarkers of HCC. We explore data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Gene Expression Profiling Interactive Analysis (GEPIA), Oncomine and Kaplan Meier plotter (KM plotter) and performed integrated analyses to evaluate the clinical significance and prognostic values of the tyrosine catabolic genes in HCC. We find that five tyrosine catabolic enzymes are downregulated in HCC compared to normal liver at mRNA and protein level. Moreover, low expression of these enzymes correlates with poorer survival in patients with HCC. Notably, we identify pathways and upstream regulators that might involve in tyrosine catabolic reprogramming and further drive HCC development. In total, our results underscore tyrosine metabolism alteration in HCC and lay foundation for incorporating these pathway components in therapeutics and preventative strategies.


Assuntos
Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica , Neoplasias Hepáticas/patologia , Tirosina/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , Mutação , Prognóstico
10.
Chimia (Aarau) ; 74(5): 391-397, 2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32482216

RESUMO

Tyramine is a health-adverse biogenic amine, which can accumulate in fermented foods like cheese by decarboxylation of the free amino acid tyrosine by either starter cultures or resident microbes such as lactic acid bacteria including Enterococcus spp., respectively. Our study aimed to show the effect of sodium chloride concentrations on tyramine production as well as to characterise bacterial strains as anti-tyramine biocontrol agents in a 2 mL micro-cheese fermentation model. The effect of sodium chloride on tyramine production was assayed with tyramine producing strains from eight different species or subspecies. Generally, an increase in sodium chloride concentration enhanced tyramine production, e.g. from 0% to 1.5% of sodium chloride resulted in an increase of tyramine of 870% with a Staphylococcus xylosus strain. In the biocontrol screening among lactic acid bacteria, a Lactobacillus plantarum JA-1199 strain was screened that could consume in successful competition with other resident bacteria tyrosine in the micro-cheese model as a source of energy gain. Thereby tyramine accumulation was reduced between 4% to 99%. The results of this study disclose a feasible strategy for decreasing tyramine concentration and increasing the safety level of fermented food. It is an example of development and application of bacterial isolates as starter or protective cultures in food, a biocontrol topic, which Oreste Ghisalba - in his project evaluation function of SNF and later on CTI - was promoting with great emphasis in our ETH Food Biotechnology research group.


Assuntos
Lactobacillus plantarum , Fermentação , Cloreto de Sódio , Tiramina , Tirosina
11.
PLoS Negl Trop Dis ; 14(5): e0008262, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469928

RESUMO

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.


Assuntos
Matriz Extracelular/parasitologia , Histonas/análise , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/análise , Trypanosoma cruzi/química , Trypanosoma cruzi/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Matriz Extracelular/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Nitrosação , Proteômica , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/imunologia
12.
Proc Natl Acad Sci U S A ; 117(22): 11916-11922, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414932

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both [Formula: see text] and [Formula: see text] as cosubstrates. In this study, the [Formula: see text] reaction with reduced Hypocrea jecorina LPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than the [Formula: see text] reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuII sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.


Assuntos
Aminoácidos/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/química , Polissacarídeos/metabolismo , Sítios de Ligação , Biocombustíveis , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hypocrea/metabolismo , Cinética , Espectroscopia de Ressonância Magnética/métodos , Oxigenases de Função Mista/metabolismo , Oxirredução , Peróxidos/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(21): 11421-11431, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393642

RESUMO

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas Intrinsicamente Desordenadas/química , Substituição de Aminoácidos , Arginina/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Microrganismos Geneticamente Modificados , Simulação de Dinâmica Molecular , Transição de Fase , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Tirosina/química
14.
Proc Natl Acad Sci U S A ; 117(23): 12657-12664, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461364

RESUMO

Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the Amblyomma cajennense tick can be posttranslationally sulfated at two tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that tyrosine sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.


