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1.
Molecules ; 28(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36615531

RESUMO

BACKGROUND: As a result of the paucity of treatment, Leishmaniasis continues to provoke about 60,000 deaths every year worldwide. New molecules are needed, and drug discovery research is oriented toward targeting proteins crucial for parasite survival. Among them, trypanothione reductase (TR) is of remarkable interest owing to its vital role in Leishmania species protozoan parasite life. Our previously identified compound 1 is a novel chemotype endowed with a unique mode of TR inhibition thanks to its binding to a formerly unknown but druggable site at the entrance of the NADPH binding cavity, absent in human glutathione reductase (hGR). METHODS: We designed and synthesized new 3-amino-1-arylpropan-1-one derivatives structurally related to compound 1 and evaluated their potential inhibition activity on TR from Leishmania infantum (LiTR). Cluster docking was performed to assess the binding poses of the compounds. RESULTS: The newly synthesized compounds were screened at a concentration of 100 µM in in vitro assays and all of them proved to be active with residual activity percentages lower than 75%. CONCLUSIONS: Compounds 2a and 2b were the most potent inhibitors found, suggesting that an additional aromatic ring might be promising for enzymatic inhibition. Further structure-activity relationships are needed to optimize our compounds activity.


Assuntos
Antiprotozoários , Leishmania infantum , Humanos , NADP/metabolismo , Modelos Moleculares , NADH NADPH Oxirredutases , Sítios de Ligação , Antiprotozoários/farmacologia
2.
Eur J Med Chem ; 244: 114878, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36332553

RESUMO

N-methylation of the triazole moiety present in our recently described triazole-phenyl-thiazole dimerization disruptors of Leishmania infantum trypanothione disulfide reductase (LiTryR) led to a new class of potent inhibitors that target different binding sites on this enzyme. Subtle structural changes among representative library members modified their mechanism of action, switching from models of classical competitive inhibition to time-dependent mixed noncompetitive inhibition. X-ray crystallography and molecular modeling results provided a rationale for this distinct behavior. The remarkable potency and selectivity improvements, particularly against intracellular amastigotes, of the LiTryR dimerization disruptors 4c and 4d reveal that they could be exploited as leishmanicidal agents. Of note, L. infantum promastigotes treated with 4c significantly reduced their low-molecular-weight thiol content, thus providing additional evidence that LiTryR is the main target of this novel compound.


Assuntos
Antiprotozoários , Leishmania infantum , Dissulfetos , Antiprotozoários/química , NADH NADPH Oxirredutases , Triazóis/farmacologia , Triazóis/metabolismo
3.
Front Endocrinol (Lausanne) ; 13: 1023194, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36387896

RESUMO

Dysregulation of decidual macrophages leads to the occurrence of recurrent spontaneous abortion (RSA). However, the role of macrophages in RSA occurrence remains unclear. In this study, we found that the expression of Grim-19 was decreased, and the expression of autophagy related proteins Beclin1, LC3B II/I and BNIP3 was markedly upregulated in decidual macrophages of RSA patients compared with the normal pregnancy group. Furthermore, we demonstrated that downregulation of GRIM-19 increased the expression of autophagy related proteins Beclin1, LC3B II/I, BNIP3 and the proinflammatory cytokines IL1B, IL6 and TNFa in uterine mononuclear cells of GRIM-19+/- mice. The proportion of CD45+CD11b+F4/80+LC3B+ cells in GRIM-19+/- mouse uteri was significantly higher than that in WT mouse uteri. In addition, we confirmed that inhibition of Grim-19 by siRNA enhanced the expression of autophagy related proteins in RAW264.7 cells and THP-1 cells. More importantly, downregulation of Grim-19 in RAW264.7 cells promoted the release of proinflammatory cytokines and promoted phagocytic activity, which could be reversed by autophagy blockade. For THP-1-derived macrophages, the results of RNA-seq suggested that Grim-19 mainly modulates immune and inflammatory-related pathways, leading to cytokine production, and thus contributing to inflammation. Therefore, our data reveal that Grim-19 deficiency influences macrophage function, characterized by enhanced proinflammatory cytokines and phagocytic activity, and this might be regulated by autophagy. This may represent a novel mechanism for the occurrence of RSA.


