Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics

Abstract

Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death.

Proteomic analysis of injured storage roots in cassava (Manihot esculenta Crantz) under postharvest physiological deterioration

“…It is a metalloenzyme involved in the catalysis of 2-phosphoglycerate to phosphoenolpyruvate in the penultimate step of glycolysis [62]. Manaa et al .…”

Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast

“…Cell vitality was assessed using FungaLight 5-carboxyfluorescein diacetate, acetoxymethyl ester (CFDA,AM)/Propidium Iodide (PI) Yeast Vitality Kit (Life Technologies) according to the manufacturer’s protocol. Protocol and LC 50 concentrations were reported in previous published manuscript [16] . Briefly, volume of culture equivalent to 10 6 cells was collected and exposed to a range of lidocaine concentrations (5-30 mM), hydrogen peroxide concentrations (1-20 mM), and vehicle control.…”

“…Previous studies assessing lidocaine toxicity using rat dorsal root ganglion neurons reported superoxide generation, mitochondrial depolarization, intracellular alkalization, and phosphatidylserine externalization [13] . Characteristically, oxidative stress causes protein carbonylation that frequently reduces protein activity [14] , [15] , [16] . In prior examination of lidocaine toxicity in S. cerevisiae we have shown carbonylation to multiple proteins involved in carbohydrate metabolism and general bioenergetics.…”

“…In prior examination of lidocaine toxicity in S. cerevisiae we have shown carbonylation to multiple proteins involved in carbohydrate metabolism and general bioenergetics. Most notably there was an increase in aconitase and glyderaldehyde-3-phosphate dehydrogenase (GAPDH) carbonylation paralleled by a decrease enzyme activity [16] . We also showed there to be a decrease in Cu-Zn superoxide dismutase [16] ; potentially providing an explanation for the increased superoxide generation observed upon lidocaine exposure in rat dorsal root ganglion neurons .…”

“…Most notably there was an increase in aconitase and glyderaldehyde-3-phosphate dehydrogenase (GAPDH) carbonylation paralleled by a decrease enzyme activity [16] . We also showed there to be a decrease in Cu-Zn superoxide dismutase [16] ; potentially providing an explanation for the increased superoxide generation observed upon lidocaine exposure in rat dorsal root ganglion neurons . Additional studies have implicated protein kinase C (PKC) and heat shock proteins (HSPs) in lidocaine toxicity [17] , [18] , [19] .…”