SIGNIFICANCE OF YNK INTERACTING PROTEINS UNDER OXIDATIVE STRESS USING Saccharomyces cerevisiae MODEL SYSTEM
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Date
41187
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Adverse environmental conditions caused by drought, temperature,
salinity are the most challenging factors that affect plant growth and survival.
Organisms exposed to such stresses tend to accumulate reactive oxygen species
(ROS) like hydrogen peroxide, superoxide etc. NDPKs are multi functional
proteins that play an important role in cellular signaling processes involving
regulation of ROS levels. Study was conducted using yeast strains like BWG7
(wild type) and ΔYNK (NDPK mutant strain). Yeast strain ΔYNK is highly
susceptible to ROS compared to BWG7 based on the serial dilution, paper disc
and Evans blue assay. High level expression of GST-YNK fusion protein using
pGEX4T-YNK construct was achieved in E.coli BL21 strain. GST -YNK fusion
protein was purified using GST sepharose affinity column and the purity was
established by SDS-PAGE. GST-YNK fusion protein was conjugated to cyanogen
bromide activated sepharose column and used for isolating the YNK binding
proteins. Drastic reduction in the soluble protein and thermostable protein
content was observed in both BWG7 and ΔYNK cells upon H2O2 treatment.
SDS-PAGE showed significant differences in the protein profile in control as
well as H2O2 treated BWG7 and ΔYNK cells. Increased number of YNK
interacting protein was observed in BWG7 cells treated with H2O2 compared to
ΔYNK. ΔYNK cells showed only one YNK interacting protein of low molecular
weight in presence of H2O2. These results put together suggest that YNK is
important for cellular metabolism, interact with several proteins in vivo,
possibly regulate their function by its kinase activity. Deletion of YNK results in
down regulation of certain proteins which could be the part of cellular signaling
cascade.