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1.
Med Gas Res ; 13(2): 72-77, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36204786

RESUMO

Diabetic peripheral neuropathy (DPN) is a complex disorder caused by long-standing diabetes. Oxidative stress was considered the critical creed in this DPN pathophysiology. Hydrogen has antioxidative effects on diabetes mellitus and related complications. However, there is still no concern on the beneficial effects of hydrogen in DPN. This paper aimed to evaluate the effects of exogenous hydrogen to reduce the severity of DPN in streptozotocin-induced diabetic rats. Compared with hydrogen-rich saline treatment, hydrogen inhalation significantly reduced blood glucose levels in diabetic rats in the 4th and 8th weeks. With regard to nerve function, hydrogen administration significantly attenuated the decrease in the velocity of motor nerve conduction in diabetic animals. In addition, hydrogen significantly attenuated oxidative stress by reducing the level of malondialdehyde, reactive oxygen species, and 8-hydroxy-2-deoxyguanosine and meaningfully enhanced the antioxidant capability by partially restoring the activities of superoxide dismutase. Further studies showed that hydrogen significantly upregulated the expression of nuclear factor erythroid-2-related factor 2 and downstream proteins such as catalase and hemeoxygenase-1 in the nerves of diabetic animals. Our paper showed that hydrogen exerts significant protective effects in DPN by downregulating oxidative stress via the pathway of nuclear factor erythroid-2-related factor 2, which suggests its potential value in clinical applications.


Assuntos
Diabetes Mellitus Experimental , Neuropatias Diabéticas , Fármacos Neuroprotetores , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Glicemia , Catalase/metabolismo , Catalase/farmacologia , Catalase/uso terapêutico , Desoxiguanosina/metabolismo , Desoxiguanosina/farmacologia , Desoxiguanosina/uso terapêutico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Hidrogênio , Malondialdeído , NAD/metabolismo , NAD/farmacologia , NAD/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio , Estreptozocina , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Superóxido Dismutase/uso terapêutico
2.
Methods Mol Biol ; 2554: 123-139, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36178624

RESUMO

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy is an established technique for detecting and characterizing the binding of small molecules, such as metabolites, to biological macromolecules like proteins and nucleic acids. STD NMR allows detection of binding in complex mixtures of potential ligands, which is often used for library screening in the pharmaceutical industry but may also be beneficial for binding studies with metabolite mixtures. The nature of the ligand is normally restricted to small molecules in terms of NMR spectroscopy, and the size of the macromolecule on the other side should be larger than 10-15 kDa. This technique is especially applicable to detecting binders of intermediate to low affinity with the dissociation constant (KD) above 1 µM. In this chapter, we focus on recent developments and the applications of STD NMR to studying interactions of natural products and metabolites, in particular. The reader is also referred to excellent reviews of the field and the literature cited therein. This chapter also provides a detailed experimental protocol for performing the STD NMR measurement based on the example of the subunit A of the Na+-transporting NADH/ubiquinone oxidoreductase (Na+-NQR) from V. cholerae interacting with its natural quinone substrate and inhibitors.


Assuntos
Produtos Biológicos , Ácidos Nucleicos , Vibrio cholerae , Misturas Complexas , Ligantes , Espectroscopia de Ressonância Magnética/métodos , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/metabolismo , Oxirredutases/metabolismo , Ligação Proteica , Proteínas/química , Ubiquinona/metabolismo , Vibrio cholerae/metabolismo
3.
J Environ Sci (China) ; 124: 952-962, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36182197

RESUMO

The long-term impact of fulvic acid (FA) on partial nitritation (PN) system was initially examined in this study. The obtained results revealed that the FA lower than 50 mg/L had negligible effect on the nitrite accumulation rate (NAR nearly 100%) and ammonium removal rate (ARR 56.85%), while FA over 50 mg/L decreased ARR from 56.85% to 0.7%. Sludge characteristics analysis found that appropriate FA (<50 mg/L) exposure promoted the settling performance and granulation of PN sludge by removing Bacteroidetes and accumulating Chloroflexi. The analysis of metagenomics suggested that the presence of limited FA (0-50 mg/L) stimulated the generation of NADH, which favors the denitrification and nitrite reduction. The negative impact of FA on the PN system could be divided into two stages. Initially, limited FA (50-120 mg/L) was decomposed by Anaerolineae to stimulate the growth and propagation of heterotrophic bacteria (Thauera). Increasing heterotrophs competed with AOB (Nitrosomonas) for dissolved oxygen, causing AOB to be eliminated and ARR to declined. Subsequently, when FA dosage was over 120 mg/L, Anaerolineae were inhibited and heterotrophic bacteria reduced, resulting in the abundance of AOB recovered. Nevertheless, the ammonium transformation pathway was suppressed because genes amoABC and hao were obviously reduced, leading to the deterioration of reactor performance. Overall, these results provide theoretical guidance for the practical application of PN for the treatment of FA-containing sewage.


