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1.
Genes (Basel) ; 10(8)2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398921

RESUMO

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe some of the molecular targets and the duration of the effects. We treated rats with 500 mg grape seed proanthocyanidin extract (GSPE)/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on a cafeteria diet, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA of the key energy metabolism enzymes from the liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. The liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2. In addition, muscle showed a higher fatty oxidation (Oxct1 and Cpt1b mRNA). However, after seventeen weeks, there was a completely different gene expression pattern. At the conclusion of the study, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl. Concomitantly there was an increase in fatty acid oxidation in liver and muscle.


Assuntos
Adiposidade/efeitos dos fármacos , Depressores do Apetite/farmacologia , Dieta da Carga de Carboidratos/efeitos adversos , Dieta Ocidental/efeitos adversos , Sobrepeso/prevenção & controle , Proantocianidinas/farmacologia , Tecido Adiposo/metabolismo , Animais , Depressores do Apetite/uso terapêutico , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Leptina/genética , Leptina/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Sobrepeso/tratamento farmacológico , Proantocianidinas/uso terapêutico , Ratos , Vitis/química
2.
J Microbiol Biotechnol ; 29(6): 839-844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154751

RESUMO

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, 185.3 µM N-methylanthranilate and 95.2 µM methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , ortoaminobenzoatos/metabolismo , Vias Biossintéticas , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/metabolismo , Ruta/enzimologia , Ruta/genética , Vitis/enzimologia , Vitis/genética , ortoaminobenzoatos/química
3.
Biochem Biophys Res Commun ; 512(4): 779-785, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30928098

RESUMO

3-oxoacid CoA-transferase 1 (OXCT1) is a key enzyme in ketone body metabolism that is expressed in adipose and other tissues. The present study addressed the function of OXCT1 in adipose tissue from Tan sheep. The 1563 bp ovine OXCT1 coding sequence was cloned from ovine adipose tissue. The OXCT1 protein sequence was highly homologous to OXCT1 from other species. OXCT1 was highly expressed in kidney and at lower levels in small intestine, lung, spleen, heart, stomach, liver, tail adipose, and cartilage, but not in longissimus muscle. OXCT1 was expressed at higher levels in perirenal and tail adipose tissues than in subcutaneous adipose tissue. OXCT1 expression levels increased during the in vitro differentiation of adipocytes, but decreased dramatically at day 8. OXCT1 knockdown in ovine adipocytes promoted lipid accumulation, whereas overexpression did the converse. This study demonstrates that OXCT1 may play a role in adipogenesis and provides new insight on adipose deposition in sheep.


Assuntos
Adipócitos/citologia , Tecido Adiposo/fisiologia , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Adipócitos/fisiologia , Tecido Adiposo/citologia , Animais , Diferenciação Celular , Clonagem Molecular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Marcadores Genéticos , Ovinos , Gordura Subcutânea/citologia , Gordura Subcutânea/fisiologia
4.
J Ind Microbiol Biotechnol ; 46(7): 899-909, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30963328

RESUMO

Lactoyl-CoA is critical for the biosynthesis of biodegradable and biocompatible lactate-based copolymers, which have wide applications. However, reports on acetyl-CoA: lactate CoA-transferases (ALCTs) are rare. To exploit novel ALCTs, amino acid sequence similarity searches based on the CoA-transferases from Clostridium propionicum and Megasphaera elsdenii were conducted. Two known and three novel enzymes were expressed, purified and characterized. Three novel ALCTs were identified, one each from Megasphaera sp. DISK 18, Clostridium lactatifermentans An75 and Firmicutes bacterium CAG: 466. ME-PCT from Megasphaera elsdenii had the highest catalytic efficiency for both acetyl-CoA (264.22 s-1 mM-1) and D-lactate (84.18 s-1 mM-1) with a broad temperature range for activity and good stability. This study, therefore, offers novel and efficient enzymes for lactoyl-CoA generation. To our best knowledge, this is the first report on the systematic mining of ALCTs, which offers valuable new tools for the engineering of pathways that rely on these enzymes.


