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1.
FASEB J ; 36(1): e22096, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34907600

RESUMO

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1ß production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1ß production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1ß production during mycobacterial infection.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , NADH NADPH Oxirredutases/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Bactérias , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamassomos/genética , Metaloproteases , Camundongos , Membranas Mitocondriais/metabolismo , Mycobacterium tuberculosis/genética , NADH NADPH Oxirredutases/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
2.
Gene ; 809: 146017, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34655725

RESUMO

Flavonoids and lignin consist of a large number of secondarymetabolites which are derived from the phenylpropanoid pathway, and they act as a significant role in plant growth, development, and stress response. However, few reports have documented that how different subbranches of phenylpropanoid metablolic pathway mutually interact. In Arabidopsis, AtCPC (AtCAPRICE) is known to play a negative role in anthocyanin accumulation. Nonetheless, whether AtCPC could control the biosynthesis of lignin is largely unknown. Additionally, whether the RrFLS and RrANR, flavonol synthase and anthocyanidin reductase, from Rosa rugosa regulate different branches of phenylpropanoid pathway is unclear. Here, we performed a series of transgenic experiments with short life cycle tobacco and RNA-Seq analysis. Finally, a series of assays related to biological, physiological, and phenotypic characteristics were undertaken. Our results indicated that ectopic expression of AtCPC in tobacco not only decreased the flavonoid compound accumulation, but also up-regulated several lignin biosynthetic genes, and significantly increased the accumulation of lignin. Our results also revealed that although they respectively improved the flavonol and proanthocyanidin contents, the overexpression of RrFLS and RrANR plays positive roles in lignin biosynthesis in transgenic tobacco plants. Our findings provide a novel insight into the mechanism underlying homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of plants.


Assuntos
Flavonoides/biossíntese , Lignina/biossíntese , Tabaco/genética , Tabaco/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flavonoides/genética , Regulação da Expressão Gênica de Plantas , Homeostase , Lignina/genética , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Rosa/genética , Fatores de Transcrição/genética
3.
Cells ; 10(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34571983

RESUMO

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell's expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.


Assuntos
Trifosfato de Adenosina/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adulto , Animais , Feminino , Humanos , Lactobacillus brevis/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD/metabolismo , Oxirredução , Linfócitos T/metabolismo
4.
Exp Cell Res ; 407(1): 112799, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34461110

RESUMO

Colorectal cancer (CRC) is the leading deadly cancer worldwide. Gene associated with retinoid-IFN-induced mortality-19 (GRIM-19), a novel tumor suppressor, has been reported to be expressed at low levels in human CRC. However, the role of GRIM-19 in CRC progression and the corresponding detailed mechanisms are unclear. The results of this study indicated that GRIM-19 expression is related to CRC progression. Overexpression of GRIM-19 was found to inhibit CRC cell proliferation and induce apoptosis in vitro and in vivo. Our results demonstrated that GRIM-19 suppresses CRC through posttranslational regulation of p53, in which SIRT7 is activated by GRIM-19 and triggers PCAF-mediated MDM2 ubiquitination, eventually stabilizing the p53 protein. We also observed that GRIM-19 enhances the effect of oxaliplatin against CRC. In conclusion, GRIM-19 plays an important role in CRC development and is a potential biomarker and therapeutic target for clinical treatment of CRC.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , NADH NADPH Oxirredutases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Sirtuínas/metabolismo , Ubiquitinação/fisiologia
5.
Cells ; 10(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34440713

RESUMO

Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the most severe disease that threatens artichoke (Cynara scolymus L.) plants. Arbuscular mycorrhizal fungi (AMF) may represent a useful biological control strategy against this pathogen attack, replacing chemical compounds that, up to now, have been not very effective. In this study, we evaluated the effect of the AMF Glomus viscosum Nicolson in enhancing the plant tolerance towards the pathogen V. dahliae. The role of the ascorbate-glutathione (ASC-GSH) cycle and other antioxidant systems involved in the complex network of the pathogen-fungi-plant interaction have been investigated. The results obtained showed that the AMF G. viscosum is able to enhance the defense antioxidant systems in artichoke plants affected by V. dahliae, alleviating the oxidative stress symptoms. AMF-inoculated plants exhibited significant increases in ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and superoxide dismutase (SOD) activities, a higher content of ascorbate (ASC) and glutathione (GSH), and a decrease in the levels of lipid peroxidation and hydrogen peroxide (H2O2). Hence, G. viscosum may represent an effective strategy for mitigating V. dahliae pathogenicity in artichokes, enhancing the plant defense systems, and improving the nutritional values and benefit to human health.


