Selenium supplementation shows protective effects against patulin-induced brain damage in mice via increases in GSH-related enzyme activity and expression

Aims

This study was designed to investigate the protective effects of selenium supplementation on patulin-induced neurotoxicity.

Main methods

Mice were subjected to patulin for 8 weeks. Sodium selenite (Na₂SeO₃) and selenium–methionine (Se–Met) were supplemented with the diet, and we investigated the effects of selenium on patulin-induced neurotoxicity. The animals were randomly divided into 4 groups containing 6–8 mice each. The first group was used as a control, and only physiological saline (0.9%) was injected. The second group was treated with patulin (1 mg/kg) intraperitoneally. The third group was treated with patulin (1 mg/kg) along with a dietary supplementation of Na₂SeO₃ (0.2 mg Se/kg of diet). The fourth group was treated with patulin (1 mg/kg) plus Se–Met (0.2 mg Se/kg of diet).

Key findings

Patulin treatment increased oxidative damage in the brain, as evidenced by a decrease in non-protein thiol and total thiol groups, along with significant increases in GSSG, reactive oxygen species, thiobarbituric acid reactive substances and protein carbonyl levels. Moreover, the activities of glutathione peroxidase (GPx) and glutathione reductase were inhibited with patulin treatment. Selenium supplementation significantly ameliorated these biological parameter changes. In addition, selenium treatments significantly increased the mRNA levels of GPx-1, GPx-4 and thioredoxin reductase.

Significance

Our data show that selenium supplementation increases the activity and expression of glutathione-related enzymes and offers significant protection against brain damage induced by patulin.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Glutathione peroxidases in different stages of carcinogenesis

Cancer cells produce high amounts of reactive oxygen species (ROS) and evade apoptosis. Hydroperoxides support proliferation, invasion, migration and angiogenesis, but at higher levels induce apoptosis, thus being pro- and anti-carcinogenic. Accordingly, glutathione peroxidases (GPxs) regulating hydroperoxide levels might have dual roles too. GPx1, clearly an antioxidant enzyme, is down-regulated in many cancer cells. Its main role would be prevention of cancer initiation by ROS-mediated DNA damage. GPx2 is up-regulated in cancer cells. GPx1/GPx2 double knockout mice develop colitis and intestinal cancer. However, GPx2 knockdown cancer cells grow better in vitro and in vivo probably reflecting the physiological role of GPx2 in intestinal mucosa homeostasis. GPx2 counteracts COX-2 expression and PGE₂ production, which explains its potential to inhibit migration and invasion of cultured cancer cells. Overexpression of GPx3 inhibits tumor growth and metastasis. GPx4 is decreased in cancer tissues. GPx4-overexpressing cancer cells have low COX-2 activity and tumors derived therefrom are smaller than from control cells and do not metastasize. Collectively, GPxs prevent cancer initiation by removing hydroperoxides. GPx4 inhibits but GPx2 supports growth of established tumors. Metastasis, but also apoptosis, is inhibited by all GPxs. GPx-mediated regulation of COX/LOX activities may be relevant to early stages of inflammation-mediated carcinogenesis.     

Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes

Background

Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances.

Scope of review

This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family.

Major conclusions

It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid–base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions.

General significance

Glutathione-dependent enzymes are excellent study objects for structure–function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Metabolic control analysis of the Trypanosoma cruzi peroxide detoxification pathway identifies tryparedoxin as a suitable drug target

Background

The principal oxidative-stress defense in the human parasite Trypanosoma cruzi is the tryparedoxin-dependent peroxide detoxification pathway, constituted by trypanothione reductase (TryR), tryparedoxin (TXN), tryparedoxin peroxidase (TXNPx) and tryparedoxin-dependent glutathione peroxidase A (GPxA). Here, Metabolic Control Analysis (MCA) was applied to quantitatively prioritize drug target(s) within the pathway by identifying its flux-controlling enzymes.

Methods

The recombinant enzymes were kinetically characterized at physiological pH/temperature. Further, the pathway was in vitro reconstituted using enzyme activity ratios and fluxes similar to those observed in the parasites; then, enzyme and substrate titrations were performed to determine their degree of control on flux. Also, kinetic characterization of the whole pathway was performed.