Assuntos
Compostos de Amônio , Esgotos , Compostos de Amônio/metabolismo , Bactérias/genética , Bactérias/metabolismo , Benzopiranos , Reatores Biológicos/microbiologia , Metagenômica , NAD/metabolismo , Nitritos/metabolismo , Nitrogênio/metabolismo , Oxirredução , Oxigênio/metabolismo , Esgotos/microbiologia
4.
Methods Mol Biol ; 2589: 95-110, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255620

RESUMO

Sirtuins are identified as NAD+-dependent class III histone deacetylases (HDAC) and are involved in a variety of cellular activities, including energy metabolism, DNA repair, epigenetics, gene expression, cell proliferation, differentiation, and survival. Using genetically modified model organisms, sirtuins are proved to be one of the most conserved aging-regulatory and longevity-promoting genes/pathways among species. Of the seven sirtuins, SIRT7 is the only sirtuin that localizes in the nucleolus. SIRT7 senses endogenous and environmental stress to maintain physiological homeostasis. Sirt7 deficient and transgenic mice provide a useful tool to understand the mechanisms of aging and related pathologies. In this chapter, we summarized the most widely applied methods to understand the physiopathological function of SIRT7 in mice.


Assuntos
Sirtuínas , Camundongos , Animais , Sirtuínas/genética , Sirtuínas/metabolismo , NAD/metabolismo , Envelhecimento/genética , Epigênese Genética , Metabolismo Energético
5.
Methods Mol Biol ; 2589: 411-428, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255640

RESUMO

Protein lysine acylation represents one of the most common post-translational modifications. Obviously, highly reactive metabolic intermediates, like thioesters and mixed anhydrides between phosphoric acid and organic acids, modify lysine residues spontaneously. Additionally, enzymes using acyl-CoAs as co-substrates transfer the acyl residue specifically to defined sequences within proteins. The counteracting enzymes are called histone deacetylases (HDACs), releasing the free lysine side chain. Such enzymatic activities are involved in different cellular processes like tumor progression, immune response, regulation of metabolism, and aging. Modulators of such enzymatic activities represent valuable tools in drug discovery. Therefore, direct and continuous assays to monitor enzymatic activity of HDACs are needed. Here we describe different assay formats allowing both monitoring of Zn2+-dependent HDACs via UV-Vis-spectroscopy and NAD+-dependent HDACs (sirtuins) by fluorescence-based assay formats. Additionally, we describe methods enabling efficient screening of HDAC-inhibitors via fluorescence displacement assays.


Assuntos
Histonas , Sirtuínas , Lisina/metabolismo , NAD/metabolismo , Histona Desacetilases/metabolismo , Sirtuínas/metabolismo , Ácidos Fosfóricos/metabolismo , Anidridos
6.
J Sci Food Agric ; 103(1): 450-456, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36205212

RESUMO

BACKGROUND: Nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide, plays an important in anti-aging and disease. Lactococcus lactis, an important probiotic lactic acid bacteria (LAB), has shown great potential for the biosynthesis of NMN, which will significantly affect the probiotic effects of the dairy products. RESULTS: We used the CRISPR/nCas9 technique to knockout nadR gene of L. lactis NZ9000 to enhance the accumulation of NMN by 61%. The nadE* gene from Francisella tularensis with codon optimization was heterologous in L. lactis NZ9000ΔnadR and has a positive effect on NMN production. Combined with optimization of the concentration of substrate nicotinamide, a final intracellular NMN titer was 2289 µmol L-1  mg-1 with 10 g L-1 nicotinamide supplement, which was 5.7-fold higher than that of the control. The transcription levels of key genes (pncA, nadD and prs1) involved in NMN biosynthesis were up-regulated by more than two-fold, indicating that the increase of NMN titer was attributed to FtnadE* heterologous expression. CONCLUSION: Our study provides a better understanding of the NMN biosynthesis pathway in L. lactis, and can facilitate NMN production in LAB via synthetic biology approaches. © 2022 Society of Chemical Industry.


