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1.
Pestic Biochem Physiol ; 170: 104704, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980065

RESUMO

Carboxylesterases have widely been used in a series of industrial applications, especially, the detoxification of pesticide residues. In the present study, EstC, a novel carboxylesterase from Streptomyces lividans TK24, was successfully heterogeneously expressed, purified and characterized. Phylogenetic analysis showed that EstC can be assigned as the first member of a novel family XIX. Multiple sequence alignment indicated that EstC has highly conserved structural features, including a catalytic triad formed by Ser155, Asp248 and His278, as well as a canonical Gly-His-Ser-Ala-Gly pentapeptide. Biochemical characterization indicated that EstC exhibited maximal activity at pH 9.0 (Tris-HCl buffer) and 55 °C. It also showed higher activity towards short-chain substrates, with the highest activity for p-nitrophenyl acetate (pNPA2) (Km = 0.31 ± 0.02 mM, kcat/Km = 1923.35 ± 9.62 s-1 mM-1) compared to other pNP esters used in this experiment. Notably, EstC showed hyper-thermostability and good alkali stability. The activity of EstC had no significant changes when it was incubated under 55 °C for 100 h and reached half-life after incubation at 100 °C for 8 h. Beyond that, EstC also showed stability at pH ranging from 6.0 to 11.0 and about 90% residual activity still reserved after treatment at pH 8.0 or 9.0 for 26 h, especially. Furthermore, EstC had outstanding potential for bioremediation of chlorpyrifos-contaminated environment. The recombinant enzyme (0.5 U mL-1) could hydrolyze 79.89% chlorpyrifos (5 mg L-1) at 37 °C within 80 min. These properties will make EstC have a potential application value in various industrial productions and detoxification of chlorpyrifos residues.


Assuntos
Carboxilesterase/genética , Clorpirifos , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/genética , Clonagem Molecular , Concentração de Íons de Hidrogênio , Filogenia , Proteínas Recombinantes/genética , Especificidade por Substrato , Temperatura
2.
Medicine (Baltimore) ; 99(36): e22084, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899083

RESUMO

RATIONALE: Dabigatran is a direct thrombin inhibitor that is widely used to prevent the formation of thrombus formation. Amiodarone can increase the plasma concentration of dabigatran. CES1 (carboxylesterase 1) and ABCB1 (ATP-binding cassette subfamily B member 1) genetic polymorphisms associate with the pharmacokinetics of dabigatran. PATIENT CONCERNS: A 62-year-old woman was admitted to the hospital due to chest tightness, fatigue, and discomfort despite long-term anticoagulation with dabigatran 110 mg twice daily for 6 months, with concomitant use of amiodarone. DIAGNOSES: Left atrial appendage thrombus formation with a history of atrial fibrillation. INTERVENTIONS: The clinician changed dabigatran to warfarin. To explore the causes of insufficient anticoagulation using dabigatran in this patient, we examined the ABCB1 and CES1 genes. Results showed that she carried ABCB1 variant alleles with 3 heterozygote single nucleotide polymorphisms (SNPs: rs4148738, rs1045642, rs2032582) and CES1 variant alleles with 2 heterozygote SNPs (rs2244613, rs4580160). OUTCOMES: The left atrial appendage thrombus disappeared. LESSONS: Multiple mutations in the ABCB1 and CES1 genes may influence the pharmacokinetics of dabigatran and could have contributed to the thrombus formation in the left atrial appendage.


Assuntos
Apêndice Atrial/patologia , Fibrilação Atrial/complicações , Hidrolases de Éster Carboxílico/genética , Trombose/etiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Antitrombinas/administração & dosagem , Antitrombinas/farmacocinética , Apêndice Atrial/diagnóstico por imagem , Dabigatrana/administração & dosagem , Dabigatrana/farmacocinética , Feminino , Humanos , Pessoa de Meia-Idade , Trombose/prevenção & controle
3.
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
4.
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
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.
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
7.
J Microbiol Biotechnol ; 30(6): 937-945, 2020 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-32160697

RESUMO

N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA, which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/ß hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency (kcat/KM) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 × 106 to 1.45 × 106 M-1 s-1, with distinctly low KM values (0.16 - 0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.


Assuntos
Hidrolases de Éster Carboxílico/genética , Percepção de Quorum , Rhodococcus/enzimologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Biofilmes/crescimento & desenvolvimento , Genes Bacterianos , Cinética , Rhodococcus/genética , Águas Residuárias/microbiologia , Sequenciamento Completo do Genoma
8.
Insect Biochem Mol Biol ; 120: 103333, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32119906