Results

Analyses of the kinetic properties indicated that TXN is the less efficient pathway enzyme derived from its high Km_app for trypanothione and low Vmax values within the cell. MCA established that the TXN–TXNPx and TXN–GPxA redox pairs controlled by 90–100% the pathway flux, whereas 10% control was attained by TryR. The Km_app values of the complete pathway for substrates suggested that the pathway flux was determined by the peroxide availability, whereas at high peroxide concentrations, flux may be limited by NADPH.

Conclusion

These quantitative kinetic and metabolic analyses pointed out to TXN as a convenient drug target due to its low catalytic efficiency, high control on the flux of peroxide detoxification and role as provider of reducing equivalents to the two main peroxidases in the parasite.

General Significance

MCA studies provide rational and quantitative criteria to select enzymes for drug-target development.     

Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (C_P). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the C_P is formed with an alleged resolving Cys (C_R) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional C_R suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which C_P both reduces H₂O₂ and oxidizes PDI, and that the C_P-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.     

A T/C polymorphism in the GPX4 3′UTR affects the selenoprotein expression pattern and cell viability in transfected Caco-2 cells

Background

Synthesis of selenoproteins such as glutathione peroxidases (GPx) requires a specific tRNA and a stem-loop structure in the 3′untranslated region (3′UTR) of the mRNA. A common single nucleotide polymorphism occurs in the GPX4 gene in a region corresponding to the 3′UTR.

Methods

The two variant 3′UTR sequences were linked to sequences from a selenoprotein reporter gene (iodothyronine deiodinase) and expressed in Caco-2 cells. Clones expressing comparable levels of deiodinase (assessed by real-time PCR) were selected and their response to tert-butyl hydroperoxide assessed by cell viability and measurement of reactive oxygen species. Selenoprotein expression was assessed by real-time PCR, enzyme activity and immunoassay.

Results

When selenium supply was low, cells overexpressing the C variant 3′UTR showed lower viability after oxidative challenge, increased levels of reactive oxygen species and lower GPx activity and SelH mRNA expression compared to cells overexpressing the T variant. After selenium supplementation, cell viability and GPx4 expression were higher in the cells overexpressing the C variant. Expression of transgenes incorporating the T/C variant GPX4 (rs713041) sequences in Caco-2 cells leads to alterations in both cell viability after an oxidative challenge and selenoprotein expression. This suggests that the two variants compete differently in the selenoprotein hierarchy.

General Significance

The data provide evidence that the T/C variant GPX4 (rs713041) alters the pattern of selenoprotein synthesis if selenium intake is low. Further work is required to assess the impact on disease susceptibility.     

Glutathione peroxidases and redox-regulated transcription factors

Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides. cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-kappaB activation by interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis. GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells. GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways. In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs.     

Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult

Background

Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1–6) that can reduce hydrogen peroxide (H₂O₂) and organic hydroperoxides to water and alcohols.

Methods

We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis.

Results

Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H₂O₂-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H₂O₂-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage.

Conclusions

These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo.

General significance

PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.     

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity

Background

Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis.

Methods

The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9 Å and 1.7 Å by synchrotron radiation and the molecular replacement method.

Results

The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H₂ into its E₂ form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity.

Conclusion

We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity.

General significance

These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.     

Peroxiredoxins as biomarkers of oxidative stress

Background

Peroxiredoxins (Prxs) are a class of abundant thiol peroxidases that degrade hydroperoxides to water. Prxs are sensitive to oxidation, and it is hypothesized that they also act as redox sensors. The accumulation of oxidized Prxs may indicate disruption of cellular redox homeostasis.

Scope of review

This review discusses the biochemical properties of the Prxs that make them suitable as endogenous biomarkers of oxidative stress, and describes the methodology available for measuring Prx oxidation in biological systems.

Major conclusions

Two Prx oxidation products accumulate in cells under increased oxidative stress: an intermolecular disulfide and a hyperoxidized form. Methodologies are available for measuring both of these redox states, and oxidation has been reported in cells and tissues under oxidative stress from external or internal sources.

General significance

Monitoring the oxidation state of Prxs provides insight into disturbances of cellular redox homeostasis, and complements the use of exogenous probes of oxidative stress. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Stabilities and structures of islet amyloid polypeptide (IAPP22–28) oligomers: From dimer to 16-mer

Background

The formation of amyloid fibrils is associated with many age-related degenerative diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood.

