First Proteomic Study of S-Glutathionylation in Cyanobacteria

Abstract

Glutathionylation, the reversible post-translational formation of a mixed disulfide between a cysteine residue and glutathione (GSH), is a crucial mechanism for signal transduction and regulation of protein function. Until now this reversible redox modification was studied mainly in eukaryotic cells. Here we report a large-scale proteomic analysis of glutathionylation in a photosynthetic prokaryote, the model cyanobacterium Synechocystis sp. PCC6803. Treatment of acellular extracts with N,N-biotinyl glutathione disulfide (BioGSSG) induced glutathionylation of numerous proteins, which were subsequently isolated by affinity chromatography on streptavidin columns and identified by nano LC-MS/MS analysis. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and mass spectrometry analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H2 production. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acids metabolism, was confirmed by biochemical studies on the purified recombinant proteins. These results suggest that glutathionylation constitutes a major mechanism of global regulation of the cyanobacterial metabolism under oxidative stress conditions.

Proteomic analysis of post translational modifications in cyanobacteria

“…Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and MS analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H 2 production [28]. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acid metabolism, was confirmed by biochemical studies on the purified recombinant proteins.…”

“…PTM events are also reported to exist in photosynthesis apparatus, including photosystem I [35], photosystem II [36], phycobiliproteins [37,38] and cytochrome b6f complex [39]. Classes of PTMs that have already been reported in cyanobacteria include protein splicing [40][41][42][43][44], phosphorylation [22,[24][25][26], acetylation [23], methylation [11], biotinylation [45][46][47], persulfide [48][49][50], ADP-ribosylation [51], glutathionylation [28], prenylation [52], palmitoylation [32], Histidine Hydroxyl Modification [53] and carboxylterminalprocessing [54,55].…”

“…These methods, varying from analysis of purified proteins in vitro to large-scale proteomic analysis in vivo, allowed identification of nearly 200 targets of glutathionylation in mammals [95]. Chardonnet and colleagues present the first systematic study of in vitro glutathionylation in Synechocystis 6803 by using biotinylatedoxidized glutathione (BioGSSG) and streptavidin-affinity purification as previously described [96,97], followed by nano LC-MS/MS analysis [28]. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and MS analysis.…”

“…Therefore, systematic investigation of PTMs could contribute to the holistic and comprehensive description of protein species and to elucidate potential biological roles of each protein species in cyanobacteria. Although the proteomics studies of PTMs carried out in cyanobacteria were limited [22][23][24][25][26][27][28], these studies provided clues to elucidate the complex signal transduction network and the biochemical processes that contribute to their ecological success. Here we provide a snapshot of the PTM proteomic studies, including the current status, the progresses have been made, and the main challenges.…”

Advances in proteomics for production strain analysis

“…Despite the enrichment strategies required for identifying these low abundance features, it is possible to assess PTMs on a systems level . Three pioneering studies have been conducted in the last two years, cataloguing system-wide PTM responses in Synechocystis and demonstrating their role in regulation of photosynthesis and central metabolic pathways [19,30,31].…”