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1.
Ecotoxicol Environ Saf ; 208: 111748, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396074

RESUMO

Microcystin-leucine arginine (MC-LR) is a kind of toxin produced by cyanobacterial, resulting in decrease of testosterone levels in serum and leading to impaired spermatogenesis. Gonadotropin-releasing hormone (GnRH) neurons play crucial roles in the regulation of testosterone release. Meanwhile, it has been demonstrated that MC-LR is capable of entering the GnRH neurons and inducing apoptosis. Nevertheless, the molecular mechanism of MC-LR induced apoptosis of GnRH neurons remains elusive. In present study, we found that MC-LR inhibited the cell viability of GT1-7 cells. In addition, we discovered apoptosis of GnRH neurons and GT1-7 cells treated with MC-LR. And increased intracellular ROS production and the release of intracellular Ca2+ were all observed following exposure to MC-LR. Furthermore, we also found the endoplasmic reticulum stress (ERs) and autophagy were activated by MC-LR. Additionally, pretreatment of the ERs inhibitor (4-Phenyl butyric acid) reduced the apoptotic rate of GT1-7 cells comparing with MC-LR exposure alone. Comparing with MC-LR treatment alone, apoptotic cell death was increased by pretreatment of GT1-7 cells with an autophagy inhibitor (3-methyladenine). Together, our data implicated that the treatment of MC-LR induced the apoptosis of GnRH neurons by activating the ERs resulting in a decrease of serum testosterone level in mice. Autophagy is a protective cellular process which was activated by ER stress and thus protected cells from apoptosis upon MC-LR exposure.


Assuntos
Estresse do Retículo Endoplasmático , Microcistinas/toxicidade , Testosterona/sangue , Animais , Apoptose , Arginina/metabolismo , Bioensaio , Sobrevivência Celular , Cianobactérias/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Leucina/metabolismo , Masculino , Toxinas Marinhas/metabolismo , Camundongos , Microcistinas/metabolismo , Neurônios/metabolismo , Testosterona/metabolismo
2.
PLoS Pathog ; 16(9): e1008918, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32997715

RESUMO

The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway promoting mitochondrial recovery and defense against infection. In C. elegans, the UPRmt is activated during infection with the pathogen Pseudomonas aeruginosa-but only transiently. As this may reflect a pathogenic strategy to target a pathway required for host survival, we conducted a P. aeruginosa genetic screen to uncover mechanisms associated with this temporary activation. Here, we find that loss of the P. aeruginosa acyl-CoA dehydrogenase FadE2 prolongs UPRmt activity and extends host survival. FadE2 shows substrate preferences for the coenzyme A intermediates produced during the breakdown of the branched-chain amino acids valine and leucine. Our data suggests that during infection, FadE2 restricts the supply of these catabolites to the host hindering host energy metabolism in addition to the UPRmt. Thus, a metabolic pathway in P. aeruginosa contributes to pathogenesis during infection through manipulation of host energy status and mitochondrial stress signaling potential.


Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Metabolismo Energético/fisiologia , Leucina/metabolismo , Mitocôndrias/metabolismo , Aminoácidos de Cadeia Ramificada/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
3.
J Pharmacol Sci ; 144(1): 16-22, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32653341

RESUMO

JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 µM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 µM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC50 value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Benzoxazóis/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Tirosina/análogos & derivados , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Leucina/metabolismo , Fatores de Tempo , Tirosina/farmacologia
4.
Nat Commun ; 11(1): 3148, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561715

RESUMO

Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.


Assuntos
Autofagia/fisiologia , Leucina/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Animais , Autofagossomos , Linhagem Celular , Proteína p300 Associada a E1A/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Cultura Primária de Células , Inanição/metabolismo
5.
Nat Commun ; 11(1): 2847, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504036

