Cold atmospheric pressure plasma causes protein denaturation and endoplasmic reticulum stress in Saccharomyces cerevisiae

Abstract

Cold atmospheric pressure plasma (CAP) does not cause thermal damage or generate toxic residues; hence, it is projected as an alternative agent for sterilization in food and pharmaceutical industries. The fungicidal effects of CAP have not yet been investigated as extensively as its bactericidal effects. We herein examined the effects of CAP on yeast proteins using a new CAP system with an improved processing capacity. We demonstrated that protein ubiquitination and the formation of protein aggregates were induced in the cytoplasm of yeast cells by the CAP treatment. GFP-tagged Tsa1 and Ssa1, an HO-responsive molecular chaperone and constitutively expressed Hsp70, respectively, formed cytoplasmic foci in CAP-treated cells. Furthermore, Tsa1 was essential for the formation of Ssa1-GFP foci. These results indicate that the denaturation of yeast proteins was caused by CAP, at least partially, in a HO-dependent manner. Furthermore, misfolded protein levels in the endoplasmic reticulum (ER) and the oligomerization of Ire1, a key sensor of ER stress, were enhanced by the treatment with CAP, indicating that CAP causes ER stress in yeast cells as a specific phenomenon to eukaryotic cells. The pretreatment of yeast cells at 37 °C significantly alleviated cell death caused by CAP. Our results strongly suggest that the induction of protein denaturation is a primary mechanism of the fungicidal effects of CAP.

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“…An increase of ROS and a rapid release of calcium from the ER into the cytosol are common features of ER stress [46]. In that respect, it is not surprising that CAP induced ER stress has been observed in yeast as well as in human cells [47,48]. Although the ER and mitochondria have distinct functions, they are physically connected via so called mitochondria-associated ER membranes (MAMs).…”

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“…This has also been observed after exposure of Saccharomyces cerevisiae yeast cells to atmospheric-pressure plasma as determined by protein quantitation and SDS-PAGE analysis. Plasmagenerated hydrogen peroxide (H 2 O 2 ) was shown to be at least partially responsible for this effect [21]. Heat shock proteins and molecular chaperones like Hsp104 Sc and Tsa1 Sc , respectively, were found to be upregulated in yeast after treatment with sublethal plasma doses emphasizing the importance of protein unfolding and aggregation under plasma-induced stress [21].…”

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“…The plasma-generated reactive species and plasma induced endogenous ROS possibly contributed to bacteria death synergistically 45. The CAP treatment of Saccharomyces cerevisiae cells caused oxidative stress responses including the nuclear accumulation of the oxidative stress responsive transcription factor Yap1, mitochondrial fragmentation, and enhanced intracellular oxidation. Yeast cells also induced the expression of heat shock protein (HSP) genes and formation of Hsp104 aggregates when treated with CAP, suggesting that CAP denatures proteins 23,46 . Plasma-generated ROS leads in S. cerevisiae to the accumulation of intracellular ROS and Ca 2+ that ultimately contribute to apoptosis and fragmentation of nuclear DNA 47 .…”

“…synergistically 22 . These oxidative stresses can cause cell death by damaging macromolecules such as deoxyribonucleic acid, proteins, lipids, and affecting the cellular metabolism [23][24][25][26] .…”