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.     

Lipoic acid prevents liver metabolic changes induced by administration of a fructose-rich diet

Background

To evaluate whether co-administration of R/S-α-lipoic acid can prevent the development of oxidative stress and metabolic changes induced by a fructose-rich diet (F).

Methods

We assessed glycemia in the fasting state and during an oral glucose tolerance test, triglyceridemia and insulinemia in rats fed with standard diet (control) and fructose without or with R/S-α-lipoic acid. Insulin resistance and hepatic insulin sensitivity were also calculated. In liver, we measured reduced glutathione, protein carbonyl groups, antioxidant capacity by ABTS assay, antioxidant enzymes (catalase and superoxide dismutase 1 and 2), uncoupling protein 2, PPARδ and PPARγ protein expressions, SREBP-1c, fatty acid synthase and glycerol-3-phosphate acyltransferase-1 gene expression, and glucokinase activity.

Results

R/S-α-lipoic acid co-administration to F-fed rats a) prevented hyperinsulinemia, hypertriglyceridemia and insulin resistance, b) improved hepatic insulin sensitivity and glucose tolerance, c) decreased liver oxidative stress and increased antioxidant capacity and antioxidant enzymes expression, d) decreased uncoupling protein 2 and PPARδ protein expression and increased PPARγ levels, e) restored the basal gene expression of PPARδ, SREBP-1c and the lipogenic genes fatty acid synthase and glycerol-3-phosphate acyltransferase, and f) decreased the fructose-mediated enhancement of glucokinase activity.

Conclusions

Our results suggest that fructose-induced oxidative stress is an early phenomenon associated with compensatory hepatic metabolic mechanisms, and that treatment with an antioxidant prevented the development of such changes.

General significance

This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced oxidative stress and to develop effective strategies to prevent and treat, at early stages, obesity and type 2 diabetes mellitus.     

High uric acid directly inhibits insulin signalling and induces insulin resistance

Background and aim

Accumulating clinical evidence suggests that hyperuricemia is strongly associated with abnormal glucose metabolism and insulin resistance. However, how high uric acid (HUA) level causes insulin resistance remains unclear. We aimed to determine the direct role of HUA in insulin resistance in vitro and in vivo in mice.

Methods

An acute hyperuricemia mouse model was created by potassium oxonate treatment, and the impact of HUA level on insulin resistance was investigated by glucose tolerance test, insulin tolerance test and insulin signalling, including phosphorylation of insulin receptor substrate 1 (IRS1) and Akt. HepG2 cells were exposed to HUA treatment and N-acetylcysteine (NAC), reactive oxygen species scavenger; IRS1 and Akt phosphorylation was detected by Western blot analysis after insulin treatment.

Results

Hyperuricemic mice showed impaired glucose tolerance with insulin resistance. Hyperuricemia inhibited phospho-Akt (Ser473) response to insulin and increased phosphor-IRS1 (Ser307) in liver, muscle and fat tissues. HUA induced oxidative stress, and the antioxidant NAC blocked HUA-induced IRS1 activation and Akt inhibition in HepG2 cells.

Conclusion

This study supplies the first evidence of HUA directly inducing insulin resistance in vivo and in vitro. Increased uric acid level may inhibit IRS1 and Akt insulin signalling and induce insulin resistance. The reactive oxygen species pathway plays a key role in HUA-induced insulin resistance.     

Antioxidant effect of vegetable oils containing conjugated linolenic acid isomers against induced tissue lipid peroxidation and inflammation in rat model

