Relationships between single nucleotide polymorphisms of antioxidant enzymes and disease

The presence and progression of numerous diseases have been linked to deficiencies in antioxidant systems. The relationships between single nucleotide polymorphisms (SNPs) arising from specific antioxidant enzymes and diseases associated with elevated oxidative stress have been studied with the rationale that they may be useful in screening for diseases. The purpose of this narrative review is to analyse evidence from these studies. The antioxidant enzyme SNPs selected for analysis are based on those most frequently investigated in relation to diseases in humans: superoxide dismutase (SOD2) Ala16Val (80 studies), glutathione peroxidise (GPx1) Pro197Leu (24 studies) and catalase C-262T (22 studies). Although the majority of evidence supports associations between the SOD2 Ala16Val SNP and diseases such as breast, prostate and lung cancers, diabetes and cardiovascular disease, the presence of the SOD2 Ala16Val SNP confers only a small, clinically insignificant reduction (if any) in the risk of these diseases. Other diseases such as bladder cancer, liver disease, nervous system pathologies and asthma have not been consistently related to this SOD SNP genotype. The GPx1 Pro197Leu and catalase C-262T SNP genotypes have been associated with breast cancer, but only in a small number of studies. Thus, currently available evidence suggests antioxidant enzyme SNP genotypes are not useful for screening for diseases in humans.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ?yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using ³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.     

Synthesis,structural characterization and biological activity of novel Knoevenagel condensates on DLD-1 human colon carcinoma

Biologically active Knoevenagel condensates (1–14) of diarylheptanoids: 1,7-bis(3-methoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione and 1,7-bis(3-ethoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione, were synthesized and structurally characterized. Compounds 1–14 exhibited cytotoxicity against colon carcinoma cells, and their antiproliferative effect was associated with a significant decrease of multidrug resistance proteins. One of the underlying mechanisms of these effects is the reduction of intracellular and extracellular SOD enzymes by compounds 1, 12 and 14, which render the tumor cells more vulnerable to oxidative stress.     

The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis

Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development. During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report the first crystal structure of hTrxR1 (Sec-->Cys) in complex with FAD and NADP(+) at a resolution of 2.8 A. From the crystals three different conformations of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three side-chain residues of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development.     

Crystal structures of a poxviral glutaredoxin in the oxidized and reduced states show redox-correlated structural changes

Glutaredoxins act as reducing agents for the large subunit of ribonucleotide reductase (R1) in many prokaryotes and eukaryotes, including humans. The same relationship has been proposed for the glutaredoxin and R1 proteins expressed by all orthopoxviruses, including vaccinia, variola, and ectromelia virus. Interestingly, the orthopoxviral proteins share 45% and 78% sequence identity with human glutaredoxin-1 (Grx-1) and R1, respectively. To study structure-function relationships of the vertebrate Grx-1 family, and reveal potential viral adaptations, we have determined crystal structures of the ectromelia virus glutaredoxin, EVM053, in the oxidized and reduced states. The structures show a large redox-induced conformational rearrangement of Tyr21 and Thr22 near the active site. We predict that the movement of Tyr21 is a viral-specific adaptation that increases the redox potential by stabilizing the reduced state. The conformational switch of Thr22 appears to be shared by vertebrate Grx-1 and may affect the strictly conserved Lys20. A crystal packing-induced structural change in residues 68-70 affects the GSH-binding loop, and our structures reveal a potential interaction network that connects the GSH-binding loop and the active site. EVM053 also exhibits a novel cis-proline (Pro53) in a loop that has been shown to contribute to R1-binding in Escherichia coli Grx-1. The cis-peptide bond of Pro53 may be required to promote electrostatic interactions between Lys52 and the C-terminal carboxylate of R1. Finally, dimethylarsenite was covalently attached to Cys23 in one reduced EVM053 structure and our preliminary data show that EVM053 has dimethylarsenate reductase activity.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

Uncoupling mechanism and redox regulation of mitochondrial uncoupling protein 1 (UCP1)

Brown adipose tissue (BAT) and brown in white (brite) adipose tissue, termed also beige adipose tissue, are major sites of mammalian nonshivering thermogenesis. Mitochondrial uncoupling protein 1 (UCP1), specific for these tissues, is the key factor for heat production. Recent molecular aspects of UCP1 structure provide support for the fatty acid cycling model of coupling, i.e. when UCP1 expels fatty acid anions in a uniport mode from the matrix, while uncoupling. Protonophoretic function is ensured by return of the protonated fatty acid to the matrix independent of UCP1. This mechanism is advantageous for mitochondrial uncoupling and compatible with heat production in a pro-thermogenic environment, such as BAT. It must still be verified whether posttranslational modification of UCP1, such as sulfenylation of Cys253, linked to redox activity, promotes UCP1 activity. BAT biogenesis and UCP1 expression, has also been linked to the pro-oxidant state of mitochondria, further endorsing a redox signalling link promoting an establishment of pro-thermogenic state. We discuss circumstances under which promotion of superoxide formation exceeds its attenuation by uncoupling in mitochondria and throughout point out areas of future research into UCP1 function.     

嗜酸氧化亚铁硫杆菌的谷胱甘肽还原酶的结构模建和底物分子对接

极端环境微生物嗜酸氧化亚铁硫杆菌的谷胱甘肽还原酶(GR)可能在它的抵抗极端酸性,有毒和氧化性的生物浸出环境中发挥至关重要的作用.通过同源模建技术和分子动力学模拟,它的一个三维结构被构建,优化和检验了.获得的结构被进一步用于搜索绑定位点,跟辅因子黄素腺嘌呤二核苷酸(FAD)和底物谷胱甘肽(GSSG)进行分子柔性对接,并以此识别关健残基.对接结果显示,位于活性残基Cys42和Cys47之间的二硫键夹在FAD的活性位点和底物GSSG的二硫键之间.它们之间的距离非常靠近,这跟底物反应机理的初始步骤的情况十分一致.相互作用能表明8个酶中残基Cys42,Cys47,GIu443B,Glu444B,His438B,Ser14,Thr447B和Lys51是固定或激活GSSG的关键残基,这跟以前的实验事实相吻合.此外,根据相互作用能我们还新发现7个重要残基(Arg449B,Pro439B,Thr440B,Thr310,Va143,Gly46 and Va148).所有这些残基在其它物种中的相应物中也都是保守的.这些结果有助于进一步的实验研究和理解其催化机理,进而揭示这种细菌的抗毒机理,服务于工业应用.