Assuntos
Acil Coenzima A/metabolismo , Coenzima A-Transferases/metabolismo , Acetilcoenzima A/metabolismo , Clostridium/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/isolamento & purificação , Ácido Láctico/metabolismo
5.
Yonsei Med J ; 60(3): 308-311, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30799594

RESUMO

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inborn error of ketone body utilization, characterized by episodic or permanent ketosis. SCOT deficiency is caused by mutations in the OXCT1 gene, which is mapped to 5p13 and consists of 17 exons. A 12-month-old girl presented with severe ketoacidosis and was treated with continuous renal replacement therapy. She had two previously unrecognized mild-form episodes of ketoacidosis followed by febrile illness. While high levels of ketone bodies were found in her blood and urine, other laboratory investigations, including serum glucose, were unremarkable. We identified novel compound heterozygous mutations in OXCT1:c.1118T>G (p.Ile373Ser) and a large deletion ranging from exon 8 to 16 through targeted exome sequencing and microarray analysis. This is the first Korean case of SCOT deficiency caused by novel mutations in OXCT1, resulting in life-threatening ketoacidosis. In patients with unexplained episodic ketosis, or high anion gap metabolic acidosis in infancy, an inherited disorder in ketone body metabolism should be suspected.


Assuntos
Acidose/genética , Coenzima A-Transferases/deficiência , Cetose/etiologia , Mutação/genética , Sequência de Bases , Coenzima A-Transferases/genética , Éxons/genética , Feminino , Heterozigoto , Humanos , Lactente , Corpos Cetônicos/sangue , Corpos Cetônicos/urina
6.
EBioMedicine ; 41: 509-516, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30796005

RESUMO

BACKGROUND: The therapeutic potential of faecal microbiota transplantation (FMT) is under investigation for a range of inflammatory conditions. While mechanisms of benefit are poorly understood, most models rely on the viability of transplanted microbes. We hypothesised that protocols commonly used in the preparation of faecal transplants will substantially reduce the number, diversity and functional potential of viable microbes. METHODS: Stools from eight screened donors were processed under strict anaerobic conditions, in ambient air, and freeze-thawed. Propidium monoazide (PMA) sample treatment was combined with quantitative PCR, 16S rRNA gene amplicon sequencing and short-chain fatty acid (SCFA) analysis to define the viable microbiota composition and functional potential. FINDINGS: Approximately 50% of bacterial content of stool processed immediately under strict anaerobic conditions was non-viable. Homogenisation in ambient air or freeze-thaw reduced viability to 19% and 23% respectively. Processing of samples in ambient air resulted in up to 12-fold reductions in the abundance of important commensal taxa, including the highly butyrogenic species Faecalibacterium prausnitzii, Subdoligranulum variable, and Eubacterium hallii. The adverse impact of atmospheric oxygen exposure on the capacity of the transplanted microbiota to support SCFA biosynthesis was demonstrated by significantly reduced butyrate and acetate production by faecal slurries processed in ambient air. In contrast, while reducing overall levels of viable bacteria, freeze-thaw did not significantly alter viable microbiota composition. INTERPRETATION: The practice of preparing material for faecal transplantation in ambient air profoundly affects viable microbial content, disproportionately reducing the abundance of anaerobic commensals and the capacity for biosynthesis of important anti-inflammatory metabolites. FUND: This work was supported by the South Australian Health and Medical Research Institute. LP is supported by a scholarship from the Flinders Foundation. GR is supported by a Matthew Flinders Research Fellowship.


Assuntos
Bactérias/metabolismo , Transplante de Microbiota Fecal , Fezes/microbiologia , Acetatos/metabolismo , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Butiratos/metabolismo , Coenzima A-Transferases/genética , Ácidos Graxos Voláteis/biossíntese , Congelamento , Microbioma Gastrointestinal , Humanos , Viabilidade Microbiana , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
7.
Biochim Biophys Acta Proteins Proteom ; 1867(3): 317-330, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30342240

RESUMO

The availability of complete genome sequence of Mycobacterium tuberculosis has provided an important tool to understand the mycobacterial biology with respect to host-pathogen interaction, which is an unmet need of the hour owing to continuous increasing drug resistance. Hypothetical proteins are often an overlooked pool though half the genome encodes for such proteins of unknown function that could potentially play vital roles in mycobacterial biology. In this context, we report the structural and functional characterization of the hypothetical protein Rv3272. Sequence analysis classifies Rv3272 as a Family III CoA transferase with the classical two domain structure and conserved Aspartate residue (D175). The crystal structure of the wild type protein (2.2 Å) demonstrated the associated inter-locked dimer while that of the D175A mutant co-crystallized with octanoyl-CoA demonstrated relative movement between the two domains. Isothermal titration calorimetry studies indicate that Rv3272 binds to fatty acyl-CoAs of varying carbon chain lengths, with palmitoyl-CoA (C16:0) exhibiting maximum affinity. To determine the functional relevance of Rv3272 in mycobacterial biology, we ectopically expressed Rv3272 in M. smegmatis and assessed that its expression encodes significant alteration in cell surface with marked differences in triacylglycerol accumulation. Additionally, Rv3272 expression protects mycobacteria from acidic, oxidative and antibiotic stress under in vitro conditions. Taken together, these studies indicate a significant role for Rv3272 in host-pathogen interaction.


