Structural Studies with Neurotoxic Phospholipases A2.

• Grant number: 13/17864-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: January 01, 2014
• End date: December 31, 2016
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcos Roberto de Mattos Fontes
• Grantee: Carlos Alexandre Henrique Fernandes
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Crotoxin, the major component of venom from Crotalus snakes, is a heterodimeric complex that contains a phospholipase A2 (PLA2) and exerts a lethal action by blocking neuromuscular transmission. Crotoxin consists of a noncovalent association of an acidic, nonezymatic and nontoxic subunit, the crotapotin (or crotoxin A; CA) and a basic and toxic PLA2 subunit, the crotoxin B (CB). Crotalus neutralization factor (CNF) is an endogenous inhibitor of PLA2s isolated from Crotalus durissus terrificus plasma blood. CNF would bind only to CB and provides a protection against snake self-envenomation. This research project proposes structural and functional studies with crotoxin, its isolated subunits (CA and CB) and CNF towards the techniques of x-ray crystallography, small angle x-ray scattering (SAXS), fluorescence spectroscopy and site-directed mutagenesis. In solution (SAXS and fluorescence) and site-directed mutagenesis experiments never has been performed before with crotoxin and can provide essential information about the mechanism of action of crotoxin B and help to develop mechanisms for its inhibition. In this research project, the applicant pretends to get more understanding of in solution structural molecular biology techniques since that he obtained great experience in x-ray protein crystallography during your master and PhD. Furthermore, this research project proposes the application insertion in functional studies due to site-directed mutagenesis experiments and subsequently in vitro validation. These experiments will provide to the applicant a great autonomy to elaboration and execution of scientific research projects. Along the master and PhD, the applicant was supported by FAPESP with an excellent academic yield. The applicant has published 14 articles in addition to an article that was submitted and another two actually in preparation.

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

FERNANDES, CARLOS A. H.; PAZIN, WALLANCE M.; DREYER, THIAGO R.; BICEV, RENATA N.; CAVALCANTE, WALTER L. G.; FORTES-DIAS, CONSUELO L.; ITO, AMANDO S.; OLIVEIRA, CRISTIANO L. P.; FERNANDEZ, ROBERTO MORATO; FONTES, MARCOS R. M.. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism. SCIENTIFIC REPORTS, v. 7, MAR 3 2017. (15/17286-0, 13/17864-8)

CAVALCANTE, WALTER L. G.; NORONHA-MATOS, JOSE B.; TIMOTEO, MARIA A.; FONTES, MARCOS R. M.; GALLACCI, MARCIA; CORREIA-DE-SA, PAULO. Neuromuscular paralysis by the basic phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit acetylcholine release and produce muscle blockage. Toxicology and Applied Pharmacology, v. 334, p. 8-17, NOV 1 2017. (13/17864-8, 13/03624-5, 12/00428-8)

MAKIYAMA, RODRIGO K.; FERNANDES, CARLOS A. H.; DREYER, THIAGO R.; MODA, BRUNO S.; MATIOLI, FABIO F.; FONTES, MARCOS R. M.; MAIA, IVAN G.. Structural and thermodynamic studies of the tobacco calmodulin-like rgs-CaM protein. International Journal of Biological Macromolecules, v. 92, p. 1288-1297, NOV 2016. (10/03001-0, 13/17864-8)

PAVANI, RAPHAEL SOUZA; DA SILVA, MARCELO SANTOS; HENRIQUE FERNANDES, CARLOS ALEXANDRE; MORINI, FLAVIA SOUZA; ARAUJO, CHRISTIANE BEZERRA; DE MATTOS FONTES, MARCOS ROBERTO; SANT'ANNA, OSVALDO AUGUSTO; MACHADO, CARLOS RENATO; CANO, MARIA ISABEL; FRAGOSO, STENIO PERDIGAO; et al. Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi. PLoS Neglected Tropical Diseases, v. 10, n. 12, DEC 2016. (14/13375-5, 14/24170-5, 13/17864-8, 14/02978-0, 13/07467-1, 15/10580-0)