Differential effects of TR ligands on hormone dissociation rates: Evidence for multiple ligand entry/exit pathways

Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here we show, that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation (T(1/2) < 2 h) of , radiolabeled T(3) vs. T(3) (T(1/2), approximate to 5-7 h). We cannot discern relationships between this effect and ligand size, activity or affinity for TR beta. One ligand, GC-24, binds the TR LBC and (weakly) to the TR beta-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T(3) dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T(3) release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T(3) release and that this effect enhances hormone dissociation. (C) 2009 Elsevier Ltd. All rights reserved.

Ligand induced interaction of thyroid hormone receptor beta with its coregulators

Thyroid hormones exert most of their physiological effects through two thyroid hormone receptor (TR) subtypes, TR alpha and TR beta, which associate with many transcriptional coregulators to mediate activation or repression of target genes. The search for selective TR beta ligands has been stimulated by the finding that several pharmacological actions mediated by TR beta might be beneficial in medical conditions such as obesity, hypercholesterolemia and diabetes. Here, we present a new methodology which employs surface plasmon resonance to investigate the interactions between TR beta ligand binding domain (LBD) complexes and peptides derived from the nuclear receptor interaction motifs of two of its coregulators, SRC2 and DAX1. The effect of several TR beta ligands, including the TR beta selective agonist GC-I and the TR beta selective antagonist NH-3, were investigated. We also determined the kinetic rate constants for the interaction of TR beta-T3 with both coregulators, and accessed the thermodynamic parameters for the interaction with DAX1. Our findings Suggest that flexibility plays an important role in the interaction between the receptor and its coregulators. and point out important aspects of experimental design that should be addressed when using TR beta LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR beta ligands. (C) 2008 Elsevier Ltd. All rights reserved.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.

Analysis of Agonist and Antagonist Effects on Thyroid Hormone Receptor Conformation by Hydrogen/Deuterium Exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 mu M. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2`-deoxyguanosine and 1,N(2)-etheno-2`-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals

The effects of nitrosative species on cyt c structure and peroxidase activity were investigated here in the presence of O(2)(center dot-) and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt c heme iron and/or molecular oxygen as electron acceptor. Far-and near-UV CD spectra of SIN1-treated cyt c revealed respectively a slight decrease of a-helix content (from 39 to 34%) and changes in the tryptophan structure accompanied by increased fluorescence. The Soret CD spectra displayed a significant decrease of the positive signal at 403 nm. EPR spectra revealed the presence of a low-spin cyt c form (S = 1/2) with g(1) = 2.736, g(2) = 2.465, and g(3) = 2.058 after incubation with SIN1. These data suggest that the concomitant presence of NO(center dot) and O(2)(center dot-) generated from dissolved oxygen, in a system containing cyt c and liposomes, promotes chemical and conformational modi. cations in cyt c, resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O(2)(center dot-) prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt c itself due to the ability of O(2)(center dot-) to reduce heme iron. Finally, lipoperoxidation measurements showed that, although it is a more efficient peroxidase, SIN1-treated cyt c is not more effective than native cyt c in promoting damage to anionic liposomes in the presence of tert-ButylOOH, probably due to loss of affinity with negatively charged lipids. (C) 2009 Elsevier Inc. All rights reserved.

Thymine hydroperoxide as a potential source of singlet molecular oxygen in DNA

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O(2) ((1)Delta(g))] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O(2) ((1)Delta(g)). Spectroscopic evidence for generation of O(2) ((1)Delta(g)) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D(2)O enhancement of O(2) ((1)Delta(g)) production and the quenching effect of NaN(3). Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O(2) ((1)Delta(g)) derived from the H(2)O(2)/HOCl system and from the thermolysis of the N,N`-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O(2) ((1)Delta(g)) generation was obtained by chemical trapping of O(2) ((1)Delta(g)) with anthracene-9,10-divinylsulfonate (AVS) and detection of the specific AVS endoperoxide by HPLC/MS/MS. The detection by HPLC/MS of 5-(hydroxymethyl)uracil and 5-formyluracil, two thymine oxidation products generated from the reaction of 5-HPMU and Ce(4+) ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O(2) ((1)Delta(g)) by the Russell mechanism and point to the involvement of O(2) ((1)Delta(g)) in thymidine hydroperoxide cytotoxicity. (C) 2009 Elsevier Inc. All rights reserved.

