Banca de DEFESA: PRICILA GOMES DA SILVA

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : PRICILA GOMES DA SILVA
DATE: 29/05/2024
TIME: 14:30
LOCAL: Sala Virtual Remota (Microsoft Teams) https://bit.ly/Def_MSc_PricilaSilva_PGBIOEXP
TITLE:

PURIFICATION OF A BOVINE LIVER CATHEPSIN D AND PARTIAL CHARACTERIZATION OF ITS INTERACTION WITH A PLA2 FROM Bothrops jararacussu.

KEY WORDS:

Aspartic Proteases, Catepsin D, phospholipases A2, Protein-protein
complex, functionalization.

PAGES: 54
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

The enzyme Cathepsin D (CatD) and Phospholipase A2 (PLA2) play an important role in
various pathophysiological conditions, being involved in processes ranging from
neurodegenerative and inflammatory conditions to the life cycle of various parasites.
Moraes et al. (2022) demonstrated the interaction of human CatD with BthTX-II, a PLA2
from Bothrops jararacussu, forming a stable complex that modulates the activity of CatD,
rendering it active at higher pH values. This phenomenon may be capable of assisting in
the elucidation of some biochemical events related to CatD in the tumor
microenvironment and Alzheimer's disease. Considering the high cost of obtaining
human CatD for the continuity of studies, the present work proposes the adaptation of a
new protocol for obtaining CatD from bovine liver (Bos indicus) and the partial evaluation
of its interaction with a BthTX-II, aiming to enable future biophysical and structural
studies. The methodology used to obtain the enzyme consisted of grinding 500g of
bovine liver, followed by two solubilization/precipitation steps and subsequent
fractionation on a Sepharose affinity column + Pepstatin A previously equilibrated with
0.1M sodium acetate pH 5.0 and eluted in a step gradient of 0 to 100% sodium acetate
pH 5.0 + 0.5M NaCl (buffer B) and 0.1M Tris-HCl pH 8.5 (buffer C) at a flow rate of
1mL/min for 40 minutes. Subsequently, the sample was subjected to reverse-phase C18
column chromatography (25 cm x 0.45 cm, Discovery) previously equilibrated with 0.1%
trifluoroacetic acid and eluted under a gradient of 0 to 70% ACN 99.9% (v/v) and 0.1%
TFA (v/v) at a flow rate of 1mL/min. The evaluation of relative mass, purity degree, and
isoelectric point was observed on 12.5% SDS-PAGE and two-dimensional
electrophoresis. The enzymatic complex was evaluated for its enzymatic activity on the
substrate casein at different pH values, subjected to reading at 660 nm in a
spectrophotometer. The predicted interaction between the proteins was observed in silico
through molecular docking using the ClusPro tool and molecular dynamics simulations
(MD) performed using the GROMACS program with the CHARMM36m force field. The
bovine liver CatD exhibited a relative molecular mass of ~32 kDa, an approximate pI of
5.5, and optimal activity on casein at pH 3 and temperature 50°C. The CatD/BthTX-II
complex showed modulation in its catalytic activity, extending its activity on the casein
substrate up to pH 6, with the optimal activity value at pH 5. In silico data highlight that
the most predominant amino acid residues related to protein binding consist of
hydrophobic residues. The complex showed little variation in its stability, suggesting
significant structural resistance. Thus, the bovine liver CatD forms the same complex
observed with its human analog and BthTX-II, enabling its use for subsequent structural
studies.

COMMITTEE MEMBERS:
Presidente - 1522184 - LEONARDO DE AZEVEDO CALDERON
Externo à Instituição - LUIS ANDRE MORAIS MARIÚBA - FIOCRUZ
Externa à Instituição - SHARON ROSE ARAGAO MACEDO OLIVEIRA - UNIR