Assuntos
Ácaros e Carrapatos/metabolismo , Proteínas de Artrópodes/metabolismo , Quimiocinas/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Saliva/metabolismo , Animais , Proteínas de Artrópodes/química , Quimiocinas/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Sulfatos/metabolismo , Tirosina/metabolismo
15.
Sci China Life Sci ; 63(5): 697-705, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32246402

RESUMO

The Hippo pathway is a newly identified pathway and evolutionarily conserved from flies to humans mainly regulating cell proliferation. Transcriptional co-activator Yes-associated protein (YAP) functions as a major downstream effector and key node of the Hippo pathway. Phosphorylation of YAP is critical to regulate YAP activity and its corresponding functions. ß-adrenergic receptor (ß-AR), a typical G protein coupled receptor (GPCR), mediates proliferation in various cell types and regulates multiple physical and pathological processes. However, the role of ß-AR in regulating YAP remains elusive. Here, we report that ß-AR can obviously stimulate YAP tyrosine phosphorylation. The mechanism is that ß-AR stimulation results in tyrosine kinase Src activation and Src phosphorylates YAP tyrosine at Y357. Further studies demonstrate that inhibition of Src kinase activity can obviously alleviate ß-AR induced YAP tyrosine phosphorylation and cell proliferation. We conclude that ß-AR can induce YAP tyrosine phosphorylation and also establish the Src/YAP pathway as a critical signaling branch downstream of GPCR.


Assuntos
Receptores Adrenérgicos beta/metabolismo , Fatores de Transcrição/metabolismo , Quinases da Família src/metabolismo , Animais , Proliferação de Células , Fibroblastos/citologia , Regulação da Expressão Gênica , Células HEK293 , Coração , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fosforilação , Ratos , Transfecção , Tirosina/metabolismo
16.
PLoS One ; 15(4): e0230618, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302317

RESUMO

PURPOSE: The aim of this study was to derive reference values of 18F-fluoro-ethyl-L-tyrosine positron emission tomography (18F-FET-PET) uptake in normal brain and head structures to allow for differentiation from tumor tissue. MATERIALS AND METHODS: We examined the datasets of 70 patients (median age 53 years, range 15-79), whose dynamic 18F-FET-PET was acquired between January 2016 and October 2017. Maximum standardized uptake value (SUVmax), target-to-background standardized uptake value ratio (TBR), and time activity curve (TAC) of the 18F-FET-PET were assessed in tumor tissue and in eight normal anatomic structures and compared using the t-test and Mann-Whitney U-test. Correlation analyses were performed using Pearson or Spearman coefficients, and comparisons between several variables with Pearson's chi-squared tests and Kruskal-Wallis tests as well as the Benjamini-Hochberg correction. RESULTS: All analyzed structures showed an 18F-FET uptake higher than background (threshold: TBR > 1.5). The venous sinuses and cranial muscles exhibited a TBR of 2.03±0.46 (confidence interval (CI) 1.92-2.14), higher than the uptake of caudate nucleus, pineal gland, putamen, and thalamus (TBR 1.42±0.17, CI 1.38-1.47). SUVmax, TBR, and TAC showed no difference in the analyzed structures between subjects with high-grade gliomas and subjects with low-grade gliomas, except the SUVmax of the pineal gland (t-tests of the pineal gland: SUVmax: p = 0.022; TBR: p = 0.411). No significant differences were found for gender and age. CONCLUSION: Normal brain tissue demonstrates increased 18F-FET uptake compared to background tissue. Two distinct clusters have been identified, comprising venous structures and gray matter with a reference uptake of up to SUVmax of 2.99 and 2.33, respectively.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Tomografia por Emissão de Pósitrons/normas , Tirosina/análogos & derivados , Adolescente , Adulto , Idoso , Transporte Biológico , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Tirosina/metabolismo , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 117(15): 8503-8514, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234784

RESUMO

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/química
18.
Nat Commun ; 11(1): 1515, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251291

RESUMO

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.


Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/enzimologia , Depuradores de Radicais Livres/metabolismo , Engenharia Metabólica , Álcool Feniletílico/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Vias Biossintéticas/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estudos de Viabilidade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Álcool Feniletílico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido Vanílico/metabolismo
19.
JAMA Netw Open ; 3(3): e201357, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32202644

