On the Mechanism of Interaction of Steroids with Human Glucose 6-Phosphate Dehydrogenase

Gordon, Geoffrey L. R.; Mackow, Mary C.; Levy, Haim

doi:10.1006/abbi.1995.1199

Cited by 118 publications

(94 citation statements)

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“…The coincubation with DPI significantly inhibited both basal and oxidative stressstimulated PPP, ruling out that DPI inhibits the PPP activity by blocking NADPH oxidase and suggesting that such inhibition is exerted at a different level, more likely at the G6PD step. Indeed, DHEA, a potent uncompetitive inhibitor of G6PD (35,36), significantly inhibited the PPP and abolished its activation by H 2 O 2 , confirming that also in N11 cells, G6PD is the rate-limiting step in the PPP and that the PPP measurement is a useful tool to monitor the actual G6PD activity in whole cells (18). GSH is the most important antioxidant agent in the cells, where it is present at mM concentrations.…”

Section: Resultsmentioning

confidence: 77%

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Riganti

Gazzano

Polimeni

et al. 2004

Journal of Biological Chemistry

176

133

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Diphenyleneiodonium (DPI) and the structurally related compound diphenyliodonium (DIP) are widely used as inhibitors of flavoenzymes, particularly NADPH oxidase. Here we report further evidence that DPI and DIP are not specific flavin binders. A 3-h incubation of N11 glial cells with DPI significantly inhibited in a dosedependent way both the pentose phosphate pathway and the tricarboxylic acid cycle. In parallel, we observed a dose-dependent increase of reactive oxygen species generation and lipoperoxidation and increased leakage of lactate dehydrogenase activity in the extracellular medium. The glutathione/glutathione disulfide ratio decreased, whereas the efflux of glutathione out of the cells increased. This suggests that DPI causes an augmented oxidative stress and exerts a cytotoxic effect in N11 cells. Indeed, the cells were protected from these events when loaded with glutathione. Similar results were observed using DIP instead of DPI and also in other cell types. We suggest that the DPI-elicited inhibition of the pentose phosphate pathway and tricarboxylic acid cycle may be mediated by the blockade of several NAD(P)-dependent enzymes, such as glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase. In light of these results, we think that some effects of DPI or DIP in in vitro and in vivo experimental models should be interpreted with caution. Diphenyleneiodonium (DPI) 1 and the structurally related compound diphenyliodonium (DIP) are widely used as uncompetitive inhibitors of flavoenzymes. Firstly identified as a hypoglycemic agent able to block gluconeogenesis and respiration in rat liver (1), DPI has been subsequently shown to inhibit the activity of NADH:ubiquinone oxidoreductase (2, 3), NADPH oxidase (4 -6), nitric-oxide synthase (7), xanthine oxidase (6), and NADPH cytochrome P450 oxidoreductase (8). DPI and other iodonium derivatives have been shown to react via a radical mechanism, whereby an electron is abstracted from FAD or FMN to form a radical, which then adds back to the flavin to form covalent, phenylated adducts (9). Also, heme groups, such as the heme b of NADPH oxidase, have been found to react with DPI and DIP (10).In most experimental works of the last years, DPI or DIP have been used as inhibitors of NADPH oxidase. NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells of mesodermal origin. The most thoroughly studied is the leukocyte isoform, which catalyzes the production of superoxide (O 2 . ) by the one-electron reduction of oxygen, using NADPH as the reducing agent (11). The O 2 .generated by NADPH oxidase serves as the starting material for the production of a vast assortment of reactive oxidants used by phagocytes to kill invading microorganisms or tumor cells (11). A low activity NADPH oxidase is present in a variety of nonphagocytic cells, wherein this enzyme is a source of second messengers. It has been postulated, for instance, that the O 2 . generated by the aorta functions as a blood ...

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Section: Resultsmentioning

confidence: 77%

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Riganti

Gazzano

Polimeni

et al. 2004

Journal of Biological Chemistry

176

133

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“…NADPH/ NADP ϩ ratio is a critical modulator of intracellular redox states and is mainly determined by the pentose phosphate pathway through the action of glucose-6-phosphate dehydrogenase (G6PD) (16,17). DHEA is an endogenous steroid (18,19), which is known to be an uncompetitive inhibitor of G6PD, among other functions (20). Inhibition of G6PD could lead to a 30-40% decrease in the NADPH/NADP ϩ ratio (21).…”

