Molecular Modeling of Cytochrome P450 1A1: Enzyme-Substrate Interactions and Substrate Binding Affinities

Abstract

Human cytochrome P450 1A1, which is present in lungs, plays an important role in the metabolic activation of chemical carcinogens, and in particular, is thought to be linked to lung cancer. The mechanism of carcinogenesis is related to the enzyme's ability to oxidize highly toxic compounds, such as polycyclic aromatic hydrocarbons (PAHs), to their carcinogenic derivatives.

In order to better understand P450 1A1 function, a homology model of this enzyme has been constructed. The model has been based on the structure of P450 2C5, the first mammalian P450 to be crystallized. The coordinates of the model have been calculated using a consensus strategy, and the resulting structure has been evaluated with the ProStat and Profiles-3D programs. P450 1A1 substrates, such as benzo[a]pyrene, ethoxyresorufin and methoxyre- sorufin, were then docked into the active site of the model, and key amino acid residues able to interact with the substrate, have been identified. The analysis of enzyme-substrate interactions indicated that hydrophobic interactions are mainly responsible for binding of these substrates in the active site. Moreover, the non-bond enzyme-substrate interaction energy for ethoxyresorufin was lower than that for methoxyresorufin, which is consistent with higher activity of 1A1 towards the former substrate. Key residue Val-382 may play an important role in these interactions. Additionally, we performed binding free energy calculations for the three substrates. The obtained values were similar to those observed experimentally, which suggests that this approach might be useful for prediction of binding constants.     

The role of juvenile hormone during larval-pupal transformation of Spodoptera littoralis: Switchover in the sensitivity of the prothoracic gland to juvenile hormone

Application of JHA to the final instar of Spodoptera littoralis larvae before they attain maximal body weight causes delay to or completely blocks the onset of metamorphosis, as indicated by the dorsal vessel exposure. When the same dose of JHA is administered to the larvae just after their attaining maximal body weight, at the time of the onset of gut-emptying, metamorphosis is substantially accelerated.By means of ligature experiments, it is shown that JHA has a direct effect on the prothoracic glands. It inhibits them at the beginning of the final instar and then stimulates them shortly before pupation. It seems that under normal conditions JH is necessary for pupal cuticle formation; those larvae which pupate without JH show adult characteristics.     

Antioxidant defense in the leaves of C3 and C4 plants under salinity stress

The effect of salt stress (50, 100 and 150 m M of NaCl) on the activity of superoxide dismutase (SOD, EC. 1.15.1.1), ascorbate peroxidase (APX, EC. 1.11.1.11), glutathione reductase (GR, EC. 1.6.4.2) enzymes and also on the rate of lipid peroxidation in terms of thiobarbituric acid-reactive substances (TBARS) content and photosynthetic capacity in two wheat (C3 plants) and two maize (C4 plants) varieties was studied. In the non-salined control plants, the antioxidant enzymes activities were significantly higher for maize than for wheat. Adding salt to the nutrient solution increased the level of antioxidants in leaves of both maize and wheat. The first substantial response to salinity was found for SOD on the 2nd day, whereas changes occurred for APX on the 4th day and for GR on the 4th/5th day of salt treatment. Although SOD activity increased considerably more in wheat (C3), it never reached as high levels as in maize (C4) grown in the same treatment combinations. The total increase in APX activity was similar for wheat and maize, whereas GR activity was higher in leaves of maize. Lipid peroxidation analyses showed an increase in TBARS contents in both plants' species grown under salinity that corresponded to the damage that occurred in secondary oxidative stress. However, as a result of advanced antioxidant defense in maize, the TBARS quantities did not elevate to as high level as in wheat. Chlorophyll fluorescence measurements revealed a considerable decrease in the efficiency of PS II and electron-transport chain (ETC). Assimilation rate of CO₂ decreased in both plant groups; however, in C4 maize, we observed a much better capacity to preserve the photosynthetic apparatus against overproduction of ROS. Results suggest that efficient antioxidant defense plays an important role in maize, the C₄ plant, resistance to environmental stresses like salinity or drought.     

Some properties of methylotrophic bacteria isolated from sewage sludges derived from mechanical and biological sewage treatment plants.

The presented studies were carried out to get more information about physiological properties of methylotrophic bacteria selected from sewage sludges derived from mechanical and biological sewage treatment plants. All the isolated bacterial strains belonged to facultative methylotrophs. The majority of them utilized glucose, starch and lipids. Moreover, most of them were also found to possess proteolytic properties and were able to hydrolyze urea but they were incapable of asparagine ammonification.     

Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Delta and pmt2Delta mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.     

Psychological Stress-Induced,IDO1-Dependent Tryptophan Catabolism: Implications on Immunosuppression in Mice and Humans

It is increasingly recognized that psychological stress influences inflammatory responses and mood. Here, we investigated whether psychological stress (combined acoustic and restraint stress) activates the tryptophan (Trp) catabolizing enzyme indoleamine 2,3-dioxygenase 1(IDO1) and thereby alters the immune homeostasis and behavior in mice. We measured IDO1 mRNA expression and plasma levels of Trp catabolites after a single 2-h stress session and in repeatedly stressed (4.5-days stress, 2-h twice a day) naïve BALB/c mice. A role of cytokines in acute stress-induced IDO1 activation was studied after IFNγ and TNFα blockade and in IDO1^−/− mice. RU486 and 1-Methyl-L-tryptophan (1-MT) were used to study role of glucocorticoids and IDO1 on Trp depletion in altering the immune and behavioral response in repeatedly stressed animals. Clinical relevance was addressed by analyzing IDO1 activity in patients expecting abdominal surgery. Acute stress increased the IDO1 mRNA expression in brain, lung, spleen and Peyer''s patches (max. 14.1±4.9-fold in brain 6-h after stress) and resulted in a transient depletion of Trp (−25.2±6.6%) and serotonin (−27.3±4.6%) from the plasma measured 6-h after stress while kynurenine levels increased 6-h later (11.2±9.3%). IDO1 mRNA up-regulation was blocked by anti-TNFα and anti-IFNγ treatment. Continuous IDO1 blockade by 1-MT but not RU486 treatment normalized the anti-bacterial defense and attenuated increased IL-10 inducibility in splenocytes after repeated stress as it reduced the loss of body weight and behavioral alterations. Moreover, kynurenic acid which remained increased in 1-MT treated repeatedly stressed mice was identified to reduce the TNFα inducibility of splenocytes in vitro and in vivo. Thus, psychological stress stimulates cytokine-driven IDO1 activation and Trp depletion which seems to have a central role for developing stress-induced immunosuppression and behavioral alteration. Since patients showed Trp catabolism already prior to surgery, IDO is also a possible target enzyme for humans modulating immune homeostasis and mood.     

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Minimum estimated germ-line transformation frequencies were approximately 13–21% per fertile G₀ individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was integrated into the Chiapas wild type strain with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3′ terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5–10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the A. suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.     

Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry

In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24 h was inhibited by 95–100% in the presence of HSA but only 0–34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid–protein interactions in vivo.     

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.