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1.
Nat Commun ; 11(1): 5078, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033266

RESUMO

Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.


Assuntos
Acetilglucosamina/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Vias Biossintéticas , Escherichia coli/crescimento & desenvolvimento , Ácido N-Acetilneuramínico/metabolismo , Bacillus subtilis/metabolismo , Proliferação de Células , Escherichia coli/metabolismo , Código Genético , Proteínas de Fluorescência Verde/metabolismo , Engenharia Metabólica , Análise do Fluxo Metabólico , Regiões Promotoras Genéticas/genética , RNA de Transferência/genética , Tirosina/metabolismo
2.
Nat Commun ; 11(1): 4820, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973160

RESUMO

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.


Assuntos
Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/metabolismo , Animais , Sistemas CRISPR-Cas , Quimiocinas/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tirosina-tRNA Ligase/química
3.
Nat Commun ; 11(1): 4333, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859933

RESUMO

Diarrhoea is one of the most burdensome and common adverse events of chemotherapeutics, and has no standardised therapy to date. Increasing evidence suggests that the gut microbiome can influence the development of chemotherapy-induced diarrhoea. Here we report findings from a randomised clinical trial of faecal microbiota transplantation (FMT) to treat diarrhoea induced by tyrosine kinase inhibitors (TKI) in patients with metastatic renal cell carcinoma (ClinicalTrials.gov number: NCT04040712). The primary outcome is the resolution of diarrhoea four weeks after the end of treatments. Twenty patients are randomised to receive FMT from healthy donors or placebo FMT (vehicle only). Donor FMT is more effective than placebo FMT in treating TKI-induced diarrhoea, and a successful engraftment is observed in subjects receiving donor faeces. No serious adverse events are observed in both treatment arms. The trial meets pre-specified endpoints. Our findings suggest that the therapeutic manipulation of gut microbiota may become a promising treatment option to manage TKI-dependent diarrhoea.


Assuntos
Carcinoma de Células Renais/complicações , Diarreia/terapia , Inibidores Enzimáticos/metabolismo , Transplante de Microbiota Fecal/métodos , Neoplasias Renais/complicações , Tirosina/metabolismo , Idoso , Método Duplo-Cego , Tratamento Farmacológico , Disbiose , Fezes , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Pessoa de Meia-Idade , Doadores de Tecidos
4.
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
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.
BMC Evol Biol ; 20(1): 73, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576155

RESUMO

BACKGROUND: Small leucine-rich repeat protein (SLRP) family members contain conserved leucine-rich repeat motifs flanked by highly variable N- and C-terminal regions. Most class II and III SLRPs have tyrosine-rich N-terminal regions and some of these are sulfated. However, the evolutionary origin and conservation of the tyrosine-rich and acidic terminal regions remain undetermined. In this study, we present the most comprehensive multiple sequence alignment (MSA) analyses of all eight class II and III SLRPs to date. Based on the level of conservation of tyrosine residues and adjacent sequences, we predict which tyrosine residues are most likely to be sulfated in the terminal regions of human class II and III SLRPs. RESULTS: Using this novel approach, we predict a total of 22 tyrosine sulfation sites in human SLRPs, of which only 8 sites had been experimentally identified in mammals. Our analyses suggest that sulfation-prone, tyrosine-rich and acidic terminal regions of the class II and III SLRPs emerged via convergent evolution at different stages of vertebrate evolution, coinciding with significant evolutionary events including the development of endochondral bones and articular cartilage, the aquatic to terrestrial transition, and the formation of an amnion. CONCLUSIONS: Our study suggests that selective pressures due to changes in life conditions led to the formation of sulfotyrosine-rich and acidic terminal regions. We believe the independent emergence and evolution of sulfotyrosine-rich and acidic N- and C-terminal regions have provided each class II and III SLRP member with novel vital functions required to develop new specialized extracellular matrices and tissues in vertebrate species.


Assuntos
Tecido Conjuntivo/metabolismo , Evolução Molecular , Proteínas/química , Proteínas/metabolismo , Sulfatos/metabolismo , Tirosina/metabolismo , Animais , Humanos , Proteoglicanas/química , Proteoglicanas/genética , Proteoglicanas/metabolismo , Vertebrados/metabolismo
7.
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
8.
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
9.
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
10.
J Med Chem ; 63(10): 5488-5500, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32337993

