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1.
Proc Natl Acad Sci U S A ; 117(27): 15895-15901, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571932

RESUMO

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, in Salmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBL was acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBL deacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Salmonella enterica/metabolismo , Sirtuínas/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Isoformas de Proteínas , Salmonella enterica/enzimologia , Espectrometria de Massas em Tandem
2.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1021-1030, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597053

RESUMO

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel ß-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.


Assuntos
Hidrolases de Éster Carboxílico , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Cristalografia , Pectinas/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato
3.
Bioresour Technol ; 310: 123476, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32402987

RESUMO

The effects of feruloyl esterase-producing Lactobacillus plantarum A1, cellulase, or their combination on the fermentation characteristics, carbohydrate composition, and enzymatic hydrolysis of mixed corn stalk and potato pulp silage were investigated. Two mixture ratios were used: a weight ratio of rehydrated corn stalk to potato pulp of 35:1 (HD) and a weight ratio of dry corn stalk to potato pulp of 5:11 (LD). No advantage was observed with the addition of strain A1 alone for lignocellulosic degradation and cellulose conversion, while its combination with cellulase enhanced the lignocellulosic degradation and preserved more fermentable carbohydrates in co-ensiled corn stalk and potato pulp. The enzymatic hydrolysis results indicated a potential benefit of pretreatment for biogas production, as the co-ensiled HD ratio mixture without additive treatment showed high glucose yield after enzymatic hydrolysis following 60 d of fermentation.


Assuntos
Celulase , Lactobacillus plantarum , Solanum tuberosum , Hidrolases de Éster Carboxílico , Celulose , Fermentação , Hidrólise , Lignina , Silagem , Zea mays
5.
PLoS One ; 15(5): e0233087, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407372

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a pathological condition caused by excess triglyceride deposition in the liver. The SMXA-5 severe fatty liver mouse model has been established from the SM/J and A/J strains. To explore the genetic factors involved in fatty liver development in SMXA-5 mice, we had previously performed quantitative trait locus (QTL) analysis, using (SM/J×SMXA-5)F2 intercross mice, and identified Fl1sa on chromosome 12 (centromere-53.06 Mb) as a significant QTL for fatty liver. Furthermore, isoamyl acetate-hydrolyzing esterase 1 homolog (Iah1) was selected as the most likely candidate gene for Fl1sa. Iah1 gene expression in fatty liver-resistant A/J-12SM mice was significantly higher than in fatty liver-susceptible A/J mice. These data indicated that the Iah1 gene might be associated with fatty liver development. However, the function of murine Iah1 remains unknown. Therefore, in this study, we created Iah1 knockout (KO) mice with two different backgrounds [C57BL/6N (B6) and A/J-12SM (A12)] to investigate the relationship between Iah1 and liver lipid accumulation. Liver triglyceride accumulation in Iah1-KO mice of B6 or A12 background did not differ from their respective Iah1-wild type mice under a high-fat diet. These results indicated that loss of Iah1 did not contribute to fatty liver. On the other hands, adipose tissue dysfunction causes lipid accumulation in ectopic tissues (liver, skeletal muscle, and pancreas). To investigate the effect of Iah1 deficiency on white adipose tissue, we performed DNA microarray analysis of epididymal fat in Iah1-KO mice of A12 background. This result showed that Iah1 deficiency might decrease adipokines Sfrp4 and Metrnl gene expression in epididymal fat. This study demonstrated that Iah1 deficiency did not cause liver lipid accumulation and that Iah1 was not a suitable candidate gene for Fl1sa.


Assuntos
Hidrolases de Éster Carboxílico/genética , Deleção de Genes , Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica/genética , Adiposidade , Animais , Peso Corporal , Hidrolases de Éster Carboxílico/metabolismo , Colesterol/sangue , Dieta Hiperlipídica , Epididimo/metabolismo , Regulação Enzimológica da Expressão Gênica , Metabolismo dos Lipídeos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triglicerídeos/sangue
6.
Annu Rev Phytopathol ; 58: 253-276, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32396762

