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1.
Proc Natl Acad Sci U S A ; 119(23): e2119719119, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35648819

RESUMO

SignificanceIdentifying the intrinsic factors that regulate leaf photosynthetic rate may pave the way toward developing new strategies to enhance carbon assimilation. While the dependence of photosynthesis on the reductive activation of the Calvin-Benson cycle enzymes is well established, the role of oxidative signals in counterbalancing the reductive activity is just beginning to be explored. By developing 2-Cys peroxiredoxin-based genetically encoded biosensors, we demonstrated the induction of photosynthetically derived oxidative signals under habitual light conditions, a phenomenon typically masked by the dominance of the reductive power. Moreover, we unraveled the simultaneous activation of reductive and oxidative signals during photosynthesis induction phase and showed that 2-Cys peroxiredoxin activity attenuates carbon assimilation rates, demonstrating the restrictions imposed on photosynthetic performance by oxidative signals.


Assuntos
Arabidopsis , Técnicas Biossensoriais , Carbono , Peroxirredoxinas , Fotossíntese , Folhas de Planta , Arabidopsis/metabolismo , Carbono/metabolismo , NADP/metabolismo , Oxirredução , Peroxirredoxinas/análise , Peroxirredoxinas/metabolismo , Fotossíntese/fisiologia , Folhas de Planta/química , Folhas de Planta/metabolismo
2.
Microb Cell Fact ; 21(1): 101, 2022 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-35643569

RESUMO

BACKGROUND: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. RESULTS: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. CONCLUSIONS: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.


Assuntos
Bacillus subtilis , Biologia Sintética , Bacillus subtilis/metabolismo , Carbono/metabolismo , Engenharia Metabólica/métodos , NAD/metabolismo , NADP/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/metabolismo
3.
Biol Pharm Bull ; 45(6): 798-802, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35650106

RESUMO

Redox-active quinones generate reactive oxygen species (ROS) through their redox cycling with electron donors. Hydrogen peroxide (H2O2) causes S-oxidation of proteins and is associated with activation of the redox signaling pathway and/or toxicity (Chem. Res. Toxicol., 30, 2017, Kumagai et al.). In the present study, we developed a convenient assay based on a combination of an enzyme-linked immunosorbent assay and a biotin-PEAC5-maleimide assay and used it to determine protein S-oxidation by ROS during redox cycling of 9,10-phenanthrenequinone (9,10-PQ) and pyrroloquinoline quinone (PQQ). S-Oxidation of proteins in a mouse liver supernatant was detected during reaction of 9,10-PQ or PQQ with electron donors such as dithiothreitol or reduced nicotinamide adenine dinucleotide phosphate (NADPH), whereas cellular protein oxidation was not observed in the absence of electron donors. These results suggest that the developed assay is useful for the detection of S-oxidation of proteins.


Assuntos
Peróxido de Hidrogênio , Quinonas , Animais , Camundongos , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
4.
Methods Mol Biol ; 2526: 97-106, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35657514

RESUMO

Pyridine nucleotides (NAD(H) and NADP(H)) are key redox carriers in cells and may also have other functions related to stress. These two molecules are crucial in linking metabolism to electron transport chains in photosynthesis and respiration, but they are also critical for ensuring redox signaling and homeostasis during episodes of stress. This is especially the case for NADPH, which must be generated from its oxidized form, NADP+, by key dehydrogenases. Here, we describe methods that can be used to assay contents and redox states of NAD(H) and NADP(H), as well as simple assays to measure the capacity of two key NADPH-generating enzymes.


Assuntos
NAD , Fotossíntese , Homeostase , NAD/metabolismo , NADP/metabolismo , Oxirredução
5.
Elife ; 112022 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-35723663

RESUMO

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.


