Silver‐induced conformational changes of polypeptides: a CD study

The role of silver ions in various pathologies, as well as their effect on peptide conformation and properties are less understood. Consequently, we synthesized several peptides with various residues in their sequence to investigate silver‐induced conformational changes at various pH values by Circular Dichroism spectroscopy. Uniquely, the glycine‐based, histidine‐containing peptide showed a severe change from a random coil and β‐turn conformation to large α‐helices during silver binding. When comparing the effect of silver ions on the conformation of bradykinin a similar tendency was found. Besides, silver ions reduced the amyloid‐β peptide tendency to aggregation. Our results suggest a specific and protective role for silver ions in brain pathologies, which is related to their high affinity toward physiologically and pharmacologically active peptides. Fourier transform infrared spectroscopy studies as well as the mass spectrometric ones support our conclusions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

An immunological study of secreted human polymeric immunoglobulins' J-peptide tissue specificity

Contents of J-peptide of secreted human polymeric immunoglobulins may vary considerably with different pathologies, reflecting the state of the adaptive immune system. In this work assessed the content of J-peptide in various tissues of healthy people to use as a baseline for studies related to the change in the content of J-peptide in pathologies.     

Selection of peptide ligands binding to fibroblast growth factor receptor 1

Inappropriate expression of fibroblast growth factors (FGFs) or activation of FGF receptors (FGFRs) could contribute to several human angiogenic pathologies. In an attempt to design antagonists of FGF, we developed a screening procedure for identifying peptide ligands binding to FGFR1. To retain the natural conformation of FGFR1 during screening, we expressed recombinant FGFR1 on the surface of Sf9 insect cells. A 6-mer phage display peptide library was then screened on the cell surface and a group of hydrophobic peptide sequences were identified. Further experiments demonstrated that the phages displaying these sequences can specifically bind to FGFR1. The docking analysis suggests that the peptide ValTyrMetSerProPhe can specifically bind to the hydrophobic surface of FGFR1. The synthetic peptide Ac-ValTyrMetSerProPhe-NH2 can inhibit mitogenic activity of aFGF and has the potential to become a therapeutic agent as an aFGF antagonist.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Fusion of phospholipid vesicles induced by an amphiphilic model peptide: close correlation between fusogenicity and hydrophobicity of the peptide in an alpha-helix.

A model peptide with 51 amino acid residues consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and a turn sequence of Asn-Pro-Gly at the center of the molecule has a random conformation at neutral pH but adopts an amphiphilic alpha-helical form in the presence of various salts or nucleotides [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396; Goto, Y., Okamura, N., & Aimoto, S. (1991) J. Biochem. (Tokyo) 109, 746-750]. The interaction of this model peptide with liposome membranes and the resulting alpha-helical conformational transition and membrane fusion as well as the effect of the nucleotide ATP on these events were examined at neutral pH. The peptide associated stoichiometrically with liposome membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) in a molar ratio of 2:1, resulting in formation of an amphiphilic alpha-helix and induction of fusion of the liposomes. However, the final fusion level was not correlated with the amount of binding or the helix content and was found to increase on an increase in hydrophobicity of the peptide in the alpha-helical form by neutralization of its positive charges by the negative charges of PS. In contrast, in the presence of ATP, the peptide bound completely to the PS/PC membranes at a lower concentration of liposome and concomitantly induced membrane fusion, indicating that ATP cooperates with PS to neutralize the charges of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.     

Oral health and frailty in the medieval English cemetery of St Mary Graces

The analysis of oral pathologies is routinely a part of bioarcheological and paleopathological investigations. Oral health, while certainly interesting by itself, is also potentially informative about general or systemic health. Numerous studies within modern populations have shown associations between oral pathologies and other diseases, such as cardiovascular disease, certain types of cancer, and pulmonary infections. This article addresses the question of how oral health was associated with general health in past populations by examining the relationship between two oral pathologies (periodontal disease and dental caries) and the risk of mortality in a cemetery sample from medieval England. The effects of periodontitis and dental caries on risk of death were assessed using a sample of 190 individuals from the St Mary Graces cemetery, London, dating to ～AD 1350–1538. The results suggest that the oral pathologies are associated with elevated risks of mortality in the St Mary Graces cemetery such that individuals with periodontitis and dental caries were more likely to die than their peers without such pathologies. The results shown here suggest that these oral pathologies can be used as informative indicators of general health in past populations. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

Protease-mediated arsenic prodrug strategy in cancer and infectious diseases: a hypothesis for targeted activation