RESUMO

The EDS1 family of structurally unique lipase-like proteins EDS1, SAG101, and PAD4 evolved in seed plants, on top of existing phytohormone and nucleotide-binding-leucine-rich-repeat (NLR) networks, to regulate immunity pathways against host-adapted biotrophic pathogens. Exclusive heterodimers between EDS1 and SAG101 or PAD4 create essential surfaces for resistance signaling. Phylogenomic information, together with functional studies in Arabidopsis and tobacco, identify a coevolved module between the EDS1-SAG101 heterodimer and coiled-coil (CC) HET-S and LOP-B (CCHELO) domain helper NLRs that is recruited by intracellular Toll-interleukin1-receptor (TIR) domain NLR receptors to confer host cell death and pathogen immunity. EDS1-PAD4 heterodimers have a different and broader activity in basal immunity that transcriptionally reinforces local and systemic defenses triggered by various NLRs. Here, we consider EDS1 family protein functions across seed plant lineages in the context of networking with receptor and helper NLRs and downstream resistance machineries. The different modes of action and pathway connectivities of EDS1 family members go some way to explaining their central role in biotic stress resilience.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis , Hidrolases de Éster Carboxílico/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas , Imunidade Vegetal
7.
PLoS One ; 15(5): e0232698, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32369496

RESUMO

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that is synthesized by a wide range of microorganisms. One of the derivatives of PHA, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) has flexible material properties and low melting temperature. We have previously demonstrated that PHBH is degradable in a freshwater environment via the formation of biofilm on the surface of the PHBH film. Undibacterium sp. KW1 and YM2, which were isolated from the biofilm present on the PHBH film in the freshwater sample, were shown to have PHBH-degrading activity. In this study, the complete genome sequence of KW1 and YM2 revealed that the extracellular PHA depolymerase gene, designated as phaZUD, was present in their chromosomes. Sequence analysis revealed that PhaZUD contained four domains: a signal peptide, catalytic domain, linker domain, and substrate-binding domain. Escherichia coli harboring a PhaZUD-expressing plasmid showed PHBH-degrading activity in LB medium containing 1 wt% PHBH powder. The recombinant His-tagged PhaZUD from KW1 and YM2 was purified from the culture supernatant and shown to have PHBH-degrading activity at the optimum temperature of 35 and 40°C, respectively. When the degradation product in the PHBH solution was treated with PhaZUD and assayed by LC-TOF-MS, we detected various oligomer structures, but no more than pentamers, which consist of 3-hydroxybutyrate and 3-hydroxyhexanoate. These results demonstrated that PhaZUD may have an endo-type extracellular PHA depolymerase activity.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Oxalobacteraceae/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Caproatos/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Oxalobacteraceae/química , Oxalobacteraceae/genética , Sequenciamento Completo do Genoma
8.
PLoS One ; 15(5): e0227591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433654

RESUMO

Plants emit high rates of methanol (meOH), generally assumed to derive from pectin demethylation, and this increases during abiotic stress. In contrast, less is known about the emission and source of acetic acid (AA). In this study, Populus trichocarpa (California poplar) leaves in different developmental stages were desiccated and quantified for total meOH and AA emissions together with bulk cell wall acetylation and methylation content. While young leaves showed high emissions of meOH (140 µmol m-2) and AA (42 µmol m-2), emissions were reduced in mature (meOH: 69%, AA: 60%) and old (meOH: 83%, AA: 76%) leaves. In contrast, the ratio of AA/meOH emissions increased with leaf development (young: 35%, mature: 43%, old: 82%), mimicking the pattern of O-acetyl/methyl ester ratios of leaf bulk cell walls (young: 35%, mature: 38%, old: 51%), which is driven by an increase in O-acetyl and decrease in methyl ester content with age. The results are consistent with meOH and AA emission sources from cell wall de-esterification, with young expanding tissues producing highly methylated pectin that is progressively demethyl-esterified. We highlight the quantification of AA/meOH emission ratios as a potential tool for rapid phenotype screening of structural carbohydrate esterification patterns.


Assuntos
Ácido Acético/metabolismo , Parede Celular/metabolismo , Metanol/metabolismo , Folhas de Planta/metabolismo , Acetilação , Atmosfera , Hidrolases de Éster Carboxílico/metabolismo , Esterificação , Metilação , Pectinas/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Populus/efeitos dos fármacos , Populus/crescimento & desenvolvimento , Populus/metabolismo , Estresse Fisiológico/genética
9.
Environ Pollut ; 263(Pt A): 114463, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32283456