Assuntos
Staphylococcus aureus , Fatores de Virulência , Animais , NAD/metabolismo , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/genética , Peixe-Zebra/metabolismo
6.
PLoS One ; 17(6): e0269130, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35696356

RESUMO

INTRODUCTION: Increased systemic oxidative stress is common in schizophrenia (SZ) patients. NADPH-oxidase 4 (NOX4) is the cell oxidoreductase, catalyzing the hydrogen peroxide formation. Presumably, NOX4 is the main oxidative stress factor in a number of diseases such as cardiovascular diseases and cancer. We hypothesized that NOX4 may be involved in the oxidative stress development caused by the disease in the schizophrenic patients' peripheral blood lymphocytes (PBL). MATERIALS AND METHODS: The SZ group included 100 patients (68 men and 32 women aged 28 ± 11 years). The control group included 60 volunteers (35 men and 25 women aged 25 ± 12 years). Flow cytometry analysis (FCA) was used for DNA damage markers (8-oxodG, É£H2AX), pro- and antiapoptotic proteins (BAX1 and BCL2) and the master-regulator of anti-oxidant response NRF2 detection in the lymphocytes of the untreated SZ patients (N = 100) and the healthy control (HC, N = 60). FCA and RT-qPCR were used for NOX4 and RNANOX4 detection in the lymphocytes. RT-qPCR was used for mtDNA quantitation in peripheral blood mononuclear cells. Cell-free DNA concentration was determined in blood plasma fluorimetrically. RESULTS: 8-oxodG, NOX4, and BCL2 levels in the PBL in the SZ group were higher than those in the HC group (p < 0.001). É£H2AX protein level was increased in the subgroup with high 8-oxodG (p<0.02) levels and decreased in the subgroup with low 8-oxodG (p <0.0001) levels. A positive correlation was found between 8-oxodG, É£H2AX and BAX1 levels in the SZ group (p <10-6). NOX4 level in lymphocytes did not depend on the DNA damage markers values and BAX1 and BCL2 proteins levels. In 15% of PBL of the HC group a small cellular subfraction was found (5-12% of the total lymphocyte pool) with high DNA damage level and elevated BAX1 protein level. The number of such cells was maximal in PBL samples with low NOX4 protein levels. CONCLUSION: Significant NOX4 gene expression was found a in SZ patients' lymphocytes, but the corresponding protein is probably not a cause of the DNA damage.


Assuntos
NADPH Oxidase 4/metabolismo , Esquizofrenia , 8-Hidroxi-2'-Desoxiguanosina , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Masculino , NADP/metabolismo , NADPH Oxidase 4/genética , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo
7.
PLoS One ; 17(6): e0269693, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35709186

RESUMO

Biocatalysts are widely used in industry, but few examples of the use of oxidoreductases, in which enzymatic function often requires electrons, have been reported. NADPH is a cofactor that supplies an electron to oxidoreductases, but is consequently inactivated and no longer able to act as an electron donor. NADP+ can not receive electrons from electrodes through straightforward electrochemistry owing to its complicated three-dimensional structure. This study reports that bipyridines effectively mediate electron transfer between an electrode and NADP+, allowing them to serve as electron mediators for NADPH production. Using bipyridines, quinones, and anilines, which have negative oxidation-reduction potentials, an electrochemical investigation was conducted into whether electrons were transferred to NADP+. Only bipyridines with a reduction potential near -1.0 V exhibited electron transfer. Furthermore, the NADPH production level was measured using spectroscopy. NADPH was efficiently produced using bipyridines, such as methyl viologen and ethyl viologen, in which the bipyridyl 1- and 1'-positions bear small substituents. However, methyl viologen caused a dehydrogenation reaction of NADPH, making it unsuitable as an electron mediator for NADPH production. The dehydrogenation reaction did not occur using ethyl viologen. These results indicated that NADP+ can be reduced more effectively using substituents that prevent a dehydrogenation reaction at the bipyridyl 1- and 1'-positions while maintaining the reducing power.


