Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.     

ATPase activity of non-ribosomal peptide synthetases

Adenylation domains of non-ribosomal peptide synthetases (NRPS) catalyse the formation of aminoacyl adenylates, and in addition synthesize mono- and dinucleoside polyphosphates. Here, we show that NRPS systems furthermore contain an ATPase activity in the range of up to 2 P(i)/min. The hydrolysis rate by apo-tyrocidine synthetase 1 (apo-TY1) is enhanced in the presence of non-cognate amino acid substrates, correlating well with their structural features and the diminishing adenylation efficiency. A comparative analysis of the functional relevance of an analogous sequence motif in P-type ATPases and adenylate kinases (AK) allowed a putative assignment of the invariant aspartate residue from the TGDLA(V)R(K) core sequence in NRPS as the Mg(2+) binding site. Less pronounced variations in ATPase activity are observed in domains with relaxed amino acid specificity of gramicidin S synthetase 2 (GS2) and delta-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), known to produce a set of substitutional variants of the respective peptide product. These results disclose new perspectives about the mode of substrate selection by NRPS.     

Radial arrangement of chromosome territories in human cell nuclei: a computer model approach based on gene density indicates a probabilistic global positioning code

Numerous investigations in the last years focused on chromosome arrangements in interphase nuclei. Recent experiments concerning the radial positioning of chromosomes in the nuclear volume of human and primate lymphocyte cells suggest a relationship between the gene density of a chromosome territory (CT) and its distance to the nuclear center. To relate chromosome positioning and gene density in a quantitative way, computer simulations of whole human cell nuclear genomes of normal karyotype were performed on the basis of the spherical 1 Mbp chromatin domain model and the latest data about sequence length and gene density of chromosomes. Three different basic assumptions about the initial distribution of chromosomes were used: a statistical, a deterministic, and a probabilistic initial distribution. After a simulated decondensation in early G1, a comparison of the radial distributions of simulated and experimentally obtained data for CTs Nos. 12, 18, 19, and 20 was made. It was shown that the experimentally observed distributions can be fitted better assuming an initial probabilistic distribution. This supports the concept of a probabilistic global gene positioning code depending on CT sequence length and gene density.     

Torque generation by the Fo motor of the sodium ATPase

Based on recent structural and functional findings, we have constructed a mathematical model for the sodium-driven Fo motor of the F1Fo-ATPase from the anaerobic bacterium Propionigenium modestum. The model reveals the mechanochemical principles underlying the Fo motor's operation, and explains all of the existing experimental data on wild-type and mutant Fo motors. In particular, the model predicts a nonmonotonic dependence of the ATP hydrolysis activity on the sodium concentration, a prediction confirmed by new experiments. To explain experimental observations, the positively charged stator residue (R227) must assume different positions in the ATP synthesis and hydrolysis directions. This work also illustrates how to extract a motor mechanism from dynamical experimental observations in the absence of complete structural information.     

Preparation and mass spectrometry of 14 pure and 18O(2)-labeled oxidation products from the phytosterols beta-sitosterol and stigmasterol

To lower cholesterol, phytosterols are currently introduced as food additives, where they may become oxidized. In addition, specific biological effects of oxyphytosterols are suggested by the recent molecular clarification of the phytosterol storage disease as a dysfunctional mutation of an active sterol reexporter potentially regulated by oxidized phytosterols. We therefore studied the hydroxybenzotriazole-mediated PbO(2)-driven oxidation of phytosterols and compared it to the oxidation of cholesterol. We prepared, identified, and purified standards of 14 oxidation products of two major phytosterols. The gas chromatographic mass spectrometric characteristics of the 7alpha- and 7beta-hydroxy-, 5alpha,6alpha-epoxy, 5beta,6beta-epoxy, 7keto-, 3beta,5alpha,6beta-trihydroxy-, 3keto-, and 7-dehydro-derivatives of beta-sitosterol and stigmasterol are presented. The method also provided a convenient access to prepare 18O-labeled oxyphytosterols of high chemical and isotopic purity and can easily be extended to further phytosterols and -stanols. This enables the gas chromatography-mass spectrometry analysis of oxyphytosterols and the study of their biological effects.     

The mechanism-based inactivation of p450 2B4 by tert-butyl 1-methyl-2-propynyl ether: structural determination of the adducts to the p450 heme

tert-Butyl 1-methyl-2-propynyl ether (tBMP) was analyzed for its ability to act as a mechanism-based inactivator of p450 2B4. tBMP inactivated p450 2B4 in a time-, concentration-, and NADPH-dependent manner. Losses in activity occurred with concurrent losses in the reduced CO spectrum and native p450 heme; however, there was a greater loss in activity than could be accounted for by reduced CO spectra or native heme loss. LC/MS analysis demonstrated that the losses in native heme were accompanied by the appearance of two modified hemes with m/z values of 705Da, consistent with tBMP adducted hemes. Both adducts had identical fragmentation patterns when analyzed by LC/MS/MS. The spectra were consistent with a tBMP molecule and an oxygen atom attached to iron-depleted heme. Proton NMR studies suggest that the two modified hemes in p450 2B1 are N-alkylated on pyrrole rings A and D.     

ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3

Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the "death" phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)-, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) overexpression results in a dramatic reduction of PM damaged neurons and nuclear condensation. In contrast, overexpression of constitutively active MEK potentiates PM damage and nuclear condensation. ERK-promoted cellular damage is independent of caspase-3. Persistent active ERK translocates to the nucleus, whereas caspase-3 remains in the cytoplasm. Antioxidants that reduced ERK activation and PM damage showed no effect on caspase-3 activation or DNA damage. These data identify ERK as an important executor of neuronal damage involving a caspase-3-independent mechanism.     

Human intestinal microvascular endothelial cells express Toll-like receptor 5: a binding partner for bacterial flagellin

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.     

Beta-tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations

The molecular mechanism of benzimidazole (BZ) resistance in cyathostomins of horses is still unclear. Previous studies revealed that the TTC or TAC polymorphism in codon 200 of the beta-tubulin isotype 1 gene is not as strictly correlated with BZ resistance as in trichostrongyles in sheep. To identify further sites of polymorphism within the beta-tubulin gene related to BZ resistance, complete complementary DNAs (cDNAs) encoding beta-tubulin of adult worms of Cylicocyclus nassatus, Cyathostomum pateratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicostephanus longibursatus, and Cylicostephanus goldi of a BZ-resistant cyathostomin population were characterized using specific primers. The cDNA sequence of each species spans 1,429 bp, encoding a protein of 448 amino acids. The interspecific identities are 95.2-99.6% at the nucleotide and 98.7-100.0% at the peptide level. The comparison of the amino acid sequences of individuals isolated from the BZ-resistant cyathostomin population with those from individuals of Cc. nassatus, Cy. coronatum, Cy. pateratum, and Cy. catinatum of a BZ-susceptible one showed differing amino acids in 11 positions. The commonness of a phenylalanine to tyrosine mutation at position 167 in all the 6 cyathostomin species isolated from a BZ-resistant population suggests its involvement in the molecular mechanism in BZ resistance.     

Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.