Methods

Here, we performed MD simulations for the NFGAILS motif of hIAPP associated with the type II diabetes to estimate the stabilities of hIAPP_22–28 protofibrils with different sizes: from 2 to 16 chains. In addition, to study the initial self-assembly stage, 4 and 8 IAPP_22–28 chains in explicit solvent were also simulated.

Results

Our results indicate that the ordered protofibrils with no more than 16 hIAPP_22–28 chains will be structurally stable in two layers, while one-layer or three-layer models are not stable as expected. Furthermore, the oligomerization simulations show that the initial coil structures of peptides can quickly aggregate and convert to partially ordered β-sheet-rich oligomers.

Conclusions

Based on the obtained results, we found that the stability of an IAPP_22–28 oligomer was not only related with its size but also with its morphology. The driving forces to form and stabilize an oligomer are the hydrophobic effects and backbone H-bond interaction. Our simulations also indicate that IAPP_22–28 peptides tend to form an antiparallel strand orientation within the sheet.

General significance

Our finding can not only enhance the understanding about potential mechanisms of hIAPP nuclei formation and the extensive structural polymorphisms of oligomers, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

Function of thyroid hormone transporters in the central nervous system

Background

Iodothyronines are charged amino acid derivatives that cannot passively cross a phospholipid bilayer. Transport of thyroid hormones across plasma membranes is mediated by integral membrane proteins belonging to several gene families. These transporters therefore allow or limit access of thyroid hormones into brain. Since thyroid hormones are essential for brain development and cell differentiation, it is expected that genetic deficiency of such transporters would result in neurodevelopmental derangements.

Scope of review

We introduce concepts of thyroid hormone transport into the brain and into brain cells. Important thyroid hormone transmembrane transporters are presented along with their expression patterns in different brain cell types. A focus is placed on monocarboxylate transporter 8 (MCT8) which has been identified as an essential thyroid hormone transporter in humans. Mutations in MCT8 underlie one of the first described X-linked mental retardation syndromes, the Allan–Herndon–Dudley syndrome.

Major conclusions

Thyroid hormone transporter molecules are expressed in a developmental and cell type-specific pattern. Any thyroid hormone molecule has to cross consecutively the luminal and abluminal membranes of the capillary endothelium, enter astrocytic foot processes, and leave the astrocyte through the plasma membrane to finally cross another plasma membrane on its way towards its target nucleus.

General significance

We can expect more transporters being involved in or contributing to in neurodevelopmental or neuropsychiatric disease. Due to their expression in cellular components regulating the hypothalamus–pituitary–thyroid axis, mutations and polymorphisms are expected to impact on negative feedback regulation and hormonal setpoints. This article is part of a Special Issue entitled Thyroid hormone signalling.     

The pathophysiological consequences of thyroid hormone transporter deficiencies: Insights from mouse models

Background

As a prerequisite for thyroid hormone (TH) metabolism and action TH has to be transported into cells where TH deiodinases and receptors are located. The trans-membrane passage of TH is facilitated by TH transporters of which the monocarboxylate transporter MCT8 has been most intensively studied. Inactivating mutations in the gene encoding MCT8 are associated with a severe form of psychomotor retardation and abnormal serum TH levels (Allan–Herndon–Dudley syndrome). In order to define the underlying pathogenic mechanisms, Mct8 knockout mice have been generated and intensively studied. Most surprisingly, Mct8 ko mice do not show any neurological symptoms but fully replicate the abnormal serum thyroid state.

Scope of review

We will summarize the findings of these mouse studies that shed light on various aspects of Mct8 deficiency and unambiguously demonstrated the pivotal role of Mct8 in mediating TH transport in various tissues. These studies have also revealed the presence of the complex interplay between different pathogenic mechanisms that contribute to the generation of the abnormal TH serum profile.

Major conclusions

Most importantly, studies of Mct8 ko mice indicated the presence of additional TH transporters that act in concert with Mct8. Interesting candidates for such a function are the L-type amino acid transporters Lat1 and Lat2 as well as the organic anion transporting polypeptide Oatp1c1.

General significance

Overall, the analysis of Mct8 deficient mice has greatly expanded our knowledge about the (patho-) physiological function of this transporter and established a sound basis for the characterization of additional TH transporter candidates. This article is part of a Special Issue entitled Thyroid hormone signalling.     