RESUMO

PFASs are highly persistent in both natural and living environment, and pose a significant risk for wildlife and human beings. The present study was carried out to determine the inhibitory behaviours of fourteen PFASs on metabolic activity of two major isoforms of carboxylesterases (CES). The probe substrates 2-(2-benzoyl-3-methoxyphenyl) benzothiazole (BMBT) for CES1 and fluorescein diacetate (FD) for CES2 were utilized to determine the inhibitory potentials of PFASs on CES in vitro. The results demonstrated that perfluorododecanoic acid (PFDoA), perfluorotetradecanoic acid (PFTA) and perfluorooctadecanoic acid (PFOcDA) strongly inhibited CES1 and CES2. The half inhibition concentration (IC50) value of PFDoA, PFTA and PFOcDA for CES1 inhibition was 10.6 µM, 13.4 µM and 12.6 µM, respectively. The IC50 for the inhibition of PFDoA, PFTA and PFOcDA towards CES2 were calculated to be 9.56 µM, 17.2 µM and 8.73 µM, respectively. PFDoA, PFTA and PFOcDA exhibited noncompetitive inhibition towards both CES1 and CES2. The inhibition kinetics parameters (Ki) were 27.7 µM, 26.9 µM, 11.9 µM, 4.04 µM, 29.1 µM, 27.4 µM for PFDoA-CES1, PFTA-CES1, PFOcDA-CES1, PFDoA-CES2, PFTA-CES2, PFOcDA-CES2, respectively. In vitro-in vivo extrapolation (IVIVE) predicted that when the plasma concentrations of PFDoA, PFTA and PFOcDA were greater than 2.77 µM, 2.69 µM and 1.19 µM, respectively, it might interfere with the metabolic reaction catalyzed by CES1 in vivo; when the plasma concentrations of PFDoA, PFTA and PFOcDA were greater than 0.40 µM, 2.91 µM, 2.74 µM, it might interfere with the metabolic reaction catalyzed by CES2 in vivo. Molecular docking was used to explore the interactions between PFASs and CES. In conclusion, PFASs were found to cause inhibitory effects on CES in vitro, and this finding would provide an important experimental basis for further in vivo testing of PFASs focused on CES inhibition endpoints.


Assuntos
Carboxilesterase , Hidrolases de Éster Carboxílico , Humanos , Simulação de Acoplamento Molecular , Isoformas de Proteínas
10.
Nature ; 580(7802): 216-219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269349

RESUMO

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização
11.
Gene ; 739: 144515, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32112987

RESUMO

Protein phosphatase methylesterase 1 has been identified as a novel gene in skeletal muscle that is upregulated in response to neurogenic atrophy in mice. Western blot analysis confirms that Ppme1 is expressed during both muscle cell proliferation and differentiation. Additionally, the Ppme1 promoter is active in muscle cells, while mutation of a conserved E-box element prevents full induction of the Ppme1 reporter gene, suggesting that Ppme1 is transcriptionally regulated by myogenic regulatory factors. Interestingly, immunofluorescence analysis indicates that Ppme1 is localized to both the cytoplasm and the nucleus, while cell fractionation shows that Ppme1 is found only in the cytoplasm. Functional studies reveal that inhibition of Ppme1 using ABL127 or AMZ30 attenuates muscle cell differentiation. Interestingly, inhibition of Ppme1 by ABL127 led to a significant increase in AP-1 reporter activity, as well as, increases in ERK1/2, c-Jun, Ppme1, and PP2A protein levels in differentiating muscle cells. In contrast, AMZ30 treated cells showed a significant decrease in AP-1 reporter activity and a decrease in ERK1/2 and p38 phosphorylation levels. Finally, co-immunoprecipitation studies show that ABL127, but not AMZ30, causes disruption of the endogenous interaction between Ppme1 and PP2A. The data in this study show for the first time that Ppme1 is expressed in skeletal muscle and is upregulated in response to neurogenic atrophy. Furthermore, these findings suggest that Ppme1 may act as a sentinel of the MAP kinase signaling pathway and may indirectly regulate the ERK1/2 and p38 branches via a non-canonical mechanism leading to inhibition of muscle cell differentiation.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Diferenciação Celular , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/genética , Linhagem Celular , Genes Reporter , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Fosforilação , Regulação para Cima
12.
Org Biomol Chem ; 18(10): 1968-1977, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32101217

