Glucose-derived Amadori compounds of glutathione

Under the chromatographic conditions used in these studies we observed time- and concentration-dependent formation of N-1-Deoxy-fructos-1-yl glutathione as the major glycation product formed in the mixtures of GSH with glucose. N-1-Deoxy-fructos-1-yl glutathione had a characteristic positively charged ion with m/z=470 Th in its LC-MS spectra. Mixtures of glutathione disulfide and glucose generated two compounds: N-1-Deoxy-fructos-1-yl GSSG (m/z=775 Th) as major adduct and bis di-N, N'-1-Deoxy-fructos-1-yl GSSG (m/z=937 Th) as the minor one. All three compounds showed a resonance signal at 55.2 ppm in the 13C-NMR spectra as C1 methylene group of deoxyfructosyl, which represents direct evidence that they are Amadori compounds. All three compounds purified from GSSG/Glc or GSH/Glc mixtures also showed LC-MS/MS fragmentation patterns identical to those of the synthetically synthesized N-1-Deoxy-fructos-1-yl glutathione, N-1-Deoxy-fructos-1-yl GSSG and bis di-N, N'-1-Deoxy-fructos-1-yl GSSG. N-1-Deoxy-fructos-1-yl glutathione was shown to be a poor substrate for glutathione peroxidase (6.7% of the enzyme's original specific activity) and glutathione-S-transferase (25.7% of the original enzyme's specific activity). Glutathione reductase failed to recycle the disulfide bond within the structure of di-substituted bis di-N, N'-1-Deoxy-fructos-1-yl GSSG. It showed only 1% of the original enzyme's specific activity, but retained its ability to reduce the disulfide bond within the structure of N-1-Deoxy-fructos-1-yl GSSG by 57% of its original specific activity. Since the GSH concentration in diabetic lens is significantly decreased and the glucose concentration can increase 10-fold and higher, the formation of Amadori products of the different forms of glutathione with this monosaccharide may be favored under these conditions and could contribute to a lowering of glutathione levels and an increase of oxidative stress observed in diabetic lens.     

Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster

Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. We present a detailed analysis of regulatory interactions involved in gap gene regulation based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. Our models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. Our models predict activation of Kr by Cad and clarify several other regulatory interactions. Our analysis suggests a central role for repressive feedback loops between complementary gap genes. We observe that repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, our models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system.     

Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation

Glycoprotein B (gB) enables the fusion of viral and cell membranes during entry of herpesviruses. However, gB alone is insufficient for membrane fusion; the gH/gL heterodimer is also required. The crystal structure of the herpes simplex virus type 1 (HSV-1) gB ectodomain, gB730, has demonstrated similarities between gB and other viral fusion proteins, leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the membranes together by refolding from its initial or prefusion form to its final or postfusion form. The only available crystal structure likely represents the postfusion form of gB; the prefusion form has not yet been determined. Previously, a panel of HSV-1 gB mutants was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite being expressed on the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions might not be accommodated in the postfusion form. Thus, we hypothesized that some insertion mutants were nonfunctional due to being “trapped” in a prefusion form. Here, we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We show that the ectodomains of all five mutants assume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall structures that are indistinguishable from those of the wild-type gB730. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, one mutant, while able to adopt the overall postfusion structure, displays significant conformational differences in the vicinity of fusion loops, relative to wild-type gB730. Moreover, this mutant has a diminished ability to associate with liposomes, suggesting that the fusion loops in this mutant have decreased functional activity. We propose that these insertions cause a fusion-deficient phenotype not by preventing conversion of gB to a postfusion-like conformation but rather by interfering with other gB functions.Herpes simplex virus type 1 (HSV-1) is the prototype of the diverse herpesvirus family that includes many notable human pathogens (26). In addition to the icosahedral capsid and the tegument that surround its double-stranded DNA genome, herpesviruses have an envelope—an outer lipid bilayer—bearing a number of surface glycoproteins. During infection, HSV-1 must fuse its envelope with a cellular membrane in order to deliver the capsid into a target host cell. Among its viral glycoproteins, only glycoprotein C (gC), gB, gD, gH, and gL participate in this entry process, and only the last four are required for fusion (28). Although gD is found only in alphaherpesviruses, all herpesviruses encode gB, gH, and gL, which constitute their core fusion machinery. Of these three proteins, gB is the most highly conserved.We recently determined the crystal structure of a nearly full-length ectodomain of HSV-1 gB, gB730 (18). The crystal structure of the ectodomain of gB from Epstein-Barr virus, another herpesvirus, has also been subsequently determined (4). The two structures showed similarities between gB and other viral fusion proteins, in particular, G from an unrelated vesicular stomatitis virus (VSV) (25), leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the viral and host cell membranes together to enable their fusion. However, gB alone is known to be insufficient for membrane fusion; the gH/gL heterodimer is also required. This insufficiency raises the question of exactly how gB functions during viral entry. Answering this question is critical for understanding the complex mechanism that herpesviruses use to enter their host cells.In acting as a viral fusogen, gB must undergo dramatic conformational changes, refolding through a series of conformational intermediates from its initial, or prefusion form, to its final, or postfusion form (15). These conformational changes are not only necessary to bring the two membranes into proximity; they are also thought to provide the energy for the fusion process. The prefusion form corresponds to the protein present on the viral surface prior to initiation of fusion. The postfusion form represents the protein after fusion of the viral and host cell membranes. The available gB structure likely represents its postfusion form, since it shares more in common with the postfusion rather than the prefusion structure of vesicular stomatitis virus (VSV) G (3, 17). However, the prefusion form has not yet been characterized.Recently, a panel of gB mutants was generated by using random linker-insertion mutagenesis (21). Of these mutants, 16 were particularly interesting because they were nonfunctional in cell-cell fusion assays despite being expressed on the cell surface at levels that indicate proper folding for transport. These observations suggested that each insertion somehow interfered with gB function. Insertions in 12 of these mutants are located within the available structure of the gB ectodomain, which allowed Lin and Spear to analyze their locations (21).The most prominent examples of such nonfunctional mutants are two mutants with insertions after residues I185 or E187, henceforth referred to as “cavity mutants” because both I185 and E187 point into a cavity inside the gB trimer (Fig. 1B and D). Although this cavity might accommodate a single 5-amino-acid insertion, it “is not large enough to accommodate three 5-amino-acid insertions” (21) that would be present in the trimer (one insertion per protomer).Open in a separate windowFIG. 1.Location of the insertion sites in the sequence of gB and the structure of the postfusion form of its ectodomain. (A) Linear diagram of the full-length gB with functional domains highlighted (as in reference 18). Domain I is shown in cyan, domain II in green, domain III in yellow, domain IV in orange, domain V in red, and the disordered region between domains II and III in purple. Regions absent from the crystal structure of gB730 are shown in gray. Sequences in the region of 5-amino-acid insertions (residues 181 to 190 and residues 661 to 680) are shown in black. Arrows mark the locations of 5-amino-acid insertions, shown as red text. (B) Crystal structure of gB730 (18). Residues preceding the 5-amino-acid insertions in mutants studied here are shown as spheres colored by domain, consistent with panel A. Boxes delineate the hinge region, enlarged in panel C, and the cavity region, enlarged in panel D. (C) Close-up view of the hinge region shown in molecular surface representation, with residues 663 to 675 displayed as sticks. Hydrophobic residues are colored orange. Residues preceding the 5-amino-acid insertions in mutants studied here are labeled with asterisks; remaining labels correspond to additional hydrophobic residues in the 663-675 region. (D) Enlarged view of the cavity region. Residues that line the cavity and are not solvent exposed are colored magenta. Residue E187 of each protomer is colored teal and shown as spheres. Fusion loops for two protomers are marked with asterisks; the third pair of fusion loops lies behind the crystal structure and is not visible. Panels B, C, and D were made by using Pymol (http://www.pymol.org/).Five other nonfunctional mutants have insertions after residues D663, T665, V667, I671, or L673, respectively. We refer to them as “hinge mutants.” These residues lie in the region located between domains IV and V, which has been termed the hinge region because it may play an important role during the conformational transition from the prefusion to the postfusion form (17). Lin and Spear proposed that insertions following these residues “would likely affect hinge regions” (21), with the implication that they may prevent gB from refolding into the postfusion conformation. Our analysis suggested that insertions after these residues could, perhaps, be sterically accommodated in the structure but would probably be energetically unfavorable by causing several buried hydrophobic side chains in the 665-673 region, such as F670, I671, and L673, to become exposed (Fig. 1B and C).In light of these observations, we hypothesized that the insertion mutants are “trapped” in a prefusion form. We decided to test this hypothesis by determining whether the ectodomain of gB containing one of these insertion mutations is able to assume the conformation seen in the crystal structure of the wild-type gB ectodomain, which we are referring to as the likely postfusion conformation. For this purpose, we chose one cavity mutant, containing an insertion after E187, and four hinge mutants, containing insertions after T665, V667, I671, or L673, respectively. We chose to test four hinge mutants because structure analysis suggested to us that insertions following the respective residues might not affect the structure in precisely the same way. We expressed the soluble ectodomain of each mutant by using a baculovirus expression system and characterized the purified proteins by using biochemical and biophysical methods. Surprisingly, we found that the ectodomains of all five mutants assume a conformation similar to that of the wild-type gB ectodomain. The four hinge mutants had biochemical properties and overall three-dimensional structures that were indistinguishable from those of the wild-type gB ectodomain. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, the cavity mutant, while able to adopt the overall postfusion structure, still displayed significant conformational differences relative to wild-type gB. Because these conformational differences are in the vicinity of fusion loops, we conclude that the fusion loops in this mutant have decreased functional activity.     

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

Sequence-dependent base pair opening in DNA double helix

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.     

A high-throughput cell-based assay for inhibitors of ABCG2 activity

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 microM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z' 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (> or = 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 microM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.     

Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti-inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor-α (TNF-α) and lipopolysaccharide- (LPS) induced inflammatory responses in the HT-29 cell line. In contrast, neither killed bifidobacterial cells, nor cell-free extracts showed such activities. Further investigations resulted in attribution of this activity to heat-stable, non-lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3 kDa. The anti-inflammatory effects were dose- and time-dependent and associated with inhibition of IκB phosphorylation and nuclear factor-κ light chain enhancer of activated B cells (NF-κB)-dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF-α, but not with CMs alone, resulted in upregulation of transforming growth factor-β1, IκBζ, and p21(CIP) mRNAs. Our data suggest certain species-specificities of the anti-inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.