RESUMO

Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and renin-angiotensin-aldosterone system, respectively. They play an important role in blood pressure regulation and reducing the risk of heart failure. Vasopeptidase inhibitors omapatrilat and sampatrilat possess dual activity against these enzymes by blocking the ACE-dependent conversion of angiotensin I to the potent vasoconstrictor angiotensin II while simultaneously halting the NEP-dependent degradation of vasodilator atrial natriuretic peptide. Here, we report crystal structures of omapatrilat, sampatrilat, and sampatrilat-ASP (a sampatrilat analogue) in complex with NEP at 1.75, 2.65, and 2.6 Å, respectively. A detailed analysis of these structures and the corresponding structures of ACE with these inhibitors has provided the molecular basis of dual inhibitor recognition involving the catalytic site in both enzymes. This new information will be very useful in the design of safer and more selective vasopeptidase inhibitors of NEP and ACE for effective treatment in hypertension and heart failure.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Desenho de Fármacos , Mesilatos/metabolismo , Neprilisina/metabolismo , Peptidil Dipeptidase A/metabolismo , Piridinas/metabolismo , Tiazepinas/metabolismo , Tirosina/análogos & derivados , Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Cristalografia por Raios X/métodos , Mesilatos/química , Neprilisina/química , Peptidil Dipeptidase A/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Piridinas/química , Tiazepinas/química , Tirosina/química , Tirosina/metabolismo
11.
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
12.
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
13.
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
14.
J Vet Sci ; 21(2): e23, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32233131

RESUMO

The identification of biomarkers that distinguish diseased from healthy individuals is of great interest in human and veterinary fields. In this research area, a metabolomic approach and its related statistical analyses can be useful for biomarker determination and allow non-invasive discrimination of healthy volunteers from breast cancer patients. In this study, we focused on the most common canine neoplasm, mammary gland tumor, and herein, we describe a simple method using ultra-high-performance liquid chromatography to determine the levels of tyrosine and its metabolites (epinephrine, 3,4-dihydroxy-L-phenylalanine, 3,4-dihydroxyphenylacetic acid, and vanillylmandelic acid), tryptophan and its metabolites (5-hydroxyindolacetic acid, indoxyl sulfate, serotonin, and kynurenic acid) in canine mammary cancer urine samples. Our results indicated significantly increased concentrations of three tryptophan metabolites, 5-hydroxyindolacetic acid (p < 0.001), serotonin, indoxyl sulfate (p < 0.01), and kynurenic acid (p < 0.05), and 2 tyrosine metabolites, 3,4-dihydroxy-L-phenylalanine (p < 0.001), and epinephrine (p < 0.05) in urine samples from the mammary gland tumor group compared to concentrations in urine samples from the healthy group. The results indicate that select urinary tyrosine and tryptophan metabolites may be useful as non-invasive diagnostic markers as well as in developing a therapeutic strategy for canine mammary gland tumors.


Assuntos
Cromatografia Líquida de Alta Pressão/veterinária , Doenças do Cão/urina , Neoplasias Mamárias Animais/urina , Tirosina/urina , Animais , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão/métodos , Cães , Feminino , Tirosina/metabolismo , Urina/química
15.
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
16.
Proc Natl Acad Sci U S A ; 117(14): 7719-7728, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213582

RESUMO

Chitin is the most abundant renewable nitrogenous material on earth and is accessible to humans in the form of crustacean shell waste. Such waste has been severely underutilized, resulting in both resource wastage and disposal issues. Upcycling chitin-containing waste into value-added products is an attractive solution. However, the direct conversion of crustacean shell waste-derived chitin into a wide spectrum of nitrogen-containing chemicals (NCCs) is challenging via conventional catalytic processes. To address this challenge, in this study, we developed an integrated biorefinery process to upgrade shell waste-derived chitin into two aromatic NCCs that currently cannot be synthesized from chitin via any chemical process (tyrosine and l-DOPA). The process involves a pretreatment of chitin-containing shell waste and an enzymatic/fermentative bioprocess using metabolically engineered Escherichia coli The pretreatment step achieved an almost 100% recovery and partial depolymerization of chitin from shrimp shell waste (SSW), thereby offering water-soluble chitin hydrolysates for the downstream microbial process under mild conditions. The engineered E. coli strains produced 0.91 g/L tyrosine or 0.41 g/L l-DOPA from 22.5 g/L unpurified SSW-derived chitin hydrolysates, demonstrating the feasibility of upcycling renewable chitin-containing waste into value-added NCCs via this integrated biorefinery, which bypassed the Haber-Bosch process in providing a nitrogen source.


