Decarbonylated cyclophilin A Cpr1 protein protects KNU5377Y when exposed to stress induced by menadione

Abstract

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.

Saccharomyces cerevisiae KNU5377 Stress Response during High-Temperature Ethanol Fermentation

“…However, if fermentation could be performed at higher temperatures, for example within 40-50°C range, several cost reductions could be applied to large-scale fuel ethanol production (Abdel-Banat et al, 2010). S. cerevisiae KNU5377 is thermotolerant and tolerant of ROS-induced oxidative stress (Kim et al, 2011). We have demonstrated that KNU5377 has a very specific stress response mechanism during fermentation at high temperature (40°C).…”

“…The pellet was then vacuum-dried and resuspended in sample buffer containing 9.5 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris, 0.1 M DTT, and 0.2% Bio-Lyte (3-10; Bio-Rad, USA) at room temperature with shaking. The sample was centrifuged at 15,000 rpm for 30 min, after which the cleared supernatant was carefully collected, and the protein concentration was measured using a modified Bradford assay (Kim et al, 2011). Next, 1 mg protein was loaded onto preparative gels and analyzed with pH 4-7 immobilized pH gradient (IPG) strips (17 cm, linear; BioRad, USA) under the following focusing conditions: 250 V for 1 h, 250-10,000 V for 6 h, and 90,000 V-h at 10,000 V. After isoelectric focusing (IEF), the gel strips were equilibrated and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 15% gels at 10 mA per gel for the first 1 h followed by 25 mA per gel, stained with Coomassie Brilliant Blue R-250 (CBB R-250; Sigma, USA), and then destained.…”

“…, -aldehyde dehydrogenase (Ald) (Rockland Chemicals Inc., USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ab Frontier, Korea), antiglucose-6-phosphate dehydrogenase (G6PDH), -Zpr1, -dinitrophenyl (DNP) (Sigma, USA), anti-Hsp104, -Hsp60 (Stressgen, Belgium), anti-thioredoxin 2 (Trx2), -Trx3, -thioredoxin reductase (Trr), -thioredoxin peroxidase (Tsa1), -heat shock protein 90 (Hsp90 or Hsp82), -Ssa, -Ssb, -Hsp42, -Hsp30, -Hsp26, -cyclophilin isoform 1 (Cpr1), -Sti, and -Grp (Kim et al, 2011). The blots were washed 4 times for 40 min with TBST, then incubated with anti-rabbit, -rat, -goat, and -mouse secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, USA) diluted with blocking buffer lacking 0.02% sodium azide for 1.5 h at room temperature.…”

“…In this regard, thermotolerant, ethanol-resistant S. cerevisiae KNU5377 isolated in spoilage is a good candidate for fuel ethanol production. S. cerevisiae KNU5377 grows at 50°C, and produces ethanol at 40°C (Kim et al, 2011). Despite these advantages, a systematic analysis of the stress response of S. cerevisiae KNU5377 during alcoholic fermentation has not been reported, making it difficult to determine which yeast strain is best for any given conditions.…”

Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae

“…Finally, protein concentrations were determined by Bradford assay using a Protein Dye Reagent (Bio-Rad). Protein extracts (20 μg) were loaded onto a 12 % SDS-PAGE gel and separated at 50 V. The proteins were then transferred to PVDF membranes (Bio-Rad), which were incubated in blocking buffer containing 5 % nonfat skim milk and 0.02 % sodium azide in TBST (0.05 % Tween-20, 10 mM Tris-HCl, pH 7.6; and 150 mM NaCl) for 1.5 h at room temperature, and then incubated overnight at 4°C with anti-Prx (Ab Frontier, Seoul, South Korea), anti-tubulin (Tub; Santa Cruz Biotechnology, Santa Cruz, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ab Frontier), and anti-Tsa1 and anti-Ahp1 (Kim et al 2011a) antibodies diluted appropriately with blocking buffer. The blots were then washed three times for 30 min with TBST, after which they were incubated with anti-rabbit or antirat secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, USA) diluted with blocking buffer lacking 0.02 % sodium azide for 1.5 h at room temperature.…”