RESUMO

A majority of cocaine users also consume alcohol. The concurrent use of cocaine and alcohol produces the pharmacologically active metabolites cocaethylene and norcocaethylene, in addition to norcocaine. Both cocaethylene and norcocaethylene are more toxic than cocaine itself. Hence, a truly valuable cocaine-metabolizing enzyme for cocaine abuse/overdose treatment should be effective for the hydrolysis of not only cocaine, but also its metabolites norcocaine, cocaethylene, and norcocaethylene. However, there has been no report on enzymes capable of hydrolyzing norcocaethylene (the most toxic metabolite of cocaine). The catalytic efficiency parameters (kcat and KM) of human butyrylcholinesterase (BChE) and two mutants (known as cocaine hydrolases E14-3 and E12-7) against norcocaethylene have been characterized in the present study for the first time, and they are compared with those against cocaine. According to the obtained kinetic data, wild-type human BChE showed a similar catalytic efficiency against norcocaethylene (kcat = 9.5 min-1, KM = 11.7 µM, and kcat/KM = 8.12 × 105 M-1 min-1) to that against (-)-cocaine (kcat = 4.1 min-1, KM = 4.5 µM, and kcat/KM = 9.1 × 105 M-1 min-1). E14-3 and E12-7 showed an improved catalytic activity against norcocaethylene compared to wild-type BChE. E12-7 showed a 39-fold improved catalytic efficiency against norcocaethylene (kcat = 210 min-1, KM = 6.6 µM, and kcat/KM = 3.18 × 107 M-1 min-1). It has been demonstrated that E12-7 as an exogenous enzyme can efficiently metabolize norcocaethylene in rats.


Assuntos
Butirilcolinesterase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cocaína/análogos & derivados , Proteínas Recombinantes/metabolismo , Animais , Biocatálise , Butirilcolinesterase/química , Butirilcolinesterase/genética , Células CHO , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Cocaína/química , Cocaína/metabolismo , Cocaína/farmacocinética , Cricetulus , Ensaios Enzimáticos , Humanos , Hidrólise , Cinética , Masculino , Simulação de Acoplamento Molecular , Mutação , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
13.
BMC Complement Med Ther ; 20(1): 47, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046706

RESUMO

BACKGROUND: The aim of this study was to investigate the effect of tannase-converted green tea extract with a high (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and gallic acid (GA) content on myotube density and fusion in normal and oxidative stress-induced C2C12 skeletal muscle cells. Although the use of green tea extract is considered beneficial, cellular and molecular mechanisms of action of tannase-converted green tea extracts that are used as potential muscle growth materials have not been thoroughly studied. METHODS: This study used histological analysis and molecular biology techniques, and compared the results with those for AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) and green tea extracts. RESULTS: The myotube density of normal and oxidative stress-induced C2C12 cells was significantly higher in the tannase-converted green tea extract-treated group than that observed in the other groups (normal cells: P < 0.01; oxidative stress-induced cells: P < 0.05). In addition, tannase-converted green tea extract and green tea extract treatments significantly upregulated the genetic expression of myogenin, Myf5, and MyoD (P < 0.05). The levels of AMP-activated protein kinase-α (AMPKα) and muscle RING-finger protein-1 (MuRF-1) in the tannase-converted green tea extract group were higher than those in the AICAR and green tea extract groups (P < 0.05). CONCLUSIONS: Taken together, our findings describe that the high levels of EC, EGC, and GA in the tannase-converted green tea extract are attributable to the morphological changes in C2C12 cells and intercellular signaling pathways. Therefore, tannase-converted green tea extract can be used in the treatment of sarcopenia.


Assuntos
Catequina/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Chá/química , Animais , Antioxidantes/metabolismo , Camellia sinensis/química , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular , Camundongos , República da Coreia
14.
Am J Physiol Renal Physiol ; 318(4): F1006-F1016, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32003596

RESUMO

Corticotropin-releasing factor (CRF) regulates diverse physiological functions, including bladder control. We recently reported that Crf expression is under genetic control of Aoah, the locus encoding acyloxyacyl hydrolase (AOAH), suggesting that AOAH may also modulate voiding. Here, we examined the role of AOAH in bladder function. AOAH-deficient mice exhibited enlarged bladders relative to wild-type mice and had decreased voiding frequency and increased void volumes. AOAH-deficient mice had increased nonvoiding contractions and increased peak voiding pressure in awake cystometry. AOAH-deficient mice also exhibited increased bladder permeability and higher neuronal firing rates of bladder afferents in response to stretch. In wild-type mice, AOAH was expressed in bladder projecting neurons and colocalized in CRF-expressing neurons in Barrington's nucleus, an important brain area for voiding behavior, and Crf was elevated in Barrington's nucleus of AOAH-deficient mice. We had previously identified aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor-γ as transcriptional regulators of Crf, and conditional knockout of AhR or peroxisome proliferator-activated receptor-γ in Crf-expressing cells restored normal voiding in AOAH-deficient mice. Finally, an AhR antagonist improved voiding in AOAH-deficient mice. Together, these data demonstrate that AOAH regulates bladder function and that the AOAH-Crf axis is a therapeutic target for treating voiding dysfunction.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Neurônios/enzimologia , Bexiga Urinária/inervação , Transtornos Urinários/enzimologia , Micção , Urodinâmica , Animais , Compostos Azo/farmacologia , Núcleo de Barrington/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Hormônio Liberador da Corticotropina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Contração Muscular , Neurônios/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Pressão , Pirazóis/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Bexiga Urinária/efeitos dos fármacos , Micção/efeitos dos fármacos , Transtornos Urinários/tratamento farmacológico , Transtornos Urinários/genética , Transtornos Urinários/fisiopatologia , Urodinâmica/efeitos dos fármacos
15.
PLoS One ; 15(2): e0226232, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32109244