RESUMO

Viviparous reproduction is characterized by maternal retention of developing offspring within the reproductive tract during gestation, culminating in live birth. In some cases, a mother will provide nutrition beyond that present in the yolk; this is known as matrotrophic viviparity. While this phenomenon is best associated with mammals, it is observed in insects such as the viviparous cockroach, Diploptera punctata. Female D. punctata carry developing embryos in the brood sac, a reproductive organ that acts as both a uterus and a placenta by protecting and providing a nutritive secretion to the intrauterine developing progeny. While the basic physiology of D. punctata pregnancy has been characterized, little is known about the molecular mechanisms underlying this phenomenon. This study combined RNA-seq analysis, RNA interference, and other assays to characterize molecular and physiological changes associated with D. punctata reproduction. A comparison of four stages of the female reproductive cycle and males revealed unique gene expression profiles corresponding to each stage and between sexes. Differentially regulated transcripts of interest include the previously identified family of milk proteins and transcripts associated with juvenile hormone metabolism. RNA interference and methoprene application experiments established the potential impacts of bothbreakdown and synthesis reduction of juvenile hormone in maintaining pregnancy in D. punctata. These studies provide the comprehensive molecular mechanisms associated with cockroach viviparity, which will be a critical resource for comparative purposes among viviparity in insect systems.


Assuntos
Hidrolases de Éster Carboxílico/genética , Baratas/fisiologia , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Metiltransferases/genética , Transcriptoma , Viviparidade não Mamífera , Animais , Hidrolases de Éster Carboxílico/metabolismo , Baratas/enzimologia , Proteínas de Insetos/metabolismo , Metiltransferases/metabolismo , Leite/metabolismo , Interferência de RNA , RNA-Seq , Reprodução , Viviparidade não Mamífera/genética
9.
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
10.
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
11.
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
12.
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
13.
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
14.
Mol Plant Microbe Interact ; 33(2): 328-335, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31702436

RESUMO

Plants have evolved mechanisms to protect themselves against pathogenic microbes and insect pests. In Arabidopsis, the immune regulator PAD4 functions with its cognate partner EDS1 to limit pathogen growth. PAD4, independently of EDS1, reduces infestation by green peach aphid (GPA). How PAD4 regulates these defense outputs is unclear. By expressing the N-terminal PAD4 lipase-like domain (PAD4LLD) without its C-terminal EDS1-PAD4 (EP) domain, we interrogated PAD4 functions in plant defense. Here, we show that transgenic expression of PAD4LLD in Arabidopsis is sufficient for limiting GPA infestation but not for conferring basal and effector-triggered pathogen immunity. This suggests that the C-terminal PAD4 EP domain is necessary for EDS1-dependent immune functions but is dispensable for aphid resistance. Moreover, PAD4LLD is not sufficient to interact with EDS1, indicating the PAD4-EP domain is required for stable heterodimerization. These data provide molecular evidence that PAD4 has domain-specific functions.


Assuntos
Afídeos , Arabidopsis , Resistência à Doença , Domínios Proteicos , Animais , Afídeos/fisiologia , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Domínios Proteicos/genética , Domínios Proteicos/fisiologia
15.
J Biosci Bioeng ; 129(2): 150-154, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31492608

RESUMO

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80-100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-ß-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30-35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2-8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.


Assuntos
Aspergillus oryzae/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Glucana 1,4-alfa-Glucosidase/genética , Regiões Promotoras Genéticas , Aspergillus oryzae/genética , Biocatálise , Hidrolases de Éster Carboxílico/genética , Eletroforese em Gel de Poliacrilamida , Cinética , Peso Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Chem Biol Interact ; 316: 108914, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31837295

RESUMO

Heroin (diamorphine) is a highly addictive opioid drug synthesized from morphine. The use of heroin and incidence of heroin associated overdose death has increased sharply in the US. Heroin is primarily metabolized via deacetylation (hydrolysis) forming the active metabolites 6-monoacetylmorphine (6-MAM) and morphine. A diminution in heroin hydrolysis is likely to cause higher drug effects and toxicities. In this study, we sought to determine the contribution of the major hepatic hydrolase carboxylesterase 1 (CES1) to heroin metabolism in the liver as well as the potential influence of one of its known genetic variants, G143E (rs71647871). Furthermore, given the potential therapeutic application of cannabidiol (CBD) for heroin addiction and the frequent co-abuse of cannabis and heroin, we also assessed the effects of CBD on heroin metabolism. In vitro systems containing human liver, wild-type CES1, and G143E CES1 S9 fractions were utilized in the assessment. The contribution of CES1 to the hydrolysis of heroin to 6-MAM was determined as 3.66%, and CES1 was unable to further catalyze 6-MAM under our assay conditions. The G143E variant showed a 3.2-fold lower intrinsic clearance of heroin as compared to the WT. CBD inhibited heroin and 6-MAM hydrolysis in a reversible manner, with IC50s of 14.7 and 12.1 µM, respectively. Our study results suggested only minor involvement of CES1 in heroin hydrolysis in the liver. Therefore, the G143E variant is unlikely to cause significant impact despite a much lower hydrolytic activity. CBD exhibited potent in vitro inhibition toward both heroin and 6-MAM hydrolysis, which may be of potential clinical relevance.


