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1.
Sci Signal ; 14(675)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758061

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Assuntos
/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADP/análogos & derivados , /fisiologia , Marcadores de Afinidade , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Química Click/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transcriptoma , Internalização do Vírus
2.
Nat Immunol ; 22(2): 193-204, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33398181

RESUMO

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.


Assuntos
Linfócitos T CD8-Positivos/enzimologia , Neoplasias do Colo/enzimologia , Metabolismo Energético , Ativação Linfocitária , Linfócitos do Interstício Tumoral/enzimologia , Melanoma Experimental/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Citotoxicidade Imunológica , Estabilidade Enzimática , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/genética , Hexoquinase/metabolismo , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Microambiente Tumoral
3.
Food Chem Toxicol ; 149: 111998, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33476691

RESUMO

Corona Virus Disease 2019 (COVID-19) has spread all over the world and brings significantly negative effects on human health. To fight against COVID-19 in a more efficient way, drug-drug or drug-herb combinations are frequently used in clinical settings. The concomitant use of multiple medications may trigger clinically relevant drug/herb-drug interactions. This study aims to assay the inhibitory potentials of Qingfei Paidu decoction (QPD, a Chinese medicine compound formula recommended for combating COVID-19 in China) against human drug-metabolizing enzymes and to assess the pharmacokinetic interactions in vivo. The results demonstrated that QPD dose-dependently inhibited CYPs1A, 2A6, 2C8, 2C9, 2C19, 2D6 and 2E1 but inhibited CYP3A in a time- and NADPH-dependent manner. In vivo test showed that QPD prolonged the half-life of lopinavir (a CYP3A substrate-drug) by 1.40-fold and increased the AUC of lopinavir by 2.04-fold, when QPD (6 g/kg) was co-administrated with lopinavir (160 mg/kg) to rats. Further investigation revealed that Fructus Aurantii Immaturus (Zhishi) in QPD caused significant loss of CYP3A activity in NADPH-generating system. Collectively, our findings revealed that QPD potently inactivated CYP3A and significantly modulated the pharmacokinetics of CYP3A substrate-drugs, which would be very helpful for the patients and clinicians to avoid potential drug-interaction risks in COVID-19 treatment.


Assuntos
/tratamento farmacológico , Citocromo P-450 CYP3A/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Interações Ervas-Drogas , Animais , Área Sob a Curva , China , Medicamentos de Ervas Chinesas/uso terapêutico , Lopinavir/farmacocinética , Masculino , Microssomos Hepáticos , NADP/metabolismo , Fitoterapia , Ratos Sprague-Dawley
4.
Nat Commun ; 12(1): 449, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469028

RESUMO

Steroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Esteroides/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/química , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Inibidores de 5-alfa Redutase/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Coenzimas/química , Coenzimas/metabolismo , Coenzimas/ultraestrutura , Cristalografia por Raios X , Desenho de Fármacos , Ligação de Hidrogênio , NADP/química , NADP/metabolismo , NADP/ultraestrutura , Oxirredução , Proteobactérias/enzimologia , Relação Estrutura-Atividade
5.
Nat Commun ; 12(1): 155, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420071

RESUMO

Dual oxidases (DUOXs) produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen. They are involved in many crucial biological processes and human diseases, especially in thyroid diseases. DUOXs are protein complexes co-assembled from the catalytic DUOX subunits and the auxiliary DUOXA subunits and their activities are regulated by intracellular calcium concentrations. Here, we report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states. These structures reveal the DUOX1 complex is a symmetric 2:2 hetero-tetramer stabilized by extensive inter-subunit interactions. Substrate NADPH and cofactor FAD are sandwiched between transmembrane domain and the cytosolic dehydrogenase domain of DUOX. In the presence of calcium ions, intracellular EF-hand modules might enhance the catalytic activity of DUOX by stabilizing the dehydrogenase domain in a conformation that allows electron transfer.


