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1.
Nat Commun ; 11(1): 3172, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576838

RESUMO

Bone marrow engraftment of the hematopoietic stem and progenitor cells (HSPCs) involves homing to the vasculatures and lodgment to their niches. How HSPCs transmigrate from the vasculature to the niches is unclear. Here, we show that loss of diaphanous-related formin mDia2 leads to impaired engraftment of long-term hematopoietic stem cells and loss of competitive HSPC repopulation. These defects are likely due to the compromised trans-endothelial migration of HSPCs since their homing to the bone marrow vasculatures remained intact. Mechanistically, loss of mDia2 disrupts HSPC polarization and induced cytoplasmic accumulation of MAL, which deregulates the activity of serum response factor (SRF). We further reveal that beta2 integrins are transcriptional targets of SRF. Knockout of beta2 integrins in HSPCs phenocopies mDia2 deficient mice. Overexpression of SRF or beta2 integrins rescues HSPC engraftment defects associated with mDia2 deficiency. Our findings show that mDia2-SRF-beta2 integrin signaling is critical for HSPC lodgment to the niches.


Assuntos
Antígenos CD18/metabolismo , Forminas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Animais , Medula Óssea/metabolismo , Modelos Animais de Doenças , Forminas/genética , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , NADPH Desidrogenase/genética , Transdução de Sinais
2.
Ecotoxicol Environ Saf ; 201: 110799, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32544743

RESUMO

Mercury (Hg) is an environmental contaminant that poses great risk to human health. However, it is still widely used in artisanal gold-mining enterprises around the world, especially in developing countries. Methylmercury (MeHg) is produced environmentally by biomethylation of inorganic Hg present in water sediments, leading to its subsequent accumulation in the aquatic food chain. Due to its high metabolic rate, the Central Nervous System (CNS) is one of the main targets of MeHg. In the present study, we investigate the impact of chronic MeHg intoxication on NADPH diaphorase (NADPH-d) activity and astrocyte mobilization in the visual cortex of the rat. After 60 days of MeHg administration by oral gavage (0.04 mg/kg/day), tissue samples containing the visual cortex were submitted to measurements of Hg levels, NADPH-d activity, and GFAP immunohistochemistry for identification of astrocytes. MeHg intoxication was associated with increased Hg deposits and with reduced NADPH-d neuropil reactivity in the visual cortex. A morphometric analysis suggested that NADPH-d-positive neurons were mostly spared from MeHg harmful action and intoxicated animals had astrocytic activation similar to the control group. The decrease in NADPH-d neuropil reactivity may be due to the negative effect of chronic MeHg poisoning on both the synthesis and transport of this enzyme in afferent pathways to the visual cortex. The relative resistance of NADPH-d-reactive neurons to chronic MeHg intoxication may be associated with peculiarities in cell metabolism or to a protective role of nitric oxide, safeguarding those neurons from Hg deleterious effects.


Assuntos
Astrócitos/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Compostos de Metilmercúrio/toxicidade , NADPH Desidrogenase/metabolismo , Neurônios/efeitos dos fármacos , Córtex Visual/efeitos dos fármacos , Animais , Astrócitos/enzimologia , Comportamento Animal/efeitos dos fármacos , Poluentes Ambientais/metabolismo , Ouro , Humanos , Masculino , Compostos de Metilmercúrio/metabolismo , Mineração , Neurônios/enzimologia , Ratos , Ratos Wistar , Córtex Visual/enzimologia , Córtex Visual/patologia
3.
Nat Commun ; 11(1): 610, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001694

RESUMO

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.


Assuntos
Complexo I de Transporte de Elétrons/química , NADPH Desidrogenase/química , Fotossíntese , Thermus/enzimologia , Sítios de Ligação , Carotenoides/química , Membrana Celular/química , Transporte de Elétrons , Complexo I de Transporte de Elétrons/ultraestrutura , Ferredoxinas/química , Ferredoxinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Modelos Moleculares , NADPH Desidrogenase/ultraestrutura , Plastoquinona/química , Plastoquinona/metabolismo , Domínios Proteicos , Subunidades Proteicas/química , Homologia Estrutural de Proteína
4.
Plant Sci ; 292: 110387, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32005392

