Non-pretreated O-acyl isopeptide of amyloid β peptide 1–42 is monomeric with a random coil structure but starts to aggregate in a concentration-dependent manner

An isopeptide of amyloid β peptide 1–42 (isoAβ42) was considered as a non-aggregative precursor molecule for the highly aggregative Aβ42. It has been applied to biological studies after several pretreatments. Here we report that isoAβ42 is monomeric with a random coil structure at 40 μM without any pretreatment. But we also found that isoAβ42 retains a slight aggregative nature, which is significantly weaker than that of the native Aβ42.     

Generation of rubella virus-neutralising antibodies by vaccination with synthetic peptides

Abstract Four short peptides from rubella virus proteins E1 and E2, predicted to contain B cell epitopes, were used to vaccinate BALB/c mice. Sera from peptide-vaccinated animals reacted with viral antigens in ELISA and three of the four induced virus-neutralising antibody (nAb) responses. Peptide PY4, in contrast to the others, induced IgG2a responses upon vaccination and stimulated spleen cells in vitro produced IFNγ in the absence of IL-5. It was reasoned that vaccination with PY4 caused Th1 subset activation, the appropriate type of response for anti-viral immunity and hence the efficient neutralising antibody response. Presentation of peptide for vaccination proved to be as important as the sequence. Similar profiles of IgG1 and IgG2a were detected in the sera of mice vaccinated with PY4 in Freund's complete adjuvant or alum; however nAb responses were not found when alum was used.     

Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.     

Membrane localisation of a UDP-sugar hydrolase, in Salmonella, is by an uncleaved N-terminal signal peptide

Abstract Most isolates of Salmonella contain two unrelated UDP-sugar hydrolases, one of which, encoded by the ushB gene, is inner membrane-associated. Previous studies showed that this enzyme contains a typical N-terminal signal peptide; the evidence also indicated, however, that this peptide is not cleaved, and serves to anchor the UshB protein in the inner membrane. In this report, we present strong evidence that this is indeed the case by using ushB'-'blaM fusions to demonstrate that this signal peptide is capable of localising β-lactamase to the inner membrane. We also present evidence that UshB is located on the exterior (periplasmic) side of the membrane, and hence has an 'N-terminus inside/C-terminus outside' membrane orientation, consistent with a role in the degradation of external substrates.     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

What limits the primary sequence space of natural proteins?

Abstract

Number of naturally occurring primary sequences of proteins is an infinitesimally small subset of the possible number of primary sequences that can be synthesized using 20 amino acids. Prevailing views ascribe this to slow and incremental mutational/selection evolutionary mechanisms. However, considering the large number of avenues available in form of diversity of emerging/evolving and/or disappearing living systems for exploring the primary sequence space over the evolutionary time scale of ～3.5 billion years, this remains a conjecture. Therefore, to investigate primary sequence space limitations, we carried out a systematic study for finding primary sequences absent in nature. We report the discovery of the smallest peptide sequence “Cysteine-Glutamine-Tryptophan-Tryptophan” that is not found in over half-a-million curated protein sequences in the Uniprot (Swiss-Prot) database. Additionally, we report a library of 83605 pentapeptides that are not found in any of the known protein sequences. Compositional analyses of these absent primary sequences yield a remarkably strong power relationship between the percentage occurrence of individual amino acids in all known protein sequences and their respective frequency of occurrence in the absent peptides, regardless of their specific position in the sequences. If random evolutionary mechanisms were responsible for limitations to the primary sequence space, then one would not expect any relationship between compositions of available and absent primary sequences. Thus, we conclusively show that stoichiometric constraints on amino acids limit the primary sequence space of proteins in nature. We discuss the possibly profound implications of our findings in both evolutionary and synthetic biology.

Communicated by Ramaswamy H. Sarma     

The mechanism of the hyperglycaemic action of synthetic peptides related to the C-terminal sequence of human growth hormone

Synthetic part sequences of human pituitary growth hormone (hGH 176–191 and hGH 177–191) corresponding to residues 176–191 or 177–191 of the hormone have been tested for their effects on glycogen and pyruvate metabolism in the rat, both in vivo and in vitro. When injected, the peptides caused transient increases in blood glucose and lactate, while decreasing the activity ratio of glycogen synthase in muscle, adipose tissue and liver and of pyruvate dehydrogenase in muscle and adipose tissue, but not in liver. These decreases were associated with the conversion of the enzymes from their active to their inactive forms, since the peptides did not affect the total amount of either the synthase or the dehydrogenase. The time course of the effect on the enzymes was similar to that for the effect on blood metabolites, and responses for synthase were produced over the range 0.07–7 nmols hGH 177–191/kg body weight. Phosphorylase activity was not affected by the peptides, nor was the capacity to dispose of injected L-lactate. Experiments with adipocytes and hepatocytes showed that the peptides also affected glycogen synthase and pyruvate dehydrogenase activities in vitro. The peptides had no effect on the overall rate of gluconeogenesis from lactate by hepatocytes. However, at times corresponding to those at which glycogen synthase was inactivated, the peptides caused increased incorporation of lactate into free glucose and decreased incorporation into glycogen. It was concluded that the peptides acted directly on their target tissues, and that the observed hyperlactataemia was the result of the inactivation of pyruvate dehydrogenase. The addition lactate increased the flux through the gluconeogenic pathway, and appeared as glucose because the peptide also inactivated glycogen synthase. Thus, the hyperglycaemia produced by hGH 177–199 and related peptides is explicable in terms of a modified Cori Cycle.     

Schistosoma mansoni: antigenic community between schistosomula surface and adult worm incubation products as a support for concomitant immunity

The use of protective monoclonal antibodies has enabled us to demonstrate antigenic community between a 38-kDa schistosomula surface molecule and a 115-kDa component derived from adult worms. Injection of adult worms in rats also led to the production of antibodies specific for the 38-kDa antigen, suggesting that the 115-kDa adult worm molecule could act as an inducer of the protective immune response raised against young invading parasites.