Assuntos
Cálcio/metabolismo , Oxidases Duais/química , Proteínas de Membrana/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Oxidases Duais/genética , Ativação Enzimática , Ensaios Enzimáticos , Flavina-Adenina Dinucleotídeo/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Modelos Moleculares , NADP/metabolismo , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
6.
Biochim Biophys Acta Gen Subj ; 1865(1): 129767, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33141062

RESUMO

BACKGROUND: The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate. When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity. METHODS: We deciphered the reasons of this disparity in studying the role of key parameters, including. cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O2 consumption rates. RESULTS: Our results show that the O2•- low permeability of the vesicle membrane as well as secondary reactions (O2•- and H2O2 disproportionations) are strong clues to shed light on this inconsistency. CONCLUSION AND GENERAL SIGNIFICANCE: These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.


Assuntos
NADPH Oxidase 2/metabolismo , Ácido Araquidônico/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , NADP/metabolismo , Neutrófilos/metabolismo , Oxirredução , Consumo de Oxigênio
7.
Food Chem ; 336: 127685, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32758803

RESUMO

Herein, we employed exogenous phytosulfokine α (PSKα) for delaying senescence and lessening decay in strawberry fruits during storage at 4 °C for 18 days. Our results showed that the strawberry fruits treated with 150 nM PSKα exhibited lower expression of poly-ADP-ribose polymerase 1 (PARP1) gene, leading to a higher intracellular NAD+ availability, beneficial for a sufficient provision of intracellular NADP+ with the activity of NAD kinase (NADK). Moreover, higher activities of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and methylenetetrahydrofolate dehydrogenase (MTHFD) may be the reason for the sufficient intracellular availability of NADPH in strawberry fruits treated with 150 nM PSKα. In addition, strawberry fruits treated with 150 nM PSKα exhibited a sufficient availability of ATP resulted from higher activities of succinate dehydrogenase (SDH) and cytochrome c oxidase (CCO). Therefore, our results indicate that exogenous PSKα could be beneficial for delaying senescence and reducing decay in strawberry fruits during cold storage.


Assuntos
Trifosfato de Adenosina/metabolismo , Armazenamento de Alimentos , Fragaria/metabolismo , Frutas/metabolismo , Espaço Intracelular/metabolismo , NADP/metabolismo , Hormônios Peptídicos/metabolismo , Proteínas de Plantas/metabolismo , Temperatura Baixa , Fragaria/enzimologia , Fatores de Tempo
8.
Artigo em Inglês | MEDLINE | ID: mdl-33010451

RESUMO

Different strategies to boost NAD+ levels are considered promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme involved in the regulation of different cell functions. In the context of systemic energy metabolism, it has been demonstrated that brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis. In this study we aim to understand the functional relevance of CD38 for NAD+ and energy metabolism in BAT and WAT, also using a CD38-/- mouse model. During cold exposure, an increase in NAD+ levels occurred in BAT of wild type mice, together with a marked downregulation of CD38, as detected at the mRNA and protein level. CD38 downregulation was observed also in WAT of cold-exposed mice, where it was accompanied by a strong increase in NADP(H) levels. Accordingly, NAD kinase and glucose-6-phosphate dehydrogenase activities were enhanced in WAT (but not in BAT). Increased NAD+ levels were observed in BAT/WAT from CD38-/- compared with wild type mice, in line with CD38 being a major NAD+-consumer in AT. CD38-/- mice kept at 6 °C had higher levels of Ucp1 and Pgc-1α in BAT and WAT, and increased levels of phosphorylated hormone-sensitive lipase in BAT, compared with wild type mice. These results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT.


Assuntos
ADP-Ribosil Ciclase 1/genética , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Glicoproteínas de Membrana/genética , NADP/metabolismo , NAD/metabolismo , RNA Mensageiro/genética , Termogênese/genética , ADP-Ribosil Ciclase 1/deficiência , Adipócitos Bege/citologia , Adipócitos Bege/metabolismo , Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/citologia , Animais , Temperatura Baixa , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Homeostase/genética , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
9.
Mol Cell ; 81(2): 355-369.e10, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33321093

RESUMO

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.