RESUMO

The browning of white adipose tissue (WAT) has got much attention for its potential beneficial effects on metabolic disorders, however, the nutritional factors and neuronal signals involved remain largely unknown. We sought to investigate whether WAT browning is stimulated by leucine deprivation, and whether the amino acid sensor, general control non-derepressible 2 (GCN2), in amygdalar protein kinase C-δ (PKC-δ) neurons contributes to this regulation. Our results show that leucine deficiency can induce WAT browning, which is unlikely to be caused by food intake, but is largely blocked by PKC-δ neuronal inhibition and amygdalar GCN2 deletion. Furthermore, GCN2 knockdown in amygdalar PKC-δ neurons blocks WAT browning, which is reversed by over-expression of amino acid responsive gene activating transcription factor 4 (ATF4), and is mediated by the activities of amygdalar PKC-δ neurons and the sympathetic nervous system. Our data demonstrate that GCN2/ATF4 can regulate WAT browning in amygdalar PKC-δ neurons under leucine deprivation.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Tecido Adiposo Branco/fisiologia , Tonsila do Cerebelo/fisiologia , Leucina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/inervação , Tonsila do Cerebelo/citologia , Animais , Técnicas de Silenciamento de Genes , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologia , Técnicas Estereotáxicas , Sistema Nervoso Simpático/fisiologia , Termogênese/fisiologia
6.
Arch Biochem Biophys ; 689: 108443, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32485152

RESUMO

Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.e. leucine-rich repeat domain (LRR), immunoglobulin domains, peroxidase domain, von Willebrand factor type C domain] of PXDN interact with the polymeric networks of the extracellular matrix (ECM), and how these interactions integrate and regulate the enzyme's cross-linking activity, without imparting oxidative damage to the ECM. In this study, we probed the interactions of four PXDN constructs with different domain compositions with components of a basement membrane extract by immunoprecipitation. Strong binding of the LRR-containing construct was detected with the major ECM protein laminin. Analysis of these interactions by surface plasmon resonance spectroscopy revealed similar kinetics and affinities of binding of the LRR-containing construct to human and murine laminin-111, with calculated dissociation constants of 1.0 and 1.5 µM, respectively. The findings are discussed with respect to the recently published in-solution structures of the PXDN constructs and the proposed biological role of this peroxidase.


Assuntos
Membrana Basal/metabolismo , Laminina/metabolismo , Peroxidases/metabolismo , Animais , Células HEK293 , Humanos , Leucina/química , Leucina/metabolismo , Camundongos , Peroxidases/química , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo
7.
J Sports Med Phys Fitness ; 60(6): 864-869, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32118385

RESUMO

BACKGROUND: In past few decades, supplementation has become very popular within professional and recreational athletes. Most interested among supplements are protein and amino acids. Therefore, the purpose of this study was to examine the effects of leucine supplementation in comparison to whey protein supplementation with placebo group on strength and body composition during 8 weeks of resistance training program. METHODS: Thirty male college athletes (mean age±SD =23.92±1.54 years) participated in this investigation and were randomly assigned to one of three groups: whey protein (WP, N.=10), leucine (LEU, N.=10) or placebo (PLA, N.=10). The WP, LEU and PLA performed resistance training for 8 weeks. Strength (1RM bench press, squat, shoulder press) and maximum pull-ups and body composition has been assessed and data was analyzed with mixed-design analysis of variance (P≤0.01). RESULTS: The WP group achieved significantly greater (P≤0.01) increases in 1RM bench press than the LEU and PLA groups (+16 kg for WP; + 7.5 kg for LEU and + 5 kg for PLA). The LEU group achieved significantly greater gains (P≤0.01) in fat free mass, muscle mass and significantly decreases (P≤0.01) in fat mass compared to WP and PLA groups. CONCLUSIONS: Whey protein supplementation in male collegiate athletes during resistance training achieved greater increase in strength than leucine and placebo groups, and leucine group achieved significantly greater improvement in body composition than whey protein and placebo groups.


Assuntos
Leucina/metabolismo , Músculo Esquelético/fisiologia , Treinamento de Resistência , Proteínas do Soro do Leite/metabolismo , Adulto , Atletas , Composição Corporal , Suplementos Nutricionais/análise , Humanos , Masculino , Força Muscular , Adulto Jovem
8.
J Dairy Sci ; 103(5): 4218-4235, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32113753