Assuntos
Antioxidantes/metabolismo , Ascomicetos/patogenicidade , Cynara scolymus/microbiologia , Fungos/fisiologia , Estresse Oxidativo , Controle Biológico de Vetores , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Ascorbato Peroxidases/metabolismo , Cynara scolymus/metabolismo , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , NADH NADPH Oxirredutases/metabolismo , Doenças das Plantas/microbiologia , Superóxido Dismutase/metabolismo
6.
ACS Chem Biol ; 16(8): 1425-1434, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34269557

RESUMO

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature's biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.


Assuntos
Antibacterianos/biossíntese , Saccharomyces cerevisiae/metabolismo , Tetraciclina/biossíntese , Oxirredutases do Álcool/metabolismo , Proteínas Fúngicas/metabolismo , Hidroxilação , Oxigenases de Função Mista/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Saccharomyces cerevisiae/enzimologia , Tetraciclinas/química , Tetraciclinas/metabolismo
7.
Int J Biol Macromol ; 185: 40-48, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34144065

RESUMO

It is well known that the chemical structure of polysaccharides is important to their final biological effect. In this study we investigated the cytotoxic effect of xyloglucan from Copaifera langsdorffii seeds (XGC) and its complex with oxovanadium (XGC:VO) on hepatocellular carcinoma cells (HepG2). After 72 h of incubation, XGC and XGC:VO (200 µg/mL) reduced cell viability in ~20% and ~40%, respectively. At same conditions, only XGC:VO increased in ~20% the LDH enzyme release. In permeabilized cells, incubated with XGC and XGC:VO (200 µg/mL) for 72 h, NADH oxidase activity was reduced by ~45% with XGC and XGC:VO. The succinate oxidase activity was reduced by ~35% with XGC and ~65% with XGC:VO, evidencing that polysaccharide complexation with vanadium could intensify its effects on the respiratory chain. According to this result, the mitochondrial membrane potential was also reduced by ~9% for XGC and ~30% for XGC:VO, when compared to the control group. Interestingly, ATP levels were more elevated for XGC:VO in respect to XGC, probably due the enhance in glycolytic flux evidenced by increased levels of lactate. These results show that the xyloglucan complexation with oxovanadium (IV/V) potentiates the cytotoxic effect of the native polysaccharide, possibly by impairment of oxidative phosphorylation.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Fabaceae/química , Glucanos/farmacologia , Neoplasias Hepáticas/metabolismo , Vanadatos/química , Xilanos/farmacologia , Antineoplásicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucanos/química , Células Hep G2 , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Oxirredutases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Xilanos/química
8.
J Med Chem ; 64(9): 6137-6160, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33945281

RESUMO

Inhibition of Leishmania infantum trypanothione disulfide reductase (LiTryR) by disruption of its homodimeric interface has proved to be an alternative and unexploited strategy in the search for novel antileishmanial agents. Proof of concept was first obtained by peptides and peptidomimetics. Building on previously reported dimerization disruptors containing an imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-triazole-based chemotype that yields noncompetitive, slow-binding inhibitors of LiTryR. Several compounds bearing (poly)aromatic substituents dramatically improve the ability to disrupt LiTryR dimerization relative to reference imidazoles. Molecular modeling studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory activity over LiTryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able to kill both extracellular and intracellular parasites in cell cultures.