Assuntos
Aborto Espontâneo , Proteínas Reguladoras de Apoptose , Autofagia , Macrófagos , NADH NADPH Oxirredutases , Animais , Feminino , Humanos , Camundongos , Gravidez , Aborto Espontâneo/genética , Proteína Beclina-1/metabolismo , Citocinas/metabolismo , Células RAW 264.7 , NADH NADPH Oxirredutases/deficiência , NADH NADPH Oxirredutases/genética , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética
4.
Int J Mol Sci ; 23(21)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36362102

RESUMO

American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against trypomastigotes of the Trypanosoma cruzi strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di-N-oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against T. cruzi trypomastigotes, with a 13% TR inhibition at 44 µM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).


Assuntos
Doença de Chagas , Tripanossomicidas , Trypanosoma cruzi , Humanos , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Quinoxalinas/química , Óxidos/farmacologia , NADH NADPH Oxirredutases , Doença de Chagas/tratamento farmacológico , Inibidores Enzimáticos/química
5.
Comput Math Methods Med ; 2022: 4117261, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36277003

RESUMO

A hand-held Raman spectroscopic device was used as a rapid nondestructive testing device to predict the metmyoglobin (MetMb) and metmyoglobin reductase activity (MRA) values on the surface layer of fresh beef. Longissimus dorsi muscles were from 10 young bulls (Holstein-Friesian) from two different cattle farms (group A = 5 and B = 5). The Raman spectra of 100 samples were correlated with the MetMb and MRA values using partial least squares regression (PLSR). Two groups could be discriminated, and the separate correlation models were better than the joint correlation model for the fresh beef. The coefficients of determination are R 2 = 0.81 (group A) and R 2 = 0.87 (group B) for MetMb and R 2 = 0.80 (group A) and R 2 = 0.85 (group B) for MRA. The results show the usefulness of Raman spectra in predicting the inner traits such as MetMb and MRA during meat storage. In conclusion, it is feasible to determine the MetMb and MRA values by Raman spectroscopy. Color is an important indicator of beef freshness and can vary depending on the age, sex, and breed of the cow. They play a very important role in human nutrition. The color of meat is an important indicator of meat freshness, and many researchers are already investigating the causes of color changes. The research was conducted in this environment.


Assuntos
Metamioglobina , NADH NADPH Oxirredutases , Bovinos , Masculino , Animais , Humanos , Metamioglobina/química , Carne/análise
6.
Biosci Rep ; 42(11)2022 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-36281795

RESUMO

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-ß peptide (Aß1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aß1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aß1-42 peptide, the aggregation kinetics of Aß1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aß1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aß1-42. Aß1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aß1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aß1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aß1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.


Assuntos
Peptídeos beta-Amiloides , Flavina-Adenina Dinucleotídeo , Animais , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/química , Flavina-Adenina Dinucleotídeo/metabolismo , NAD/genética , NAD(P)H Desidrogenase (Quinona)/química , Mutação , Benzoquinonas , NADH NADPH Oxirredutases/genética
7.
Eur J Med Chem ; 243: 114675, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36075146

RESUMO

Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypanothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 µM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 µM and a selectivity index of 35.


Assuntos
Amida Sintases , Antiprotozoários , Animais , Amida Sintases/metabolismo , NADH NADPH Oxirredutases , Sítios de Ligação , Oxirredução , Antiprotozoários/farmacologia , Antiprotozoários/química , Mamíferos/metabolismo
8.
Nat Commun ; 13(1): 5021, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-36028482

RESUMO

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.


