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1.
Biochem Soc Trans ; 48(1): 281-290, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31872231

RESUMO

The post-translational modification protein S-acylation (commonly known as palmitoylation) plays a critical role in regulating a wide range of biological processes including cell growth, cardiac contractility, synaptic plasticity, endocytosis, vesicle trafficking, membrane transport and biased-receptor signalling. As a consequence, zDHHC-protein acyl transferases (zDHHC-PATs), enzymes that catalyse the addition of fatty acid groups to specific cysteine residues on target proteins, and acyl proteins thioesterases, proteins that hydrolyse thioester linkages, are important pharmaceutical targets. At present, no therapeutic drugs have been developed that act by changing the palmitoylation status of specific target proteins. Here, we consider the role that palmitoylation plays in the development of diseases such as cancer and detail possible strategies for selectively manipulating the palmitoylation status of specific target proteins, a necessary first step towards developing clinically useful molecules for the treatment of disease.


Assuntos
Aciltransferases/metabolismo , Antígeno B7-H1/metabolismo , Lipoilação/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptor Tipo 1 de Melanocortina/metabolismo , Proteínas ras/metabolismo , Animais , Cisteína/metabolismo , Descoberta de Drogas/métodos , Humanos , Lipoilação/fisiologia , Camundongos , Neoplasias/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Processamento de Proteína Pós-Traducional
2.
Nat Commun ; 10(1): 2798, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243268

RESUMO

Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.


Assuntos
Cálcio/metabolismo , Biblioteca Gênica , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Animais , Catálise , Células Cultivadas , Técnicas de Química Combinatória , Drosophila/fisiologia , Imagem por Ressonância Magnética , Masculino , Membranas Artificiais , Camundongos , Proteínas Sensoras de Cálcio Neuronal/genética , Neurônios/metabolismo , Palmitoil-CoA Hidrolase , Permeabilidade , Conformação Proteica , Proteínas
3.
Rev Med Virol ; 29(5): e2057, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31179598

RESUMO

During human immunodeficiency virus (HIV) infection, Nef viral protein plays a crucial role in viral pathogenesis and progression of acquired immunodeficiency syndrome. Nef is expressed in the early stages of infection and alters the cellular environment increasing infectivity, viral replication, and the evasion of host immune response through several mechanisms. Nef has numerous functional domains that allow it to interact with a number of proteins, interfering with intracellular traffic. Among these proteins, human peroxisomal thioesterase 8, ACOT8, has been shown to be an important cellular partner of Nef. It has been suggested that this interaction may be involved in Nef-dependent endocytosis and also in the modulation of lipid composition in membrane rafts. However, the actual role of this interaction, as well as the mechanisms involved, has not yet been fully elucidated. In this review, we focused on the interplay between Nef and ACOT8 proteins, highlighting the possible physiological relevance in HIV infection.


Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Palmitoil-CoA Hidrolase/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Biomarcadores , Humanos , Ligação Proteica
4.
Nat Commun ; 10(1): 2756, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227702

RESUMO

Flight loss in birds is as characteristic of the class Aves as flight itself. Although morphological and physiological differences are recognized in flight-degenerate bird species, their contributions to recurrent flight degeneration events across modern birds and underlying genetic mechanisms remain unclear. Here, in an analysis of 295 million nucleotides from 48 bird genomes, we identify two convergent sites causing amino acid changes in ATGLSer321Gly and ACOT7Ala197Val in flight-degenerate birds, which to our knowledge have not previously been implicated in loss of flight. Functional assays suggest that Ser321Gly reduces lipid hydrolytic ability of ATGL, and Ala197Val enhances acyl-CoA hydrolytic activity of ACOT7. Modeling simulations suggest a switch of main energy sources from lipids to carbohydrates in flight-degenerate birds. Our results thus suggest that physiological convergence plays an important role in flight degeneration, and anatomical convergence often invoked may not.


Assuntos
Evolução Biológica , Aves/fisiologia , Metabolismo Energético/genética , Voo Animal/fisiologia , Genoma/genética , Animais , Metabolismo dos Carboidratos/fisiologia , Genômica/métodos , Lipase/genética , Lipase/metabolismo , Lipólise/fisiologia , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/metabolismo , Filogenia
5.
Am J Physiol Endocrinol Metab ; 317(5): E941-E951, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31039008