RESUMO

The cyclic electron flow (CEF) around photosystem I (PSI) plays a crucial role in photosynthesis and also functions in plant tolerance of abiotic environmental stress. However, the role of PGR5/PGRL1- and NDH-dependent CEF in tomato under hightemperature (HT) is poorly understood. Here, we assessed the photoprotective effect of these pathways in tomato leaves under HT by using antimycin A (AA) and rotenone (R), which are chemical inhibitors of PGR5/PGRL1- and NDH-dependent CEF, respectively. The results showed that AA treatment caused significantly greater inhibition of CEF under HT compared to R treatment. Moreover, AA treatment caused a greater decrease in maximal photochemistry efficiency (Fv/Fm) and increased damage to the donor and acceptor side of photosystem II (PSII); however, the limitation of the acceptor side in PSI [Y(NA)] was significantly increased. In addition, thylakoid membrane integrity was compromised and reactive oxygen species, proton gradient (ΔpH), antioxidant enzyme activity, and the expression of photosystem core subunit genes were significantly decreased under AA treatment. These findings indicate that PGR5/PGRL1-dependent CEF protects PSII and PSI from photooxidative damage through the formation of ΔpH while maintaining thylakoid membrane integrity and normal gene expression levels of core photosystem components. This study demonstrates that PGR5/PGRL1-dependent CEF plays a major role in HT response in tomato.


Assuntos
Elétrons , Temperatura Alta , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Expressão Gênica , Lycopersicon esculentum/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Força Próton-Motriz
5.
Appl Microbiol Biotechnol ; 104(5): 2051-2066, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31930452

RESUMO

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.


Assuntos
Extremófilos/enzimologia , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Alcenos/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cianobactérias/enzimologia , Cianobactérias/genética , Cianobactérias/metabolismo , Bases de Dados Genéticas , Estabilidade Enzimática , Extremófilos/genética , Extremófilos/metabolismo , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , NADP/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/isolamento & purificação , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodófitas/enzimologia , Rodófitas/genética , Especificidade por Substrato
6.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140303, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678192

RESUMO

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.


Assuntos
Mononucleotídeo de Flavina/química , NADPH Desidrogenase/química , Bacillus subtilis/enzimologia , Catálise , Cicloexanonas/química , Escherichia coli/genética , Mononucleotídeo de Flavina/efeitos da radiação , NADPH Desidrogenase/genética , NADPH Desidrogenase/efeitos da radiação , Oxirredução , Fotoquímica , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação
7.
Bull Exp Biol Med ; 167(5): 694-697, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31630304

RESUMO

Reaction of mast cells of adult male Wistar rats (n=15) in the zone of polypropylene mesh fixation was studied by histochemical, immunohistochemical, and traditional morphological methods on days 1, 5, 10, and 30 after implantation. Immediately after the intervention, mast cells stimulated the processes aimed at wound healing. Secretion of mast cells was clearly regulatory. These cells migrated to the zone of injury for subsequent activation of their function. The number of cNOS+ mast cells near the polypropylene mesh was maximum on day 1 and the number of iNOS+ mast cells peaked on day 5 of the experiment, which probably represented a compensatory reaction. Presumably, stimulation of fibrillogenesis was largely due to the activatory effect of mast cells on the fibroblast function, but not to collagen production by these mast cells.


Assuntos
Materiais Biocompatíveis/farmacologia , Expressão Gênica/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Polipropilenos/farmacologia , Telas Cirúrgicas , Parede Abdominal/cirurgia , Animais , Catecolaminas/imunologia , Catecolaminas/metabolismo , Movimento Celular/efeitos dos fármacos , Colágeno/genética , Colágeno/imunologia , Inflamação , Masculino , Mastócitos/imunologia , NADPH Desidrogenase/genética , NADPH Desidrogenase/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/imunologia , Ratos , Ratos Wistar , Cicatrização/efeitos dos fármacos , Cicatrização/imunologia
8.
BMC Genomics ; 20(1): 680, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462217

RESUMO

BACKGROUND: Fermentation completion is a major prerequisite in many industrial processes involving the bakery yeast Saccharomyces cerevisiae. Stuck fermentations can be due to the combination of many environmental stresses. Among them, high temperature and ethanol content are particularly deleterious especially in bioethanol and red wine production. Although the genetic causes of temperature and/or ethanol tolerance were widely investigated in laboratory conditions, few studies investigated natural genetic variations related to stuck fermentations in high gravity matrixes. RESULTS: In this study, three QTLs linked to stuck fermentation in winemaking conditions were identified by using a selective genotyping strategy carried out on a backcrossed population. The precision of mapping allows the identification of two causative genes VHS1 and OYE2 characterized by stop-codon insertion. The phenotypic effect of these allelic variations was validated by Reciprocal Hemyzygous Assay in high gravity fermentations (> 240 g/L of sugar) carried out at high temperatures (> 28 °C). Phenotypes impacted were mostly related to the late stage of alcoholic fermentation during the stationary growth phase of yeast. CONCLUSIONS: Our findings illustrate the complex genetic determinism of stuck fermentation and open new avenues for better understanding yeast resistance mechanisms involved in high gravity fermentations.