RESUMO

Starch digestion in the small intestine in ruminants is relatively lower compared with that in monogastric animals, likely due to low pancreatic α-amylase secretion. Previous studies suggested that leucine could increase pancreatic α-amylase secretion in the small intestine of heifers cannulated with abomasal, duodenal, and ileal catheters. However, the surgical procedures probably have an effect on pancreatic function. Thus, we used rumen-protected leucine (RP-Leu) to explore its effect on small intestinal digestion of starch in calves without any surgery in 3 experiments. The first experiment was to explore whether RP-Leu could improve post-ruminal starch digestion in 5-mo-old calves (158 ± 19 kg body weight ± standard deviation). We found that RP-Leu did not affect rumen fermentation profile or whole-tract starch digestibility, but it increased blood glucose concentration and fecal pH and decreased fecal propionate molar proportion. Additionally, RP-Leu increased fibrolytic genera Ruminiclostridium and Pseudobutyrivibrio and decreased the amylolytic genus of Faecalibacterium. The second experiment compared RP-Leu and rumen-protected lysine (RP-Lys) for their effects on post-ruminal starch digestion in 6-mo-old calves (201 ± 24 kg body weight). The responses of blood glucose concentration, fecal pH, fecal propionate proportion, and starch digestibility to RP-Leu supplementation were similar to those observed in experiment 1. Cellulolytic family Ruminococcaceae and Bacteroidales BS11 gut group tended to be increased by RP-Leu. In contrast, RP-Lys showed no significant influence on the above measurements. The third experiment determined the interaction between RP-Leu and rumen-escape starch (RES) on the small intestinal digestion of starch in 8-mo-old calves (289 ± 26 kg body weight). An interaction between RP-Leu and RES levels was observed in fecal butyrate concentration and the relative abundance of family Bacteroidaceae, and genera Ruminococcaceae UCG-005 and Bacteroides. We found that RP-Leu tended to increase the abundance of fecal Firmicutes and decrease Spirochaetae. In conclusion, RP-Leu, but not RP-Lys, increased blood glucose concentration and decreased the amount of starch fermented in the hindgut in a RES dose-dependent manner, suggesting that RP-Leu might stimulate starch digestion in the small intestine.


Assuntos
Ração Animal , Glicemia/metabolismo , Bovinos , Intestino Delgado/metabolismo , Leucina/farmacologia , Amido/metabolismo , Abomaso/metabolismo , Animais , Animais Recém-Nascidos , Dieta/veterinária , Digestão , Fezes/microbiologia , Fermentação , Leucina/metabolismo , Masculino , Nitrogênio/metabolismo , Rúmen/metabolismo
9.
Food Chem ; 320: 126619, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32203836

RESUMO

The fermentation of mare's milk into koumiss produces many beneficial functional compounds depending on the metabolism of the initial microbial flora. In this study, metabolites found in mare's milk and resulting koumiss were identified. Major metabolic pathways in the fermentation were also identified using an UPLC-Q-TOF-MS-based metabolomics method. In total, 354 metabolites were identified: 61 were up-regulated and 105 were down-regulated. Metabolic pathway analyses revealed that c-5-branched dibasic acid metabolism, valine, leucine and isoleucine degradation, arginine and proline metabolism, valine, leucine and isoleucine biosynthesis, vascular smooth muscle contraction, aminoacyl-tRNA biosynthesis and ß-alanine metabolism showed significant increases. A hierarchical cluster analysis of metabolites indicated a clear grouping pattern in which the relative concentrations of p-pyruvate, 20-HETE, 4-aminobutanoate, uracil, acetoacetate, and γ-linolenic acid differed significantly between milk and koumiss. This study provides reference values for metabolic isolates and bioactive compounds purification in mare's milk and koumiss.


Assuntos
Kumis/análise , Metabolômica , Leite/química , Animais , Arginina/química , Arginina/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Fermentação , Cavalos , Leucina/química , Leucina/metabolismo , Espectrometria de Massas , Leite/metabolismo , Valina/química , Valina/metabolismo
10.
Cancer Cell ; 37(2): 147-156, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32049045

RESUMO

Metabolic pathways must be adapted to support cell processes required for transformation and cancer progression. Amino acid metabolism is deregulated in many cancers, with changes in branched-chain amino acid metabolism specifically affecting cancer cell state as well as systemic metabolism in individuals with malignancy. This review highlights key concepts surrounding the current understanding of branched-chain amino acid metabolism and its role in cancer.


Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/metabolismo , Epigenômica/métodos , Humanos , Isoleucina/metabolismo , Leucina/metabolismo
11.
Food Funct ; 11(2): 1304-1311, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32016208

RESUMO

Branched-chain amino acids (BCAAs) are reduced in various protein restricted models, while the detailed role of BCAAs in protein restricted response is still obscure. Thus, the current study mainly investigated the amino acid metabolism in protein restricted piglets and the effects of BCAA balance in a low-protein diet on growth performance, amino acid metabolism, intestinal structure, and gut microbiota with focus on which BCAAs contributed to the protein restricted response. The results showed that protein restriction increased serum Ser, Thr, Ala, Lys, and Trp but reduced His, Cys, Val, and Ile levels. Intestinal amino acid transporters mainly mediated the mechanism of amino acid uptake. The BCAA balance refreshed the serum BCAA pool, which further improved growth performance in protein restricted piglets. Leu, Val, and Ile balances increased serum BCAA concentrations, respectively, and Leu and Val but not Ile enhanced the feed intake and weight gain in protein restricted piglets. In addition, protein restriction impaired the villus structure and increased the number of goblet cells in the ileum. Also, gut microbiota (Spirochaetales, Gammaproteobacteria, Lactobacillales at the order level) were altered in protein restricted pigs, while the BCAA balance markedly improved Gammaproteobacteria, Lactobacillales, and Aeromonadales proliferation, which might mediate growth promotion and amino acid metabolism. In conclusion, protein restriction markedly affected the host amino acid metabolism (i.e., Ser, Thr, Lys, His, BCAAs). The BCAA balance (especially for supplementation with Leu and Val) improved the amino acid metabolism, growth performance, and gut microbiota communities.


Assuntos
Ração Animal , Dieta com Restrição de Proteínas/veterinária , Leucina/metabolismo , Modelos Animais , Valina/metabolismo , Animais , Animais Recém-Nascidos , Suínos
12.
Nat Commun ; 11(1): 1005, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081981

RESUMO

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Leucina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Substituição de Aminoácidos , Bactérias/química , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Conformação Proteica
13.
J Med Chem ; 63(6): 2814-2832, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32069402

RESUMO

Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Because S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine, and spermine. Importantly, we found that this methionine-depletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using 3H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine but instead facilitated the efflux of intracellular leucine pools.


Assuntos
Transporte Biológico/efeitos dos fármacos , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Poliaminas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Benzoxazóis/farmacologia , Células CHO , Cricetulus , Descoberta de Drogas , Humanos , Leucina/metabolismo , Metionina/metabolismo , Putrescina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Espermidina/metabolismo , Espermina/metabolismo , Tirosina/análogos & derivados , Tirosina/farmacologia
14.
Amino Acids ; 52(3): 477-486, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32108266

RESUMO

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0-0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.


Assuntos
Desoxiglucose/metabolismo , Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mioblastos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
15.
Biosens Bioelectron ; 150: 111948, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31929085

RESUMO

Leucine aminopeptidase (LAP) is an essential proteolytic enzyme and potential biomarker for liver malignancy. Overexpression of LAP is directly linked with some fatal physiological and pathological disorders. In this regard, we have designed an activity based electrochemical substrate leucine-benzyl ferrocene carbamate (Leu-FC) for selective profiling of LAP activity in live cells. In practice, LAP instantaneously hydrolyze the Leu residue of the substrate Leu-FC to eliminate the unmasked electrochemical reporter amino ferrocene via predefined self-immolative cascade. The electrochemical signal is distinctly specific for LAP and free of other electroactive biological interference. The substrate Leu-FC empowered sensor displayed broad dynamic range with admirable detection limits. On top of this, the probe Leu-FC was employed in real-time active profiling of cellular LAP activity in HepG2 cells and effect of LAP inhibitor. In extent, the substrate Leu-FC can effectively monitor cisplatin induced overexpression of LAP activity in HepG2 cells in presence and absence of bestatin. The sensor showcased an excellent reliability towards monitoring cellular LAP activity in HepG2 cells. Unlike the traditional antibody-based immunoassays, our approach is capable of monitoring in-situ activity of LAP in live cells.