Assuntos
2,2'-Dipiridil , Elétrons , Eletrodos , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Paraquat , Viologênios
8.
Int J Mol Sci ; 23(10)2022 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-35628596

RESUMO

The IDH1R132H mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP+ were described, the consequences for the NAD+ synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1R132H on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1wt) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD+ in IDH1R132H glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD+ synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1R132H glioblastoma cells. Given this dependence of IDH1R132H cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1R132H and IDH1wt cells. Altogether, our results indicate that targeting the NAD+ synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose.


Assuntos
Neoplasias Encefálicas , Citocinas , Glioblastoma , Glioma , Isocitrato Desidrogenase , Nicotinamida Fosforribosiltransferase , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , NAD/metabolismo , NADP/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Interferência de RNA
9.
Mol Genet Metab ; 136(2): 101-110, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35637064

RESUMO

The NAD(P)HX repair system is a metabolite damage repair mechanism responsible for restoration of NADH and NADPH after their inactivation by hydration. Deficiency in either of its two enzymes, NAD(P)HX dehydratase (NAXD) or NAD(P)HX epimerase (NAXE), causes a fatal neurometabolic disorder characterized by decompensations precipitated by inflammatory stress. Clinical findings include rapidly progressive muscle weakness, ataxia, ophthalmoplegia, and motor and cognitive regression, while neuroimaging abnormalities are subtle or nonspecific, making a clinical diagnosis challenging. During stress, nonenzymatic conversion of NAD(P)H to NAD(P)HX increases, and in the absence of repair, NAD(P)H is depleted, and NAD(P)HX accumulates, leading to decompensation; however, the contribution of each to the metabolic derangement is not established. Herein, we summarize the clinical knowledge of NAXE deficiency from 30 cases and lessons learned about disease pathogenesis from cell cultures and model organisms and describe a metabolomics signature obtained by untargeted metabolomics analysis in one case at the time of crisis and after initiation of treatment. Overall, biochemical findings support a model of acute depletion of NAD+, signs of mitochondrial dysfunction, and altered lipidomics. These findings are further substantiated by untargeted metabolomics six months post-crisis showing that niacin supplementation reverses primary metabolomic abnormalities concurrent with improved clinical status.


Assuntos
Doenças Metabólicas , NAD , Humanos , Doenças Metabólicas/patologia , NADP/metabolismo , Racemases e Epimerases
10.
Sci Rep ; 12(1): 7264, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508502

RESUMO

Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections.


Assuntos
Nematospiroides dubius , Parasitos , Animais , Camundongos , NAD/metabolismo , NADP/metabolismo , Imagem Óptica , Parasitos/metabolismo
11.
Redox Biol ; 53: 102337, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35584568

RESUMO

Recent studies demonstrate that redox imbalance of NAD+/NADH and NADP+/NADPH pairs due to impaired respiration may trigger two "hidden" metabolic pathways on the crossroad between mitochondrial dysfunction, senescence, and proliferation: "ß-oxidation shuttle" and "hydride transfer complex (HTC) cycle". The "ß-oxidation shuttle" induces NAD+/NADH redox imbalance in mitochondria, while HTC cycle maintains the redox balance of cytosolic NAD+/NADH, increasing the redox disbalance of NADP+/NADPH. Senescence appears to depend on high cytoplasmic NADH but low NADPH, while proliferation depends on high cytoplasmic NAD+ and NADPH that are under mitochondrial control. Thus, activating or deactivating the HTC cycle can be crucial to cell fate - senescence or proliferation. These pathways are a source of enormous cataplerosis. They support the production of large amounts of NADPH and intermediates for lipid synthesis and membrane biogenesis, as well as for DNA synthesis.