Assuntos
Proteínas de Bactérias/fisiologia , Coenzima A-Transferases/fisiologia , Mycobacterium tuberculosis/fisiologia , Estresse Fisiológico/fisiologia , Acil Coenzima A/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Parede Celular/química , Coenzima A-Transferases/química , Concentração de Íons de Hidrogênio , Ligantes , Metabolismo dos Lipídeos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Estresse Oxidativo
8.
Artigo em Inglês | MEDLINE | ID: mdl-29793055

RESUMO

Channeling carbohydrates and fatty acids to thermogenic tissues, including brown and beige adipocytes, have garnered interest as an approach for the management of obesity-related metabolic disorders. Mitochondrial fatty acid oxidation (ß-oxidation) is crucial for the maintenance of thermogenesis. Upon cellular fatty acid uptake or following lipolysis from triglycerides (TG), fatty acids are esterified to coenzyme A (CoA) to form active acyl-CoA molecules. This enzymatic reaction is essential for their utilization in ß-oxidation and thermogenesis. The activation and deactivation of fatty acids are regulated by two sets of enzymes called acyl-CoA synthetases (ACS) and acyl-CoA thioesterases (ACOT), respectively. The expression levels of ACS and ACOT family members in thermogenic tissues will determine the substrate availability for ß-oxidation, and consequently the thermogenic capacity. Although the role of the majority of ACS and ACOT family members in thermogenesis remains unclear, recent proceedings link the enzymatic activities of ACS and ACOT family members to metabolic disorders and thermogenesis. Elucidating the contributions of specific ACS and ACOT family members to trafficking of fatty acids towards thermogenesis may reveal novel targets for modulating thermogenic capacity and treating metabolic disorders.


Assuntos
Ácidos Graxos/fisiologia , Termogênese , Animais , Coenzima A Ligases/fisiologia , Coenzima A-Transferases/fisiologia , Humanos , Doenças Metabólicas/fisiopatologia
9.
ACS Synth Biol ; 7(11): 2686-2697, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30346720

RESUMO

Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of target proteins. The FENIX device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. Protein production is triggered via addition of an orthogonal inducer ( i.e., 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively redirected into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h-1) with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.


Assuntos
Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Engenharia Metabólica/métodos , Poliésteres/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise
10.
J Microbiol ; 56(10): 760-771, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30136260

RESUMO

The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia-associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer's disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer-related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.


Assuntos
Doença de Alzheimer/microbiologia , Bactérias/classificação , Butiratos/metabolismo , Fezes/microbiologia , Microbioma Gastrointestinal , Acil Coenzima A/genética , Idoso de 80 Anos ou mais , Bactérias/isolamento & purificação , Bactérias/metabolismo , Proteínas de Bactérias/genética , Coenzima A-Transferases/genética , Humanos , Japão , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética
11.
Virulence ; 9(1): 1019-1035, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30052120

RESUMO

Lipid mediators, derived from arachidonic acid metabolism, play an important role in immune regulation. The functions of bioactive eicosanoids range from modulating cytokine signaling and inflammasome formation to anti-inflammatory and pro-resolving activities. Human pathogenic fungi such as Candida albicans, Candida parapsilosis, Cryptococcus neoformans and Aspergillus fumigatus have been shown to produce such lipid mediators, associated with their virulence. To date, investigations into the molecular mechanisms of fungal eicosanoid biosynthesis in different species have revealed that several genes are associated with prostaglandin production. However, these routes remain uncharacterized in C. parapsilosis with early results suggesting it uses pathways distinct from those found in C. albicans. Therefore, we aimed to identify and characterize C. parapsilosis genes involved in eicosanoid biosynthesis. Following arachidonic acid treatment of C. parapsilosis cells, we identified several genes interfering with prostaglandin production. Out of the identified genes, homologues of a multi copper oxidase (FET3), an Acyl-CoA thiolase (POT1) and an Acyl-CoA oxidase (POX1-3) were found to play a significant role in prostaglandin synthesis. Furthermore, all three genes were confirmed to enhance C. parapsilosis pathogenicity, as the corresponding deletion mutants were cleared more efficiently by human macrophages and induced higher levels of pro-inflammatory cytokines. In addition, the mutants were less virulent than the wild-type strain in a mouse model of systemic infection. Taken together, we identified three genes that regulate eicosanoid biosynthesis in C. parapsilosis and impact the fungus' virulence.


