Protective effect of a protease inhibitor from Agaricus bisporus on Saccharomyces cerevisiae cells against oxidative stress

Abstract

Protease inhibitors are known to resist damage to host organisms against external threats, hence form a part of their defense system. This property of protease inhibitors was studied on protecting oxidatively stressed Saccharomyces cerevisiae yeast cells. The protease inhibitor was extracted from Agaricus bisporus, an edible mushroom. The inhibitor showed the presence of antioxidant activity as the purified inhibitor fraction gave an IC₅₀ value of 45.13 ± 0.88 µg/mL and 33.30 ± 1.5 µg/mL when checked, respectively, by 2, 2-diphenyl-1-picrylhydrazyl, DPPH and 2, 2′-azo-bis(3-ethylbenzthiazoline-6- sulfonic acid), ABTS•+ scavenging activity. The yeast cells’ survival rate (%), was determined through 3-(4, 5-dimethylthiazol-2-yl) - 2, 5-diphenyltetrazolium bromide, MTT assay, and it was found that in the presence of 2 mM H₂O₂ cell survival decreased to 26.33%, whereas when the experiment was conducted in the presence of protease inhibitor and 2 mM H₂O₂ cell survival percentage rose to 74%. The protease inhibitor’s effect on the oxidatively stressed yeast cells was further studied by using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Confocal Microscopy to understand the morphological changes. The viable and non-viable cell populations were quantified using Fluorescence Assorted Cell Sorting (FACS) using propidium iodide, PI, 4′, 6-diamidino-2-phenylindole, DAPI and 2′, 7′-dichlorofluorescein, DCF dyes.

Keywords:

• Agaricus bisporus
• flow cytometry
• hydrogen peroxide
• oxidative stress
• protease inhibitor
• Saccharomyces cerevisiae

Acknowledgments

Authors thank DBT-BHU Interdisciplinary School of Life Science (ISLS), BHU for providing confocal microscopy and FACS facilities and Central Instrument Facility (CIF), IIT-BHU, Varanasi for providing SEM and AFM facilities.

Additional information

Funding

Authors also thanks to the financial support provided by Indian Institute of Technology Banaras Hindu University, Varanasi.