Assuntos
Desenho de Fármacos , Leishmania infantum/enzimologia , NADH NADPH Oxirredutases/química , Multimerização Proteica/efeitos dos fármacos , Tiazóis/química , Tiazóis/farmacologia , Triazóis/química , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Humanos , Leishmania infantum/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade
9.
Toxicol In Vitro ; 74: 105158, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33823240

RESUMO

BACKGROUND: Leishmaniasis is a parasitosis with a wide incidence in developing countries. The drugs which are indicated for the treatment of this infection usually are able to promote high toxicity. PURPOSE: A combination of limonene and carvacrol, monoterpenes present in plants with antiparasitic activity may constitute an alternative for the treatment of these diseases. METHODS: In this study, the antileishmania activity against Leishmania major, cytotoxicity tests, assessment of synergism, parasite membrane damage tests as well as molecular docking and immunomodulatory activity of limonene-carvacrol (Lim-Car) combination were evaluated. RESULTS: The Lim-Car combination (5:0; 1:1; 1:4; 2:3; 3:2; 4:1 and 0:5) showed potential antileishmania activity, with mean inhibitory concentration (IC50) ranging from 5.8 to 19.0 µg.mL-1. They demonstrated mean cytotoxic concentration (CC50) ranging from 94.1 to 176.0 µg.mL-1, and did not show significant hemolytic effect. In the investigation of synergistic interaction, the 4:1 Lim-Car combination showed better fractional inhibitory concentration (FIC) index as well as better activity on amastigotes and IS. The samples caused considerable damage to the parasite membrane this monoterpene activity seems to be more related to Trypanothione Reductase (TryR) enzyme interaction, demonstrated in the molecular docking assay. In addition, the 4:1 Lim-Car combination stimulated macrophage activation, and showed at was the best association, with reduction of infection and infectivity of parasitized macrophages. CONCLUSION: The 4:1 Lim-Car combination appears to be a promising candidate as a monotherapeutic antileishmania agent.


Assuntos
Antiprotozoários/toxicidade , Cimenos/toxicidade , Fatores Imunológicos/toxicidade , Leishmania major/efeitos dos fármacos , Limoneno/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Simulação de Acoplamento Molecular , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Ovinos
10.
Mol Vis ; 27: 151-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907370

RESUMO

PURPOSE: Recent reports linking HDAC6 to mitochondrial turnover and neurodegeneration led us to hypothesize that an inhibitor such as Vorinostat (suberoylanilide hydroxamic acid, SAHA) may reduce mitochondrial damage found in retinitis pigmentosa (RP), a progressive neurodegenerative disease of the eye. Here we tested the efficacy of SAHA for its ability to protect photoreceptors in in-vitro and in-situ models of RP. As the stressor, we focused on calcium overload. Calcium is one of the main drivers of cell death, and is associated with rod loss in the rd1 mouse retina, which harbors a mutation in the Pde6b gene similar to that found in human patients suffering from autosomal recessive RP. METHOD: Murine photoreceptor cell line (661W) were exposed to agents that led to calcium stress. Cell survival and redox capacity were measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, real-time changes in cellular metabolism were assessed using the Seahorse Biosciences XF24 analyzer, and mitochondrial fission-fusion using imaging. In-situ, neuroprotection was assessed in RPE/retina organ cultures of the rd1 mouse. SAHA effects on cell survival were compared in 661W cells with those of the specific HDAC6 inhibitor tubastatin A, and those on protein acetylation by Western blotting. RESULTS: In stressed 661W cells, SAHA was found to increase cell survival that was associated with improved mitochondrial respiration and reduced mitochondrial fission. The protective effects of SAHA were also observed on photoreceptor cell survival in whole retinal organ explants of the rd1 mouse. Even though tubastatin A was ineffective in increasing cell survival in 661W cells, HDAC6 activity was confirmed in 661W cells after SAHA treatment with protein acetylation specific for HDAC6, defined by an increase in tubulin, but not histone acetylation. CONCLUSIONS: SAHA was found to protect mitochondria from damage, and concomitantly reduced photoreceptor cell death in cell and organ cultures. The lack of activity of tubastatin A suggests that there must be an additional mechanism of action involved in the protective mechanism of SAHA that is responsible for its neuroprotection. Overall, SAHA may be a useful treatment for the prevention of photoreceptor degeneration associated with human RP. The results are discussed in the context of the effects of inhibitors that target different classes and members of the HDAC family and their effects on rod versus cone survival.


