• Molecules and Cells
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• 제42권8호
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• pp.597-603
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• 2019

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al. published the detailed correlation between GAPDH and di- or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at $2.29{\AA}$. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between $NAD^+$-bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the $NAD^+$ cosubstrate due to steric hindrance of the nicotinamide portion of the $NAD^+$ molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.

• BMB Reports
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• 제52권1호
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• pp.24-34
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• 2019

Sirtuin is an essential factor that delays cellular senescence and extends the organismal lifespan through the regulation of diverse cellular processes. Suppression of cellular senescence by Sirtuin is mainly mediated through delaying the age-related telomere attrition, sustaining genome integrity and promotion of DNA damage repair. In addition, Sirtuin modulates the organismal lifespan by interacting with several lifespan regulating signaling pathways including insulin/IGF-1 signaling pathway, AMP-activated protein kinase, and forkhead box O. Although still controversial, it is suggested that the prolongevity effect of Sirtuin is dependent with the level of and with the tissue expression of Sirtuin. Since Sirtuin is also believed to mediate the prolongevity effect of calorie restriction, activators of Sirtuin have attracted the attention of researchers to develop therapeutics for age-related diseases. Resveratrol, a phytochemical rich in the skin of red grapes and wine, has been actively investigated to activate Sirtuin activity with consequent beneficial effects on aging. This article reviews the evidences and controversies regarding the roles of Sirtuin on cellular senescence and lifespan extension, and summarizes the activators of Sirtuin including Sirtuin-activating compounds and compounds that increase the cellular level of nicotinamide dinucleotide.

• BMB Reports
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• 제56권6호
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• pp.353-358
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• 2023

In the present study, to determine the efficacy of oral supplementation of ginseng berry extracts in augmenting exercise performance and exercise-associated metabolism, male mice were given orally 200 and 400 mg/kg of body weight (BW) of GBC for nine weeks. Although there are no differences in pre-exercise blood lactate levels among (1) the control group that received neither exercise nor GBC, (2) the group that performed only twice-weekly endurance exercise, and (3) and (4) the groups that combined twice-weekly endurance exercise with either 200 or 400 mg/kg GBC, statistically significant reductions in post-exercise blood lactate levels were observed in the groups that combined twice-weekly endurance exercise with oral administration of either 200 or 400 mg/kg GBC. Histological analysis showed no muscle hypertrophy, but transcriptome analysis revealed changes in gene sets related to lactate metabolism and mitochondrial function. GBC intake increased nicotinamide adenine dinucleotide levels in the gastrocnemius, possibly enhancing the mitochondrial electron transport system and lactate metabolism. Further molecular mechanisms are needed to confirm this hypothesis.

• Toxicological Research
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• 제32권3호
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• pp.207-213
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• 2016

Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an $IC_{50}$ value of 6.9, 16.8, and $43.1{\mu}g/mL$, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2.

• Asian-Australasian Journal of Animal Sciences
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• 제30권6호
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• pp.857-864
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• 2017

Objective: The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK) activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. Methods: A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-${\beta}$-D-ribofuranoside (AICAR, a specific activator of AMPK), AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II) and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases). After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. Results: Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. Conclusion: Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

• Molecules and Cells
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• 제41권4호
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• pp.331-341
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• 2018

L-pipecolic acid is a non-protein amino acid commonly found in plants, animals, and microorganisms. It is a well-known precursor to numerous microbial secondary metabolites and pharmaceuticals, including anticancer agents, immunosuppressants, and several antibiotics. Lysine cyclodeaminase (LCD) catalyzes ${\beta}$-deamination of L-lysine into L-pipecolic acid using ${\beta}$-nicotinamide adenine dinucleotide as a cofactor. Expression of a human homolog of LCD, ${\mu}$-crystallin, is elevated in prostate cancer patients. To understand the structural features and catalytic mechanisms of LCD, we determined the crystal structures of Streptomyces pristinaespiralis LCD (SpLCD) in (i) a binary complex with $NAD^+$, (ii) a ternary complex with $NAD^+$ and L-pipecolic acid, (iii) a ternary complex with $NAD^+$ and L-proline, and (iv) a ternary complex with $NAD^+$ and L-2,4-diamino butyric acid. The overall structure of SpLCD was similar to that of ornithine cyclodeaminase from Pseudomonas putida. In addition, SpLCD recognized L-lysine, L-ornithine, and L-2,4-diamino butyric acid despite differences in the active site, including differences in hydrogen bonding by Asp236, which corresponds with Asp228 from Pseudomonas putida ornithine cyclodeaminase. The substrate binding pocket of SpLCD allowed substrates smaller than lysine to bind, thus enabling binding to ornithine and L-2,4-diamino butyric acid. Our structural and biochemical data facilitate a detailed understanding of substrate and product recognition, thus providing evidence for a reaction mechanism for SpLCD. The proposed mechanism is unusual in that $NAD^+$ is initially converted into NADH and then reverted back into $NAD^+$ at a late stage of the reaction.