Assuntos
Lactococcus lactis , Mononucleotídeo de Nicotinamida , Mononucleotídeo de Nicotinamida/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , NAD/metabolismo , Niacinamida/metabolismo
7.
Enzyme Microb Technol ; 162: 110122, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36103798

RESUMO

ß-Nicotinamide mononucleotide (NMN) is an important precursor in the synthesis of nicotinamide adenine dinucleotide (NAD+) and confers multiple health benefits, resulting in the rapid growth of NMN market capacity in the fields of food and health care. To overcome the drawbacks of NMN production by the existing chemical or microbial fermentation method, there is an urgent need to develop a prospective NMN production strategy with low cost, low pollution, and high yield. In this study, we demonstrated an artificial in vitro multi-enzyme cascade biocatalysis using starch and nicotinamide (Nam) as substrates for the synthesis of NMN in one-pot. This multi-enzyme cascade reaction was optimized in terms of pH value, buffer concentration, inorganic phosphate concentration, enzyme composition, and phosphoenolpyruvate concentration. Under optimized conditions, a high molar yield of 87.8% for NMN was achieved using 3.2 mM Nam as substrate, and a molar yield of 55.37% for NMN was also achieved under the initial Nam concentration of 9.21 mM. This in vitro enzymatic platform provides an environmental friendliness biomanufacturing technology for the production of NMN, showing a highly promising alternative approach for NMN production.


Assuntos
Niacinamida , Mononucleotídeo de Nicotinamida , Mononucleotídeo de Nicotinamida/metabolismo , Biocatálise , Amido , Estudos Prospectivos , NAD/metabolismo
8.
Sci Total Environ ; 856(Pt 1): 159147, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36183769

RESUMO

Denitrification is the key driving force of nitrogen cycle in surface water and plays an important role in eutrophication water remediation. Compared with some other common carbon sources, such as glucose and sodium acetate, polyhydroxyalkanoates (PHAs) were found to have the distinguished advantages in screening specific denitrifying bacteria of natural surface water bodies. In this study, the large ensembles of taxa were obtained from surface water samples and then sub-cultured with PHA or glucose as the sole carbon source. The microbial community that could be screened by PHA was identified, and the environmental functions of these bacteria were analyzed. At the genus level, the main communities regulated by PHA included Pseudomonas (56.30 %), Acinetobacter (27.75 %), Flavobacterium (10.19 %) and Comamonas (3.14 %), which all had good denitrification ability. The changes in carbon source, nitrogen source and biomass (expressed by DNA) were simultaneously monitored when culturing the model strain (P. stuzeri) with PHA or glucose. Compared with the glucose group, less PHA was consumed to remove the same amount of nitrate within a shorter incubation time, and there was no significant difference in bacterial growth with PHA or glucose as the carbon source (glucose:ΔN:ΔC:ΔDNA = 1:18:0.072; PHA:ΔN:ΔC:ΔDNA = 1:11:0.063). PHA improved the denitrification efficiency by increasing the expression of NarGHI, NirB, NirK and NorB, i.e., the key enzymes in the denitrification process. In addition, PHA accelerated the assimilating rate of extracellular nitrate by bacteria through increasing the expression of NarK. Finally, PHA-regulated electron transfer during denitrification was studied by observing the changes in NADH and NAD+. PHA could use a large proportion of NADH to offer electrons for denitrification, which increased the rate of denitrification. Improved mechanistic insights into the PHA-enhanced denitrification capacity of the microbial community can provide novel options for the in-situ remediation of eutrophic surface water.


Assuntos
Microbiota , Poli-Hidroxialcanoatos , Poli-Hidroxialcanoatos/metabolismo , Desnitrificação , Elétrons , Nitratos , NAD/metabolismo , Nitrogênio , Carbono/metabolismo , Bactérias/metabolismo , Glucose , Água
9.
Commun Biol ; 5(1): 1232, 2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371562

RESUMO

Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and ß cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 ß cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and ß cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and ß cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and ß cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power.


