Defensive roles of Sdu1, a PPPDE superfamily member with ubiquitin C-terminal hydrolase activity, against thermal stress in Schizosaccharomyces pombe

Abstract

The sdu1+ gene encodes Sdu1, a PPPDE superfamily member of deubiquitinating enzymes (DUBs) in Schizosaccharomyces pombe. Sdu1 was previously shown to contain an actual ubiquitin C-terminal hydrolase (UCH) activity using the recombinant plasmid pYSTP which harbors the sdu1 + gene. This work was designed to assess a thermotolerant role of Sdu1 against high incubation temperatures. In the temperature-shift experiments, the S. pombe cells harboring pYSTP grew much better after the shifts to 37˚C and 42˚C, when compared with the vector control cells. After being shifted to 37˚C and 42˚C for 6 h, the S. pombe cells harboring pYSTP contained lower reactive oxygen species (ROS) levels, compared with the vector control cells. The nitric oxide (NO) levels of the S. pombe cells harboring pYSTP were slightly lower than those of the vector control cells in the absence or presence of the temperature shifting. The total glutathione (GSH) levels of the S. pombe cells harboring pYSTP were significantly higher than those of the vector control cells. Total superoxide dismutase (SOD) and GSH peroxidase activities were also higher in the S. pombe cells harboring pYSTP after the temperature shifts than in the vector control cells. In brief, the S. pombe Sdu1 plays a thermotolerant role against high incubation temperature through the down-regulation of ROS and NO and the up-regulation of total GSH content, total SOD and GSH peroxidase activities.Key words: Schizosaccharomyces pombe, glutathione, reactive oxygen species, Sdu1, thermal stress In the previous work, the sdu1 + gene encoding Sdu1, belonging to the PPPDE superfamily, was cloned from S. pombe, and its gene product was shown to contain the UCH activity which is involved in the defense against oxidative and nitrosative stresses in S. pombe (Kim et al., 2013). In the present work, we further demonstrate that Sdu1 plays a defensive role against thermal stress in S. pombe. Materials and Methods Chemicals Preparation of cellular extractsThe desired number of the yeast cells was obtained by centrifugation. They were re-suspended in 20 mM Tris buffer (pH 8.0) with 2 mM EDTA and disrupted using glass beads and ultrasonication. The cellular extracts, taken after centrifugation, were used for total GSH, SOD and glutathione peroxidase activities, and protein determinations detailed below. Protein content in cellular extracts was determined by Bradford's