Banca de QUALIFICAÇÃO: Julia Freitas Daltro Vidal

Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.
STUDENT : Julia Freitas Daltro Vidal
DATE: 18/01/2024
TIME: 09:00
LOCAL: Videoconferência
TITLE:

"Evolutionary and biochemical study of serine proteases from toxicoferan venoms."

KEY WORDS:

Serine protease, venom toxin, ancestral sequence reconstruction

PAGES: 110
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

The serine proteases from the venom of reptiles from the Toxicofera clade are toxins that act on the prey's hemostatic system. Its versions present in snakes have been extensively studied for decades. On the other hand, little is known biochemically about lizards’ enzymes. Despite their high sequence homology, these enzymes act on a range of substrates, presenting, among others, activity similar to kallikrein and thrombin. The evolution that resulted in this diversification can be studied through the ancestral sequences reconstruction technique. In this approach, ancestral sequences are reconstructed from modern sequences, allowing us to understand the properties of extinct proteins. This work aims to study the evolution of venom serine proteases through the reconstruction of the ancestral sequence of Viperidae and its characterization, in addition to the characterization of a serine protease from the venom of the lizard Varanus komodoensis. The phylogeny of these enzymes and the ancestral sequence of Viperidae were built using the MEGA-X software. Both enzymes were expressed in Komagataella phaffii and purified by affinity chromatography (Ni2+). Proteins were deglycosylated using the PNGase F enzyme. Enzymatic tests were performed using both glycosylated and deglycosylated proteins. The reconstructed sequence showed good statistical values. The expressed enzymes were different in size than expected, which was confirmed to be caused by N-glycosylations, a posttranslational modification prevalent in these toxins. Activity tests showed that the ancestral enzyme presents activity against fibrinogen only when deglycosylated, while that of V. komodoensis only when glycosylated. Fibrinogen cleavage did not generate clot formation in either enzyme, indicating that both act in fibrinogen depletion. The ancestral enzyme showed activity against a synthetic kallikrein substrate when it was both glycosylated and deglycosylated, in agreement with the belief that kallikrein is the the ancestor of venom serine proteases. Structural characterization by circular dichroism and fluorescence is being finalized to comprehend the structural characteristics of these enzymes. With this study, we hope to expand the knowledge about the diversity and functional and structural evolution of these toxins

COMMITTEE MEMBERS:
Externa à Instituição - RENATA VIEIRA BUENO
Presidente - 2222088 - ELIANE FERREIRA NORONHA
Externo ao Programa - 2128128 - NAPOLEAO FONSECA VALADARES - nullExterno ao Programa - 1354514 - OSMINDO RODRIGUES PIRES JUNIOR - null