Animal toxins: structure, function and biotechnological applications

Abstract

Researches on animal toxins have contributed significantly with the development of Biological Sciences. An extensive study involving evaluation of the effects of venoms and their toxins upon different biological systems, relating them with the structure of active components, will be able to allow the development of new experimental tools and for important therapeutic agents inc1uding neuroprotectors, neurotoxins, immunomodulators and c10tting or anticoagulant factors. These studies will also be able to help in the elucidation of the c1inical picture produced by envenoming, opening the way for a more effective therapeutic strategy. hemostasis unbalance. Gene Cox-2, strictly involved with the inflammatory process, codes for the cyc10xigenase enzyme which catalyzes the transformation of arachidonic acid to prostaglandins. On basis of these considerations, our project is aimed at the investigation of the behavior of the PC 12 cell line regarding its cell cyc1e, apoptosis and expression of genes involved with signaling of injuries and inflammatory process, by means of PCR at real time, resulting from treatment with different toxins. Analysis of the activation pathways of COX-2 through study of gene expression will lead to a better understanding of this path in response to treatment with these toxins. In addition, the proteome analysis of these cells will be carried out in order to detect alterations in the protein synthesis induced by the toxins. Isolation of the biologically active components will be performed with use of c1assical chromatographic procedures, namely gel filtration, ionic exchange, hydrophobic interaction, bioaffinity and CLAE (reverse phase). This stage of the project is fundamental for all other subsequent stages, since it will provide the active components which will be, together with the crude venom, the purpose of study of this work. Enzymatic activities (hyaluronidase, proteolytic, PLA2, nuc1eotidase, L-aminoacid oxidase), biological (hemorrhagic, myotoxic, edema inducing, coagulant, anticoagulant, platelet aggregating), immunomodulatory (effect upon the complement system, anti-inflammatory, cell activation marking, apoptoic potential of the venom components), microbicide and cytotoxic (leishmanicide, trypanosomicidal, bactericidal, antitumoral, antiviral and fungicidal) and lectin activity of venoms or their isolated components will be studied. All these activities were chosen because they are venom targets and highly relevant in physiological processes. Within the different systems to be evaluated, the structure of isolated toxins will be the basis for elucidation of the structure-function relationship. The structural characterization of the toxins and their interactions with ligands and receptors will be performed through direct sequencing of the proteins, together with the cloning and sequencing of their corresponding DNA, X-ray crystallography and molecular modeling. The yet not elucidated structure of a protein can be predicted by modeling and homology, using known structures of other proteins with which it relates along evolution through a common ancestral. In the last years, modeling by molecular homology was shown to be highly important in Medicinal Chemistry, specifically plaining the development of new drugs. Keeping in mind the number of venoms and their active components to be studied, as well as the proposed assays for the characterization of their effects and structures, we have to consider this work as multidisciplinary, extensive and promising. Finally, results of this project certainly will broad the knowledge on structure-function relationship of toxins in biological systems, with probable applications in medical clinics and basic research. (AU)