Influence of alpha-helicity, amphipathicity and D-amino acid incorporation on the peptide-induced mast cell activation

Mast cell activation by polycationic substances is believed to result from a direct activation of G protein alpha subunits and it was suggested that the adaption of amphipathic, alpha-helical conformations would allow the peptide to reach the cytosolic compartment to interact with G proteins (Mousli et al., 1994, Immunopharmacology 27, 1, for review). We investigated the histamine-releasing activity of model peptides as well as analogues of magainin 2 amide and neuropeptide Y with different amphipathicities and alpha-helix content on rat peritoneal mast cells. Amphipathic helicity is not a prerequisite for mast cell activation. Moreover, non-helical magainin peptides with high histamine-releasing activity were less active in the liberation of carboxyfluoresceine from negatively charged liposomes, indicating that peptide-induced mast cell activation and peptide-induced membrane perturbation do not correlate. In contrast to the negligible influence of the secondary structure, amino acid configuration may exert a striking influence on peptide-induced mast cell activation. Thus histamine-release by substance P was markedly impaired when the L-amino acids in the positively charged N-terminal region were replaced by D-amino acids, with [D-Arg(1)]substance P being the most inactive substance P diastereoisomer.

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha(1)-adrenergic receptor activation: Evidence for a salt bridge as the initiating process

alpha(1)-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (Porter et al., 1996). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha(1)-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha(1b)-AR and in Rat-1 fibroblasts stably transfected with the human alpha(1a)-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha(1)-AR agonists and this effect was fully inhibited by the alpha(1)-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha(1)-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha(1)-ARs with a K-i of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha(1)-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha(1)-AR activation.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Autosomal dominant Alport syndrome linked to the type IV collage alpha 3 and alpha 4 genes (COL4A3 and COL4A4)

Alport syndrome is a hereditary nephritis that may lead to end-stage renal disease (ESRD) in young adult life and is often associated with sensorineural deafness and/or ocular abnormalities. The majority of families are X-linked due to mutations in the COL4A5 gene at Xq22. Autosomal forms of the disease are also recognized with recessive disease, having been shown to be due to mutations in the COL4A3 and COL4A4 genes on chromosome 2. Familial benign haematuria has also been mapped to this region in some families.