Assuntos
Membrana Celular/química , Sistema Enzimático do Citocromo P-450/genética , Citocromo-B(5) Redutase/genética , Ácidos Graxos Insaturados/metabolismo , Ferroptose/genética , NADP/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Concanavalina A/farmacologia , Sistema Enzimático do Citocromo P-450/deficiência , Citocromo-B(5) Redutase/deficiência , Transporte de Elétrons/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Oxigênio/metabolismo , Compostos de Fenilureia/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Sorafenibe/farmacologia
10.
Nat Commun ; 11(1): 5430, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110062

RESUMO

Human steroid 5α-reductase 2 (SRD5A2) is an integral membrane enzyme in steroid metabolism and catalyzes the reduction of testosterone to dihydrotestosterone. Mutations in the SRD5A2 gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride, as SRD5A2 inhibitors, are widely used antiandrogen drugs for benign prostate hyperplasia. The molecular mechanisms underlying enzyme catalysis and inhibition for SRD5A2 and other eukaryotic integral membrane steroid reductases remain elusive due to a lack of structural information. Here, we report a crystal structure of human SRD5A2 at 2.8 Å, revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the transmembrane domain. Structural analysis together with computational and mutagenesis studies reveal the molecular mechanisms of the catalyzed reaction and of finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region that regulate NADPH/NADP+ exchange. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and may facilitate drug development.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/química , Antagonistas de Androgênios/química , Finasterida/química , Proteínas de Membrana/química , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Motivos de Aminoácidos , Dutasterida/química , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , NADP/química , NADP/metabolismo
11.
PLoS One ; 15(9): e0239372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32966327

RESUMO

As a ubiquitous enzyme, succinic semialdehyde dehydrogenase contributes significantly in many pathways including the tricarboxylic acid cycle and other metabolic processes such as detoxifying the accumulated succinic semialdehyde and surviving in nutrient-limiting conditions. Here the cce4228 gene encoding succinic semialdehyde dehydrogenase from Cyanothece sp. ATCC51142 was cloned and the homogenous recombinant cce4228 protein was obtained by Ni-NTA affinity chromatography. Biochemical characterization revealed that cce4228 protein displayed optimal activity at presence of metal ions in basic condition. Although the binding affinity of cce4228 protein with NAD+ was about 50-fold lower than that of cce4228 with NADP+, the catalytic efficiency of cce4228 protein towards succinic semialdehyde with saturated concentration of NADP+ is same as that with saturated concentration of NAD+ as its cofactors. Meanwhile, the catalytic activity of cce4228 was competitively inhibited by succinic semialdehyde substrate. Kinetic and structural analysis demonstrated that the conserved Cys262 and Glu228 residues were crucial for the catalytic activity of cce4228 protein and the Ser157 and Lys154 residues were determinants of cofactor preference.


Assuntos
Cyanothece/enzimologia , Succinato-Semialdeído Desidrogenase/química , Succinato-Semialdeído Desidrogenase/metabolismo , Sequência de Aminoácidos , Cinética , Modelos Moleculares , Mutação , NAD/metabolismo , NADP/metabolismo , Conformação Proteica , Especificidade por Substrato , Succinato-Semialdeído Desidrogenase/genética
12.
J Oleo Sci ; 69(9): 1061-1075, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879197