Assuntos
Quitina/química , Nitrogênio/química , Resíduos/análise , Acetilglucosamina/metabolismo , Animais , Carbono/farmacologia , Quitosana/química , Crustáceos , Escherichia coli/genética , Engenharia Genética , Glucose/metabolismo , Hidrólise , Levodopa/metabolismo , Minerais/química , Nitrogênio/farmacologia , Polimerização , Tirosina/metabolismo
17.
Nanoscale Horiz ; 5(3): 523-529, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32118213

RESUMO

Super-resolution imaging technology has been a powerful tool for revealing fine biological structures and functions. Its high-quality imaging always needs highly accurate labeling. Here, by exploiting the high specificity and affinity of natural substrates to transporters, we developed one set of substrate-based small molecule fluorescent probes for labeling membrane transporters. A glucose-based probe (Glu-probe) and tyrosine-based probe (Tyr-probe) were synthesized as two examples. Confocal imaging showed that the Glu-probe could label glucose transporters on live cells by being stuck into the binding site. Compared with antibody-probe labeling, the labeling advantages of the Glu-probe were revealed. High specificity of the Glu-probe or Tyr-probe was examined by a colocalization experiment and glucose replacement or amino acid (AA) blocking. The synthetic probes were also tested on imaging HeLa cells to confirm their wide labeling application. Additionally, we found that membrane transporters were mostly in the clustered state on cellular membranes, changing their assembly pattern to regulate the transport effectiveness. These results suggest that the substrate-based probes can serve as valuable tools for investigating the spatial information of membrane transporters.


Assuntos
Corantes Fluorescentes/química , Proteínas de Membrana Transportadoras/análise , Imagem Molecular/métodos , Glucose/metabolismo , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Especificidade por Substrato , Tirosina/metabolismo
18.
Life Sci ; 250: 117546, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32184125

RESUMO

AIM: The enzyme 3-phosphoinositide-dependent protein kinase-1 (PDK1) is associated with cardiac and pathological remodeling and ion channel function regulation. However, whether it regulates hyperpolarization-activated cyclic nucleotide-modulated channels (HCNs) remains unclear. MAIN METHODS: In the atrial myocytes of heart-specific PDK1 "knockout" mouse model and neonatal mice, protein kinase B (AKT)-related inhibitors or agonists as well as knockdown or overexpression plasmids were used to study the relationship between PDK1 and HCNs. KEY FINDINGS: HCN1 expression and AKT phosphorylation at the Thr308 site were significantly decreased in atrial myocytes after PDK1 knockout or inhibition; in contrast, HCN2 and HCN4 levels were significantly increased. Also, a similar trend of HCNs expression has been observed in cultured atrial myocytes after PDK1 inhibition, as further demonstrated via immunofluorescence and patch-clamp experiments. Moreover, these results of PDK1 overexpression indicate an opposite trend compared with the previous experimental results. However, the results of PDK1 inhibition or overexpression could be reversed by activating or inhibiting AKT, respectively. SIGNIFICANCE: These results indicate that the PDK1-AKT signaling pathway is involved in the regulation of HCN mRNA transcription, protein expression, HCN current density, and cell membrane location.


Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Regulação Enzimológica da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Células Cultivadas , Feminino , Deleção de Genes , Átrios do Coração/citologia , Masculino , Camundongos , Camundongos Knockout , Células Musculares/citologia , Técnicas de Patch-Clamp , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tirosina/metabolismo
19.
Nature ; 578(7796): 627-630, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025030

RESUMO

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.


Assuntos
Microscopia Crioeletrônica , Tireoglobulina/química , Tireoglobulina/ultraestrutura , Proteínas de Bactérias/química , Células HEK293 , Humanos , Proteínas Ligantes de Maltose/química , Modelos Moleculares , Mutação , Reprodutibilidade dos Testes , Solventes/química , Tireoglobulina/genética , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/metabolismo , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
20.
Biochim Biophys Acta Bioenerg ; 1861(5-6): 148176, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061653

RESUMO

Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the QX absorption of PheD1 differs, a band-shift in the QX region of PheD2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of TyrD. In contrast, a band-shift due to the formation of either QA•- or TyrZ• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to PheD1 and at ~ 544 nm as expected with Q130 H-bonded to PheD1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O2-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of PD1 is blue shifted and in the PsbA3/T179H mutant. Upon TyrZ•QA•- formation the Soret band of PD1 is red shifted and the Soret band of ChlD1 is blue shifted. In contrast, only PD1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to PD1 is biphasic for all the S-state transitions except for S1 to S2, and shows that: i) the proton release in S0 to S1 occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S2 to S3, in T. elongatus, are significantly faster than often considered. The nature of S2TyrZ• is discussed in view of the models in the literature involving intermediate states in the S2 to S3 transition.


Assuntos
Elétrons , Complexo de Proteína do Fotossistema II/metabolismo , Clorofila/metabolismo , Luz , Modelos Moleculares , Oxirredução , Complexo de Proteína do Fotossistema II/química , Synechococcus/metabolismo , Tirosina/metabolismo
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