RESUMO

Neurons uniquely antagonize fatty acid utilization by hydrolyzing the activated form of fatty acids, long chain acyl-CoAs, via the enzyme acyl-CoA thioesterase 7, Acot7. The loss of Acot7 results in increased fatty acid utilization in neurons and exaggerated stimulus-evoked behavior such as an increased startle response. To understand the contribution of Acot7 to seizure susceptibility, we generated Acot7 knockout (KO) mice and assayed their response to kainate-induced seizures. Acot7 KO mice exhibited potentiated behavioral and molecular indices of seizure severity following kainic acid administration, suggesting that fatty acid metabolism in neurons can be a critical regulator of neuronal activity. These data are consistent with the presentation of seizures in a human with genomic deletion of ACOT7 demonstrating the conservation of function across species. To further understand the metabolic complications arising from a deletion in Acot7, we subjected Acot7 KO mice to a high-fat diet. While the loss of Acot7 did not result in metabolic complications following a normal chow diet, a high-fat diet induced greater body weight gain, adiposity, and glucose intolerance in Acot7 KO mice. These data demonstrate that Acot7, a fatty acid metabolic enzyme highly enriched in neurons, regulates both brain-specific metabolic processes related to seizure susceptibility and the whole body response to dietary lipid.


Assuntos
Doenças Metabólicas/genética , Palmitoil-CoA Hidrolase/genética , Convulsões/genética , Adiposidade , Animais , Comportamento Animal , Dieta Hiperlipídica , Agonistas de Aminoácidos Excitatórios , Feminino , Intolerância à Glucose/genética , Ácido Caínico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Gravidez , Convulsões/induzido quimicamente , Convulsões/psicologia , Ganho de Peso
6.
Appl Microbiol Biotechnol ; 103(9): 3693-3704, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30834961

RESUMO

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.


Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Acil Coenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Tioléster Hidrolases/genética , Ácido 3-Hidroxibutírico/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Halomonas/enzimologia , Engenharia Metabólica , Palmitoil-CoA Hidrolase/genética , Tioléster Hidrolases/metabolismo
7.
Chem Res Toxicol ; 32(2): 255-264, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30667213

RESUMO

Orlistat has been proved to be an effective fatty acid synthase inhibitor that is able to inhibit the proliferation and induce apoptosis in many cancer cell types. However, the anticancer effects of orlistat on hepatocellular carcinoma are undefined. We found that orlistat inhibited cell growth and induced G0/G1 cell cycle arrest with increased cyclin D, cyclin E, and p21 expression in human hepatoma Hep3B cells. Furthermore, protein expression of cyclin A, cyclin B, Cdk1, Cdk2, and Cdk4 was reduced by orlistat. This study investigated the role of lipid metabolism on orlistat-induced human hepatoma Hep3B cell death. The decrease in the expression of key enzymes in fatty acid metabolism, including FASN, ACOT8, PPT1, FABP1, CPT1 and CPT2, was observed after orlistat treatment. We also demonstrated that peroxisomal activity was involved in the orlistat-induced Hep3B cell death. In this study, we established an in vitro model to investigate the effect of orlistat on lipid accumulation. We found that orlistat significantly inhibited the cellular lipid content when administered in fatty acid overload conditions in Hep3B cells. Combination treatment of orlistat and paclitaxel was able to induce a synergistic effect on growth inhibition and cell apoptosis in Hep3B cells. Our data suggested that orlistat displays antitumor activity and enhances the efficacy of paclitaxel in Hep3B cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Orlistate/farmacologia , Paclitaxel/farmacologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/patologia , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/metabolismo , Peroxissomos/metabolismo
8.
Biomed Res Int ; 2019: 2916243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31930115

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is characterized by a spectrum of liver pathologies, from simple steatosis to steatohepatitis. Recent studies have increasingly noted the aberrant expression of microRNAs closely related to NAFLD pathologies. We have previously shown the presence of increased levels of microRNA-27b (miR-27b) in patients with NAFLD. In this study, we investigated the role of miR-27b in NAFLD by examining the impact of up-regulated miR-27b on the differentiation of preadipocytes into mature adipocytes. We found that miR-27b-3p remarkably enhances the adipocyte differentiation of 3T3-L1 cells associated with lipid accumulation and intracellular triglyceride contents. Furthermore, we have demonstrated not only that miR-27b-3p induces acyl-CoA thioesterase 2 (ACOT2) expression in 3T3-L1 cells, but also that the knockdown of ACOT2 suppresses lipid accumulation and adipocyte differentiation in both the presence and absence of miR-27b-3p treatment. Our data strongly suggest that the miR-27b-ACOT2 axis is an important pathway in adipocyte differentiation and may play a role in the pathogenesis of NAFLD.


