Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

Global iron-dependent gene regulation in Escherichia coli - A new mechanism for iron homeostasis

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur ( ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron- transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron- rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron- containing proteins is repressed under iron- restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of Fe-55-labeled E. coli proteins revealed a marked decrease in iron- protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron- sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O-2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of Fe-B(2+) to Fe-C(2+) enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.

Inhibition of E2F abrogates the development of cardiac myocyte hypertrophy

Growth of the post- natal mammalian heart occurs primarily by cardiac myocyte hypertrophy. Previously, we and others have shown that a partial re- activation of the cell cycle machinery occurs in myocytes undergoing hypertrophy such that cells progress through the G(1)/ S transition. In this study, we have examined the regulation of the E2F family of transcription factors that are crucial for the G(1)/ S phase transition during normal cardiac development and the development of myocyte hypertrophy in the rat. Thus, mRNA and protein levels of E2F- 1, 3, and 4 and DP- 1 and DP- 2 were down- regulated during development to undetectable levels in adult myocytes. Interestingly, E2F- 5 protein levels were substantially up- regulated during development. In contrast, an induction of E2F- 1, 3, and 4 and the DP- 1 protein was observed during the development of myocyte hypertrophy in neonatal myocytes treated with serum or phenylephrine, whereas the protein levels of E2F- 5 were decreased with serum stimulation. E2F activity, as measured by a cyclin E promoter luciferase assay and E2F- DNA binding activity, increased significantly during the development of hypertrophy with serum and phenylephrine compared with non- stimulated cells. Inhibiting E2F activity with a specific peptide that blocks E2F- DP heterodimerization prevented the induction of hypertrophic markers ( atrial natriuretic factor and brain natriuretic peptide) in response to serum and phenylephrine, reduced the increase in myocyte size, and inhibited protein synthesis in stimulated cells. Thus, we have shown that the inhibition of E2F function prevents the development of hypertrophy. Targeting E2F function might be a useful approach for treating diseases that cause pathophysiological hypertrophic growth.

Mapping CD55 function: the structure of two pathogen-binding domains at 1.7 A

Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells. It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection. We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement. We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

Synthesis and crystal structure of the thiophene-3-acetonitrile complex fac-[Mo(CO)(3){NCCH2(C4H3S-3)}(3)]

Reaction of fac-[ Mo( CO)(3)( NCMe)(3)] with three equivalents of NCCH2(C4H3S- 3) in acetonitrile gives the tris(thiophene- 3- acetonitrile) complex, fac-[Mo(CO)(3){NCCH2(C4H3S-3)}(3)] (1) in 7% yield. Complex 1 crystallizes out in the orthorhombic space group Pnma with a = 12.714( 17), b = 16.41( 2), c = 11.304(16) Angstrom, Z = 4. The structure has crystallographic m symmetry and the metal is in an almost perfect octahedral environment, with a facial arrangement of carbonyl and thiophene- 3- acetonitrile groups. The thiophene rings are disordered.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Solid state annular tautomerism in a molecule containing two imidazole moieties

The X-ray crystal structure of the 1:2 condensate (1) of hydrazine hydrate and 4-methyl-imidazole-5-carboxaldehyde has been determined. The molecule is centrosymmetric crystallising in the space group Fddd with cell dimensions: a = 10.557(14), b = 17.062(22), c = 24.759(27) angstrom. Fourier map shows that the NH hydrogen atom of each imidazole moiety has equal possibility of occupying any of its two ring N atoms. This poses the possibility of finding three tautomers in 1 in the solid state. Consideration of the H-bonding pattern observed in 1 and related B3LYP/6-311+G(2d, p) calculations show that only two tautomers are present in the solid state. The situation is compared with that in the structure of 4(5)-nitro-5(4)-methoxy-imidazole reported previously by Kubicki.

Influence of a halide ion on ribbons of water pentamer

Reactions of CuF2, CuCl2 center dot 2H(2)O and CuBr2 with 2,2'-dipyridylamine (HDPA) in water at room temperature using Cu: HDPA = 2: 1 mol yield [Cu(HDPA) (H2O)(2)F]F center dot 3H(2)O (1), Cu(HDPA) Cl-2 (2) and [Cu(HDPA) Br-2 (3) respectively. The structures of 2 and 3 are isostructural in spacegroup C-2 with cell dimensions; for 2, a = 14.702(8), b = 7.726(2), c = 4.829(6) angstrom, beta = 96.68(8)degrees and for 3, a = 14.2934(8), b = 7.9057(6), c = 5.1982(5) angstrom, beta = 94.049(7)degrees. In the X-ray crystal structure, the complex 1 is found to contain tapes of water pentamers. Our DFT calculations at the B3LYP/LanL2DZ level show that the reaction Cu(HDPA)X-2 + 2H(2)O = [Cu(HDPA)(H2O)(2)X]X is most exothermic in the gas phase when X- = F-, i.e., the tendency of water uptake is maximum for Cu(HDPA) F-2. It seems that the exothermicities of the aquations of Cu(HDPA) Cl-2 and Cu(HDPA) Br-2 are not sufficient to stabilise the type of ribbons of water observed in 1 and consequently water is eschewed when X- = Cl- or Br-.

Description of Enterococcus canis sp nov from dogs and reclassification of Enterococcus porcinus Teixeira et al. 2001 as a junior synonym of Enterococcus villorum Vancanneyt et al. 2001

Strains from anal swabs and chronic otitis externa in dogs were shown to be phylogenetically related to the Enterococcus faecium species group. They shared a number of phenotypic characteristics with these species, but they could be easily differentiated by biochemical reactions. In addition, the canine strains were unusual in their nearly complete failure to grow on sodium azide-containing enterococci-selective media and in their Voges-Proskauer reactions (usually negative). By using 16S rRNA sequencing and DNA-DNA hybridization of representative strains, as well as tDNA interspacer gene PCR and SDS-PAGE of whole-cell proteins, the group of canine strains was shown to constitute a novel enterococcal species. The name Enterococcus canis sp. nov. is proposed for this species, with LMG 12316(T) (= CCUG 46666(T)) as the type strain. Concurrently, the taxonomic situation and nomenclatural position of Enterococcus porcinus were investigated. As no phenotypic or genotypic differences were found between this species and Enterococcus villorum, the name E. porcinus is considered to be a junior synonym of E. villorum.

Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg(39) and Arg(85), which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg(10), while Arg(33) appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg(39) and Arg(85) are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.