Assuntos
NADH NADPH Oxirredutases , NAD , Escherichia coli , NADP , Oxirredução
9.
Biol Reprod ; 107(4): 956-966, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-35908189

RESUMO

The processes underlying adenomyosis are similar to those of tumor metastasis, and it is defined as progressive invasion by the endometrium and the subsequent creation of ectopic lesions. GRIM-19 regulates cell death via the mitochondrial respiratory chain. Stress following oxygen deprivation can induce tumor cell autophagy, leading to cell invasion and migration. Here, we revealed that GRIM-19 negatively regulates autophagy, and, at least in adenomyosis, decreased expression of GRIM-19 is accompanied by an increased level of autophagy and 5'-adenosine monophosphate-activated protein kinase-Unc-51 like autophagy activating kinase 1 (AMPK-ULK1) activation. Upregulation of GRIM-19 expression in human primary endometrial cells and ISHIKAWA cells inhibits autophagy via the AMPK-ULK1 pathway and helps control cell invasion and migration. In addition, we also identified increased expression of AMPK and ULK1, and higher levels of autophagy in the uterine tissues of GRIM-19+/- mice. Importantly, the function of the GRIM-19-AMPK-ULK1 axis in regulating autophagy in adenomyosis is similar to that of tumor tissues, which may help elucidate the regulation of adenomyosis tumor-like behavior, and is expected to help identify novel targets for the diagnosis and treatment of adenomyosis.


Assuntos
Proteínas Quinases Ativadas por AMP , Adenomiose , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adenomiose/genética , Monofosfato de Adenosina , Animais , Proteínas Reguladoras de Apoptose , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , NADH NADPH Oxirredutases , Oxigênio , Transdução de Sinais
10.
ACS Infect Dis ; 8(8): 1687-1699, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35880849

RESUMO

Leishmania spp. are responsible for up to 1 million new cases each year. The current therapeutic arsenal against Leishmania is largely inadequate, and there is an urgent need for better drugs. Trypanothione reductase (TR) represents a druggable target since it is essential for the parasite and not shared by the human host. Here, we report the optimization of a novel class of potent and selective LiTR inhibitors realized through a concerted effort involving X-ray crystallography, synthesis, structure-activity relationship (SAR) investigation, molecular modeling, and in vitro phenotypic assays. 5-Nitrothiophene-2-carboxamides 3, 6e, and 8 were among the most potent and selective TR inhibitors identified in this study. 6e and 8 displayed leishmanicidal activity in the low micromolar range coupled to SI > 50. Our studies could pave the way for the use of TR inhibitors not only against leishmaniasis but also against other trypanosomatidae due to the structural similarity of TR enzymes.


Assuntos
Leishmania , Leishmaniose , Descoberta de Drogas , Humanos , Leishmaniose/tratamento farmacológico , NADH NADPH Oxirredutases
11.
PLoS One ; 17(7): e0272090, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35905076

RESUMO

NAD(P)H quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes two-electron reduction of quinone to hydroquinone by using nicotinamide adenine dinucleotide (NADPH), and functions as a scavenger for reactive oxygen species (ROS). The function of NQO1 in the immune response is not well known. In the present study, we demonstrated that Nqo1-deficient T cells exhibited reduced induction of T helper 17 cells (Th17) in vitro during Th17(23)- and Th17(ß)- skewing conditions. Nqo1-deficient mice showed ameliorated symptoms in a Th17-dependent autoimmune Experimental autoimmune encephalomyelitis (EAE) model. Impaired Th17-differentiation was caused by overproduction of the immunosuppressive cytokine, IL-10. Increased IL-10 production in Nqo1-deficient Th17 cells was associated with elevated intracellular Reactive oxygen species (ROS) levels. Furthermore, overproduction of IL-10 in Th17 (ß) cells was responsible for the ROS-dependent increase of c-avian musculoaponeurotic fibrosarcoma (c-maf) expression, despite the lack of dependency of c-maf in Th17(23) cells. Taken together, the results reveal a novel role of NQO1 in promoting Th17 development through the suppression of ROS mediated IL-10 production.


Assuntos
NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD , Células Th17 , Animais , Antioxidantes , Interleucina-10 , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NADH NADPH Oxirredutases , Quinonas , Espécies Reativas de Oxigênio/metabolismo , Células Th17/metabolismo
12.
J Biol Chem ; 298(8): 102210, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35780837

RESUMO

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.