Assuntos
Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Leucil Aminopeptidase/metabolismo , Neoplasias/enzimologia , Resistencia a Medicamentos Antineoplásicos , Técnicas Eletroquímicas/métodos , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Células Hep G2 , Humanos , Leucina/análogos & derivados , Leucina/metabolismo , Limite de Detecção , Metalocenos/química , Metalocenos/metabolismo , Neoplasias/tratamento farmacológico
16.
Nat Commun ; 11(1): 174, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924757

RESUMO

mTORC1 is an important regulator of muscle mass but how it is modulated by oxygen and nutrients is not completely understood. We show that loss of the prolyl hydroxylase domain isoform 1 oxygen sensor in mice (PHD1KO) reduces muscle mass. PHD1KO muscles show impaired mTORC1 activation in response to leucine whereas mTORC1 activation by growth factors or eccentric contractions was preserved. The ability of PHD1 to promote mTORC1 activity is independent of its hydroxylation activity but is caused by decreased protein content of the leucyl tRNA synthetase (LRS) leucine sensor. Mechanistically, PHD1 interacts with and stabilizes LRS. This interaction is promoted during oxygen and amino acid depletion and protects LRS from degradation. Finally, elderly subjects have lower PHD1 levels and LRS activity in muscle from aged versus young human subjects. In conclusion, PHD1 ensures an optimal mTORC1 response to leucine after episodes of metabolic scarcity.


Assuntos
Leucina-tRNA Ligase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Adulto , Idoso , Envelhecimento/metabolismo , Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Leucina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular , Músculos/patologia , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Transdução de Sinais
17.
Mol Genet Metab ; 129(3): 193-206, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31980395

RESUMO

Over the past three decades, we studied 184 individuals with 174 different molecular variants of branched-chain α-ketoacid dehydrogenase activity, and here delineate essential clinical and biochemical aspects of the maple syrup urine disease (MSUD) phenotype. We collected data about treatment, survival, hospitalization, metabolic control, and liver transplantation from patients with classic (i.e., severe; n = 176), intermediate (n = 6) and intermittent (n = 2) forms of MSUD. A total of 13,589 amino acid profiles were used to analyze leucine tolerance, amino acid homeostasis, estimated cerebral amino acid uptake, quantitative responses to anabolic therapy, and metabolic control after liver transplantation. Standard instruments were used to measure neuropsychiatric outcomes. Despite advances in clinical care, classic MSUD remains a morbid and potentially fatal disorder. Stringent dietary therapy maintains metabolic variables within acceptable limits but is challenging to implement, fails to restore appropriate concentration relationships among circulating amino acids, and does not fully prevent cognitive and psychiatric disabilities. Liver transplantation eliminates the need for a prescription diet and safeguards patients from life-threatening metabolic crises, but is associated with predictable morbidities and does not reverse pre-existing neurological sequelae. There is a critical unmet need for safe and effective disease-modifying therapies for MSUD which can be implemented early in life. The biochemistry and physiology of MSUD and its response to liver transplantation afford key insights into the design of new therapies based on gene replacement or editing.


Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Biomarcadores/sangue , Leucina/sangue , Transplante de Fígado , Doença da Urina de Xarope de Bordo/dietoterapia , Doença da Urina de Xarope de Bordo/terapia , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/fisiopatologia , Estudos de Coortes , Dieta , Feminino , Homozigoto , Humanos , Lactente , Leucina/metabolismo , Masculino , Doença da Urina de Xarope de Bordo/genética , Doença da Urina de Xarope de Bordo/metabolismo , Transtornos Mentais/metabolismo , Transtornos Mentais/fisiopatologia , Pessoa de Meia-Idade , Fenótipo
18.
Infect Immun ; 88(4)2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-31964747

RESUMO

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.