A strategy for the selective in vivo activation of prodrugs by proteases is presented. The approach is based on the design of polythiol peptides able to neutralize the toxicity of As(III) through chelation, and contemporarily to be recognized as substrates of a disease-linked specific protease. Enzyme digestion implies conversion of such polythiol peptides into monothiol fragments with irreversible loss of the ability to chelate the metalloid, thus triggering the release in its free and pharmacologically effective form. The proteases whose activity appears dramatically up-regulated in various pathologies, ranging from cancer to infectious diseases, can be conveniently employed as prodrug activators in the disease microenvironment. The design of the representative peptide shown here has been assisted by molecular modeling in order to fulfill the dual characteristic to be an efficient As(III) chelator and simultaneously a substrate of the matrix metalloproteinase-9 (MMP-9) whose activity results dramatically increased at the surface of cells affected by several pathologies.     

Adenosine A2A receptor activation and hyaluronan fragment inhibition reduce inflammation in mouse articular chondrocytes stimulated with interleukin-1β

Small hyaluronan (HA) fragments produced from native HA during inflammation contribute greatly to cell injury in many pathologies. HA oligosaccharides increase proinflammatory cytokine levels by activating both CD44 and toll-like receptor (TLR)-4. Stimulation of CD44 and TLR-4 then activates nuclear factor-κB, which induces the production of proinflammatory cytokines. The adenosine 2A receptor (A(2A)R) is also involved in several inflammation pathologies, and the nucleoside adenosine acts as a potent endogenous inhibitor of inflammation in various tissues by interacting with this receptor. The aim of this study was to investigate the effects of an HA-blocking peptide that inhibits the proinflammatory action of HA oligosaccharides produced during inflammation, together with a specific A(2A)R agonist in a model of normal mouse articular chondrocytes stimulated with interleukin (IL)-1β. IL-1β stimulation significantly increased mRNA expression and the related protein production of TLR-4, TLR-2, CD44 and A(2A)R in articular chondrocytes. The induced nuclear factor-κB activation was also associated with increased levels of inflammatory cytokines, including tumor necrosis factor-α and IL-6, and other inflammatory mediators, such as matrix metalloprotease-13 and inducible nitric oxide synthase. Treatment of chondrocytes with the HA-blocking peptide Pep-1 and/or a specific A(2A)R agonist (CGS-21680) significantly reduced all of the inflammatory parameters upregulated by IL-1β. These results suggest that the inflammatory response may be reduced either by blocking oligosaccharides from HA degradation or by A(2A)R stimulation.     

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.     

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.     

Emile Brumpt's contribution to the characterization of parasitic diseases in Brazil, 1909-1914

During the first thirty years of the XXth century, parasitologists and epidemiologists who were at the origin of the nosography and etiology of parasitic diseases were faced with several overlapping problems. A person can be infected simultaneoulsy by several different parasites. The delineation of clinical signs is an essential step, in the field and without the help of the laboratory, to identify the various parasitic pathologies and to propose the most likely diagnoses. The use of photography as a nosographic tool enabled the French parasitologist Emile Brumpt (1877-1951) to set up clinical pictures, given the multiplicity of pathologies on a given patient (for instance, goitre and ankylostomiasis, ankylostomiasis and myxoedema, "sylvan" leishmaniasis and palpebral oedema, goitre and Chagas' disease). We will set the paper on a part of Brumpt's photographic archives which he constitued during his missions to South America between 1913 and 1914.     

New insights in wound response and repair of epithelium

Epithelial wounds usually heal relatively quickly, but repair may be impaired by environmental stressors, such as hypoxic or diabetic states, rendering patients vulnerable to a number of corneal pathologies. Though this response appears simple, at first, years of research have uncovered the complicated biochemical pathways coordinating the wound healing response. Here, we investigate signaling cascades and individual proteins involved in the corneal epithelium's self‐repair. We will explore how an epithelial cell migrates across the wound bed and attaches itself to its new post‐injury surroundings, including its neighboring cells and the basement membrane, through focal adhesions and hemidesmosomes. We will also discuss how the cell coordinates this motion physiologically, through calcium signaling and protein phosphorylation, focusing on the communication through purinergic, glutamatergic, and growth factor receptors. Many of these aspects reflect and can be extended to similar epithelial surfaces, and can be used to facilitate wound healing in patients with various underlying pathologies. The collective library of laboratory and clinical research done around the world has demonstrated how important precise regulation of these processes is in order for the injured corneal epithelium to properly heal. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence

A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.     