Assuntos
Etanol/farmacologia , Fermentação , Saccharomyces cerevisiae/genética , Temperatura , Alelos , Mapeamento Cromossômico , Etanol/metabolismo , NADPH Desidrogenase/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Locos de Características Quantitativas , Saccharomyces cerevisiae/metabolismo , Açúcares/metabolismo , Sequenciamento Completo do Genoma , Vinho
9.
Eur J Med Chem ; 180: 213-223, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31306908

RESUMO

Reactions of Ni(II) and Pd(II) precursors with S-benzyl-N-(ferrocenyl)methylenedithiocarbazate (HFedtc) led to the formation of heterobimetallic complexes of the type [MII(Fedtc)2] (M = Ni and Pd). The characterization of the compounds involved the determination of melting point, FTIR, UV-Vis, 1H NMR, elemental analysis and electrochemical experiments. Furthermore, the crystalline structures of HFedtc and [NiII(Fedtc)2] were determined by single crystal X-ray diffraction. The compounds were evaluated against the intracellular form of Trypanosoma cruzi (Tulahuen Lac-Z strain) and the cytotoxicity assays were assessed using LLC-MK2 cells. The results showed that the coordination of HFedtc to Ni(II) or Pd(II) decreases the in vitro trypanocidal activity while the cytotoxicity against LLC-MK2 cells does not change significantly. [PdII(Fedtc)2] showed the greater potential between the two complexes studied, showing an SI value of 8.9. However, this value is not better than that of the free ligand with an SI of 40, a similar value to that of the standard drug benznidazole (SI = 48). Additionally, molecular docking simulations were performed with Trypanosoma cruzi Old Yellow Enzyme (TcOYE), which predicted that HFedtc binds to the protein, almost parallel to the flavin mononucleotide (FMN) prosthetic group, while the [NiII(Fedtc)2] complex was docked into the enzyme binding site in a significantly different manner. In order to confirm the hypothetical interaction, in vitro experiments of fluorescence quenching and enzymatic activity were performed which indicated that, although HFedtc was not processed by the enzyme, it was able to act as a competitive inhibitor, blocking the hydride transfer from the FMN prosthetic group of the enzyme to the menadione substrate.


Assuntos
Compostos de Benzil/farmacologia , Complexos de Coordenação/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrazinas/farmacologia , Metalocenos/farmacologia , NADPH Desidrogenase/antagonistas & inibidores , Níquel/farmacologia , Paládio/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Hidrazinas/química , Macaca mulatta , Metalocenos/química , Simulação de Acoplamento Molecular , Estrutura Molecular , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Níquel/química , Níquel/metabolismo , Paládio/química , Paládio/metabolismo , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/metabolismo , Trypanosoma cruzi/metabolismo
10.
Acta Histochem ; 121(6): 690-694, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31227177

RESUMO

Leptin, a hormone mainly produced by adipocytes in proportion to fat mass, is a key component in the regulation of energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. Leptin binds to the leptin receptor, which is expressed throughout the central nervous system but particularly in neurons of several nuclei of the hypothalamus, such as the arcuate nucleus (ARC) and paraventricular nucleus (PVN). It has been found that nitric oxide (NO) plays an important role in mediating effects of leptin. Since PVN and ARC neurons are known to express leptin receptors, we investigated the effects of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the PVN and ARC of male Wistar rats. Our results have shown that systemic administration of leptin resulted in increased NADPH-d positive cell number in the PVN and ARC, suggesting that both the PVN and ARC may be important centers in the hypothalamus for the leptin action, mediated by increased NO production. In addition, we have also observed that hypothalamic tanycytes in the ventral portion of the third ventricle were NADPH-d positive. We speculate that leptin may affect the release of neurohormones and hypothalamic neurogenesis by activating nitric oxide synthase in hypothalamic tanycytes.