Assuntos
Glucose , Ilhotas Pancreáticas , Humanos , Glucose/metabolismo , NAD/metabolismo , NADP/metabolismo , Ilhotas Pancreáticas/metabolismo , Insulina/metabolismo
10.
Nat Commun ; 13(1): 7031, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396642

RESUMO

An enhanced NADH/NAD+ ratio, termed reductive stress, is associated with many diseases. However, whether a downstream sensing pathway exists to mediate pathogenic outcomes remains unclear. Here, we generate a soluble pyridine nucleotide transhydrogenase from Escherichia coli (EcSTH), which can elevate the NADH/NAD+ ratio and meantime reduce the NADPH/NADP+ ratio. Additionally, we fuse EcSTH with previously described LbNOX (a water-forming NADH oxidase from Lactobacillus brevis) to resume the NADH/NAD+ ratio. With these tools and by using genome-wide CRISPR/Cas9 library screens and metabolic profiling in mammalian cells, we find that accumulated NADH deregulates PRPS2 (Ribose-phosphate pyrophosphokinase 2)-mediated downstream purine biosynthesis to provoke massive energy consumption, and therefore, the induction of energy stress. Blocking purine biosynthesis prevents NADH accumulation-associated cell death in vitro and tissue injury in vivo. These results underscore the pathophysiological role of deregulated purine biosynthesis in NADH accumulation-associated disorders and demonstrate the utility of EcSTH in manipulating NADH/NAD+ and NADPH/NADP+.


Assuntos
Escherichia coli , NAD , Animais , NADP/metabolismo , NAD/metabolismo , Oxirredução , Escherichia coli/metabolismo , Morte Celular , Mamíferos/metabolismo
11.
Cell Death Dis ; 13(11): 944, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36351893

RESUMO

High expression of CD38 in tissues is a characteristic of aging, resulting in a decline in nicotinamide adenine dinucleotide (NAD) and increasing cellular reactive oxygen species (ROS). However, whether CD38 increases susceptibility to ferroptosis remains largely unexplored. Our previous study showed that CD38 overexpression decreased dihydrofolate reductase (DHFR). In the present study, we confirmed that high expression of CD38 increased ROS levels and induced DHFR degradation, which was prevented by nicotinamide mononucleotide (NMN) replenishment. We further revealed that ROS-mediated sulfonation on Cys7 of DHFR induced its degradation via the autophagy and non-canonical proteasome pathways. Mutation of Cys7 to alanine abolished ROS-induced DHFR degradation. Moreover, oxidative degradation of DHFR was responsible for the increased ferroptosis susceptibility of cells in which CD38 was highly expressed. We also found that CD38 expression was higher in bone-marrow-derived macrophages (BMDMs) from aged mice than those from young mice, while the DHFR level was lower. Consequently, we demonstrated that BMDMs from aged mice were more susceptible to ferroptosis that can be reverted by NMN replenishment, suggesting that CD38 high expression rendered cells more susceptible to ferroptosis. Taken together, these results indicated that CD38-mediated NAD+ decline promoted DHFR oxidative degradation, thus resulting in increased cellular susceptibility to ferroptosis and suggesting that NMN replenishment may protect macrophages from ferroptosis in aged mice.


Assuntos
Ferroptose , NAD , Animais , Camundongos , NAD/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Tetra-Hidrofolato Desidrogenase/genética
12.
Biochem Biophys Res Commun ; 636(Pt 1): 89-95, 2022 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-36332487