Assuntos
Modelos Animais de Doenças , Inibidores de Histona Desacetilases/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Retinite Pigmentosa/tratamento farmacológico , Vorinostat/uso terapêutico , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/prevenção & controle , NADH NADPH Oxirredutases/metabolismo , Técnicas de Cultura de Órgãos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/patologia
11.
Biochem J ; 478(6): 1241-1259, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33650635

RESUMO

HtrA2 (high-temperature requirement A2) and GRIM-19 (gene associated with retinoic and interferon-induced mortality 19 protein) are involved in various biological functions with their deregulation leading to multiple diseases. Although it is known that the interaction between GRIM-19 with HtrA2 promotes the pro-apoptotic activity of the latter, the mechanistic details remained elusive till date. Moreover, designing allosteric modulators of HtrA2 remains obscure due to lack of adequate information on the mode of interaction with its natural substrates cum binding partners. Therefore, in this study, we have unfolded the interaction between HtrA2 and GRIM-19 so as to understand its subsequent functional repercussions. Using in silico analyses and biochemical assays, we identified the region in GRIM-19 that is involved in protein-protein interaction with HtrA2. Furthermore, we have presented a comprehensive illustration of HtrA2's cleavage site specificity. Quantitative analysis using enzyme kinetics underscored the role of GRIM-19 in significant allosteric activation of HtrA2. Overall, this is an extensive study that not only defines HtrA2-GRIM-19 interaction, but also creates a framework for developing strategies toward allosteric regulation of HtrA2 for future therapeutic interventions.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Serina Peptidase 2 de Requerimento de Alta Temperatura A/química , Serina Peptidase 2 de Requerimento de Alta Temperatura A/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Domínios PDZ , Regulação Alostérica , Sítios de Ligação , Humanos , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
12.
N Biotechnol ; 63: 19-28, 2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-33640482

RESUMO

The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid by-product. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.


Assuntos
Álcool Desidrogenase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Imino Piranoses/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Biocatálise , Imino Piranoses/química , Cinética , Estrutura Molecular
13.
Cells ; 10(1)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467683

RESUMO

Obesity, a condition characterized by excessive accumulation of body fat, is a metabolic disorder related to an increased risk of chronic inflammation. Obesity is mediated by signal transducer and activator of transcription (STAT) 3, which is regulated by genes associated with retinoid-interferon-induced mortality (GRIM) 19, a protein ubiquitously expressed in various human tissues. In this study, we investigated the role of GRIM19 in diet-induced obese C57BL/6 mice via intravenous or intramuscular administration of a plasmid encoding GRIM19. Splenocytes from wild-type and GRIM19-overexpressing mice were compared using enzyme-linked immunoassay, real-time polymerase chain reaction, Western blotting, flow cytometry, and histological analyses. GRIM19 attenuated the progression of obesity by regulating STAT3 activity and enhancing brown adipose tissue (BAT) differentiation. GRIM19 regulated the differentiation of mouse-derived 3T3-L1 preadipocytes into adipocytes, while modulating gene expression in white adipose tissue (WAT) and BAT. GRIM19 overexpression reduced diet-induced obesity and enhanced glucose and lipid metabolism in the liver. Moreover, GRIM19 overexpression reduced WAT differentiation and induced BAT differentiation in obese mice. GRIM19-transgenic mice exhibited reduced mitochondrial superoxide levels and a reciprocal balance between Th17 and Treg cells. These results suggest that GRIM19 attenuates the progression of obesity by controlling adipocyte differentiation.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , NADH NADPH Oxirredutases/metabolismo , Linfócitos T Reguladores/citologia , Células Th17/citologia , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Feminino , Regulação da Expressão Gênica , Inflamação , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Obesidade/metabolismo , Fator de Transcrição STAT3/metabolismo , Baço/citologia
14.
N Biotechnol ; 62: 18-25, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33460816