Assuntos
Canabidiol/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Hepatócitos/efeitos dos fármacos , Heroína/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Cromatografia Líquida de Alta Pressão , Hepatócitos/citologia , Hepatócitos/metabolismo , Heroína/análise , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas em Tandem , Ácido Valproico/farmacologia
17.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140315, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740410

RESUMO

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.


Assuntos
Proteínas de Bactérias/metabolismo , Plásticos Biodegradáveis/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Lipase/metabolismo , Poliésteres/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Plásticos Biodegradáveis/química , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Domínio Catalítico , Lipase/química , Lipase/genética , Poliésteres/química , Engenharia de Proteínas
18.
Xenobiotica ; 50(1): 92-100, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31601149

RESUMO

The carboxylesterase drug hydrolysis pathway has been used extensively to improve the oral availability of drugs under the assumption that the high capacity and low substrate specificity of hydrolytic enzymes would ensure rapid, complete, and consistent conversion of prodrugs to their active metabolite. However, a growing body of literature indicates that drug hydrolysis is usually catalyzed by one primary enzyme, either carboxylesterase-1 or carboxlylesterase-2, and that there is wide variability in enzyme activity affecting the metabolism of prodrugs to their active metabolites.This review identifies carboxylesterase substrates and describes our current understanding of the influence of genetic polymorphisms on substrate disposition and clinical effects. Several polymorphisms are described in the literature and included in the personalized medicine database PharmGKB, but there are no carboxylesterase genotypes referenced in Food and Drug Administration approved drug labeling. The limited validation of metabolic pathways for drugs undergoing hydrolysis, and the small number of studies evaluating genotype-drug interactions confirm that this is an emerging field of drug metabolism research.The dependence of prodrugs, many with low therapeutic indexes, on carboxylesterase-mediated hydrolysis indicate that genetic variation plays an important role in prodrug activation, and that carboxylesterase genotyping will become an important component of personalized medicine.


Assuntos
Hidrolases de Éster Carboxílico/genética , Medicina de Precisão , Hidrolases de Éster Carboxílico/metabolismo , Interações Medicamentosas , Genótipo , Humanos , Hidrólise , Inativação Metabólica/genética , Taxa de Depuração Metabólica , Polimorfismo Genético , Pró-Fármacos , Especificidade por Substrato
19.
Biochem Biophys Res Commun ; 523(2): 416-422, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31870548

RESUMO

Poplar is a superior forestation species with high adaptability. The woody tissue of poplar is mainly derived from cell wall. Cell wall formation determines cell shape and woody growth. Pectin is rich in primary cell wall, but it is also involved in the regulation of wood formation. In our study, we cloned a gene from poplar (Populus tomentos), designed as PtoPME35, which encodes a putative pectin methylesterase. PtoPME35 has higher sequence similarity with Arabidopsis AtPME35. Gene expression analysis shows that PtoPME35 has a constitutive expression pattern in multiple tissues, with the highest expression in stem. Subcellular localization result indicates that PtoPME35 is localized to the cell wall. To elucidate the biological function of PtoPME35 in vivo, we generated overexpression plants in poplar and Arabidopsis. The degree of pectin methylesterification is decreased in PtoPME35-overexpressing transgenic poplar, although no obvious phenotypes were displayed. In PtoPME35-overexpressing Arabidopsis plants, stomatal opening is inhibited and water loss rate is decreased under the drought condition. Moreover, the expression levels of drought-stress responsive genes were higher with mannitol treatment in PtoPME35-overexpressing Arabidopsis plants than in wild type controls. Accordingly, these results suggest that PtoPME35 may regulate osmotic stress responses by modulating stomatal functions.


Assuntos
Arabidopsis/fisiologia , Hidrolases de Éster Carboxílico/genética , Estômatos de Plantas/fisiologia , Populus/genética , Arabidopsis/genética , Secas , Regulação da Expressão Gênica de Plantas , Osmose/fisiologia , Pectinas/genética , Pectinas/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Estômatos de Plantas/genética , Plantas Geneticamente Modificadas
20.
Cancer Invest ; 38(1): 37-51, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31868021

RESUMO

Regucalcin plays a pivotal role as a suppressor of human carcinogenesis, and downregulation of regucalcin expression may contribute to the promotion of human osteosarcoma. Overexpression of regucalcin suppressed the proliferation of Saos-2 human osteosarcoma cells in vitro and decreased the protein levels of multiple signaling components, transcription factors, and tumor suppressors. Interestingly, extracellular regucalcin repressed colony formation and proliferation of Saos-2 cells, and reduced the protein levels of multiple signaling components, cell cycle inhibitor, and various transcription factors. Thus, regucalcin suppressed the growth of human osteosarcoma cells, providing a novel strategy with the gene therapy for treatment of osteosarcoma.


Assuntos
Neoplasias Ósseas/terapia , Proteínas de Ligação ao Cálcio/metabolismo , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/terapia , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado , Osteossarcoma/genética , Osteossarcoma/patologia , Prognóstico , Ratos , Transfecção
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