RESUMO

Nicotinamide adenine dinucleotide (NAD+), a biological molecule integral to redox reactions involved in multiple cellular processes, has the potential to treat nonalcoholic fatty liver diseases (NAFLDs) and nonalcoholic steatohepatitis (NASH). Nicotinamide mononucleotide adenylyltransferase (Nmnat1), one of the NAD+ biosynthesizing enzymes, plays a central role in all NAD+ metabolic pathways and it is vital to embryonic development. However, the function of Nmnat1 in metabolic pathology and, specifically, in the development and progression of NAFLD and NASH remains unexplored. First, we generated hepatic Nmnat1 knockout (H-Nmnat1-/-) mice to investigate the physiological function of Nmnat1 and found that NAD+ levels were significantly lower in H-Nmnat1-/- mice than control mice. However, H-Nmnat1-/- mice appeared normal with comparable metabolic activity. Next, we used three different diet-induced NASH models to assess the pathophysiological role of Nmant1 in metabolic disorders and discovered that hepatic loos of Nmnat1 decreased 35%-40% of total NAD+ in an obese state. Nevertheless, our analysis of phenotypic variations found comparable body composition, gene expression, and liver histology in all NASH models in H-Nmnat1-/- mice. We also found that aged H-Nmnat1-/- mice exhibited comparable liver phenotypes with control mice. These findings suggest that Nmnat1 has a redundancy to the pathophysiology of obesity-induced hepatic disorders.


Assuntos
Nicotinamida-Nucleotídeo Adenililtransferase , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , NAD/metabolismo , Fígado/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Obesidade/metabolismo , Dieta , Camundongos Endogâmicos C57BL
13.
Sci Rep ; 12(1): 19040, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36352014

RESUMO

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for numerous enzymes involved in energy metabolism. Because decreasing NAD levels is a common hallmark of the aging process in various tissues and organs, maintaining NAD levels has recently been of interest for the prevention of aging and age-related diseases. Although placental extract (PE) are known to possess several anti-aging effects, the NAD-boosting activity of PE remains unknown. In this study, we found that porcine PE (PPE) significantly increased intracellular NAD levels in normal human epidermal keratinocytes (NHEKs). PPE also attenuated the NAD depletion induced by FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Interestingly, only the fraction containing nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) restored NAD content in NHEKs in the absence of NAMPT activity. These results suggest that PPE increases intracellular NAD by providing NAD precursors such as NMN, NR, and NAM. Finally, we showed that the application of PPE to the stratum corneum of the reconstructed human epidermis significantly ameliorated FK866-induced NAD depletion, suggesting that topical PPE may be helpful for increasing skin NAD levels. This is the first study to report the novel biological activity of PE as an NAD booster in human epidermal cells.


Assuntos
NAD , Extratos Placentários , Gravidez , Humanos , Animais , Feminino , Suínos , NAD/metabolismo , Placenta/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Epiderme/metabolismo , Queratinócitos/metabolismo
14.
Pharm Biol ; 60(1): 2145-2154, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36373991

RESUMO

CONTEXT: Veratramine may have a potential therapeutic effect for diabetic peripheral neuropathy (DPN). OBJECTIVE: To evaluate whether veratramine ameliorates neuropathic pain in a rat diabetic model. MATERIALS AND METHODS: Sprague-Dawley rats were used for a diabetic model induced by a streptozotocin + high-fat diet. Two months after the induction of the diabetic model, the rats with DPN were screened according to the mechanical pain threshold. The rats with DPN were divided into a model group (n = 12) and a treated group (n = 12). Rats with diabetes, but without peripheral neuropathy, were used in the vehicle group (n = 9). The treatment group received 50 µg/kg veratramine via the tail vein once a day for 4 weeks. During modelling and treatment, rats in all three groups were fed a high-fat diet. RESULTS: The mechanical withdrawal threshold increased from 7.5 ± 1.9 N to 17.9 ± 2.6 N in DPN rats treated with veratramine. The tolerance time of the treated group to hot and cold ectopic pain increased from 11.8 ± 4.2 s and 3.4 ± 0.8 s to 20.4 ± 4.1 s and 5.9 ± 1.7 s, respectively. Veratramine effectively alleviated L4-L5 spinal cord and sciatic nerve pathological injury. Veratramine inhibited the expression of SIGMAR1 and the phosphorylation of the N-methyl-d-aspartate receptor (NMDAR) Ser896 site in spinal cord tissue, as well as inhibited the formation of SIGMAR1-NMDAR and NMDAR-CaMKII complexes. DISCUSSION AND CONCLUSIONS: Veratramine may alleviate the occurrence of pain symptoms in rats with DPN by inhibiting activation of the SIGMAR1-NMDAR pathway.