Assuntos
Células Ependimogliais/enzimologia , Leptina/farmacologia , NADPH Desidrogenase/metabolismo , Núcleo Hipotalâmico Paraventricular/enzimologia , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/enzimologia , Células Ependimogliais/citologia , Masculino , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Ratos , Ratos Wistar , Receptores para Leptina/metabolismo
11.
Appl Microbiol Biotechnol ; 103(12): 5015-5022, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31044312

RESUMO

Old Yellow Enzymes play key roles in several cellular processes and have become an important family of enzymes with biotechnological potential. One of the major challenges of biotechnology consists of the bioremediation of co-polluted soils with organic and inorganic compounds. In co-contaminated areas, chromium normally exists in its more toxic and carcinogenic form Cr(VI). Microorganisms can reduce this metal to the insoluble and less toxic Cr(III). Streptomyces sp. M7 is a strain able to efficiently bioremediate polluted soils with γ-hexachlorocyclohexane and Cr(VI). The complete degradation pathway for γ-hexachlorocyclohexane was recently elucidated in this strain. In the present work, we confirmed the ability of Streptomyces sp. M7 to eliminate a high percentage of Cr(VI) from a synthetic culture medium. After a transcriptional study in the presence of Cr(VI), we also report the molecular cloning of a gene coding for an Old Yellow Enzyme with chromate reductase activity. Our results suggest that the elimination of Cr(VI) by Streptomyces sp. M7 is directly related to the activity of this Old Yellow Enzyme. The importance of our work is in identifying for the first time an Old Yellow Enzyme with chromate reductase activity in Streptomyces and Actinobacteria. Finding this enzyme helps understand chromium homeostasis in Streptomyces sp. M7, in addition to opening a new research window related to Old Yellow Enzymes from Actinobacteria.


Assuntos
Biodegradação Ambiental , Cromo/metabolismo , Meios de Cultura/química , NADPH Desidrogenase/metabolismo , Streptomyces/enzimologia , Redes e Vias Metabólicas , NADPH Desidrogenase/genética , Oxirredução , Oxirredutases/metabolismo , Microbiologia do Solo , Streptomyces/genética
12.
Molecules ; 24(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889828

RESUMO

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal-a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.


Assuntos
Aldeídos/metabolismo , Engenharia Metabólica/métodos , Monoterpenos/metabolismo , NADPH Desidrogenase/química , Saccharomyces cerevisiae/enzimologia , Monoterpenos Acíclicos , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Modelos Moleculares , Mutagênese/genética , Proteínas Mutantes/metabolismo , NADPH Desidrogenase/metabolismo , Oxirredução , Estereoisomerismo
13.
Phys Chem Chem Phys ; 21(22): 11589-11598, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30801593

RESUMO

Biodegradation is a cost-effective and environmentally friendly alternative to removing 2,4,6-trinitrotoluene (TNT) pollution. However, mechanisms of TNT biodegradation have been elusive. To enhance the understanding of TNT biotransformation by the Old Yellow Enzyme (OYE) family, we investigated the crucial first-step hydrogen-transfer reaction by molecular dynamics simulations, docking technologies and empirical valence bond calculations. We revealed the significance of the π-π stacking conformation between the substrate TNT and the reduced flavin mononucleotide (FMNH2) cofactor, which is a prerequisite for the aromatic ring reduction of TNT. Under the π-π stacking conformation, the barrier of the hydrogen-transfer reaction in the aromatic ring reduction is about 16 kcal mol-1 lower than that of nitro group reduction. Then, we confirmed the mechanism of controlling the π-π stacking, that is, the π-π interaction competition mechanism. It indicates that the π-π stacking of TNT and FMNH2 occurs only when the π-π interaction between FMNH2 and TNT is stronger than that between TNT and several key residues with aromatic rings. Finally, based on the competition mechanism, the formation of π-π stacking of TNT and FMNH2 can be successfully enabled by removing the aromatic ring of those key residues in enzymes that originally only transform TNT through the nitro group reduction. This testified the validity of the π-π interaction competition mechanism. This work theoretically clarifies the molecular mechanism of the first-step hydrogen-transfer reaction for the biotransformation of TNT by the OYE family. It is helpful to obtain the enzymes that can biodegrade TNT through the aromatic ring reduction.