RESUMO

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.


Assuntos
Controle Biológico de Vetores , Pseudomonas chlororaphis/genética , Percepção de Quorum/genética , Regulon/genética , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/deficiência , Movimento Celular/genética , Quitinases/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Mutantes , RNA-Seq , Sideróforos/genética , Transativadores/genética , Transcriptoma
16.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101840

RESUMO

The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The Vmax and Km values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g-1 min-1 and 5.7 10-4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Doenças das Plantas/microbiologia , Polímeros/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Ácidos Graxos/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solanum tuberosum/microbiologia , Streptomyces/genética
17.
Anal Chim Acta ; 1097: 176-185, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910958

RESUMO

Hepatocellular carcinoma (HCC) is a common and lethal cancer. New serum markers for detecting HCC are urgently needed. Human carboxylesterase 1 (hCE1) is an important member of the serine hydrolase superfamily and is closely related to the occurrence of HCC. It can be used as a good serum marker for early diagnosis of HCC. Here, we developed a surface enhanced Raman scattering (SERS)- based magnetic immunosensor that specifically recognizes and detects trace amounts of hCE1 in human serum via a sandwich structure consisting of a SERS tags, magnetic supporting substrates, and target antigen (hCE1). The SERS tags are 4-mercaptobenzoic acid (4-MBA)-labeled AgNPs, and the SERS supporting substrates are composed of a raspberry-like morphology of Fe3O4@SiO2@AgNPs magnetic nanocomposites surface-functionalized with a hCE1 antibody. The prepared SERS magnetic immunosensor exhibits excellent selectivity and extremely high sensitivity for hCE1 detection. The SERS signal and logarithm of hCE1 concentration presented a wide linear response range of 0.1 ng mL-1 to 1.0 mg mL-1, and the detection limit of hCE1 was 0.1 ng mL-1. The results indicate that the immunosensor can be used for the rapid determination of hCE1 in human serum without a complicated sample pre-treatment. Furthermore, the immunosensor has good reproducibility and stability, and has a promising prospect for the quantitative detection of other tumor markers in early clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Hidrolases de Éster Carboxílico/sangue , Imunoensaio , Neoplasias Hepáticas/química , Nanopartículas de Magnetita/química , Hidrolases de Éster Carboxílico/metabolismo , Voluntários Saudáveis , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/metabolismo , Dióxido de Silício/química , Prata/química , Análise Espectral Raman
18.
Plant Sci ; 291: 110357, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31928671

RESUMO

Phospholipase PLA1-Iγ2 or otherwise DAD1-LIKE LIPASE 3 (DALL3) is a member of class I phospholipases and has a role in JA biosynthesis. AtDALL3 was previously identified in a yeast two-hybrid screening as an interacting protein of the Arabidopsis Selenium Binding Protein 1 (SBP1). In this work, we have studied AtDALL3 as an interacting partner of the Arabidopsis Selenium Binding Protein 1 (SBP1). Phylogenetic analysis showed that DALL3 appears in the PLA1-Igamma1, 2 group, paired with PLA1-Igammma1. The highest level of expression of AtDALL3 was observed in 10-day-old roots and in flowers, while constitutive levels were maintained in seedlings, cotyledons, shoots and leaves. In response to abiotic stress, DALL3 was shown to participate in the network of genes regulated by cadmium, selenite and selenate compounds. DALL3 promoter driven GUS assays revealed that the expression patterns defined were overlapping with the patterns reported for AtSBP1 gene, indicating that DALL3 and SBP1 transcripts co-localize. Furthermore, quantitative GUS assays showed that these compounds elicited changes in activity in specific cells files, indicating the differential response of DALL3 promoter. GFP::DALL3 studies by confocal microscopy demonstrated the localization of DALL3 in the plastids of the root apex, the plastids of the central root and the apex of emerging lateral root primordia. Additionally, we confirmed by yeast two hybrid assays the physical interaction of DALL3 with SBP1 and defined a minimal SBP1 fragment that DALL3 binds to. Finally, by employing bimolecular fluorescent complementation we demonstrated the in planta interaction of the two proteins.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Proteínas de Ligação a Selênio/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Ligação a Selênio/química , Proteínas de Ligação a Selênio/metabolismo , Alinhamento de Sequência
19.
Int J Food Microbiol ; 320: 108508, 2020 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31986350