Assuntos
Candida parapsilosis/enzimologia , Candida parapsilosis/patogenicidade , Candidíase/microbiologia , Eicosanoides/biossíntese , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Animais , Vias Biossintéticas , Candida parapsilosis/genética , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Oxirredutases/genética , Oxirredutases/metabolismo , Virulência
12.
Appl Environ Microbiol ; 84(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29728392

RESUMO

The recently revived Clostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology. C. acetobutylicum has been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad, ctfA, ctfB, and adc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, "degenerate" cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion of solB resulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact on adc mRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reduced adhE1 and ctfA-ctfB mRNA levels, and abolished the ctfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of the ctfA transcript, and this was verified by expressing variants of the ctfA-ctfB genes to demonstrate the importance of SolB for acetone formation.IMPORTANCE Small noncoding RNAs regulate many important metabolic and developmental programs in prokaryotes, but their role in anaerobes has been explored minimally. Regulation of solvent formation in the important industrial organism C. acetobutylicum remains incompletely understood. While the genes for solvent formation and their promoters are known, the means by which this organism tunes the ratios of key solvents, notably the butanol/acetone ratio to balance its electron resources, remains unknown. Significantly, the roles of several coding and noncoding genes in the sol locus in tuning the solvent formation ratios have not been explored. Here we show that the small RNA SolB fine-tunes the expression of solvents, with acetone formation being a key target, by regulating the translation of the acetone formation rate-limiting enzyme, the coenzyme A transferase (CoAT). It is notable that SolB expressed at very low levels enables CoAT translation, while at high, nonphysiological expression levels, it leads to degradation of the corresponding transcript.


Assuntos
Proteínas de Bactérias/genética , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , RNA/fisiologia , Solventes/metabolismo , Acetona/metabolismo , Sequência de Bases , Reatores Biológicos , Biotecnologia , Butanóis/metabolismo , Carboxiliases/genética , Clostridium acetobutylicum/crescimento & desenvolvimento , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Etanol/metabolismo , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Estabilidade de RNA , RNA não Traduzido/fisiologia
13.
J Lipid Res ; 59(4): 625-634, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29414764

RESUMO

The ketogenic diet (KD) is a high-fat, very-low-carbohydrate diet that triggers a fasting state by decreasing glucose and increasing ketone bodies, such as ß-hydroxybutyrate (ßHB). In experimental models and clinical trials, the KD has shown anti-tumor effects, possibly by reducing energy supplies to cells, which damage the tumor microenvironment and inhibit tumor growth. Here, we determined expression levels of genes encoding the ketolytic enzymes 3-hydroxybutyrate dehydrogenase 1 (BDH1) and succinyl-CoA: 3-oxoacid CoA transferase 1 (OXCT1) in 33 human cancer cell lines. We then selected two representative lines, HeLa and PANC-1, for in vivo examination of KD sensitivity in tumors with high or low expression, respectively, of these two enzymes. In mice with HeLa xenografts, the KD increased tumor growth and mouse survival decreased, possibly because these tumors actively consumed ketone bodies as an energy source. Conversely, the KD significantly inhibited growth of PANC-1 xenograft tumors. ßHB added to each cell culture significantly increased proliferation of HeLa cells, but not PANCI-1 cells. Downregulation of both BDH1 and OXCT1 rendered HeLa cells sensitive to the KD in vitro and in vivo. Tumors with low ketolytic enzyme expression may be unable to metabolize ketone bodies, thus predicting a better response to KD therapy.


Assuntos
Coenzima A-Transferases/metabolismo , Dieta Cetogênica , Hidroxibutirato Desidrogenase/metabolismo , Corpos Cetônicos/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Proliferação de Células , Coenzima A-Transferases/genética , Humanos , Hidroxibutirato Desidrogenase/genética , Masculino , Camundongos , Camundongos Nus , Neoplasias/patologia , Células Tumorais Cultivadas
14.
Eur J Med Genet ; 61(7): 388-392, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29421601