RESUMO

Importance: Alkaptonuria is an autosomal recessive disorder caused by pathogenic variants in the HGD gene. Deficiency of the HGD enzyme leads to tissue deposition of homogentisic acid (HGA), causing severe osteoarthropathies and cardiac valve degeneration. Although HGD is vital for the catabolism of tyrosine, which provides the basis for thyroid hormone synthesis, the prevalence of thyroid dysfunction in alkaptonuria is unknown. Objective: To assess thyroid structure and function in patients with alkaptonuria. Design, Setting, and Participants: A single-center cohort study was conducted in a tertiary referral center including patients with alkaptonuria followed up for a median of 93 (interquartile range, 48-150) months between February 1, 2000, and December 31, 2018. The alkaptonuria diagnosis was based on clinical presentation and elevated urine HGA levels. A total of 130 patients were considered for participation. Main Outcomes and Measures: Prevalence of thyroid dysfunction in adults with alkaptonuria compared with the general population. Thyrotropin and free thyroxine levels were measured by immunoassay and repeated in each patient a median of 3 (interquartile range, 2-22) times. Neck ultrasonographic scans were analyzed in a subset of participants. Logistic regression was used to test the association of thyroid dysfunction with age, sex, thyroid peroxidase (TPO) antibodies, serum tyrosine levels, and urine HGA levels. Results: Of the 130 patients, 5 were excluded owing to thyroidectomy as the cause of hypothyroidism. The study cohort consisted of 125 patients; the median age was 45 (interquartile range, 35-51) years. Most of the patients were men (72 [57.6%]). The prevalence of primary hyperthyroidism was 0.8% (1 of 125 patients), similar to 0.5% observed in the general population (difference, 0.003; 95% CI, -0.001 to 0.04; P = .88). The prevalence of primary hypothyroidism was 16.0% (20 of 125 patients), which is significantly higher than 3.7% reported in the general population (difference, 0.12; 95% CI, 0.10-0.24; P < .001). Women were more likely to have primary hypothyroidism than men (odds ratio, 10.99; 95% CI, 3.13-38.66; P < .001). Patients with TPO antibodies had a higher likelihood of primary hypothyroidism than those without TPO antibodies (odds ratio, 7.36; 95% CI, 1.89-28.62; P = .004). There was no significant difference in the prevalence of thyroid nodules between patients in this study (29 of 49 [59.2%]) vs the general population (68%) (difference, 0.088; 95% CI, -0.44 to 0.73; P = .20) or of cancer (7% vs 5%; difference, 0.01; 95% CI, -0.01 to 0.17; P = .86). Conclusions and Relevance: The high prevalence of primary hypothyroidism noted in patients with alkaptonuria in this study suggests that serial screening in this population should be considered and prioritized.


Assuntos
Alcaptonúria/metabolismo , Hipotireoidismo/epidemiologia , Adulto , Alcaptonúria/complicações , Alcaptonúria/genética , Autoanticorpos/sangue , Autoantígenos/imunologia , Estudos de Coortes , Feminino , Ácido Homogentísico/urina , Humanos , Hipertireoidismo/epidemiologia , Hipertireoidismo/genética , Hipotireoidismo/genética , Iodeto Peroxidase/imunologia , Proteínas de Ligação ao Ferro/imunologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Prevalência , Testes de Função Tireóidea , Glândula Tireoide/enzimologia , Tireotropina/sangue , Tiroxina/sangue , Tirosina/sangue
20.
Biochim Biophys Acta Biomembr ; 1862(6): 183260, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142822

RESUMO

Because of their potential as novel antibiotic agents, antimicrobial peptides (AMPs) have generated considerable interest. The mechanism of bacterial toxicity of AMPs often involves the disruption and/or permeabilization of the bacterial membrane; even those that act intracellularly first have to traverse the membrane. In this work we have explored the incorporation of the fluorinated aromatic amino acids fluoro-Phe and fluoro-Tyr into the Trp- and Arg-rich AMP tritrpticin, and investigated their role in the membrane binding properties and the antimicrobial activity of the peptide. Fluorinated peptides were obtained with good yield by recombinant expression of tritrpticin as a calmodulin-fusion protein in Escherichia coli. Cells were grown in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, and the peptides were released by proteolysis from the purified fusion protein. By using SDS micelles, as a simplified model of the bacterial cytoplasmic membrane, we could study the peptide-membrane interactions and the preferred location of individual fluorinated residues in the micelles by 19F NMR spectroscopy. Solvent-perturbation 19F NMR measurements revealed that para-fluoro-Phe residues are embedded deeply in the hydrophobic region of the micelles. On the other hand, 3-fluoro-Tyr residues introduced in tritrpticin were located near the surface of the micelles with high solvent exposure, while 2-fluoro-Tyr sidechains were less solvent exposed. In combination with the outcome of determinations of their antimicrobial activity, our 19F NMR results indicate that the higher solvent exposure of Tyr residues correlates with a decrease of the antimicrobial potency. This different role of Tyr can likely be extended from tritrpticin to other cationic AMPs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Flúor , Espectroscopia de Ressonância Magnética/métodos , Oligopeptídeos/química , Tirosina/fisiologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Membrana Celular/metabolismo , Micelas , Oligopeptídeos/metabolismo , Dodecilsulfato de Sódio
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