Assuntos
Adipócitos/patologia , Diferenciação Celular/genética , MicroRNAs/genética , Palmitoil-CoA Hidrolase/genética , Regulação para Cima/genética , Células 3T3-L1 , Adipogenia/genética , Animais , Linhagem Celular , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Interferência de RNA/fisiologia , Ativação Transcricional/genética
9.
Sci Rep ; 8(1): 13967, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30228369

RESUMO

Increased lipid metabolism in muscle is associated with insulin resistance and therefore, many strategies have been employed to alter fatty acid metabolism and study the impact on insulin action. Metabolism of fatty acid requires activation to fatty acyl CoA by Acyl CoA synthases (ACSL) and fatty acyl CoA can be hydrolysed by Acyl CoA thioesterases (Acot). Thioesterase activity is low in muscle, so we overexpressed Acot7 in muscle of chow and high-fat diet (HFD) rats and investigated effects on insulin action. Acot7 overexpression modified specific phosphatidylcholine and phosphatidylethanolamine species in tibialis muscle of chow rats to levels similar to those observed in control HFD muscle. The changes in phospholipid species did not alter glucose uptake in tibialis muscle under hyperinsulinaemic/euglycaemic clamped conditions. Acot7 overexpression in white extensor digitorum longus (EDL) muscle increased complete fatty acid oxidation ex-vivo but was not associated with any changes in glucose uptake in-vivo, however overexpression of Acot7 in red EDL reduced insulin-stimulated glucose uptake in-vivo which correlated with increased incomplete fatty acid oxidation ex-vivo. In summary, although overexpression of Acot7 in muscle altered some aspects of lipid profile and metabolism in muscle, this had no major effect on insulin-stimulated glucose uptake.


Assuntos
Glucose/metabolismo , Resistência à Insulina , Insulina/farmacologia , Lipídeos/análise , Músculo Esquelético/enzimologia , Palmitoil-CoA Hidrolase/metabolismo , Fosfolipídeos/metabolismo , Acil Coenzima A/metabolismo , Animais , Humanos , Hipoglicemiantes/farmacologia , Masculino , Oxirredução , Palmitoil-CoA Hidrolase/genética , Ratos , Ratos Wistar
10.
J Am Heart Assoc ; 7(13)2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29945911

RESUMO

BACKGROUND: Carboxyl-terminal modulator protein (CTMP) has been implicated in cancer, brain injury, and obesity. However, the role of CTMP in pathological cardiac hypertrophy has not been identified. METHODS AND RESULTS: In this study, decreased expression of CTMP was observed in both human failing hearts and murine hypertrophied hearts. To further explore the potential involvement of CTMP in pathological cardiac hypertrophy, cardiac-specific CTMP knockout and overexpression mice were generated. In vivo experiments revealed that CTMP deficiency exacerbated the cardiac hypertrophy, fibrosis, and function induced by pressure overload, whereas CTMP overexpression alleviated the response to hypertrophic stimuli. Consistent with the in vivo results, adenovirus-mediated gain-of-function or loss-of-function experiments showed that CTMP also exerted a protective effect against hypertrophic responses to angiotensin II in vitro. Mechanistically, CTMP ameliorated pathological cardiac hypertrophy through the blockade of the protein kinase B signaling pathway. Moreover, inhibition of protein kinase B activation with LY294002 rescued the deteriorated effect in aortic banding-treated cardiac-specific CTMP knockout mice. CONCLUSIONS: Taken together, these findings imply, for the first time, that increasing the cardiac expression of CTMP may be a novel therapeutic strategy for pathological cardiac hypertrophy.


Assuntos
Proteínas de Transporte/metabolismo , Hipertrofia Ventricular Esquerda/prevenção & controle , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Humanos , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Miócitos Cardíacos/patologia , Palmitoil-CoA Hidrolase , Ratos Sprague-Dawley , Transdução de Sinais , Tioléster Hidrolases/metabolismo
11.
Plant Physiol Biochem ; 127: 104-118, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29571003

RESUMO

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and ß-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 ß-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/ß-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities.