Assuntos
Flavoproteínas/metabolismo , NADPH Desidrogenase/metabolismo , Trinitrotolueno/metabolismo , Animais , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biotransformação , Domínio Catalítico , Mononucleotídeo de Flavina/química , Flavoproteínas/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Himenópteros/enzimologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , NADPH Desidrogenase/química , Oxirredução , Ligação Proteica , Saccharomyces/enzimologia , Eletricidade Estática , Trinitrotolueno/química
14.
Invest Ophthalmol Vis Sci ; 60(2): 473-487, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30707220

RESUMO

Purpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, ß-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. Methods: Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Results: The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed ß-actin, nestin, and CD44, but they did not express CD31 and α-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-ß). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. Conclusions: A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease.


Assuntos
Células Endoteliais/patologia , Hipertensão/patologia , Retinopatia Hipertensiva/patologia , Células Intersticiais de Cajal/patologia , Artéria Retiniana/patologia , Actinas/metabolismo , Animais , Anoctamina-1/metabolismo , Pressão Arterial , Arteríolas/patologia , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Histocitoquímica , Receptores de Hialuronatos/metabolismo , Retinopatia Hipertensiva/metabolismo , Células Intersticiais de Cajal/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , NADPH Desidrogenase/metabolismo , Nestina/metabolismo
15.
Nature ; 566(7744): 411-414, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742075

RESUMO

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.


Assuntos
Microscopia Crioeletrônica , Cianobactérias/química , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/ultraestrutura , NADPH Desidrogenase/química , NADPH Desidrogenase/ultraestrutura , Oxigênio/metabolismo , Fotossíntese , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Coenzimas/química , Coenzimas/metabolismo , Cianobactérias/enzimologia , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Ferredoxinas/metabolismo , Modelos Biológicos , Modelos Moleculares , NADPH Desidrogenase/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema I/metabolismo , Plastoquinona/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
16.
Chembiochem ; 20(12): 1569-1577, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-30758121

RESUMO

Many drug candidate molecules contain at least one chiral centre, and consequently, the development of biocatalytic strategies to complement existing metal- and organocatalytic approaches is of high interest. However, time is a critical factor in chemical process development, and thus, the introduction of biocatalytic steps, even if more suitable, is often prevented by the limited availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ene reductases, we screened 19 bacterial strains for double bond reduction activity by using the model substrates cyclohexanone and carvone. Overall, we identified 47 genes coding for putative ene reductases. Remarkably, bioinformatic analysis of all genes and the biochemical characterization of four representative novel ene reductases led us to propose the existence of two new Old Yellow Enzyme subclasses, which we named OYE class III and class IV. Our results demonstrate that although, on a DNA level, each new OYE subclass features a distinct combination of sequence motifs previously known from the classical and the thermophilic-like group, their substrate scope more closely resembles the latter subclass.


Assuntos
Bactérias/enzimologia , NADPH Desidrogenase , Biocatálise , NADPH Desidrogenase/química , NADPH Desidrogenase/classificação , Oxirredução
17.
Acta Histochem ; 121(3): 268-276, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30642627

RESUMO

Dp71 is the major form of dystrophins (Dp) in the supraoptic nucleus (SON) and in the neural lobe of hypophysis (NL/HP). Dp71-null mice exhibit a hypo-osmolar status attributed to an altered osmosensitivity of the SON and to a perturbed vasopressinergic axis. Because oxytocin (OT) is implicated in osmoregulation via natriuresis, this study explored the oxytocinergic axis in Dp71-null mice after salt-loading (SL). Under normosmolar conditions, OT-mRNA expression was higher in the Dp71-null SON compared to wild-type (wt) and the OT peptide level has not changed. Dp-immunostaining was localized in astrocytes end-feet surrounding vessels in wt SON. This distribution changed in Dp71-null SON, Dp being detected in OT-soma of MCNs. nNOS and NADPH-diaphorase levels increased in the OT area of the Dp71-null SON compared to wt. In the NL/HP, OT level reduced in Dp71-null mice and Dp localization changed from pituicytes end-feet in wt SON to OT terminals in Dp71-null SON. Salt-Loading resulted in an increase of OT-mRNA and peptide levels in wt SON but had no effect in Dp71-null SON. In the NL/HP, OT content was reduced after SL. For Dp71-null mice, OT level, already low in control, was not modified by SL. Dp level was not affected by SL in the SON nor in the NL/HP. Our data confirmed the importance of Dp71 for the SON functionality in osmoregulation. The localization of Dp71 at the glial-vascular interface could be associated with SON osmosensitivity, leading to an adequate OT synthesis in the SON and release from the NL/HP upon plasmatic hyperosmolality.