RESUMO

Trichothiodystrophy type 4 is a rare autosomal recessive and ectodermal disorder, characterized by dry, brittle, sparse and sulfur-deficient hair and other features like intellectual disability, ichthyotic skin and short stature, caused by a homozygous mutation in MPLKIP gene. Glutaric aciduria type 3 is caused by a homozygous mutation in SUGCT gene with no distinctive phenotype. Both genes are localized on chromosome 7 (7p14). We report an 8-year-old female with short stature, microcephaly, development delay, intellectual disability and hair characterized for dark, short, coarse, sparse and brittle associated to classical trichorrhexis microscopy pattern. Chromosome microarray analysis showed a 125 kb homozygous pathogenic deletion, which includes genes MPLKIP and SUGCT, not described before. This is the first case described in Peru of a novel contiguous gene deletion of Trichothiodystrophy type 4 and Glutaric aciduria type 3 performed by chromosome microarray analysis, highlighting the contribution and importance of molecular technologies on diagnosis of rare genetic conditions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Coenzima A-Transferases/genética , Oxirredutases/deficiência , Síndromes de Tricotiodistrofia/genética , Criança , Feminino , Deleção de Genes , Humanos , Análise em Microsséries , Oxirredutases/genética , Peru
15.
Nat Commun ; 9(1): 79, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29311546

RESUMO

Aromatic polyesters are widely used plastics currently produced from petroleum. Here we engineer Escherichia coli strains for the production of aromatic polyesters from glucose by one-step fermentation. When the Clostridium difficile isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (HadA) and evolved polyhydroxyalkanoate (PHA) synthase genes are overexpressed in a D-phenyllactate-producing strain, poly(52.3 mol% 3-hydroxybutyrate (3HB)-co-47.7 mol% D-phenyllactate) can be produced from glucose and sodium 3HB. Also, various poly(3HB-co-D-phenyllactate) polymers having 11.0, 15.8, 20.0, 70.8, and 84.5 mol% of D-phenyllactate are produced from glucose as a sole carbon source by additional expression of Ralstonia eutropha ß-ketothiolase (phaA) and reductase (phaB) genes. Fed-batch culture of this engineered strain produces 13.9 g l-1 of poly(61.9 mol% 3HB-co-38.1 mol% D-phenyllactate). Furthermore, different aromatic polyesters containing D-mandelate and D-3-hydroxy-3-phenylpropionate are produced from glucose when feeding the corresponding monomers. The engineered bacterial system will be useful for one-step fermentative production of aromatic polyesters from renewable resources.


Assuntos
Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , Engenharia Metabólica/métodos , Poliésteres/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Acetil-CoA C-Aciltransferase/genética , Acetil-CoA C-Aciltransferase/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium difficile/enzimologia , Clostridium difficile/genética , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Cupriavidus necator/enzimologia , Cupriavidus necator/genética , Escherichia coli/genética , Lactatos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Polietilenotereftalatos/metabolismo , Polímeros/metabolismo
16.
New Phytol ; 218(1): 81-93, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29315591

RESUMO

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.


Assuntos
Biomassa , Parede Celular/metabolismo , Coenzima A-Transferases/genética , Ácidos Cumáricos/metabolismo , Genes de Plantas , Setaria (Planta)/enzimologia , Setaria (Planta)/genética , Supressão Genética , Ácidos/metabolismo , Brachypodium/genética , Metabolismo dos Carboidratos , Coenzima A-Transferases/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Hidrólise , Lignina/metabolismo , Espectroscopia de Ressonância Magnética , Tamanho do Órgão , Filogenia , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Sementes/anatomia & histologia , Sementes/crescimento & desenvolvimento , Transcriptoma/genética , Xilanos/metabolismo
17.
J Crohns Colitis ; 12(2): 204-216, 2018 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-29373727

RESUMO

Background and Aims: Alterations in short chain fatty acid metabolism, particularly butyrate, have been reported in inflammatory bowel disease, but results have been conflicting because of small study numbers and failure to distinguish disease type, activity or other variables such as diet. We performed a comparative assessment of the capacity of the microbiota for butyrate synthesis, by quantifying butyryl-CoA:acetate CoA-transferase [BCoAT] gene content in stool from patients with Crohn's disease [CD; n = 71], ulcerative colitis [UC; n = 58] and controls [n = 75], and determined whether it was related to active vs inactive inflammation, microbial diversity, and composition and/or dietary habits. Methods: BCoAT gene content was quantified by quantitative polymerase chain reaction [qPCR]. Disease activity was assessed clinically and faecal calprotectin concentration measured. Microbial composition was determined by sequencing 16S rRNA gene. Dietary data were collected using an established food frequency questionnaire. Results: Reduced butyrate-synthetic capacity was found in patients with active and inactive CD [p < 0.001 and p < 0.01, respectively], but only in active UC [p < 0.05]. In CD, low BCoAT gene content was associated with ileal location, stenotic behaviour, increased inflammation, lower microbial diversity, greater microbiota compositional change, and decreased butyrogenic taxa. Reduced BCoAT gene content in patients with CD was linked with a different regimen characterised by lower dietary fibre. Conclusions: Reduced butyrate-synthetic capacity of the microbiota is more evident in CD than UC and may relate to reduced fibre intake. The results suggest that simple replacement of butyrate per se may be therapeutically inadequate, whereas manipulation of microbial synthesis, perhaps by dietary means, may be more appropriate.


