Conformation of the cecropin-melittin hybrid, cecropin A(1-8)-melittin (1-18) antibacterial Peptide

Cecropin A (1-8)-Melittin (1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. The primary sequence of the peptide is as follows: KWKLPKKIGIGAVLKVLTTGLPALIS-NH2. 1H and 13C 2D NMR techniques were used to deduce the conformational parameters of chemical shift, 3JNHalpha coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter-residue nOe's. NMR studies were carried out in water (pH 6.0) and hexafluoroacetone (HFA). The peptide was found in a beta-pleated structure in water, and in HFA it adopts a right-handed alpha-helix conformation. Solution structures generated using restrained molecular dynamics simulations were refined by Mardigras to R factors ranging from 0.5 to 0.6.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Conformation of the peptide antibiotic — histatin 8 in aqueous and non aqueous media

Histatin 8 (Lys¹-Phe-His-Glu-Lys⁵-His-His-Ser-His-Arg¹⁰-Gly-Tyr¹²)belongs to a group of related neutral and basic histidine richpeptides present in human salivary secretions that possess fungicidal and bactericidal activities. The conformation of thispeptide has been examined by ¹H and ¹³C 2D-NMR in DMSO-d₆, water (pH 4.0) and 40% HFA solutions. MD simulations incorporating NMR data was used to generate the solution conformations. The structures were refined by MARDIGRAS employing the RANDMARDI approach. In both DMSO-d₆ and water, the peptide is seen to adopt a -pleated sheet, while HFA induces an -helix structure. The role of these structures in its mechanism of action has been explained.     

Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion.

Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.     

Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049–13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.     

Identification of a chemotactic,MCP-1-like protein from Mycobacterium avium

In the immunocompetent host, Mycobacterium avium is responsible for chronic localized pulmonary disease, which is characterized by the presence of increased numbers of activated T cells and macrophages in the lungs. M. avium organisms as well as sonic extracts of M. avium were found to act as chemoattractants for THP-1 cells as well as monocytes, monocyte-derived macrophages and alveolar macrophages obtained from normal human donors in an in vitro chemotaxis assay, where a significantly higher number of cells were found in wells containing M. avium compared to control wells. Proteolytic treatment of M. avium sonicate resulted in significant loss (50%) of chemotactic activity. Monoclonal antibodies against recombinant human monocyte chemoattractant protein-1 (MCP-1) were found to cross-react with a 34-kDa protein of M. avium sonicate on Western blot and inhibit M. avium sonicate-mediated chemotaxis of THP-1 cells (47%). These data suggest the presence of an 'MCP-1 like' molecule on M. avium. Recruitment of host immune regulatory cells to the site of infection by pathogens may be involved in generating a local immune response or may be a bacterial strategy for survival within the host by recruiting the cells that they infect, i.e. macrophages.     

Crystallization and structure determination of goat lactoferrin at 4.0 A resolution: a new form of packing in lactoferrins with a high solvent content in crystals

Lactoferrin was purified from fresh samples of goat colostrums, saturated with Fe3+ and CO3(2-) ions and crystallized by microdialysis method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with a=104.6 A, b=153.8 A, c=155.1 A and Z=4. The quality of crystals was poor, thus the intensity data were restricted to 4.0 A resolution only. The structure was determined by molecular replacement method using diferric buffalo lactoferrin as a model. The solution clearly indicated the presence of one molecule in the asymmetric unit, which corresponds to a Vm value of 7.1 A3/Da. The structure was refined with stringent constraints to an R-factor of 0.246 using all the reflections 15,870 to 4.0 A resolution. The overall structure of goat lactoferrin is essentially similar to those of buffalo and bovine lactoferrins. However, the iron-binding environment in goat lactoferrin is somewhat different, in which 2 CO3(2-). ions have low occupancies. The solvent content of approximately 84% was very high in the present case which explains the fragility of the crystals of goat lactoferrin. In a way, it is very surprising that the crystals grow at all, although crystals with solvent as high as 89% have been reported.     

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

An antiparallel beta-sheet and a beta-turn characterize the structure of antiviral HIV-1 peptide T140, as revealed by 2D NMR and MD Simulations

The polyphemusins present in the hemocytes of the horsechoe crab and their structurally modified analogs have been shown to exhibit activity against HIV-1. Among the many variants, T22 ([Tyr(5,12), Lys(7)]-polyphemusin II), and its shorter and more potent analog, T140 [Arg(1)-Arg-2-Nal-Cys-Tyr(5)-Arg-Lys-D-Lys-Pro-Tyr(10)-Arg-Cit-Cys-Arg(14)] (Polyphemusin II-derived peptide), affect the HIV-cell fusion process and inhibit the T-cell line-tropic (T-tropic) HIV-1 infection. Conformational studies of polyphemusin II derived peptide have been carried out by (1)H and (13)C 2D-NMR and MD simulations in water and HFA (40%). The NMR parameters of chemical shift, temperature coefficients of the NH chemical shifts, (3)JNHalpha coupling constants and the pattern of nOe's were used to deduce the structural characteristics. Solution structures were generated using dihedral and distance restraints by MD simulations. The structures are characterized by a dominant family possessing an anti-parallel beta-pleated sheet that is constrained by the disulphide bridge between Cys4 and Cys13. The two strands of the beta-sheet are joined by a Type II' beta-turn spanning the residues Lys(7)-D-Lys(8)-Pro(9)-Tyr(10). This conformation is present in both water and HFA. The only difference in the two structures is that the beta-strands are more cohesive in HFA being firmly held by H-bonds. The solution structures generated from MD simulations were refined by MARDIGRAS to R-factors of 0.44 and 0.57 in water and HFA respectively. The conformation deduced for T140 is very similar to that reported for T22 and is thought to be associated with their anti HIV activity.