Assuntos
Rubredoxinas , Trichomonas vaginalis , Flavinas/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Rubredoxinas/genética , Rubredoxinas/metabolismo , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo , Água/metabolismo
13.
J Exp Med ; 219(8)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35819408

RESUMO

In certain instances, Th17 responses are associated with severe immunopathology. T cell-intrinsic mechanisms that restrict pathogenic effector functions have been described for type 1 and 2 responses but are less well studied for Th17 cells. Here, we report a cell-intrinsic feedback mechanism that controls the pathogenicity of Th17 cells. Th17 cells produce IL-24, which prompts them to secrete IL-10. The IL-10-inducing function of IL-24 is independent of the cell surface receptor of IL-24 on Th17 cells. Rather, IL-24 is recruited to the inner mitochondrial membrane, where it interacts with the NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 13 (also known as Grim19), a constituent of complex I of the respiratory chain. Together, Grim19 and IL-24 promote the accumulation of STAT3 in the mitochondrial compartment. We propose that IL-24-guided mitochondrial STAT3 constitutes a rheostat to blunt extensive STAT3 deflections in the nucleus, which might then contribute to a robust IL-10 response in Th17 cells and a restriction of immunopathology in experimental autoimmune encephalomyelitis.


Assuntos
Citocinas/imunologia , Interleucina-10 , Células Th17 , Animais , Diferenciação Celular , Interleucina-10/metabolismo , Camundongos , NADH NADPH Oxirredutases/metabolismo , Transdução de Sinais , Virulência
14.
Chembiochem ; 23(15): e202200121, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35593146

RESUMO

Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications.


Assuntos
Elétrons , NADH NADPH Oxirredutases , Compostos Azo/química , Corantes/química , NADH NADPH Oxirredutases/metabolismo , Nitrorredutases
15.
J Med Chem ; 65(11): 7746-7769, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35640078

RESUMO

Targeting NAD+ metabolism has emerged as an effective anticancer strategy. Inspired by the synergistic antitumor effect between NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates increasing the NAD consumption and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors hampering the NAD synthesis, first-in-class small molecules simultaneously targeting NQO1 and NAMPT were identified through structure-based design. In particular, compound 10d is an excellent NQO1 substrate that is processed faster than TSA by NQO1 and exhibited a slightly decreased NAMPT inhibitory potency than that of FK866. It can selectively inhibit the proliferation of NQO1-overexpressing A549 cells and taxol-resistant A549/taxol cells and also induce cell apoptosis and inhibit cell migration in an NQO1- and NAMPT-dependent manner in A549/taxol cells. Significantly, compound 10d demonstrated excellent in vivo antitumor efficacy in the A549/taxol xenograft models with no significant toxicity. This proof-of-concept study affirms the feasibility of discovering small molecules that target NQO1 and NAMPT simultaneously, and it also provides a novel, effective, and selective anticancer strategy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , NAD/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADH NADPH Oxirredutases , Nicotinamida Fosforribosiltransferase/metabolismo , Paclitaxel , Quinonas
16.
Appl Environ Microbiol ; 88(11): e0051922, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35612301

RESUMO

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here, we produced and characterized one such protein encoded in the Vibrio harveyi genome (GenBank ID: AIV07243) and found it to be a specific NADH:acrylate oxidoreductase (ARD). This previously unknown enzyme is formed by the OYE-like, FMN_bind, and FAD_binding_2 domains and contains covalently bound flavin mononucleotide (FMN) and noncovalently bound flavin adenine dinucleotide (FAD) and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to the FMN_bind domain. Quantitative reverse transcription PCR (RT-qPCR) and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically without acrylate was higher than that in aerobic cultures and increased only 2-fold in the presence of acrylate. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. IMPORTANCE The benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the product of the V. harveyi ard gene exhibits a yet-undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.