Assuntos
Mitocôndrias , NAD , Proliferação de Células , Mitocôndrias/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredução
12.
Brain Res Bull ; 185: 162-173, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35588962

RESUMO

Subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke, is a neurological emergency with high morbidity and mortality. Early brain injury (EBI) after SAH is the leading cause of poor prognosis in SAH patients. TRX system is a NADPH-dependent antioxidant system which is composed of thioredoxin reductase (TRXR), thioredoxin (TRX). The pentose phosphate pathway (PPP), a pathway through which glucose can be metabolized, is a major source of NADPH. Thioredoxin 1 (TRX1) is a member of thioredoxin system mainly located in cytoplasm. Serine/threonine kinases ataxia telangiectasia mutated (ATM) is an important oxidative stress receptor, and TRX1 can regulate ATM phosphorylation and then affect the activity of PPP key enzyme glucose 6-phosphate dehydrogenase (G6PD). However, whether TRX1 is involved in the regulation of PPP pathway after subarachnoid hemorrhage remains unclear. The results showed that after SAH, the level of TRX1 and phosphor-ATM decreased while the level of TRXR1 increased. G6PD protein level remained unchanged but the activity decreased, and the NADPH contents decreased. Overexpression of TRX1 by lentivirus upregulates the level of phosphor-ATM, G6PD activity and NADPH content. TRX1 overexpression improved short-term and long-term neurobehavioral outcomes and alleviated neuronal impairment in rats. Nissl staining showed that upregulation of TRX1 reduced cortical neuron injury. Our study shows that TRX1 participates in the PPP pathway by regulating phosphorylation ATM, which is accomplished by affecting G6PD activity. TRX1 may be an important target for EBI intervention after SAH.


Assuntos
Ataxia Telangiectasia , Hemorragia Subaracnóidea , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Glucose , Humanos , NADP/metabolismo , Via de Pentose Fosfato/fisiologia , Fosforilação , Ratos , Tiorredoxinas/metabolismo
13.
Cell Calcium ; 104: 102594, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35561646

RESUMO

Intracellular Ca2+ signaling via changes or oscillation in cytosolic Ca2+ concentration controls almost every aspect of cellular function and physiological processes, such as gene transcription, cell motility and proliferation, muscle contraction, and learning and memory. Two-pore channels (TPCs) are a class of eukaryotic cation channels involved in intracellular Ca2+ signaling, likely present in a multitude of organisms from unicellular organisms to mammals. Accumulated evidence indicates that TPCs play a critical role in Ca2+ mobilization from intracellular stores mediated by the second messenger molecule, nicotinic acid adenine dinucleotide phosphate (NAADP). In recent years, significant progress has been made regarding our understanding of the structures and function of TPCs, including Cryo-EM structure determination of mammalian TPCs and characterization of a plastid TPC in a single-celled parasite.. The recent identification of Lsm12 and JPT2 as NAADP-binding proteins provides a new molecular basis for understanding NAADP-evoked Ca2+ signaling. In this review, we summarize basic structural and functional aspects of TPCs and highlight the most recent studies on the newly discovered TPC in a parasitic protozoan and the NAADP-binding proteins LSM12 and JPT2 as new key players in NAADP signaling.


Assuntos
Canais de Cálcio , Sinalização do Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Lisossomos/metabolismo , Mamíferos/metabolismo , NADP/análogos & derivados , NADP/metabolismo
14.
Cells ; 11(9)2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35563771

RESUMO

Two-pore channels (TPCs) are ligand-gated cation-selective ion channels that are preserved in plant and animal cells. In the latter, TPCs are located in membranes of acidic organelles, such as endosomes, lysosomes, and endolysosomes. Here, we focus on the function of these unique ion channels in mast cells, which are leukocytes that mature from myeloid hematopoietic stem cells. The cytoplasm of these innate immune cells contains a large number of granules that comprise messenger substances, such as histamine and heparin. Mast cells, along with basophil granulocytes, play an essential role in anaphylaxis and allergic reactions by releasing inflammatory mediators. Signaling in mast cells is mainly regulated via the release of Ca2+ from the endoplasmic reticulum as well as from acidic compartments, such as endolysosomes. For the crosstalk of these organelles TPCs seem essential. Allergic reactions and anaphylaxis were previously shown to be associated with the endolysosomal two-pore channel TPC1. The release of histamine, controlled by intracellular Ca2+ signals, was increased upon genetic or pharmacologic TPC1 inhibition. Conversely, stimulation of TPC channel activity by one of its endogenous ligands, namely nicotinic adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), were found to trigger the release of Ca2+ from the endolysosomes; thereby improving the effect of TPC1 on regulated mast cell degranulation. In this review we discuss the importance of TPC1 for regulating Ca2+ homeostasis in mast cells and the overall potential of TPC1 as a pharmacological target in anti-inflammatory therapy.


