Fluorescent hydrophobic peptide fragments of emerimicin. Models for the study of peptide aggregation and interactions with lipids and proteins.

The fluorescent amino terminal fragments of emerimicin, dansyl-Phe-Aib-Aib-OMe, dansyl-Phe-Aib-Aib-Aib-Val-OMe and dansyl-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-OMe and the corresponding peptide acids have been synthesised. The nonapeptide ester aggregates at concentrations greater than 8 μM whereas the tri and pentapeptide esters do not. The peptide acids also do not aggregate. The esters bind to lipid dispersions with the largest changes in fluorescence observed for the nonapeptide, whereas the acids interact very weakly. The acids show changes in fluorescence in the presence of bovine serum albumin, in contrast to the esters with the shorter peptides showing larger effects.     

Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes

Indolicidin is a 13-residue broad-spectrum antibacterial peptide isolated from bovine neutrophils. The primary structure of the peptide ILPWKWPWWPWRR-amide (IL) reveals an unusually high percentage of tryptophan residues. IL and its analogues where proline residues have been replaced by alanine (ILA) and trp replaced by phe (ILF) show comparable antibacterial activitieso While IL and ILA are haemolytic, ILF does not have this property. Since aromatic residues would strongly favour partitioning of the peptide into the lipid bilayer interface, the biological activities of IL and its analogues could conceivably arise due perturbation of the lipid bilayer of membranes. We have therefore investigated the interaction of IL and its analogues with lipid vesicles. Peptides IL and ILA bind to lipid vesicles composed of phosphatidylcholine and phosphatidylethanol amine: phosphatidyl glycerol: cardiolipin. The position of λ_max and I^- quenching experiments suggest that the trp residues are localized at the membrane interface and not associated with the hydrophobic core of the lipid bilayer in both the peptides. Hence, membrane permeabilization is likely to occur due to deformation of the membrane surface rather than formation of transmembrane channels by indolicidin and its analogues. Peptides ILA, IL and ILF cause the release of entrapped carboxyfluorescein from phosphatidyl choline vesicles. The peptide-lipid ratios indicate that ILF is less effective than IL and ILA in permeabilizing lipid vesicles, correlating with their haemolytic activities. An erratum to this article is available at .     

Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca²⁺) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes. Deletion of the two phospholipid methyltransferases CHO2 and OPI3 modulated PC, TAG levels and the lipid droplets with cadmium exposure. Interestingly, we noticed an increase in the calcium levels upon Cd exposure in the mutant cells. This study concluded that Cd interrupted calcium homeostasis-induced lipid dysregulation leading to ER stress.     

No apparent cost of evolved immune response in Drosophila melanogaster

Maintenance and deployment of the immune system are costly and are hence predicted to trade‐off with other resource‐demanding traits, such as reproduction. We subjected this longstanding idea to test using laboratory experimental evolution approach. In the present study, replicate populations of Drosophila melanogaster were subjected to three selection regimes—I (Infection with Pseudomonas entomophila), S (Sham‐infection with MgSO₄), and U (Unhandled Control). After 30 generations of selection flies from the I regime had evolved better survivorship upon infection with P. entomophila compared to flies from U and S regimes. However, contrary to expectations and previous reports, we did not find any evidence of trade‐offs between immunity and other life history related traits, such as longevity, fecundity, egg hatchability, or development time. After 45 generations of selection, the selection was relaxed for a set of populations. Even after 15 generations, the postinfection survivorship of populations under relaxed selection regime did not decline. We speculate that either there is a negligible cost to the evolved immune response or that trade‐offs occur on traits such as reproductive behavior or other immune mechanisms that we have not investigated in this study. Our research suggests that at least under certain conditions, life‐history trade‐offs might play little role in maintaining variation in immunity.     

Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs

Detergents are indispensable solubilizing agents in the purification and analysis of membrane proteins. For mass spectrometric identification of proteins, it is essential that detergents are removed prior to analysis, necessitating an in-gel digestion step. Here, we report a procedure that allows use of detergents and in-solution digestion of proteins. Crude membrane preparations from mouse brain were solubilized with Triton X-100, CHAPS, or SDS, and the detergents were depleted from the membrane proteins using a desalting column equilibrated with 8 M urea. Following digestion with endoproteinase Lys-C, the resulting peptides were analyzed by LC-MS/MS on Linear ion trap-Orbitrap instrument. Applying stringent identification criteria, in single-LC-MS-runs, 1059 +/- 108 proteins, including 797 +/- 43 membrane proteins, were mapped from mouse brain. The identified proteins represented a broad spectrum of neurotransmitter receptors and other ion channels. The general applicability of the method is demonstrated by profiling of membrane proteins from four other mouse organs. Single-run analyses of eye, liver, spleen, and skeletal muscle allowed identification of 522 +/- 9, 610 +/- 7, 777 +/- 8, and 307 +/- 7 membrane proteins. Our results demonstrate that membrane proteins can be analyzed as efficiently as soluble proteins.     

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His–Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.     

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.