The purpose of the present study was to examine the antioxidant activity of two typical oils obtained from two vegetables, bitter gourd seed and snake gourd seed, containing two different isomers of conjugated linolenic acid (CLnA) against oxidative stress induced by sodium arsenite in relation to tissue lipid peroxidation and inflammation. Male albino rats were taken as subject and divided into six groups: Group 1 was control and Group 2 was treated with sodium arsenite (Sa; 10mg/Kg BW); Groups 3-6 were orally treated with different doses of seed oils maintaining definite concentration of CLnA isomers (0.5% and 1.0% of total lipid for each CLnA isomer) along with sodium arsenite. There was significant increase in lipid peroxidation, pro-oxidant enzyme activity and decrease in antioxidant enzyme activity in brain due to Sa administration. Decrease in total protein content was also observed in plasma, liver and brain of Sa treated group. Significant decrease in phospholipid content and increase in total lipid content and cholesterol content were observed in arsenite treated group. There was significant increase in relative organ weight of liver due to Sa administration. Fatty acid profile of liver and brain lipid shows significant (P<0.05) reduction in most of the polyunsaturated fatty acids and increase in arachidonic acid (20:4n-6) (75.23%) due to inflammation after arsenite treatment. Administration of experimental oils made almost complete restoration of those altered parameters. Overall, these two oils were effective in protecting tissue lipid profiles which were altered due to oxidative stress.     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.     

Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury

Background

Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low.

Methods

Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl₄).

Results

TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT.

Conclusions

Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury.

General significance

The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.     

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress

Background

Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes.

Methods

Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35 mg/kg, i.p.) (control + L and fructose + L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration.

Results

Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91^phox and p22^phox) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration.

Conclusions

Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration.

General significance

Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.     

Insulin sensitization via partial agonism of PPARγ and glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt signaling pathway by embelin in type 2 diabetic rats

Background

The present study was aimed at isolating an antidiabetic molecule from a herbal source and assessing its mechanism of action.

Methods

Embelin, isolated from Embelia ribes Burm. (Myrsinaceae) fruit, was evaluated for its potential to regulate insulin resistance, alter β-cell dysfunction and modulate key markers involved in insulin sensitivity and glucose transport using high-fat diet (HFD) fed-streptozotocin (STZ) (40 mg/kg)-induced type 2 diabetic rats. Molecular-dockings were performed to investigate the binding modes of embelin into PPARγ, PI3K, p-Akt and GLUT4 active sites.

Results

Embelin (50 mg/kg b wt.) reduced body weight gain, blood glucose and plasma insulin in treated diabetic rats. It further modulated the altered lipid profiles and antioxidant enzymes with cytoprotective action on β-cell. Embelin significantly increased the PPARγ expression in epididymal adipose tissue compared to diabetic control group; it also inhibited adipogenic activity; it mildly activated PPARγ levels in the liver and skeletal muscle. It also regulated insulin mediated glucose uptake in epididymal adipose tissue through translocation and activation of GLUT4 in PI3K/p-Akt signaling cascade. Embelin bound to PPARγ; it disclosed stable binding affinities to the active sites of PI3K, p-Akt and GLUT4.

Conclusions

These findings show that embelin could improve adipose tissue insulin sensitivity without increasing weight gain, enhance glycemic control, protect β-cell from damage and maintain glucose homeostasis in adipose tissue.

General significance

Embelin can be used in the prevention and treatment of type 2 diabetes mellitus caused due to obesity.     

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid,uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: A potential drug for bipolar disorder

Background

Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation–reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain long-chain acyl-CoA synthetase (Acsl)4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl4 catalyzed acylation, and thus have a potential anti-BD action.

Methods

Rat Acsl4-flag protein was expressed in Escherichia coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis–Menten kinetics.

Results

Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a K_i of 11.4 mM compared to a published K_i of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect.

Conclusions

PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients.     

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.     

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer

Background

Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling.

Methods

Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots.

Results

Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~ 1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity.

Conclusion

In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies.

General significance

Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.     

Oxidized albumin. The long way of a protein of uncertain function

Background

Proteins are extremely reactive to oxidants and should represent a potential target of instable reactive oxygen. This may represent a problem for plasma proteins since they may be directly modified in vivo in a compartment where antioxidant enzymatic systems are scarcely represented. On the other hand, it is possible that some plasma components have evolved over time to guarantee protection, in which case they can be considered as anti-oxidants.

Scope of review

To present and discuss main studies which addressed the role of albumin in plasma antioxidant activity mainly utilizing in vitro models of oxidation. To present some advances on structural features of oxidized albumin deriving from studies carried out on in vitro models as well as albumin purified in vivo from patients affected by clinical conditions characterized by oxidative stress.