Assuntos
Adaptação Fisiológica , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Triptofano/metabolismo , Linhagem Celular , Chlamydia trachomatis/citologia , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/enzimologia , Chlamydophila pneumoniae/citologia , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/enzimologia , Inibidores Enzimáticos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Indóis/metabolismo , Leucina/metabolismo , Modelos Biológicos , Transcrição Genética
19.
Med Sci Sports Exerc ; 52(1): 37-48, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31389908

RESUMO

PURPOSE: Studies suggest ketogenic diets (KD) produce favorable outcomes (health and exercise performance); however, most rodent studies have used a low-protein KD, which does not reflect the normal- to high-protein KD used by humans. Liver has an important role in ketoadaptation due to its involvement in gluconeogenesis and ketogenesis. This study was designed to test the hypothesis that exercise training (ExTr) while consuming a normal-protein KD (NPKD) would induce additive/synergistic responses in liver metabolic pathways. METHODS: Lean, healthy male C57BL/6J mice were fed a low-fat control diet (15.9% kcal protein, 11.9% kcal fat, 72.2% kcal carbohydrate) or carbohydrate-deficient NPKD (16.1% protein, 83.9% kcal fat) for 6 wk. After 3 wk on the diet, half were subjected to 3-wk treadmill ExTr (5 d·wk, 60 min·d, moderate-vigorous intensity). Upon conclusion, metabolic and endocrine outcomes related to substrate metabolism were tested in liver and pancreas. RESULTS: NPKD-fed mice had higher circulating ß-hydroxybutyrate and maintained glucose at rest and during exercise. Liver of NPKD-fed mice had lower pyruvate utilization and greater ketogenic potential as evidenced by higher oxidative rates to catabolize lipids (mitochondrial and peroxisomal) and ketogenic amino acids (leucine). ExTr had higher expression of the gluconeogenic gene, Pck1, but lower hepatic glycogen, pyruvate oxidation, incomplete fat oxidation, and total pancreas area. Interaction effects between the NPKD and ExTr were observed for intrahepatic triglycerides, as well as genes involved in gluconeogenesis, ketogenesis, mitochondrial fat oxidation, and peroxisomal markers; however, none were additive/synergistic. Rather, in each instance the interaction effects showed the NPKD and ExTr opposed each other. CONCLUSIONS: An NPKD and an ExTr independently induce shifts in hepatic metabolic pathways, but changes do not seem to be additive/synergistic in healthy mice.


Assuntos
Dieta Cetogênica , Fígado/metabolismo , Condicionamento Físico Animal/fisiologia , Ácido 3-Hidroxibutírico/sangue , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Cetonas/metabolismo , Leucina/metabolismo , Metabolismo dos Lipídeos , Glicogênio Hepático/metabolismo , Masculino , Redes e Vias Metabólicas , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial , Oxirredução , Pâncreas/metabolismo , Hormônios Pancreáticos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Peroxissomos/metabolismo , Triglicerídeos/metabolismo
20.
J Biotechnol ; 307: 69-76, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31689468

RESUMO

Branched chain fatty acids (BCFA) are an appealing biorefinery-driven target of fatty acid (FA) production. BCFAs typically have lower melting points compared to straight chain FAs, making them useful in lubricants and biofuels. Actinobacteria, especially Streptomyces species, have unique secondary metabolism that are capable of producing not only antibiotics, but also high percentage of BCFAs in their membrane lipids. Since biosynthesis of polyketide (PK) and FA partially share common pathways to generate acyl-CoA precursors, in theory, Streptomyces sp. with high levels of PK antibiotics production can be easily manipulated into strains producing high levels of BCFAs. To increase the percentage of the BCFA moieties in lipids, we redirected acyl-CoA precursor fluxes from PK into BCFAs using S. coelicolor M1146 (M1146) as a host strain. In addition, 3-ketoacyl acyl carrier protein synthase III and branched chain α-keto acid dehydrogenase were overexpressed to push fluxes of branched chain acyl-CoA precursors towards FA synthesis. The maximum titer of 354.1 mg/L BCFAs, 90.3% of the total FA moieties, was achieved using M1146dD-B, fadD deletion and bkdABC overexpression mutant of M1146 strain. Cell specific yield of 64.4 mg/L/gcell was also achieved. The production titer and specific yield are the highest ever reported in bacterial cells, which provides useful insights to develop an efficient host strain for BCFAs.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Ácidos Graxos/metabolismo , Streptomyces coelicolor/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Acil Coenzima A/metabolismo , Antibacterianos/metabolismo , Biocombustíveis , Ácidos Graxos/análise , Expressão Gênica , Leucina/metabolismo , Mutação , Policetídeos/metabolismo , Metabolismo Secundário , Streptomyces coelicolor/genética
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