The ubiquitin-proteasome pathway protects Chlamydomonas reinhardtii against selenite toxicity, but is impaired as reactive oxygen species accumulate

Abstract

Plants subjected to stress imposed by their environment often accumulate misfolded proteins, that would be cytotoxic if not properly removed by the ubiquitin-proteasome pathway (UPP). During severe stress, the UPP becomes impaired, but the mechanisms that damage it are not well understood in plants. In this study, the effects of mild and severe stress selenium on the UPP were analyzed using the unicellular green algae Chlamydomonas. Mild selenium stress increased proteasome activity. However, inhibition of the UPP caused by severe selenium stress was associated with reactive oxygen species, including mitochondrial superoxide. Additionally, this is the first time that proteasome activity has been reported in lower plants.

Stuck between a ROS and a hard place: Analysis of the ubiquitin proteasome pathway in selenocysteine treated Brassica napus reveals different toxicities during selenium assimilation

“…To determine proteasome activity, root tissue from 6 plants was harvested 0, 4, and 24 h after treatment with or without Sec and ground in liquid nitrogen, as described previously (Vallentine et al, 2014). Briefly, non-denatured proteins were extracted in proteasome extract buffer (50 mM potassium-phosphate buffer -pH 7.4, 5% glycerol, 10 mM ATP, and 5 mM beta-mercaptoethanol), and protein concentration was determined using the Bradford assay.…”

“…Activity was determined after 30 min as the fluorescence per microgram of protein in reactions, and is the difference in reactions with or without MG132 to account for non-proteasomal proteolysis. Proteasome activity is reported as the percentage relative to control samples at 0 h. Levels of ubiquitinated proteins were determined in 3 pooled plants growing in 100 mL of media containing 50 M of MG132 treated with or without Sec for 4 and 24 h. Briefly, 50 g of denatured protein were separated on an 8% SDS-PAGE gel, and high molecular weight poly-ubiquitinated proteins were detected using an ubiquitin antibody raised in mouse as described (Vallentine et al, 2014). In a parallel experiment, 10 g of protein from each sample were loaded onto a 15% SDS-PAGE gel to detect levels of free ubiquitin (8 kDa).…”

“…Proteins were extracted and precipitated with 10% trichloroacetic acid, as previously described (Vallentine et al, 2014). Proteins were digested in nitric acid at 95 • C overnight.…”

“…Se toxicity in plants occurs through two different modes (Van Hoewyk, 2013). It is well established that inorganic selenium, such as selenate and selenite, deplete glutathione and cause oxidative stress (Hugouvieux et al, 2009;Łabanowska et al, 2012).…”

Recent advances in the mechanism of selenoamino acids toxicity in eukaryotic cells

“…Accumulation of superoxides and hydrogen peroxide was observed in various eukaryotic cell lines (80,(93)(94)(95)(96)(97)(98), associated to a decrease of the GSH/GSSG ratio and to an increase of protein oxidation and lipid peroxidation (94,(99)(100)(101). Transcriptome analyses, in S. cervisiae (102) and Caenorhabditis elegans (103), revealed that selenite treatment up-regulated genes involved in the oxidative stress response.…”