Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-alpha Using Oriented Peptide Library Screening

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Orpotrin: A novel vasoconstrictor peptide from the venom of the Brazilian Stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97-105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms. (c) 2006 Elsevier B.V. All rights reserved.

Gastroprotective mechanisms of Citrus lemon (Rutaceae) essential oil and its majority compounds limonene and beta-pinene: Involvement of heat-shock protein-70, vasoactive intestinal peptide, glutathione, sulfhydryl compounds, nitric oxide and prostaglandin E-2

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Involvement of Glutathione, Sulfhydryl Compounds, Nitric Oxide, Vasoactive Intestinal Peptide, and Heat-Shock Protein-70 in the Gastroprotective Mechanism of Croton cajucara Benth. (Euphorbiaceae) Essential Oil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A synthetic peptide selectively kills only virulent Paracoccidioides brasiliensis yeasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH2 (MW 1611.98Da) and IDWLKLGKMVMDVL-NH2 (MW 1658.98Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of poly-morphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging. Copyright (C) 2004 John Wiley Sons, Ltd.

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes pallipes

The venom of the neotropical social wasp Agelaia pallipes pallipes was fractionated by RP-HPLC resulting in the elution of seven fractions; the last two were re-fractionated under RP-HPLC by using isocratic elution conditions and the purity of the fractions were confirmed by using ESI-MS analysis. Both fractions are constituted of peptide components, which were sequenced by Edman degradation chemistry, resulting in the following sequences:Protonectin I-L-G-T-I-L-G-L-L-K-G-L-NH2Agelaia-MP I-N-W-L-K-L-G-K-A-I-I-D-A-L-NH2Both peptides are manually synthesized on solid-phase and functionally characterized by using Wistar rats cells. Protonectin is a non-hemolytic chemotactic peptide for polymorphonucleated leukocytes (PMNL), presenting some mast cell degranulating activity and potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Agelaia-MP was characterized as a hemolytic mast cell degranulator toxin, presenting a poor antimicrobial action and no chemotaxis for PMNL. (C) 2004 Elsevier Ltd. All rights reserved.

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inflammation and apoptosis induced by mastoparan Polybia-MPII on skeletal muscle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The neurotoxicological effects of mastoparan Polybia-MPII at the murine neuromuscular junction: an ultrastructural and immunocytochemical study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH2), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO-) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO-, a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH2 and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO- leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH2 exists mainly as a rigid structure, EMP-AF-COO- presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

Formyl-peptide receptor is not involved in the protection afforded by annexin 1 in murine acute myocardial infarct

Recent interest in the annexin 1 field has come from the notion that specific G-protein-coupled receptors, members of the formyl-peptide receptor (FPR) family, appear to mediate the anti-inflammatory actions of this endogenous mediator. Administration of the annexin 1 N-terminal derived peptide Ac2-26 to mice after 25 min ischemia significantly attenuated the extent of acute myocardial injury as assessed 60 min postreperfusion. Evident at the dose of 1 mg/kg (similar to9 nmol per animal), peptide Ac2-26 cardioprotection was intact in FPR null mice. Similarly, peptide Ac2-26 inhibition of specific markers of heart injury (specifically myeloperoxidase activity, CXC chemokine KC contents, and endogenous annexin 1 protein expression) was virtually identical in heart samples collected from wild-type and FPR null mice. Mouse myocardium expressed the mRNA for FPR and the structurally related lipoxin A(4) receptor, termed ALX; thus, comparable equimolar doses of two ALX agonists (W peptide and a stable lipoxin A4 analog) exerted cardioprotection in wild-type and FPR null mice to an equal extent. Curiously, marked (>95%) blood neutropenia produced by an anti-mouse neutrophil serum did not modify the extent of acute heart injury, whereas it prevented the protection afforded by peptide Ac2-26. Thus, this study sheds light on the receptor mechanism(s) mediating annexin 1-induced cardioprotection and shows a pivotal role for ALX and circulating neutrophil, whereas it excludes any functional involvement of mouse FPR. These mechanistic data can help in developing novel therapeutics for acute cardioprotection.