Assuntos
Diabetes Mellitus , Neuropatias Diabéticas , Neuralgia , Traumatismos dos Nervos Periféricos , Ratos , Animais , Neuropatias Diabéticas/tratamento farmacológico , Ratos Sprague-Dawley , NAD/metabolismo , Neuralgia/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo
15.
Int J Mol Sci ; 23(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36361883

RESUMO

Nephrotoxicity is the dose-limiting factor of cisplatin treatment. Magnesium isoglycyrrhizinate (MgIG) has been reported to ameliorate renal ischemia-reperfusion injury. This study aimed to investigate the protective effect and possible mechanisms of MgIG against cisplatin-induced nephrotoxicity from the perspective of cellular pharmacokinetics. We found that cisplatin predominantly accumulated in mitochondria of renal tubular epithelial cells, and the amount of binding with mitochondrial DNA (mtDNA) was more than twice that with nuclear DNA (nDNA). MgIG significantly lowered the accumulation of cisplatin in mitochondria and, in particular, the degree of target-binding to mtDNA. MgIG notably ameliorated cisplatin-induced changes in mitochondrial membrane potential, morphology, function, and cell viability, while the magnesium donor drugs failed to work. In a mouse model, MgIG significantly alleviated cisplatin-caused renal dysfunction, pathological changes of renal tubules, mitochondrial ultrastructure variations, and disturbed energy metabolism. Both in vitro and in vivo data showed that MgIG recovered the reduction of NAD+-related substances and NAD+-dependent deacetylase sirtuin-3 (SIRT3) level caused by cisplatin. Furthermore, SIRT3 knockdown weakened the protective effect of MgIG on mitochondria, while SIRT3 agonist protected HK-2 cells from cisplatin and specifically reduced platinum-binding activity with mtDNA. In conclusion, MgIG reduces the target-binding amount of platinum to mtDNA and exerts a protective effect on cisplatin-induced renal injury through SIRT3, which may provide a new strategy for the treatment of cisplatin-induced nephrotoxicity.


Assuntos
Cisplatino , Sirtuína 3 , Camundongos , Animais , Cisplatino/efeitos adversos , Cisplatino/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , DNA Mitocondrial/metabolismo , Platina/metabolismo , NAD/metabolismo , Mitocôndrias/metabolismo , Túbulos Renais/metabolismo
16.
Mitochondrion ; 67: 59-64, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36367519

RESUMO

The low cerebral metabolic rate of oxygen despite the relatively preserved perfusion in Alzheimer's disease (AD) patients' medial temporal lobes suggest histotoxic hypoxia due to mitochondrial dysfunction that is independent of, but could precede, insulin resistance. Neuropathological, metabolomic, and preclinical evidence are consistent with the notion that this mitochondrial dysfunction may be contributed to by oxidative stress and DNA damage, leading to poly-(ADP-ribose)-polymerase-1 (PARP1) activation and consequent AMP accumulation, clogging of mitochondrial adenine nucleotide transporters (ANTs), matrix ADP deprivation, and ATP synthase inhibition. Complementary mechanisms may include mitochondrial-protein poly-ADP-ribosylation and mitochondrial-biogenesis suppression via PARPs outcompeting Sirtuin-1 (SIRT1) for nicotinamide-adenine-dinucleotide (NAD+).


Assuntos
Doença de Alzheimer , Poli(ADP-Ribose) Polimerases , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Estresse Oxidativo , NAD/metabolismo , Dano ao DNA , Hipóxia , Trifosfato de Adenosina/metabolismo , Monofosfato de Adenosina , Difosfato de Adenosina/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo
17.
Extremophiles ; 26(3): 37, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36416985

RESUMO

2-Keto-3-deoxy- D-gluconate (KDG) is an important intermediate found in various sugars, sugar acids and polysaccharide catabolic pathways. Here, we report that a functionally uncharacterized type-2 malate/L-lactate dehydrogenase family protein (TTHB078) from Thermus thermophilus HB8 catalyzes a novel reaction, NAD(P)H-dependent reductase activity on KDG. This enzyme, designated KdgG, utilizes both NADH and NADPH as electron donors, but higher activity was observed with NADH. Analysis of the reaction product revealed that KdgG catalyzes reversible reduction of KDG to form 3-deoxy-D-mannonate. Molecular phylogenetic analysis indicated that KdgG and its homologs distributed in the genus Thermus form a novel clade among type-2 malate/L-lactate dehydrogenase family proteins.