RESUMO

L-Tagatose, a promising building block in the production of many value-added chemicals, is generally produced by chemical routes with a low yield, which may not meet the increasing demands. Synthesis of l-tagatose by enzymatic oxidation of d-galactitol has not been applied on an industrial scale because of the high cofactor costs and the lack of efficient cofactor regeneration methods. In this work, an efficient and environmentally friendly enzymatic method containing a galactitol dehydrogenase for d-galactitol oxidation and a water-forming NADH oxidase for regeneration of NAD+ was first designed and used for l-tagatose production. Supplied with only 3 mM NAD+, subsequent reaction optimization facilitated the efficient transformation of 100 mM of d-galactitol into l-tagatose with a yield of 90.2 % after 12 h (obtained productivity: 7.61 mM.h-1). Compared with the current chemical and biocatalytic methods, the strategy developed avoids by-product formation and achieves the highest yield of l-tagatose with low costs. It is expected to become a cleaner and more promising route for industrial biosynthesis of l-tagatose.


Assuntos
Hexoses/biossíntese , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Hexoses/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Temperatura
15.
J Microbiol Biotechnol ; 31(3): 464-474, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33397832

RESUMO

Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.


Assuntos
Proteínas Recombinantes/metabolismo , Esteroide Hidroxilases/metabolismo , Esteroides/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzeno/metabolismo , Catálise , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidroxilação , Cinética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Filogenia , Proteínas Recombinantes/genética , Esteroide Hidroxilases/genética , Streptomyces/genética , Especificidade por Substrato , Temperatura
16.
J Biol Chem ; 296: 100186, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33310705

RESUMO

The substrates O2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH2 to reduce the ferric iron, which forms the stable ferric-superoxide-TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M-1 s-1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.


Assuntos
Di-Hidropteridina Redutase/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxigenases/metabolismo , Regulação Alostérica , Di-Hidropteridina Redutase/química , Escherichia coli/química , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Humanos , Modelos Moleculares , NADH NADPH Oxirredutases/química , Óxido Nítrico/metabolismo , Oxigenases/química , Conformação Proteica
17.
J Med Chem ; 63(24): 15621-15638, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33296601

RESUMO

Since inception, the magic bullets developed against leishmaniasis traveled a certain path and then dropped down due to either toxicity or the emergence of resistance. The route of administration is also an important concern. We developed a series of water-soluble ferrocenylquinoline derivatives, targeting Leishmania donovani, among which CQFC1 showed the highest efficacy even in comparison to other drugs, in use or used, both in oral and intramuscular routes. It did not induce any toxicity to splenocytes and on hematopoiesis, induced protective cytokines, and did not hamper the drug-metabolizing enzymes in hosts. It acts through the reduction and the inhibition of parasites' survival enzyme trypanothione reductase of replicating amastigotes in hosts' reticuloendothelial tissues. Unlike conventional drugs, this molecule did not induce the resistance-conferring genes in laboratory-maintained resistant L. donovani lines. Experimentally, this easily bioavailable preclinical drug candidate overcame all of the limitations causing the discontinuation of the other conventional antileishmanial drugs.


Assuntos
Antiprotozoários/química , Leishmania donovani/enzimologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Quinolinas/química , Administração Oral , Animais , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Sítios de Ligação , Modelos Animais de Doenças , Desenho de Fármacos , Resistência a Medicamentos/efeitos dos fármacos , Compostos Ferrosos/química , Meia-Vida , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Metalocenos/química , Camundongos , Simulação de Acoplamento Molecular , Sistema Fagocitário Mononuclear/metabolismo , Sistema Fagocitário Mononuclear/parasitologia , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Solubilidade , Relação Estrutura-Atividade
18.
ACS Appl Mater Interfaces ; 12(50): 56027-56038, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33275418