Assuntos
Arabidopsis/enzimologia , Clorófitas/enzimologia , Ginkgo biloba/enzimologia , Palmitoil-CoA Hidrolase/química , Proteínas de Plantas/química , Arabidopsis/genética , Clorófitas/genética , Ginkgo biloba/genética , Palmitoil-CoA Hidrolase/genética , Proteínas de Plantas/genética , Especificidade da Espécie , Especificidade por Substrato/fisiologia
12.
J Ind Microbiol Biotechnol ; 45(4): 281-291, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29442208

RESUMO

Thioesterases (TEs) play an essential role in the metabolism of fatty acids (FAs). To explore the role of TEs in mediating intracellular lipid metabolism in the oleaginous fungus Mortierella alpina, the acyl-CoA thioesterase ACOT8I was overexpressed. The contents of total fatty acids (TFAs) were the same in the recombinant strains as in the wild-type M. alpina, whilst the production of free fatty acids (FFAs) was enhanced from about 0.9% (wild-type) to 2.8% (recombinant), a roughly threefold increase. Linoleic acid content in FFA form constituted about 9% of the TFAs in the FFA fraction in the recombinant strains but only about 1.3% in the wild-type M. alpina. The gamma-linolenic acid and arachidonic acid contents in FFA form accounted for about 4 and 25%, respectively, of the TFAs in the FFA fraction in the recombinant strains, whilst neither of them in FFA form were detected in the wild-type M. alpina. Overexpression of the TE ACOT8I in the oleaginous fungus M. alpina reinforced the flux from acyl-CoAs to FFAs, improved the production of FFAs and tailored the FA profiles of the lipid species.


Assuntos
Ácido Araquidônico/química , Ácidos Graxos não Esterificados/química , Metabolismo dos Lipídeos , Mortierella/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Biomassa , Cromatografia em Camada Delgada , DNA/química , DNA Fúngico/genética , Fermentação , Glucose/química , Concentração de Íons de Hidrogênio , Ácido Linoleico/química , Lipídeos/química , Consumo de Oxigênio , Isoformas de Proteínas , Proteínas Recombinantes/química , Ácido gama-Linolênico/química
13.
J Lipid Res ; 59(2): 368-379, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29208699

RESUMO

Thioesterase superfamily member 1 (Them1) is an acyl-CoA thioesterase that is highly expressed in brown adipose tissue, where it functions to suppress energy expenditure. Lower Them1 expression levels in the liver are upregulated in response to high-fat feeding. Them1-/- mice are resistant to diet-induced obesity, hepatic steatosis, and glucose intolerance, but the contribution of Them1 in liver is unclear. To examine its liver-specific functions, we created conditional transgenic mice, which, when bred to Them1-/- mice and activated, expressed Them1 exclusively in the liver. Mice with liver-specific Them1 expression exhibited no changes in energy expenditure. Rates of fatty acid oxidation were increased, whereas hepatic VLDL triglyceride secretion rates were decreased by hepatic Them1 expression. When fed a high-fat diet, Them1 expression in liver promoted excess steatosis in the setting of reduced rates of fatty acid oxidation and preserved glycerolipid synthesis. Liver-specific Them1 expression did not influence glucose tolerance or insulin sensitivity, but did promote hepatic gluconeogenesis in high-fat-fed animals. This was attributable to the generation of excess fatty acids, which activated PPARα and promoted expression of gluconeogenic genes. These findings reveal a regulatory role for Them1 in hepatocellular fatty acid trafficking.


Assuntos
Ácidos Graxos/metabolismo , Fígado/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Palmitoil-CoA Hidrolase/deficiência , Palmitoil-CoA Hidrolase/genética
14.
Metab Eng ; 42: 59-65, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28587908

RESUMO

Acyl-CoAs are essential intermediates in the biosynthetic pathways of a number of industrially and pharmaceutically important molecules. When these pathways are reconstituted in a heterologous microbial host for metabolic engineering purposes, the acyl-CoAs may be subject to undesirable hydrolysis by the host's native thioesterases, resulting in a waste of cellular energy and decreased intermediate availability, thus impairing bioconversion efficiency. 4-hydroxycoumarin (4HC) is a direct synthetic precursor to the commonly used oral anticoagulants (e.g. warfarin) and rodenticides. In our previous study, we have established an artificial pathway for 4HC biosynthesis in Escherichia coli, which involves the thioester intermediate salicoyl-CoA. Here, we utilized the 4HC pathway as a demonstration to examine the negative effect of salicoyl-CoA degradaton, identify and inactivate the responsible thioesterase, and eventually improve the 4HC production. We screened a total of 16 E. coli thioesterases and tested their hydrolytic activity towards salicoyl-CoA in vitro. Among all the tested candidate enzymes, YdiI was found to be the dominant contributor to the salicoyl-CoA degradation in E. coli. Remarkably, the ydiI knockout strain carrying the 4HC pathway exhibited an up to 300% increase in 4HC production. An optimized 4HC pathway construct introduced in the ydiI knockout strain led to the accumulation of 935mg/L of 4HC in shake flasks, which is about 1.5 folds higher than the wild-type strain. This study demonstrates a systematic strategy to alleviate the undesirable hydrolysis of thioester intermediates, allowing production enhancement for other biosynthetic pathways with similar issues.