Assuntos
Ácido Butírico/metabolismo , Clostridiales/isolamento & purificação , Coenzima A-Transferases/genética , Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologia , DNA Bacteriano/análise , Microbioma Gastrointestinal/genética , Adulto , Estudos de Casos e Controles , Clostridiales/genética , Dieta , Fibras na Dieta , Fezes/química , Feminino , Frutas , Microbioma Gastrointestinal/fisiologia , Humanos , Complexo Antígeno L1 Leucocitário/análise , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/análise , Verduras
18.
J Biosci Bioeng ; 125(3): 295-300, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29173967

RESUMO

The discovery of the lactate-polymerizing enzyme (LPE) enabled the biosynthesis of a polyhydroxyalkanoate (PHA) containing 2-hydroxyalkanoate (2HA). Amino acids are potential precursors of 2HA with various side chain structures if appropriate enzymes are used to convert amino acids to 2HA-coenzyme A (CoA) as the substrate for LPE. In this study, the suitability and utility of (R)-2-hydroxy-4-methylvalerate (2H4MV) dehydrogenase (LdhA) and 2H4MV-CoA transferase (HadA) from Clostridium difficile as 2HA-CoA-supplying enzymes were investigated. By expressing LPE, LdhA, and HadA in Escherichia coli DH5α, we successfully produced poly(3-hydroxybutyrate-co-2HA) [P(3HB-co-2HA)] from a related or unrelated carbon source. The 2HA units incorporated into PHA from unrelated carbon sources were primarily 2H4MV and 2-hydroxy-3-phenylpropionate (2H3PhP), which were assumed to be derived from endogenous leucine and phenylalanine, respectively. Furthermore, P(3HB-co-22 mol% 2HA) synthesis was demonstrated by means of saccharified sugars, which are an abundant and renewable feedstock for polymer production from hemicellulosic biomass (Japanese cedar) as the carbon source. Our study shows that several types of 2HA units such as 2H4MV and 2H3PhP are endogenous monomers for PHA biosynthesis in E. coli expressing LdhA and HadA.


Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Carbono/metabolismo , Ácidos Pentanoicos/metabolismo , Fenilpropionatos/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Ácido 3-Hidroxibutírico/química , Clostridium difficile/enzimologia , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Ácido Láctico/metabolismo , Ácidos Pentanoicos/química , Fenilpropionatos/química , Poliésteres/metabolismo , Poli-Hidroxialcanoatos/química
19.
Biochemistry ; 56(44): 5920-5930, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29068219

RESUMO

In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-µL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.


Assuntos
Alcaloides/metabolismo , Biocatálise , Coenzima A-Transferases/metabolismo , Paclitaxel/análogos & derivados , Paclitaxel/síntese química , Taxoides/metabolismo , Antineoplásicos Fitogênicos/síntese química , Cinética
20.
Bioresour Technol ; 245(Pt A): 560-566, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28898856

RESUMO

The acetate-assisted cultivation of Eubacterium limosum KIST612 was found to provide a way for enhancing cell mass, the carbon monoxide (CO) consumption rate, and butyrate production using CO as an electron and energy source. Cell growth (146%), µmax (121%), and CO consumption rates (151%) increased significantly upon the addition of 30mM acetate to microbial cultures. The main product of CO fermentation by E. limosum KIST612 shifted from acetate to butyrate in the presence of acetate, and 5.72mM butyrate was produced at the end of the reaction. The resting cell experimental conditions indicated acetate uptake and an increase in the butyrate concentration. Three routes to acetate assimilation and energy conservation were suggested based on given experimental results and previously genome sequencing data. Acetate assimilation via propionate CoA-transferase (PCT) was expected to produce 1.5mol ATP/mol butyrate, and was thus anticipated to be the most preferred route.


Assuntos
Butiratos , Eubacterium , Fermentação , Monóxido de Carbono , Coenzima A-Transferases
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