Assuntos
Anafilaxia , Canais de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Endossomos/metabolismo , Histamina , Homeostase , NADP/metabolismo
15.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 38(4): 374-377, 2022 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-35583068

RESUMO

Neutrophil extracellular traps (NETs) are characterized by a extracellular fibrous network structure produced by neutrophils with DNA and proteins. NETs can be formed by NADPH-dependent NETosis and NADPH-independent NETosis. After formation, it is not only hydrolyzed by DNase in plasma but can also be degraded intracellularly and extracellularly by macrophages and dendritic cells. Recent researches on NETs have reported the increased expression of NETs in a variety of hematological diseases and its immunomodulatory effect on blood system diseases. For example, NETs are closely linked to infections related to leukemia treatment, which affects the treatment and prognosis of leukemia; NETs are also involved in the progression of multiple myeloma, promote the progression of diffuse large B-cell lymphoma, and mediate the thrombotic events of chronic myeloproliferative tumors.


Assuntos
Armadilhas Extracelulares , Doenças Hematológicas , Leucemia , Armadilhas Extracelulares/metabolismo , Doenças Hematológicas/metabolismo , Humanos , NADP/metabolismo , Neutrófilos/metabolismo
16.
Biosci Rep ; 42(5)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35502767

RESUMO

Brown adipose tissue (BAT) is a promising weapon to combat obesity and metabolic disease. BAT is thermogenic and consumes substantial amounts of glucose and fatty acids as fuel for thermogenesis and energy expenditure. To study BAT function in large human longitudinal cohorts, safe and precise detection methodologies are needed. Although regarded a gold standard, the foray of PET-CT into BAT research and clinical applications is limited by its high ionizing radiation doses. Here, we show that brown adipocytes release exosomes in blood plasma that can be utilized to assess BAT activity. In the present study, we investigated circulating protein biomarkers that can accurately and reliably reflect BAT activation triggered by cold exposure, capsinoids ingestion and thyroid hormone excess in humans. We discovered an exosomal protein, methylene tetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L), to be overexpressed and detectable in plasma for all three modes of BAT activation in human subjects. This mitochondrial protein is packaged as a cargo within multivesicular bodies of the endosomal compartment and secreted as exosomes via exocytosis from activated brown adipocytes into the circulation. To support MTHFD1L as a conserved BAT activation response in other vertebrates, we examined a rodent model and also proved its presence in blood of rats following BAT activation by cold exposure. Plasma concentration of exosomal MTHFD1L correlated with human BAT activity as confirmed by PET-MR in humans and supported by data from rats. Thus, we deduce that MTHFD1L appears to be overexpressed in activated BAT compared to BAT in the basal nonstimulated state.


Assuntos
Tecido Adiposo Marrom , Exossomos , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético , Exossomos/metabolismo , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , NADP/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ratos , Tetra-Hidrofolato Desidrogenase/metabolismo
17.
Ticks Tick Borne Dis ; 13(4): 101943, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35381468

RESUMO

Redox metabolism is crucial in host defense. Previously, it was shown that Borrelia burgdorferi induces the antioxidative metabolism in primary human monocytes. In this study, we explore how B. burgdorferi affects the anti-oxidative arm of redox metabolism, i.e. the generation of reactive oxygen species (ROS). Peripheral blood mononuclear cells (PBMCs) were exposed to B. burgdorferi and generation of ROS was determined both after acute stimulation and after re-stimulation with a secondary stimulus. Though the spirochete induces very low levels of ROS itself, it dramatically decreases the long-term capacity of PBMCs to generate ROS in response to serum-opsonized zymosan (SOZ). This was followed by a compensatory overshoot in ROS generation at later time points. The PI3K/Akt pathway and intracellular levels of methionine play an important regulatory role in this process. Dysregulation of oxidative metabolism may be a novel mechanism by which the spirochete modulates the human immune system and evades killing.