Section: Resultsmentioning

confidence: 99%

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

Zheng¹,

Dai²,

Zhao³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Rossmann fold ͉ signal transduction N AD and its phosphorylated form, NADP, is a universal cofactor in a large number of redox reactions carried out by a variety of enzymes, especially dehydrogenases. It is widely recognized that NAD(P) is an essential molecule in energy metabolism in all organisms. More recently, mounting evidence suggests that, in addition to its central role in energy metabolism, NAD(P) is also involved in signaling pathways that regulate fundamental processes of cellular functions, including gene transcription and apoptosis (1,2). This realization generated a link between the energy metabolism and the regulated networks of biological processes.One of the NAD signaling mechanisms is through gene silencer proteins known as sirtuins, such as Sir2 in yeast, a NAD-dependent histone deacetylase. Increased activities of Sir2 induced by an increase in NAD production extended lifespan (3). It was shown that a product of NAD-dependent deacetylation by Sir2, nicotinamide, strongly inhibits the activity of Sir2 (4, 5). Similar mechanisms are present in other eukaryotes, including humans (6). NAD is also directly related to transcription regulation. PolyADP-ribose polymerases (PARPs) can modify the acceptor proteins by synthesizing a polyADP-ribose molecule using NAD ϩ as the substrate. The transcription factors that can be modified by PARPs include p53, YY1, NF-B, and TATA-binding protein (1). In other cases, transcription regulation is not carried out by any enzymatic reaction. For instance, the C-terminal-binding protein CtBP is a corepressor that has an increased affinity to its partners, such as adenovirus E1A or cellular repressor ZEB, when NADH binds to CtBP (7). Crystal structures showed that NADH binding to CtBP induced a conformational switch that stabilizes the dimerization of CtBP, which in turn promotes its binding to the repressors (8). In the case of the negative transcriptional regulator NmrA, NAD ϩ binding to NmrA controls the rate of nuclear entry of the GATA transcription-activating protein AreA (9, 10). NmrA has a similar structure to short-chain dehydrogenase/reductase (SDR) family proteins but no enzyme activities, because of the lack of conserved active-site residues (9). NAD(P) exerts its functions by association with proteins. The protein fold that binds NAD(P) was discovered by Rossmann when the crystal structure of lactate dehydrogenase was determined (11). The Rossmann fold is the most common fold, based on its predicted occurrence from the genes known today (12). This motif consists of six ␤-strands connected by ␣-helices with the NAD(P) molecule bound at the top [supporting information (SI) Fig. 5]. The Rossmann fold has always been present as a rigid-body domain in all other known crystal structures until the structure of HSCARG was determined. We found that the common ␣E that connects ␤5 to ␤6 in the Rossmann fold is deformed as an extended loop when NADP is not bound with HSCARG. This allows the formation of an asymmetric dimer between a subunit with NADP bound and an emp...

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“…Namely, DHEA could inhibit G6PDH directly 34 or indirectly 35 ; this is important because Leopold et al 36 showed that G6PDH inhibition blocks the Akt axis, at least in bovine endothelial cells. G6PDH is the first enzyme of the pentose-phosphate pathway, a major producer of NADPH (an important regulator of the cellular redox status) and ribulose 5-P (the precursor of ribose 5-P, ie, a critical building block of nucleic acids).…”

Section: Discussionmentioning

confidence: 99%

Dehydroepiandrosterone Reverses Systemic Vascular Remodeling Through the Inhibition of the Akt/GSK3-β/NFAT Axis

et al. 2009

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Background-The remodeled vessel wall in many vascular diseases such as restenosis after injury is characterized by proliferative and apoptosis-resistant vascular smooth muscle cells. There is evidence that proproliferative and antiapoptotic states are characterized by a metabolic (glycolytic phenotype and hyperpolarized mitochondria) and electric (downregulation and inhibition of plasmalemmal K ϩ channels) remodeling that involves activation of the Akt pathway. Dehydroepiandrosterone (DHEA) is a naturally occurring and clinically used steroid known to inhibit the Akt axis in cancer. We hypothesized that DHEA will prevent and reverse the remodeling that follows vascular injury. Methods and Results-We used cultured human carotid vascular smooth muscle cell and saphenous vein grafts in tissue culture, stimulated by platelet-derived growth factor to induce proliferation in vitro and the rat carotid injury model in vivo. DHEA decreased proliferation and increased vascular smooth muscle cell apoptosis in vitro and in vivo, reducing vascular remodeling while sparing healthy tissues after oral intake. Using pharmacological (agonists and antagonists of Akt and its downstream target glycogen-synthase-kinase-3␤ [GSK-3␤]) and molecular (forced expression of constitutively active Akt1) approaches, we showed that the effects of DHEA were mediated by inhibition of Akt and subsequent activation of GSK-3␤, leading to mitochondrial depolarization, increased reactive oxygen species, activation of redox-sensitive plasmalemmal voltage-gated K ϩ channels, and decreased [Ca 2ϩ ] i . These functional changes were accompanied by sustained molecular effects toward the same direction; by decreasing [Ca 2ϩ ] i and inhibiting GSK-3␤, DHEA inhibited the nuclear factor of activated T cells transcription factor, thus increasing expression of Kv channels (Kv1.5) and contributing to sustained mitochondrial depolarization. These results were independent of any steroidrelated effects because they were not altered by androgen and estrogen inhibitors but involved a membrane G protein-coupled receptor. Conclusions-We suggest that the orally available DHEA might be an attractive candidate for the treatment of systemic vascular remodeling, including restenosis, and we propose a novel mechanism of action for this important hormone and

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On the Mechanism of Interaction of Steroids with Human Glucose 6-Phosphate Dehydrogenase

Cited by 118 publications

References 0 publications

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

Dehydroepiandrosterone Reverses Systemic Vascular Remodeling Through the Inhibition of the Akt/GSK3-β/NFAT Axis

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