RESUMO

Understanding how the immobilization of enzymes on solid carriers affects their performance is paramount for the design of highly efficient heterogeneous biocatalysts. An efficient supply of substrates onto the solid phase is one of the main challenges to maximize the activity of the immobilized enzymes. Herein, we apply advanced single-particle analysis to decipher the optimal design of an immobilized NADH oxidase (NOX) whose activity depends both on O2 and NADH concentrations. Carrier physicochemical properties and its functionality along with the enzyme distribution across the carrier were implemented as design variables to study the effects of the intraparticle concentration of substrates (O2 and NADH) on the activity. Intraparticle O2-sensing analysis revealed the superior performance of the enzyme immobilized at the outer surface in terms of effective supply of O2. Furthermore, the co-immobilization of NADH and NOX within the tuned surface of porous microbeads increases the effective concentration of NADH in the surroundings of the enzyme. As a result, the optimal spatial organization of NOX and its confinement with NADH allow a 100% recovery of the activity of the soluble enzyme upon the immobilization process. By engineering these variables, we increase the NADH oxidation activity of the heterogeneous biocatalyst by up to 650% compared to NOX immobilized under suboptimal conditions. In conclusion, this work highlights the rational design and engineering of the enzyme-carrier interface to maximize the efficiency of heterogeneous biocatalysts.


Assuntos
Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , NAD/metabolismo , Oxigênio/metabolismo , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Complexos Multienzimáticos/química , NADH NADPH Oxirredutases/química , Sefarose/química , Especificidade por Substrato , Thermus thermophilus/enzimologia
19.
Sci Rep ; 10(1): 20440, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33235245

RESUMO

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived ß-sitosterol (ß-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC-MS). The inhibitory efficacy of this ß-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). ß-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of ß-sitosterolCCL was validated by enzyme inhibition and an in silico study in which ß-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, ß-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.


Assuntos
Antiprotozoários/farmacologia , Corchorus/química , Leishmania donovani/enzimologia , NADH NADPH Oxirredutases/metabolismo , Sitosteroides/farmacologia , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Sítios de Ligação/efeitos dos fármacos , Fracionamento Químico , Leishmania donovani/efeitos dos fármacos , Membranas Mitocondriais , Modelos Moleculares , Simulação de Acoplamento Molecular , NADH NADPH Oxirredutases/química , Extratos Vegetais/química , Folhas de Planta/química , Conformação Proteica , Proteínas de Protozoários/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Sitosteroides/química , Sitosteroides/isolamento & purificação
20.
Genes (Basel) ; 11(10)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007888

RESUMO

To investigate the effect of light intensity on flavonoid biosynthesis, grapevine calluses were subjected to high light (HL, 250 µmol m-2 s-1) and dark (0 µmol m-2 s-1) in comparison to 125 µmol m-2 s-1 under controlled conditions (NL). The alteration of flavonoid profiles was determined and was integrated with RNA sequencing (RNA-seq)-based transcriptional changes of the flavonoid pathway genes. Results revealed that dark conditions inhibited flavonoid biosynthesis. Increasing light intensity affected flavonoids differently-the concentrations of flavonols and anthocyanins as well as the expressions of corresponding genes were less affected, whereas flavan-3-ol concentrations were predominantly increased, which caused enhanced trans-flavan-3-ol concentrations. Moreover, genes encoding leucoanthocyanidin reductase (LAR) exhibited different response patterns to light intensity changes-VviLAR1 expression increased with an increased light intensity, whereas VviLAR2 expression was insensitive. We further confirmed that the known transcription factors (TFs) involved in regulating flavan-3-ol biosynthesis utilized VviLAR1 as a target gene in grapevine calluses. In addition, VviLAR1 promoter activity was more sensitive to light intensity changes than that of VviLAR2 as determined using a transgenic Arabidopsis leaf system. These results suggested that light intensity had the most prominent effect on trans-flavan-3-ols in grapevine calluses and demonstrated that the two LAR genes had different response patterns to light intensity changes.


Assuntos
Flavonoides/biossíntese , Luz , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Regiões Promotoras Genéticas , Vitis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Flavonoides/análise , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Plantas , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vitis/crescimento & desenvolvimento , Vitis/metabolismo
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