Major conclusions

There are different interaction with HOCl and chloramines. In the former case, HOCl produces an extensive alteration of ²³⁸Trp and ¹⁶²Tyr, ⁴²⁵Tyr, ⁴⁷Tyr, while thiol groups are only partially involved. Chloramines are extremely reactive with the unique free SH group of albumin (³⁴Cys) with the formation of sulfenic and sulfinic acid as intermediates and sulfonic acid as end-product. Oxidized albumin has a modified electrical charge for the addition of an acidic residue and presents α-helix and random coil reorganization with subtle changes in domain orientation.

General significance

Albumin, is the major antioxidants in plasma with a concentration (0.8 mM) higher than other antioxidants by an exponential factor. Functional and protective roles in the presence of oxidative stress must be defined. This article is part of a Special Issue entitled Serum Albumin.     

Identification of a naturally occurring retinoid X receptor agonist from Brazilian green propolis

Background

Brazilian green propolis (BGP), a resinous substance produced from Baccharis dracunculifolia by Africanized honey bees (Apis mellifera), is used as a folk medicine. Our present study explores the retinoid X receptor (RXR) agonistic activity of BGP and the identification of an RXR agonist in its extract.

Methods

RXRα agonistic activity was evaluated using a luciferase reporter gene assay. Isolation of the RXRα agonist from the ethanolic extract of BGP was performed using successive silica gel and a reversed phase column chromatography. The interaction between the isolated RXRα agonist and RXRα protein was predicted by a receptor–ligand docking simulation. The nuclear receptor (NR) cofactor assay was used to estimate whether the isolated RXRα agonist bound to various NRs, including RXRs and peroxisome proliferator-activated receptors (PPARs). We further examined its effect on adipogenesis in 3T3-L1 fibroblasts.

Results

We identified drupanin as an RXRα agonist with an EC₅₀ value of 4.8 ± 1.0 μM. Drupanin activated three RXR subtypes by a similar amount and activated PPARγ moderately. Additionally, drupanin induced adipogenesis and elevated aP2 mRNA levels in 3T3-L1 fibroblasts.

Conclusions

Drupanin, a component of BGP, is a novel RXR agonist with slight PPARγ agonistic activity.

General significance

This study revealed for the first time that BGP activates RXR and drupanin is an RXR agonist in its extract.     

Effects of passive smoking on breast cancer risk in pre/post-menopausal women as modified by polymorphisms of PARP1 and ESR1

Objectives

The association between passive smoking and breast cancer risk differs in pre- and post-menopausal women. We aimed to explore the modification effects of PARP1 rs1136410 and ESR1 rs2234693 on the association between passive smoking and breast cancer risk among pre- and post-menopausal women.

Design and methods

A case–control study of 839 breast cancer cases and 863 controls was conducted. The gene–environment interactions were tested after adjusting for potential breast cancer risk factors with unconditional logistic regression models.

Results

We found that the effect of passive smoking was modified by the genotypes in both pre- and post-menopausal women, but in opposite directions. The combination of the TC/CC genotypes of ESR1 rs2234693 and passive smoking significantly increased the risk of breast cancer [OR (95%CI): 2.06 (1.39–3.05)] in pre-menopausal women. A significant association was observed between TT genotype and passive smoking [OR (95%CI): 2.40 (1.27–4.53)] in postmenopausal women. For PARP1 rs1136410, similar differential associations were observed, but the interactions were not significant.

Conclusions

These results imply that the risk of breast cancer from passive smoking may be influenced by genetic factors, and that the association may differ depending on menopausal status.     

Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates

Background

Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.

Methods

RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.

Results

RALDH3 oxidized all-trans retinal with high catalytic efficiency (V_max/K_m = 77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (V_max/K_m = 27.4), and oxidized 13-cis retinal with lower catalytic efficiency (V_max/K_m = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl₂ activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.

Conclusions

These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.

General significance

The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori

Background

Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues.

Methods

The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu.

Results

We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features.

Conclusions

This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function.

General Significance

Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.     

Jacaric acid,a linolenic acid isomer with a conjugated triene system,has a strong antitumor effect in vitro and in vivo

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.