RESUMO

Laphet is a traditional fermented food in Myanmar, made from tea leaves (Camellia sinensis) by fermentation with limited air passage. We performed microbial diversity analyses on 14 Laphet products collected from different locations in Myanmar. Amplicon-based sequencing results revealed Lactobacillus and Acetobacter were abundant bacteria and Candida, Pichia, Cyberlindnera, and Debaryomyces were abundant yeast. Using selective media, eight species of lactic acid bacteria and nine species of yeast were isolated; Lactobacillus plantarum and L. collinoides were dominant bacteria and Pichia manshurica, Candida boidinii, and Cyberlindnera jadinii were major yeasts. PCR-DGGE analysis confirmed that most of the dominant bacterial and yeast species found in culture dependent analysis were present in Laphet samples. Microbial diversity and pH of Laphet were different between samples from tea plantation area and local markets due to possible differences in incubation time periods. When tannase activity was tested, 23 among 29 bacterial isolates and two among 36 yeast isolates showed positive activities. These findings provide new insights into microbial diversity of Laphet and increased our understanding of the core bacterial and yeast species involved in the manufacture of Laphet.


Assuntos
Bactérias/isolamento & purificação , Camellia sinensis/microbiologia , Alimentos e Bebidas Fermentados/microbiologia , Microbiologia de Alimentos , Fungos/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Biodiversidade , Hidrolases de Éster Carboxílico/metabolismo , Fermentação , Fungos/classificação , Fungos/genética , Fungos/metabolismo , Mianmar , Folhas de Planta/microbiologia
20.
BMC Plant Biol ; 20(1): 13, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31914938

RESUMO

BACKGROUND: Pectin methylesterase (PME) is a hydrolytic enzyme that catalyzes the demethylesterification of homogalacturonans and controls pectin reconstruction, being essential in regulation of cell wall modification. During fruit ripening stage, PME-mediated cell wall remodeling is an important process to determine fruit firmness and softening. Strawberry fruit is a soft fruit with a short postharvest life, due to a rapid loss of firm texture. Hence, preharvest improvement of strawberry fruit rigidity is a prerequisite for extension of fruit refreshing time. Although PME has been well characterized in model plants, knowledge regarding the functionality and evolutionary property of PME gene family in strawberry remain limited. RESULTS: A total of 54 PME genes (FvPMEs) were identified in woodland strawberry (Fragaria vesca 'Hawaii 4'). Phylogeny and gene structure analysis divided these FvPME genes into four groups (Group 1-4). Duplicate events analysis suggested that tandem and dispersed duplications effectively contributed to the expansion of the PME family in strawberry. Through transcriptome analysis, we identified FvPME38 and FvPME39 as the most abundant-expressed PMEs at fruit ripening stages, and they were positively regulated by abscisic acid. Genetic manipulation of FvPME38 and FvPME39 by overexpression and RNAi-silencing significantly influences the fruit firmness, pectin content and cell wall structure, indicating a requirement of PME for strawberry fruit softening. CONCLUSION: Our study globally analyzed strawberry pectin methylesterases by the approaches of phylogenetics, evolutionary prediction and genetic analysis. We verified the essential role of FvPME38 and FvPME39 in regulation of strawberry fruit softening process, which provided a guide for improving strawberry fruit firmness by modifying PME level.


Assuntos
Hidrolases de Éster Carboxílico/genética , Fragaria , Frutas/metabolismo , Pectinas/metabolismo , Ácido Abscísico/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/metabolismo , Fragaria/genética , Fragaria/metabolismo , Frutas/genética , Perfilação da Expressão Gênica , Genes de Plantas , Filogenia , Interferência de RNA
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