Assuntos
Distrofina/deficiência , Hipotálamo/metabolismo , Osmorregulação/fisiologia , Ocitocina/metabolismo , Animais , Distrofina/metabolismo , Camundongos Knockout , NADPH Desidrogenase/metabolismo , Neurônios/metabolismo , Ocitocina/genética , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Supraóptico/metabolismo
18.
Mol Biol Cell ; 30(5): 646-657, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625038

RESUMO

Dendritic spines (DS) are actin-rich postsynaptic terminals of neurons that are critical for higher-order brain functions. Maturation of DS is accompanied by a change in actin architecture from linear to branched filamentous structures. Presumably, the underlying cause of this is a switch in a mode of actin assembly from formin-driven to Arp2/3-mediated via an undefined mechanism. Here we present data suggesting that neuron-specific actin-binding drebrin A may be a part of such a switch. It is well documented that DS are highly enriched in drebrin A, which is critical for their plasticity and function. At the same time, mDia2 is known to mediate the formation of filopodia-type (immature) spines. We found that neuronal drebrin A directly interacts with mDia2 formin. Drebrin inhibits formin-mediated nucleation of actin and abolishes mDia2-induced actin bundling. Using truncated protein constructs we identified the domain requirements for drebrin-mDia2 interaction. We hypothesize that accumulation of drebrin A in DS (that coincides with spine maturation) leads to inhibition of mDia2-driven actin polymerization and, therefore, may contribute to a change in actin architecture from linear to branched filaments.


Assuntos
Actinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Animais , Camundongos , Proteínas Associadas aos Microtúbulos/química , NADPH Desidrogenase/química , Neuropeptídeos/química , Ligação Proteica , Domínios Proteicos , Coelhos
19.
Biosci Biotechnol Biochem ; 83(3): 456-462, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30445889

RESUMO

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.


Assuntos
Candida/enzimologia , Cetonas/química , Cetonas/metabolismo , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Pichia/enzimologia , Sequência de Aminoácidos , Biocatálise , Modelos Moleculares , Oxirredução , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
20.
Metab Eng ; 52: 77-86, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30458240

RESUMO

Increasing the availability of NADPH is commonly used to improve lysine production by Corynebacterium glutamicum since 4 mol of NADPH are required for the synthesis of 1 mol of lysine. Alternatively, engineering of enzymes in lysine synthesis pathway to utilize NADH directly can also be explored for cofactor balance during lysine overproduction. To achieve such a goal, enzyme mining was used in this study to quickly identify a full set of NADH-utilizing dehydrogenases, namely aspartate dehydrogenase from Pseudomonas aeruginosa (PaASPDH), aspartate-semialdehyde dehydrogenase from Tistrella mobilis (TmASADH), dihydrodipicolinate reductase from Escherichia coli (EcDHDPR), and diaminopimelate dehydrogenase from Pseudothermotoga thermarum (PtDAPDH). This allowed us to systematically perturb cofactor utilization of lysine synthesis pathway of C. glutamicum for the first time. Individual overexpression of PaASPDH, TmASADH, EcDHDPR, and PtDAPDH in C. glutamicum LC298, a basic lysine producer, increased the production of lysine by 30.7%, 32.4%, 17.4%, and 36.8%, respectively. Combinatorial replacement of NADPH-dependent dehydrogenases in C. glutamicum ATCC 21543, a lysine hyperproducer, also resulted in significantly improved lysine production. The highest increase of lysine production (30.7%) was observed for a triple-mutant strain (27.7 g/L, 0.35 g/g glucose) expressing PaASPDH, TmASADH, and EcDHDPR. A quadruple-mutant strain expressing all of the four NADH-utilizing enzymes allowed high lysine production (24.1 g/L, 0.30 g/g glucose) almost independent of the oxidative pentose phosphate pathway. Collectively, our results demonstrated that a combination of enzyme mining and cofactor engineering was a highly efficient approach to improve lysine production. Similar strategies can be applied for the production of other amino acids or their derivatives.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Lisina/biossíntese , Engenharia Metabólica/métodos , NAD/biossíntese , Meios de Cultura , Redes e Vias Metabólicas/genética , Modelos Moleculares , Mutação/genética , NADH Desidrogenase/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Via de Pentose Fosfato , Plasmídeos/genética
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