• Toxicological Research
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• 제17권
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• pp.329-333
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• 2001

Apoptosis is the normal physiological process of cell death essential for the maintenance of homeostasis. The function of nicotinamide adenine dinucleotide (NAD) and adenine diphosphate (ADP) ribosylation (transfer of ADP-ribose to proteins) reactions in modifying apoptosis have recently been of great interest. Recently. CD38. a type 2 transmembrane glycoprotein expressed in hematopoietic and non hematopoietic cell lines. has been reported to possess NAD glycohydrolase activity (Han. 1999) and PC-1 and CD38 NADase regulates T cells by inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycan (Hozada et al., 1999). Sindhuphak et al. (2000) has reported that cervical cancer cells can be differentiated from normal cells by using FTIR (Fourier-Transformed Infrared) technique. which has characterized shifts to be due to the phosphodiester bond in nucleic acid. protein amide I&II. carbohydrate and glycogen bands. Mechanisms how phosphodiester bond shift in cervical cancer cells as compared to control cells remain to be elucidated. Suramana et al. (2000) as well as Lohitnavy and Sinhaseni (1998) have studied methomyl and colchicine effects in rat splenocytes. Lactate Dehydroge-nase Isozymes 3 (LDH3) and LDH4 were observed to increase transiently and subsided in plasma of rats exposed to 6~8 mg/kg methomyl after 48 hours. Phosphodiester bond shift of nucleic acid. detected by FTIR. was also reported (Suramana et al., 2000). We report here, after analysis of bond shift patterns. a similar bond shifts detected by FTIR spectrum observed in human cervical cells and splenocytes of rats exposed orally to 2~8 mg/kg methomyl as well as rats exposed to colchicine 2~6 mg/kg orally.

• 한국미생물·생명공학회지
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• 제47권4호
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• pp.536-545
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• 2019

Cytochrome P450 (P450 or CYP) is involved in the metabolism of endogenous and exogenous compounds in most organisms. P450s have great potential as biocatalysts in the pharmaceutical and fine chemical industries because they catalyze diverse oxidative reactions using a wide range of substrates. The high-cost nicotinamide cofactor, NADPH, is essential for P450 reactions. Glucose-6-phosphate dehydrogenase (G6PDH) has been commonly used in NADPH-generating systems (NGSs) to provide NADPH for P450 reactions. Currently, only two G6PDHs from Leuconostoc mesenteroides and Saccharomyces cerevisiae can be obtained commercially. To supply high-cost G6PDH cost-effectively, we cloned the cytosolic G6PDH gene of Solanum lycopersicum (tomato) with 6xHis tag, expressed it in Escherichia coli, and purified the recombinant G6PDH (His-G6PDH) using affinity chromatography. In addition, enzymatic properties of His-G6PDH were investigated, and the His-G6PDH-coupled NGS was optimized for P450 reactions. His-G6PDH supported CYP102A1-catalyzed hydroxylation of omeprazole and testosterone by NADPH generation. This result suggests that tomato His-G6PDH could be a cost-effective enzyme source for NGSs for P450-catalyzed reactions as well as other NADPH-requiring reactions.

• 한국산학기술학회논문지
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• 제15권9호
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• pp.5594-5601
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• 2014

본 연구는 fomalin으로 유발한 실험동물의 안면부 통증 모델에서 사물탕의 항염증 작용과 항산화 작용을 확인하고자 수행되었다. Formalin 주입 전 사물탕추출물(3.5 mg/1 ml)을 3일간 복강 투여하였고, 3일차 사물탕 주입 30분 후 5% formalin($50{\mu}{\ell}$)을 실험동물의 오른쪽 수염부 피하에 주입하여 안면부 통증을 유발하였다. 사물탕 투여에 따른 안면부 통증행위반응을 측정하였다. 통증신호를 매개하는 염증과 산화작용의 지표로 실험동물의 뇌와 연수에서 p38 MAPK, iNOS, NOX4의 발현을 단백정량분석법으로 평가하였다. 사물탕은 안면부 통증행위반응을 유의하게 감소시켰을 뿐만 아니라, 염증성 매개 인자인 p38 MAPK, iNOS의 발현을 억제하였고 NOX4의 조절을 통한 항산화작용을 나타내었다. 이러한 결과를 통하여 사물탕의 투여가 안면부 통증 조절에 중요한 역할을 담당할 것으로 생각된다.

• Nutrition Research and Practice
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• 제11권5호
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• pp.430-434
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• 2017

BACKGROUND/OBJECTIVE: Chronic hyperglycemia induces oxidative stress via accumulation of reactive oxygen species (ROS) and contributes to diabetic complications. Hyperglycemia induces mitochondrial superoxide anion production through the increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This study aimed to determine whether fisetin and luteolin treatments suppress the oxidative stress by modulating the expression of sirtuins (SIRTs) and forkhead box O3a (FOXO3a) under hyperglycemic conditions in human monocytes. MATERIALS/METHODS: Human monocytic cells (THP-1) were cultured under osmotic control (14.5 mmol/L mannitol), normoglycemic (NG, 5.5 mmol/L glucose), or hyperglycemic (HG, 20 mmol/L glucose) conditions, in the absence or presence of fisetin and luteolin for 48 h. To determine the effect of fisetin and luteolin treatments on high glucose-induced oxidative stress, western blotting and intracellular staining were performed. RESULTS: Hyperglycemic conditions increased the ROS production, as compared to normoglycemic condition. However, fisetin and luteolin treatments inhibited ROS production under hyperglycemia. To obtain further insight into ROS production in hyperglycemic conditions, evaluation of p47phox expression revealed that fisetin and luteolin treatments inhibited p47phox expression under hyperglycemic conditions. Conversely, the expression levels of SIRT1, SIRT3, SIRT6, and FOXO3a were decreased under high glucose conditions compared to normal glucose conditions, but exposure to fisetin and luteolin induced the expression of SIRT1, SIRT3, SIRT6, and FOXO3a. The above findings suggest that fisetin and luteolin inhibited high glucose-induced ROS production in monocytes through the activation of SIRTs and FOXO3a. CONCLUSIONS: The results of our study supports current researches that state fisetin and luteolin as potential agents for the development of novel strategies for diabetes.