Assuntos
Mononucleotídeo de Flavina , Vibrio , Acrilatos , Sequência de Aminoácidos , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Fumaratos , NAD/metabolismo , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Vibrio/metabolismo
17.
Protein J ; 41(2): 230-244, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35364760

RESUMO

With the necessity to develop antileishmanial drugs with substrate specificity, trypanothione reductase (TryR) has gained popularity in parasitology. TryR is unique to be present only in trypanosomatids and is functionally similar to glutathione in mammals. It protects against oxidative stress exerted by the host defense mechanism. The TryR enzyme is essential for the survival of Leishmania parasites in the host as it reduces trypanothione and aids in neutralizing hydrogen peroxide produced by the host macrophages during infection. Henceforth, it becomes vital to decipher their functional stability and behaviour in the presence of denaturants. Our study is focused on structural, functional and behavioural stability aspects of TryR with different concentrations of Urea, Guanidinium chloride, alcohol based compounds followed by extensive molecular dynamics simulations in a lipid bilayer system. The results obtained from the study reveal an interesting insight into the possible mechanisms of modulation of the structure, function and stability of the TryR protein.


Assuntos
NADH NADPH Oxirredutases , Estresse Oxidativo , Animais , Mamíferos/metabolismo , Simulação de Dinâmica Molecular , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo
18.
BMC Vet Res ; 18(1): 126, 2022 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-35366872

RESUMO

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear. RESULTS: In this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains. CONCLUSIONS: NOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.


Assuntos
Mycoplasma hyopneumoniae , Animais , Complexos Multienzimáticos , Mycoplasma hyopneumoniae/genética , NAD , NADH NADPH Oxirredutases , Oxirredutases/metabolismo , Suínos , Virulência
19.
Arterioscler Thromb Vasc Biol ; 42(5): e117-e130, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35354309

RESUMO

BACKGROUND: Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling. METHODS: Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes' respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. RESULTS: Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-Gßγ-PI3Kγ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gαq/11-PLC (phospholipase C)-PKCß (protein kinase C ß) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. CONCLUSIONS: NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.


Assuntos
Angiotensina II , Cálcio , NADPH Oxidase 1/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Pressão Sanguínea , Cálcio/metabolismo , Camundongos , Músculo Liso Vascular/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo
20.
Reproduction ; 163(6): 365-377, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35312628

RESUMO

Abnormal sperm parameters such as oligospermia, asthenospermia, and teratozoospermia result in male factor infertility. Previous studies have shown that mitochondria play an important role in human spermatozoa motility. But the related pathogenesis is far from elucidated. The aim of this study was to investigate the association between gene associated with retinoid-interferon-induced mortality 19 (GRIM19) and asthenospermia. In this study, Grim19 knockout model (Grim19+/- mouse) was created through genome engineering. We showed that compared with WT mice, the sperm count and motility of Grim19+/- mice were significantly reduced. Grim19 may contribute to sperm count and vitality by influencing the mitochondrial membrane potential, intracellular reactive oxygen species production, and increasing cell apoptosis. The spermatogenic cells of all levels in the lumen of the seminiferous tubules were sparsely arranged, and the intercellular space became larger in the testis tissue of Grim19+/- mice. The serum testosterone concentration is significantly reduced in Grim19+/- mice. The expression of steroid synthesis-related proteins STAR, CYP11A1, and HSD3B was decreased in Grim19+/- mice. To further confirm whether changes in testosterone biosynthesis were due to Grim19 downregulation, we validated this result using Leydig cells and TM3 cells. We also found that Notch signaling pathway was involved in Grim19-mediated testosterone synthesis to some extent. In conclusion, we revealed a mechanism underlying Grim19 mediated spermatozoa motility and suggested that Grim19 affected the synthesis of testosterone and steroid hormones in male mouse partly through regulating Notch signal pathways.


Assuntos
Astenozoospermia , Oligospermia , Animais , Astenozoospermia/metabolismo , Humanos , Masculino , Camundongos , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oligospermia/metabolismo , Túbulos Seminíferos/metabolismo , Motilidade Espermática , Espermatozoides/metabolismo , Testículo/metabolismo , Testosterona/biossíntese
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