Banca de DEFESA: Giulia Causin Vieira

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : Giulia Causin Vieira
DATE: 19/09/2023
TIME: 10:00
LOCAL: Microsoft Teams:
TITLE:

In silico characterization and evaluation of the anti-hemostatic activity of antigen-5 from the saliva of Rhodnius neglectus, vector of Chagas disease

KEY WORDS:

Insect; Hematophagy; Hemostasis; Protein structural bioinformatics; Recombinant protein

PAGES: 99
BIG AREA: Ciências da Saúde
AREA: Fonoaudiologia
SUMMARY:

Triatomines are hematophagous insects that transmit the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Their saliva is rich in compounds that counteract host hemostatic and immune responses. Antigen 5 (Ag5), a member of the CAP superfamily, composed of proteins rich in cysteine residues of cysteine (CRISP - cysteine rich secretory proteins), Antigen 5 and proteins related to pathogenicity in plants (Pathogenesis-related - PR1), has been reported in arthropod saliva as responsible for causing strong allergic responses. The Ag5 activity already found in triatomines is correlated to neutrophil oxidative burst inhibition and prevention of collagen-induced platelet aggregation. This study aimed to use bioinformatics approaches to characterize R. neglectus salivary antigen 5 (RnAg5), produce the recombinant RnAG5 protein and evaluate its inhibitory potential for platelet aggregation and blood clotting. In silico analyzes were performed to predict the putative signal peptide, cellular localization and post-translational modifications. The 3D structure of RnAg5 was obtained by artificial intelligence, through the Alphafold server, and the amino acids that compose B cell epitopes were predicted with the ElliPro, BepiPred and SeRenDIP servers. In order to identify promising ligands, molecular docking calculations were performed using the DockThor web server against small molecules to identify promising ligands. The Ag5 sequence was cloned into the pET100/D-TOPO vector, expressed in Escherichia coli Rosetta(DE3)pLysS with 1 mM isopropyl B-D-thiogalactopyranoside at 37ºC, with a molecular mass of approximately 31 kDa. It was possible to obtain RnAg5 in soluble fraction and in inclusion bodies, and this recombinant protein was purified by immobilized metal ion affinity chromatography. RnAg5 has a signal peptide at the N-terminus and can be secreted by the classical pathway. It contains 25 putative phosphorylation sites and 10 potential glycosylation residues. The ability of Ag5 to interact with lipids is associated with a conserved region in CAP superfamily, the binding site has two parallel alpha-helices, which form a long hydrophobic cavity. However, despite the predicted 3D model reflects this described structure, it does not form the binding cavity. Though, another potential hydrophobic binding site was predicted in our model. Docking results showed that platelet-activating factor, leukotriene D4 and prostaglandin can bind to RnAg5 with docking score of -8,72; -8,16 and -7,80 Kcal·mol-1, respectively. In addition, 176 residues that form linear epitopes and 201 residues that form conformational epitopes were predicted. These results were extremely relevant, since Ag5 proteins are allergens already characterized in other species of arthropods and may become a possible marker of exposure to triatomine bites. Understanding the role of salivary Ag5 on the triatomine-host-pathogen interface may lead to development of new strategies to control Chagas disease transmission.

COMMITTEE MEMBERS:
Presidente - 2088007 - CARLA NUNES DE ARAUJO
Externo ao Programa - ***.367.691-** - LUCAS SILVA DE OLIVEIRA - UnB
Externa à Instituição - MELINA MOTTIN - UFG
Interno - 2376346 - OTAVIO DE TOLEDO NOBREGA