RESUMO

Our previous studies have shown that α-eleostearic acid (α-ESA; cis-9, trans-11, trans-13 (c9,t11,t13)-conjugated linolenic acid (CLnA)) is converted into c9,t11-conjugated linoleic acid (CLA) in rats. Furthermore, we have demonstrated that the conversion of α-ESA into CLA is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymatic reaction, which occurs mostly in the rat liver. However, the precise metabolic pathway and enzyme involved have not been identified yet. Therefore, in this study we aimed to determine the role of cytochrome P450 (CYP) in the conversion of α-ESA into c9,t11-CLA using an in vitro reconstitution system containing mouse hepatic microsomes, NADPH, and α-ESA. The CYP4 inhibitors, 17-ODYA and HET0016, performed the highest level of inhibition of CLA formation. Furthermore, the redox partner cytochrome P450 reductase (CPR) inhibitor, 2-chloroethyl ethyl sulfide (CEES), also demonstrated a high level of inhibition. Thus, these results indicate that the NADPH-dependent CPR/CYP4 system is responsible for CLA formation. In a correlation analysis between the specific activity of CLA formation and Cyp4 family gene expression in tissues, Cyp4a14 and Cyp4f13 demonstrated the best correlations. However, the CYP4F substrate prostaglandin A1 (PGA1) exhibited the strongest inhibitory effect on CLA formation, while the CYP4A and CYP4B1 substrate lauric acid had no inhibitory effect. Therefore, we conclude that the CYP4F13 enzyme is the major enzyme involved in CLA formation. This pathway is a novel pathway for endogenous CLA synthesis, and this study provides insight into the potential application of CLnA in functional foods.


Assuntos
Família 4 do Citocromo P450/farmacologia , Ácidos Linoleicos Conjugados/metabolismo , Ácidos Linolênicos/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Família 4 do Citocromo P450/fisiologia , Expressão Gênica , Técnicas In Vitro , Camundongos Endogâmicos ICR , NADP/metabolismo
13.
Nat Commun ; 11(1): 3802, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732903

RESUMO

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Desdobramento de Proteína , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Estresse Mecânico , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , NADP/metabolismo , Transporte Proteico , Proteínas SecA/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
14.
Toxicol Lett ; 332: 7-13, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32615244

RESUMO

Root canal sealers are commonly used to endodontically treat teeth with periapical infections. Some root canal sealers based on epoxy resin contain bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE). The presence of these chemicals is of concern due to the close contact to the blood stream at the apex and the long setting times of up to 24 h. These chemicals, or any of their degradation products or metabolites, can then exert their toxic effects before being excreted. This study aimed to identify the phase I in vitro biotransformation products of BADGE and BFDGE using human liver microsomes. During incubation with microsomal fractions, the epoxides were rapidly hydrolysed in a NADPH independent manner resulting in the formation of BADGE.2H2O and BFDGE.2H2O. Further, oxidative reactions, such as hydroxylation and carboxylation, generated other BADGE metabolites, such as BADGE.2H2O-OH and BADGE.H2O.COOH, respectively. For BFDGE, further oxidation of BFDGE.2H2O led to the newly reported carboxylic acid, BFDGE.H2O.COOH. In total, three specific metabolites have been identified which can serve in future human biomonitoring studies of BADGE and BFDGE.


Assuntos
Compostos Benzidrílicos/farmacocinética , Compostos de Epóxi/farmacocinética , Fígado/metabolismo , Materiais Restauradores do Canal Radicular/farmacocinética , Compostos Benzidrílicos/toxicidade , Biotransformação , Ácidos Carboxílicos/metabolismo , Compostos de Epóxi/toxicidade , Feminino , Humanos , Hidroxilação , Masculino , Microssomos Hepáticos/metabolismo , NADP/metabolismo , Oxirredução , Materiais Restauradores do Canal Radicular/toxicidade
15.
Biochim Biophys Acta Bioenerg ; 1861(10): 148256, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32622739

