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Comparative studies regarding the phospholipids lisis of crotoxin and its subunits at DMPC and DMPG vesicles

Grant number: 17/01682-9
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): April 01, 2017
Effective date (End): December 31, 2017
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal Investigator:Roberto Morato Fernandez
Grantee:Carlos Roberto Natal Junior
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Crotoxin is a toxin isolated from Crotalus durissus terrificus venom that displays neurotoxic and myotoxic activities, being responsible for a great number of deaths in snake venom envenomation. Besides, recently it was showed new actions of crotoxin with potential therapeutical applications, because its immunodulatory, anti-inflammatory, analgesic and anti-tumor activities. Crotoxin is a heterodimer composed by a noncovalent association between crotoxin A (CA) and crotoxin B (CB). CB is a phospholipase A2 with drastic neuromuscular blockade by presynaptic activity. Despite of the crystal structures of crotoxin complex and isolated CB had been solved, the role of phospholipids lysis on neurotoxicity action is not clear and there is little information about crotoxin interaction with presynaptic membrane. Furthermore, neither the exact mechanism of action nor the amino acid residues involved in the neurotoxicity of CTX, or of other presynaptic toxins, have been fully understood. In this context, we propose in this project the study of crotoxin, as well as its isolated subunits, with model membranes of biological membranes 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) and 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC). In order to evaluate the interaction between crotoxin and those membranes, it will be used the static and time-resolved fluorescence spectroscopy and the fluorescence microscopy techniques with fluorescent probes incorporated to membranes marked in different positions of hydrocarbon chains. Moreover, these analyses will be carried on using the intrinsic fluorescent probes of the proteins (tryptophan residues located at CA/CB interface) also. These experiments will be also realized in the presence of Ca2+ ions and phospholipases A2 inhibitors in order to evaluate the impact of these molecules on the interaction of crotoxin and its isolated subunits with membrane models. The results of this project will provide relevant information to analysis of interaction mechanism of crotoxin with biological membranes. These results could be helpful to elucidation of protein regions involved in crotoxin neurotoxic action. The applicant has been performing this project in the last months and already presents promising preliminary results. Finally, this project is an outcome of a collaborative activity between different laboratories and researchers that had been published articles in this subject with high impact factors in the last years. (AU)