Assuntos
L-Lactato Desidrogenase , Thermus thermophilus , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Malatos , Ácido Láctico , NAD/metabolismo , Filogenia
18.
Proc Natl Acad Sci U S A ; 119(48): e2207965119, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36417431

RESUMO

Nucleobase-containing coenzymes are hypothesized to be relics of an early RNA-based world that preceded the emergence of proteins. Despite the importance of coenzyme-protein synergisms, their emergence and evolution remain understudied. An excellent target to address this issue is the Rossmann fold, the most catalytically diverse and abundant protein architecture in nature. We investigated two main Rossmann lineages: the nicotinamide adenine dinucleotide phosphate (NAD(P)) and the S-adenosyl methionine (SAM)- binding superfamilies. To identify the evolutionary changes that lead to a coenzyme specificity switch on these superfamilies, we performed structural and sequence-based Hidden Markov model analysis to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed that through insertions and deletions (InDels) and a residue substitution, the ancient ß1-loop-α1 coenzyme-binding structure of NAD(P) could be reshaped into the SAM-binding ß1-loop-α1 structure. To experimentally prove this obsevation, we removed three amino acids from the NAD(P)-binding pocket and solved the structure of the resulting mutant, revealing the characteristic loop features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD binding and acquiring SAM binding. Our results uncovered how nature may have utilized insertions and deletions to optimize the different coenzyme-binding pockets and the distinct functionalities observed for Rossmann superfamilies. This work also proposes a general mechanism by which protein templates could have been recycled through the course of evolution to adopt different coenzymes and confer distinct chemistries.


Assuntos
Coenzimas , NAD , NAD/metabolismo , Proteínas/química , NADP/metabolismo , S-Adenosilmetionina
19.
Biosensors (Basel) ; 12(11)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36421150

RESUMO

A new spectroelectrochemical two-enzyme sensor system has been developed for the detection of acetaldehyde in wine. A combination of spectroscopy and electrochemistry improves the analytical features of the electrochemical sensor because the optical information collected with this system is only associated with acetaldehyde and avoids the interferents also present in wines as polyphenols. Spectroelectrochemical detection is achieved by the analysis of the optical properties of the K3[Fe(CN)6]/K4[Fe(CN)6] redox couple involved in the enzymatic process: aldehyde dehydrogenase catalyzes the aldehyde oxidation using ß-nicotinamide adenine dinucleotide hydrate (NAD+) as a cofactor and, simultaneously, diaphorase reoxidizes the NADH formed in the first enzymatic process due to the presence of K3[Fe(CN)6]. An analysis of the characteristic UV-vis bands of K3[Fe(CN)6] at 310 and 420 nm allows the detection of acetaldehyde, since absorption bands are only related to the oxidation of this substrate, and avoids the contribution of other interferents.


Assuntos
Acetaldeído , Vinho , Acetaldeído/análise , Vinho/análise , NAD/análise , NAD/química , NAD/metabolismo , Eletroquímica , Oxirredução
20.
Nat Commun ; 13(1): 6121, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253417

RESUMO

In our previous study, we reported that sirtuin5 (SIRT5), a member of the NAD+-dependent class III histone deacetylase family, is highly expressed in colorectal cancer (CRC). Herein we show that SIRT5 knockdown impairs the production of ribose-5-phosphate, which is essential for nucleotide synthesis, resulting in continuous and irreparable DNA damage and consequently leading to cell cycle arrest and enhanced apoptosis in CRC cells. These SIRT5 silencing-induced effects can be reversed by nucleoside supplementation. Mechanistically, SIRT5 activates transketolase (TKT), a key enzyme in the non-oxidative pentose phosphate pathway, in a demalonylation-dependent manner. Furthermore, TKT is essential for SIRT5-induced malignant phenotypes of CRC both in vivo and in vitro. Altogether, SIRT5 silencing induces DNA damage in CRC via post-translational modifications and inhibits tumor growth, suggesting that SIRT5 can serve as a promising target for CRC treatment.


Assuntos
Neoplasias Colorretais , Dano ao DNA , Sirtuínas , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Histona Desacetilases/genética , NAD/metabolismo , Nucleosídeos , Nucleotídeos , Sirtuínas/genética , Sirtuínas/metabolismo , Transcetolase
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