RESUMO

Flavodiiron proteins (FDPs) of photosynthetic organisms play a photoprotective role by reducing oxygen to water and thus avoiding the accumulation of excess electrons on the photosystem I (PSI) acceptor side under stress conditions. In Synechocystis sp. PCC 6803 grown under high CO2, both FDPs Flv1 and Flv3 are indispensable for oxygen reduction. We performed a detailed in vivo kinetic study of wild-type (WT) and Δflv1/3 strains of Synechocystis using light-induced NADPH fluorescence and near-infrared absorption of iron-sulfur clusters from ferredoxin and the PSI acceptors (FAFB), collectively named FeS. These measurements were performed under conditions where the Calvin-Benson cycle is inactive or poorly activated. Under such conditions, the NADPH decay following a short illumination decays in parallel in both strains and exhibits a time lag which is correlated to the presence of reduced FeS. On the contrary, reduced FeS decays much faster in WT than in Δflv1/3 (13 vs 2 s-1). These data unambiguously show that reduced ferredoxin, or possibly reduced FAFB, is the direct electron donor to the Flv1/Flv3 heterodimer. Evidences for large reduction of (FAFB) and recombination reactions within PSI were also provided by near-infrared absorption. Mutants lacking either the NDH1-L complex, the homolog of complex I of respiration, or the Pgr5 protein show no difference with WT in the oxidation of reduced FeS following a short illumination. These observations question the participation of a significant cyclic electron flow in cyanobacteria during the first seconds of the induction phase of photosynthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Ferroproteínas não Heme/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Transporte de Elétrons , Cinética , Mutação , NADP/metabolismo , Ferroproteínas não Heme/genética , Espectrometria de Fluorescência
16.
PLoS One ; 15(7): e0236188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701995

RESUMO

Microalgae and cyanobacteria are considered as important model organisms to investigate the biology of photosynthesis; moreover, they are valuable sources of biomolecules for several biotechnological applications. Understanding the species-specific traits of photosynthetic electron transport is extremely important, because it contributes to the regulation of ATP/NADPH ratio, which has direct/indirect links to carbon fixation and other metabolic pathways and thus overall growth and biomass production. In the present work, a cuvette-based setup is developed, in which a combination of measurements of dissolved oxygen, pH, chlorophyll fluorescence and NADPH kinetics can be performed without disturbing the physiological status of the sample. The suitability of the system is demonstrated using a model cyanobacterium Synechocystis sp. PCC6803, as well as biofuel-candidate microalgae species, such as Chlorella sorokiniana, Dunaliella salina and Nannochloropsis limnetica undergoing inorganic carbon (Ci) limitation. Inorganic carbon limitation, induced by photosynthetic Ci uptake under continuous illumination, caused a decrease in the effective quantum yield of PSII (Y(II)) and loss of oxygen-evolving capacity in all species investigated here; these effects were largely recovered by the addition of NaHCO3. Detailed analysis of the dark-light and light-dark transitions of NADPH production/uptake and changes in chlorophyll fluorescence kinetics revealed species- and condition-specific responses. These responses indicate that the impact of decreased Calvin-Benson cycle activity on photosynthetic electron transport pathways involving several sections of the electron transport chain (such as electron transfer via the QA-QB-plastoquinone pool, the redox state of the plastoquinone pool) can be analyzed with high sensitivity in a comparative manner. Therefore, the integrated system presented here can be applied for screening for specific traits in several significant species at different stages of inorganic carbon limitation, a condition that strongly impacts primary productivity.


Assuntos
Carbono/farmacologia , Cianobactérias/fisiologia , Compostos Inorgânicos/farmacologia , Microalgas/fisiologia , Fotossíntese , Chlorella/efeitos dos fármacos , Chlorella/fisiologia , Clorofila/metabolismo , Cianobactérias/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Fluorescência , Cinética , Microalgas/efeitos dos fármacos , NADP/metabolismo , Oxigênio/metabolismo , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Teoria Quântica , Synechocystis/efeitos dos fármacos , Synechocystis/fisiologia
17.
Eur J Endocrinol ; 183(3): 275-284, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32567554