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

Sodium Tungstate on Some Biochemical Parameters of the Parotid Salivary Gland of Streptozotocin-Induced Diabetic Rats: A Short-Term Study

Several studies have shown the antidiabetic properties of sodium tungstate. In this study, we evaluated some biochemical parameters of the parotid salivary gland of streptozotocin-induced diabetic rats treated with sodium tungstate solution (2 mg/ml). The studied groups were: untreated control (UC), treated control (TC), untreated diabetic (UD), and treated diabetic (TD). After 2 and 6 weeks of treatment, parotid gland was removed and total protein and sialic acid (free and total) concentration and amylase and peroxidase activities were determined. Data were compared by variance analysis and Tukey test (p < 0.05). The sodium tungstate treatment modestly decreased the glycemia of streptozotocin-induced diabetic rats. At week 2 of the study, parotid gland of diabetic rats presented a reduction of total protein concentration (55%) and an increase of amylase (120%) and peroxidase (160%) activities, free (150%) and total (170%) sialic acid concentration. No alteration in the evaluated parameters at week 6 of the study was observed. Sodium tungstate presented no significant effect in parotid gland. Our results suggest that diabetes causes initial modification in biochemical composition of parotid. However, this gland showed a recovery capacity after 6 week of the experimental time. Sodium tungstate has no effect in peripheral tissues, such as salivary glands.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

Inhibition of in vivo leishmanicidal mechanisms by tempol: Nitric oxide down-regulation and oxidant scavenging

Tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) has long been known to protect experimental animals from the injury associated with oxidative and inflammatory conditions. In the latter case, a parallel decrease in tissue protein nitration levels has been observed. Protein nitration represents a shift in nitric oxide actions from physiological to pathophysiological and potentially damaging pathways involving its derived oxidants such as nitrogen dioxide and peroxynitrite. In infectious diseases, protein tyrosine nitration of tissues and cells has been taken as evidence for the involvement of nitric oxide-derived oxidants in microbicidal mechanisms. To examine whether tempol inhibits the microbicidal action of macrophages, we investigated its effects on Leishmania amazonensis infection in vitro (RAW 264.7 murine macrophages) and in vivo (C57B1/6 mice). Tempol was administered in the drinking water at 2 mM throughout the experiments and shown to reach infected footpads as the nitroxide plus the hydroxylamine derivative by EPR analysis. At the time of maximum infection (6 weeks), tempol increased footpad lesion size (120%) and parasite burden (150%). In lesion extracts, tempol decreased overall nitric oxide products and expression of inducible nitric oxide synthase to about 80% of the levels in control animals. Nitric oxide-derived products produced by radical mechanisms, such as 3-nitrotyrosine and nitrosothiol, decreased to about 40% of the levels in control mice. The results indicate that tempol worsened L. amazonensis infection by a dual mechanism involving down-regulation of iNOS expression and scavenging of nitric oxide-derived oxidants. Thus, the development of therapeutic strategies based on nitroxides should take into account the potential risk of altering host resistance to parasite infection. (c) 2008 Elsevier Inc. All rights reserved.

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance. (C) 2011 Elsevier Inc. All rights reserved.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

Generation and suppression of singlet oxygen in hair by photosensitization of melanin

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen ((1)O(2)). Irradiation of hair shafts (lambda(ex)>400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by (1)H NMR. After 532-nm excitation, all hair shafts presented the characteristic (1)O(2) emission (lambda(em) = 1270 nm), whose intensity varied inversely with the melanin content. (1)O(2) lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a (1)O(2) suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for (1)O(2) in the solvents in which the hair shafts were suspended, indicating that (1)O(2) is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress (1)O(2), with similar efficiencies. The higher amount of (1)O(2) generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of (1)O(2) in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin. 2011 Published by Elsevier Inc.

Melatonin triggers PKA activation in the rodent malaria parasite Plasmodium chabaudi

Calcium (Ca2+) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra-erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca2+. Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca2+ and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca2+ within the parasite, because buffering Ca2+ changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca2+ signals within the parasite, as did the application of a membrane-permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca2+ and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca2+ evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.