Reactive Oxygen Species and Human Inflammatory Periodontal Diseases

Reactive oxygen species (ROS) have emerged as important signaling molecules in the regulation of various cellular processes. They can be generated by the mitochondrial electron transport chain in mitochondria and activation of polymorphonuclear leukocytes (PMN) during inflammatory conditions. Excessive generation of ROS may result in attack of and damage to most intracellular and extracellular components in a living organism. Moreover, ROS can directly induce and/or regulate apoptotic and necrotic cell death. Periodontal pathologies are inflammatory and degenerative diseases. Several forms of periodontal diseases are associated with activated PMN. Damage of tissues in inflammatory periodontal pathologies can be mediated by ROS resulting from the physiological activity of PMN during the phagocytosis of periodontopathic bacteria.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 751–761.Original Russian Text Copyright © 2005 by Canakci, Cicek, Canakci.     

A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth.

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.     

Structural features of glutamine substrates for transglutaminases. Role of extended interactions in the specificity of human plasma factor XIIIa and of the guinea pig liver enzyme

Amino acid residues at several locations in close primary vicinity to a substrate glutamine residue have been recognized as important determinants for the specificities of human plasma factor XIIIa and guinea pig liver transglutaminase (Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-2715). The present studies measure the influence on transglutaminase specificity of some changes in amino acid side chains in a small synthetic glutamine peptide amide, Leu-Gly-Leu-Gly-Gln-Gly-Lys-Val-Leu-GlyNH2, which was designed to contain most of the known elements needed for enzyme recognition. The results are in agreement with previous findings and show that full catalytic activity of each enzyme may be retained upon replacement of the lysine residue by certain other amino acid residues. Evidence is provided that serine in place of glycine at one or more positions causes a significant increase in specificity with factor XIIIa, but not with liver enzyme. The effective substrate property for factor XIIIa seen with the model peptide amide is lost upon reversal of the sequence Val-Leu. This is not the case with the liver enzyme even though replacement of either of these amino acids by alanine causes a pronounced loss in activity with this enzyme. These differences and the effects of various other substitutions in the model peptide amide on the enzymes' specificities points up the relatively stringent structural requirements of factor XIIIa and the rather broad requirements for liver transglutaminase.     

Structural features of glutamine substrates for transglutaminases. Specificities of human plasma factor XIIIa and the guinea pig liver enzyme toward synthetic peptides

Studies on the glutamine substrate specificities of human plasma factor XIIIa and guinea pig liver transglutaminase have been made using variants of the synthetic peptide substrate, Ser-Val-Leu-Ser-Leu-Ser-Gln-Ser-Lys-Val-Leu-Pro-Val-Pro-Glu. The sequence of this effective peptide substrate corresponds to the primary site of factor XIIIa-catalyzed amine incorporation into beta-casein, the most sensitive known macromolecular substrate for this enzyme (Gorman, J.J., and Folk, J.E. (1980) J. Biol. Chem. 255, 419-427). Variations in specificity observed with factor XIIIa for peptides containing single substitutions and multiple substitutions in this sequence are indications that several important determinants for enzyme recognition are contained therein. Among these are several of the hydrophobic amino acid residues and the lysine residue. Less pronounced changes in specificity occur with the liver enzyme and the differences in effects of the various substitutions reveal important differences in specificity requirements of factor XIIIa and the liver enzyme. Comparisons of the activities of the enzymes toward the synthetic peptides to their activities toward macromolecular substrates suggest that higher order macromolecular structural features contribute to specificity.     

Solvent Microenvironments and Copper Binding Alters the Conformation and Toxicity of a Prion Fragment

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu²⁺ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu²⁺ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu²⁺ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu²⁺ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.     

Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide.

Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata. CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryIIIA, whose crystal structure has been determined elsewhere [J. Li, J. Carrol, and D. J. Ellar, Nature (London) 353:815-821, 1991]. A clone limited to the putative 7-alpha-helical bundle domain I peptide of CryIIIB2 was constructed by PCR. The truncated protein was expressed at high levels in Escherichia coli. Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes. The results showed that CryIIIB2 domain I peptide is sufficient for ion channel formation and promotes ion efflux. Both native CryIIIB2 toxin and domain I peptide were inefficient channel-forming proteins that produced noisy ion channels of various conductance states. In ion efflux assays, native toxin promoted greater ion efflux from synthetic vesicles than did the truncated peptide.