Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH ... OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH ... OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.

Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-RI) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis, In previous studies, me have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis, We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Pour of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors, (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-RI and more so for the TRAIL decoy receptors TRAIL-R3 and -R4, Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-RI and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-RI and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

Metal-centred versus ligand-centred luminescence quenching of a macrocyclic copper(II) complex

The new macrocyclic ligand trans-6-(9-anthracenylmethylamino)-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecan-13-amine has been synthesized and characterised as its copper(II) complex and the crystal structure of this complex has been determined. Fluorescence of the anthracenyl group of the macrocycle is quenched in its free base form and when complexed with Cu-II. Fluorescence returns when Lewis acids such as H+ and Zn-II are added to solutions of the ligand, indicating that photoinduced electron transfer from the amine lone pairs is responsible for fluorescence quenching in the free base form. By contrast, fluorescence of the complex is quenched by intramolecular electronic energy transfer.

Identification of a new ligand binding domain in the al subunit of the inhibitory glycine receptor

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed loop A ligand binding domain; Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, I93A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, I-max values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile(93) and Asn(102), as contributing to the four-loop model of ligand binding.

Monooxygenases play only a minor role in resistance to synthetic pyrethroids in the cattle tick, Boophilus microplus

We investigated the role of monooxygenases in resistance to synthetic pyrethroids (SPs) in the cattle tick, Boophilus microplus. We found that monooxygenases play only a minor role in resistance to SPs in both resistant and susceptible strains of B. microplus. We blocked the monooxygenases with piperonyl butoxide (PBO) and simultaneously applied the SPs, flumethrin and cypermethrin to larval B. microplus. PBO increased the effect of flumethrin (synergism ratios 2.7-8.9) more than it increased the effect of cypermethrin (synergism ratios 1.9-3.1). Of the four strains tested, Parkhurst, which is resistant to SPs, was the least affected by the addition of PBO (synergism ratios after cypermethrin was applied 1.9; after flumethrin 2.7) whereas N.R.F.S., the strain susceptible to SPs, was the most affected by synergism between PBO and SPs (synergism ratio after cypermethrin was applied 3.1; after flumethrin 8.9). We hypothesize that B. microplus lacks monooxygenases capable of conferring resistance to SPs because it and its recent ancestors were blood-feeders rather than herbivores.

Increased ovulation rate in Merino ewes immunized against small synthetic peptide fragments of the inhibin alpha subunit

Four experiments were carried out in Merino ewes during a period of 4 years to determine the long-term effects of immunization against different synthetic peptides mimicking the amine terminal of the or subunit of porcine inhibin. Peptides were conjugated to human serum albumin and 100-200 mu g emulsified in Freund's complete adjuvant for the primary immunization. Usually two booster injections were given at monthly intervals with 50-100 mu g conjugated peptide using either incomplete Freund's adjuvant or Montanide : Marcel. In some experiments a further immunization was carried in the next year. Blood samples were taken 10 days after each immunization, during the luteal phase, for estimation of gonadotrophin concentrations and determination of inhibin antibody titres. One day after blood sampling cloprostenol was used to induce luteolysis and laparoscopy was performed in the subsequent oestrous cycle. Immunization of ewes with synthetic peptides 1-32, 1-26, 7-26 and 8-30 resulted in large increases in the ovulation rate (OR). An approximately two-fold increase in OR was observed following the first booster immunization with these peptides and a three- to five-fold increase after the second booster immunization. Immunization with these large peptides resulted in a sustained increase in OR for a period of at least 1 year after the second booster immunization. Of the shorter peptides, peptides 10-26 and 13-26 gave a reasonable ovulatory response, although it was more difficult to obtain a response with peptides 1-16, 8-22, 13-25, 8-19 and 10-19; peptides 7-13 and 1-6 gave no response (but were examined for one breeding season only). The smaller peptides led to lower inhibin antibody titres that were not necessarily associated with increased follicle-stimulating hormone (FSH) or OR. More intensive blood sampling in one experiment showed that following primary immunization against peptide 1-32 there was a transient increase in plasma FSH which did not lead to an increased OR. Moreover, a prolonged period of raised FSH after the first booster was significantly correlated with increased OR. In these animals antibody titres were only slightly increased after primary immunization, but after the first booster immunization higher titres were observed that were significantly correlated with trough FSH values and the subsequent OR. These results are interpreted as showing that (1) to obtain an increase in OR peptides 1-32, 1-26 and 7-26 are suitable as immunogens; (2) smaller peptides are less reliable, often require multiple injections, and the response may be delayed; and (3) an extended period of raised plasma FSH is needed to give a large ovulatory response.

Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells

Induction of apoptosis in cells by TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is believed to be regulated by expression of two death-inducing and two inhibitory (decoy) receptors on the cell surface. In previous studies we found no correlation between expression of decoy receptors and susceptibility of human melanoma cells to TRAIL-induced apoptosis, In view of this, we studied the localization of the receptors in melanoma cells by confocal microscopy to better understand their function. We show that the death receptors TRAIL-R1 and R2 are located in the trans-Golgi network, whereas the inhibitory receptors TRAIL-R3 and -R4 are located in the nucleus. After exposure to TRAIL, TRAIL-R1 and -R2 are internalized into endosomes, whereas TRAIL-R3 and -R4 undergo relocation from the nucleus to the cytoplasm and cell membranes. This movement of decoy receptors was dependent on signals from TRAIL-R1 and -R2, as shown by blocking experiments with Abs to TRAIL-R1 and -R2, The location of TRAIL-R1, -R3, and -R4 in melanoma cells transfected with cDNA for these receptors was similar to that in nontransfected cells, Transfection of TRAIL-R3 and -R4 increased resistance of the melanoma lines to TRAIL-induced apoptosis even in melanoma lines that naturally expressed these receptors. These results indicate that abnormalities in decoy receptor location or function may contribute to sensitivity of melanoma to TRAIL-induced apoptosis and suggest that further studies are needed on the functional significance of their nuclear location and TRAIL-induced movement within cell.

Ni-II and Zn-II complexes of the hexadentate macrocyclic ligand cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetra- decane-6,13-diamine

The title pendent-arm macrocyclic hexaamine ligand binds stereospecifically in a hexadentate manner, and we report here its isomorphous Ni-II and Zn-II complexes (both as perchlorate salts), namely (cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine-kappa(6)N)nickel(II) diperchlorate, [Ni(C12H30N6)](ClO4)(2), and (cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine-kappa(6)N)zinc(II) diperchlorate, [Zn(C-12 H30N6)](ClO4)(2). Distortion of the N-M-N valence angles from their ideal octahedral values becomes more pronounced with increasing metal-ion size and the present results are compared with other structures of this ligand.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by Smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/ DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with downregulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 and Fe(S-Cys)(3)(OH) centres

The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)(4) active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli. The C42A protein expresses with a (Fe2S2)-S-III cluster in place. In contrast, the other proteins are isolated in colourless forms, although a (Fe2S2)-S-III cluster may be assembled in the C42G protein via incubation with Fe-III and sulfide. The four mutant proteins were isolated as stable mononuclear Hg-II forms which were converted to unstable mononuclear Fe-III preparations that contain both holo and apo protein. The Fe-III systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant Fe-III form in the C9A preparation is a Fe(S-Cys)(3)(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)(3)(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe-III form in which a ligand appears to be supplied by the protein chain. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-0020355-1.

Effect of chloride concentration on the electrochemical treatment of a synthetic tannery wastewater

The electrochemical treatment of a synthetic tannery wastewater prepared with 30 compounds used in animal skin processing was studied. Electrolyses were performed in a one-compartment flow cell at a current density of 20 mA cm(-2), using a dimensionally stable anode (DSA (R)) of composition Ti/Ir(0.10)Sn(0.90)O(2) as the working electrode. Effects of chloride concentration and presence of sulfate were evaluated. Variation in the concentration of phenolic compounds as a function of electrolysis time revealed a first-order exponential decay; faster phenol removals were obtained with increasing chloride concentration in the wastewater. Lower phenol removals were obtained in the presence of sulfate. Higher chloride concentrations led to a faster decrease in total organic carbon (TOC), chemical oxygen demand (COD), and absorbance values at 228 nm. Faster wastewater color removal, higher current efficiency and lower energy consumption were also obtained. This electrochemical treatment was also able to reduce the wastewater toxicity for Daphnia similis. (C) 2008 Elsevier Ltd. All rights reserved.