Assuntos
Borrelia burgdorferi , Borrelia burgdorferi/fisiologia , Humanos , Leucócitos Mononucleares , NADP/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo
18.
Pharm Res ; 39(5): 837-850, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35484370

RESUMO

PURPOSE: Ulotaront (SEP-363856) is a TAAR1 agonist with 5-HT1A agonist activity currently in clinical development for the treatment of schizophrenia. The objectives of the current study were to characterize the in vitro ADME properties, preclinical PK, and to evaluate the DDI potential of ulotaront and its major metabolite SEP-383103. METHODS: Solubility, permeability, plasma protein binding, CYP inhibition and induction, transporter inhibition and uptake studies were conducted in vitro. Phenotyping studies were conducted using recombinant human CYPs and FMOs, human liver microsomes and human liver homogenates. Preclinical plasma and brain pharmacokinetics were determined after a single intraperitoneal, intravenous, and oral administration of ulotaront. RESULTS: Ulotaront is a compound of high solubility, high permeability, and low binding to plasma proteins. Ulotaront metabolism is mediated via both NADPH-dependent and NADPH-independent pathways, with CYP2D6 as the major metabolizing enzyme. Ulotaront is an inducer of CYP2B6, and an inhibitor of CYP2D6, OCT1 and OCT2, while SEP-383103 is neither a CYP inducer nor a potent inhibitor of CYPs and human transporters. Ulotaront exhibits rapid absorption, greater than 70% bioavailability, approximately 3.5 L/kg volume of distribution, 1.5-4 h half-life, 12-43 ml/min/kg clearance, and good penetration across the blood-brain barrier in preclinical species. CONCLUSIONS: Ulotaront has been designated as a BCS1 compound by US FDA. The ability of ulotaront to penetrate the blood-brain barrier for CNS targeting has been demonstrated in mice and rats. The potential for ulotaront and SEP-383103 to act as perpetrators of CYP and transporter-mediated DDIs is predicted to be remote.


Assuntos
Receptor 5-HT1A de Serotonina , Esquizofrenia , Animais , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , NADP/metabolismo , NADP/farmacologia , Preparações Farmacêuticas/metabolismo , Ratos , Receptor 5-HT1A de Serotonina/metabolismo , Esquizofrenia/tratamento farmacológico
19.
Biophys J ; 121(10): 1799-1812, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35443926

RESUMO

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR's efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP's flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR's higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR's dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR's higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.


Assuntos
Nêutrons , Oxirredutases , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Sulfito Redutase (NADPH)/química , Sulfito Redutase (NADPH)/metabolismo , Enxofre
20.
Cell Calcium ; 104: 102582, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35462080

RESUMO

Ca2+ signalling is of prime importance in controlling numerous cell functions in the brain. Endolysosomes are acidic organelles currently emerging as important Ca2+ stores in astrocytes, microglia, endothelial cells, and neurons. In neurons, these acidic Ca2+ stores are found in axons, soma, dendrites, and axon endings and could provide local sources of Ca2+ to control synaptic transmission, neuronal plasticity, and autophagy to name a few. This review will address how acidic Ca2+ stores are recruited in response to cell stimulation. We will focus on the role of the endolysosomal two-pore channels (TPCs) and their physiological agonist nicotinic acid adenine dinucleotide phosphate (NAADP) and how they interact with cyclic ADP-ribose and ryanodine receptors from the endoplasmic reticulum. Finally, this review will describe new pharmacological tools and animal mutant models now available to explore acidic Ca2+ stores as key elements in brain function and dysfunction.


Assuntos
Sinalização do Cálcio , Cálcio , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , NADP/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
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