Thyroid hormone receptors: The challenge of elucidating isotype-specific functions and cell-specific response

Background

Thyroid hormone receptors TRα1, TRβ1 and TRβ2 are broadly expressed and exert a pleiotropic influence on many developmental and homeostatic processes. Extensive genetic studies in mice precisely defined their respective function.

Scope of review

The purpose of the review is to discuss two puzzling issues:

–

The isoform specificity problem: the different functions of TRα1, TRβ1 and TRβ2 might reflect either their different distribution in tissues or differences in the receptor intrinsic properties.

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The cell-specificity problem: one would expect that different cell types share a common repertoire of TR target genes, but current knowledge does not support this assumption. How TR function is affected by the cellular context is an unsolved question.

Major conclusions

Mouse genetics support a balanced contribution of expression pattern and receptor intrinsic properties in defining the receptor respective functions. The molecular mechanisms sustaining cell specific response remain hypothetical and based on studies performed with other nuclear receptors.

General significance

The isoform-specificity and cell-specificity questions have many implications for clinical research, drug development, and endocrine disruptor studies. This article is part of a Special Issue entitled Thyroid hormone signalling.     

3D NMR structure of a complex between the amyloid beta peptide (1–40) and the polyphenol ε-viniferin glucoside: Implications in Alzheimer's disease

Background

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. There is a consensus that Aβ is a pathologic agent and that its toxic effects, which are at present incompletely understood, may occur through several potential mechanisms. Polyphenols are known to have wide-ranging properties with regard to health and for helping to prevent various diseases like neurodegenerative disorders. Thus inhibiting the formation of toxic Aβ assemblies is a reasonable hypothesis to prevent and perhaps treat AD

Methods

Solution NMR and molecular modeling were used to obtain more information about the interaction between the Aβ_1–40 and the polyphenol ε-viniferin glucoside (EVG) and particularly the Aβ residues involved in the complex.

Results

The study demonstrates the formation of a complex between two EVG molecules and Aβ_1–40 in peptide characteristic regions that could be in agreement with the inhibition of aggregation. Indeed, in previous studies, we reported that EVG strongly inhibited in vitro the fibril formation of the full length peptides Aβ_1–40 and Aβ_1–42, and had a strong protective effect against PC12 cell death induced by these peptides.

Conclusion

For the full length peptide Aβ_1–40, the binding sites observed could explain the EVG inhibitory effect on fibrillization and thus prevent amyloidogenic neurotoxicity.

General significance

Even though this interaction might be important at the biological level to explain the protective effect of polyphenols in neurodegenerative diseases, caution is required when extrapolating this in vitro model to human physiology.     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

Selenoproteins and selenium status in bone physiology and pathology

Background

Emerging evidence supports the view that selenoproteins are essential for maintaining bone health.

Scope of review

The current state of knowledge concerning selenoproteins and Se status in bone physiology and pathology is summarized.

Major conclusions

Antioxidant selenoproteins including glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), as a whole, play a pivotal role in maintaining bone homeostasis and protecting against bone loss. GPx1, a major antioxidant enzyme in osteoclasts, is up-regulated by estrogen, an endogenous inhibitor of osteoclastogenesis. TrxR1 is an immediate early gene in response to 1α,25-dihydroxyvitamin D3, an osteoblastic differentiation agent. The combination of 1α,25-dihydroxyvitamin D3 and Se generates a synergistic elevation of TrxR activity in Se-deficient osteoblasts. Of particular concern, pleiotropic TrxR1 is implicated in promoting NFκB activation. Coincidentally, TrxR inhibitors such as curcumin and gold compounds exhibit potent osteoclastogenesis inhibitory activity. Studies in patients with the mutations of selenocysteine insertion sequence-binding protein 2, a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins have clearly established a causal link of selenoproteins in bone development. Se transport to bone relies on selenoprotein P. Plasma selenoprotein P concentrations have been found to be positively correlated with bone mineral density in elderly women.

General significance

A full understanding of the role and function of selenoproteins and Se status on bone physiology and pathology may lead to effectively prevent against or modify bone diseases by using Se.     

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Background

Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course.

Methods

Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H₂O₂. Our focus was the characterization of products and the study of features of the reaction.

Results

We found that N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H₂O₂ as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG.

Conclusions

These results show, for the first time, that melatonin radical is able to oxidize GSH.

General significance

We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.