Assuntos
4-Hidroxicumarinas/biossíntese , Escherichia coli/metabolismo , Palmitoil-CoA Hidrolase/biossíntese , Escherichia coli/genética , Palmitoil-CoA Hidrolase/genética
15.
J Lipid Res ; 58(6): 1174-1185, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28416579

RESUMO

Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥.


Assuntos
Macrófagos/metabolismo , Palmitoil-CoA Hidrolase/biossíntese , Fosfolipídeos/metabolismo , Animais , Citocinas/biossíntese , Dinoprostona/metabolismo , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glicerofosfolipídeos/metabolismo , Inflamação/enzimologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Palmitoil-CoA Hidrolase/deficiência , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/metabolismo
16.
Biochim Biophys Acta Gen Subj ; 1861(8): 2112-2118, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28454735

RESUMO

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.


Assuntos
Vaga-Lumes/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Luciferases/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Animais , Luminescência , Estereoisomerismo
17.
Epigenomics ; 9(4): 407-418, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28322575

RESUMO

AIM: Epigenetic mechanisms are critical for normal immune development and epigenetic alterations might therefore be possible contributors to immune diseases. To investigate if DNA methylation in whole blood is associated with total and allergen-specific IgE levels. METHODS: We performed an epigenome-wide association study to investigate the association between DNA methylation and IgE level, allergen-specific IgE and self-reported immune diseases and allergies in 728 individuals. RESULTS: We identified and replicated 15 CpG sites associated with IgE, mapping to biologically relevant genes, including ACOT7, ILR5A, KCNH2, PRG2 and EPX. A total of 331 loci were associated with allergen-specific IgE, but none of these CpG sites were associated with self-reported allergies and immune diseases. CONCLUSION: This study shows that IgE levels are associated with DNA methylation levels at numerous CpG sites, which might provide new leads for investigating the links between IgE and allergic inflammation.


Assuntos
Metilação de DNA , Epigenômica/métodos , Estudo de Associação Genômica Ampla/métodos , Hipersensibilidade/genética , Imunoglobulina E/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Ilhas de CpG , Canal de Potássio ERG1/genética , Proteína Básica Maior de Eosinófilos/genética , Feminino , Humanos , Hipersensibilidade/imunologia , Masculino , Pessoa de Meia-Idade , Palmitoil-CoA Hidrolase/genética , Proteoglicanas/genética , Autorrelato , Adulto Jovem
18.
Biochemistry ; 56(10): 1460-1472, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28156101

RESUMO

Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Mycobacterium avium/enzimologia , Mycobacterium smegmatis/enzimologia , Palmitoil-CoA Hidrolase/química , Acil Coenzima A/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Estudos de Associação Genética , Hidrólise , Isoenzimas/química , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mutação , Mycobacterium avium/genética , Mycobacterium smegmatis/genética , Palmitoil-CoA Hidrolase/classificação , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
19.
Protein Pept Lett ; 23(11): 1032-1037, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27653630

RESUMO

Many polyketides show biological activities and have thus been applied in clinics, as food additives, and in agriculture. Type II thioesterases (TEIIs) play an important role in polyketide biosynthesis. Most TEIIs belong to α/ß-hydrolase family and usually contain a catalytic triad Ser-His-Asp. In polyketide biosynthesis, TEIIs can play an editing role by removal of aberrant non-extendable acyl units in elongation steps, a starter unit selection role by removal of unfavored starter acyl units in initiation steps, and a releasing role by removal of final product in termination steps. Complementation of TEIIs has been observed and applied.


Assuntos
Ácido Graxo Sintases/metabolismo , Policetídeos/metabolismo , Streptomyces/metabolismo , Tioléster Hidrolases/metabolismo , Sequência de Aminoácidos , Palmitoil-CoA Hidrolase/metabolismo , Policetídeo Sintases/metabolismo , Streptomyces/enzimologia
20.
Biochem Biophys Res Commun ; 474(4): 753-760, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27166156

RESUMO

Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-ß1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-ß1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-ß1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-ß1 and α-SMA expression in diabetic mice.


Assuntos
Proteínas de Transporte/metabolismo , Nefropatias Diabéticas/metabolismo , Matriz Extracelular/metabolismo , Rim/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Actinas/metabolismo , Animais , Matriz Extracelular/patologia , Rim/patologia , Masculino , Camundongos , Palmitoil-CoA Hidrolase
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