RESUMO

Objective: To evaluate the pathogenic role of a few benign variants and hypomorphic pathogenic variants in SRD5A2. Design and methods: We retrospectively analyzed phenotypes and genotypes in 23 Indian patients with genetically proven steroid 5α-reductase 2 (SRD5A2) deficiency. The interactions of the SRD5A2 enzymes resulting due to the most common benign variant (p.Val89Leu), the most common (hypomorphic) pathogenic variant (p.Arg246Gln) and the double variants (p.Val89Leu and p.Arg246Gln) in SRD5A2 were compared with that of the wild type (WT) enzyme by molecular dynamics (MD) simulation. Results: The majority (n = 19, 82.61%) of patients presented for atypical genitalia and had male gender identity (16/20). Including the two novel ones (p.Leu83Pro, p.Ala28Leufs*103), a total of nine different pathogenic variants were observed. p.Arg246Gln was the most common pathogenic variant (n = 12). Homozygous p.Arg246Gln (n = 9) variant was associated with milder undervirilization (Sinnecker score of ≤3a: 8/9 vs 6/14, P = 0.04) and had concurrent homozygous p.Val89Leu in all patients. Interestingly, asymptomatic fathers of two index patients were homozygous for p.Arg246Gln which questioned the pathogenicity of the variation as a sole factor. Unlike all symptomatic homozygous p.Arg246Gln patients who were also homozygous for p.Val89Leu, asymptomatic homozygous p.Arg246Gln fathers were heterozygous for p.Val89Leu. On MD simulation SRD5A2 p.Val89Leu-Testeosterone (T) and SRD5A2 p.Arg246Gln-T complexes, but not SRD5A2 p.Val89Leu and p.Arg246Gln-T complex, demonstrated close interaction between NADPH and T as that of SRD5A2 WT-T. Conclusions: p.Arg246Gln may not be pathogenic as a sole variation even in the homozygous state; additional contribution of homozygous p.Val89Leu variant may be essential for the pathogenicity of p.Arg246Gln in SRD5A2.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/deficiência , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Transtornos do Desenvolvimento Sexual/enzimologia , Homozigoto , Adolescente , Adulto , Criança , Pré-Escolar , Transtornos do Desenvolvimento Sexual/genética , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Identidade de Gênero , Genótipo , Humanos , Índia , Lactente , Recém-Nascido , Masculino , Simulação de Dinâmica Molecular , Mutação/genética , NADP/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos , Adulto Jovem
18.
Nat Commun ; 11(1): 3238, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591540

RESUMO

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/NAD+ ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/NAD+ ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle.


Assuntos
Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Luz , Proteínas Luminescentes/metabolismo , NADP/metabolismo , NAD/metabolismo , Fotossíntese/efeitos da radiação , Respiração Celular/efeitos da radiação , Cloroplastos/metabolismo , Citosol/metabolismo , Transporte de Elétrons/efeitos da radiação , Malatos/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Oxirredução , Peroxissomos/metabolismo , Plântula/metabolismo , Plântula/efeitos da radiação
19.
Nat Commun ; 11(1): 2882, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513940

RESUMO

Complex polyketides are typically associated with microbial metabolism. Here, we report that animals also make complex, microbe-like polyketides. We show there is a widespread branch of fatty acid synthase- (FAS)-like polyketide synthase (PKS) proteins, which sacoglossan animals use to synthesize complex products. The purified sacogolassan protein EcPKS1 uses only methylmalonyl-CoA as a substrate, otherwise unknown in animal lipid metabolism. Sacoglossans are sea slugs, some of which eat algae, digesting the cells but maintaining functional chloroplasts. Here, we provide evidence that polyketides support this unusual photosynthetic partnership. The FAS-like PKS family represents an uncharacterized branch of polyketide and fatty acid metabolism, encoding a large diversity of biomedically relevant animal enzymes and chemicals awaiting discovery. The biochemical characterization of an intact animal polyketide biosynthetic enzyme opens the door to understanding the immense untapped metabolic potential of metazoans.


Assuntos
Fotossíntese , Policetídeos/metabolismo , Acil Coenzima A/metabolismo , Animais , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Gastrópodes/classificação , NADP/metabolismo , Filogenia , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Policetídeos/química , Propionatos/química , Propionatos/metabolismo
20.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140462, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32485238

RESUMO

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.


Assuntos
Acetilcoenzima A/metabolismo , Coenzima A/metabolismo , Malato Desidrogenase/metabolismo , Malatos/metabolismo , NADP/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Malato Desidrogenase/genética , Fosfato Acetiltransferase/metabolismo
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