Identification of Plant Glutaredoxin Targets

Abstract

Glutaredoxins (Grxs) are small ubiquitous proteins of the thioredoxin (Trx) family, which catalyze dithiol-disulfide exchange reactions or reduce protein-mixed glutathione disulfides. In plants, several Trx-interacting proteins have been isolated from different compartments, whereas very few Grx-interacting proteins are known. We describe here the determination of Grx target proteins using a mutated poplar Grx, various tissular and subcellular plant extracts, and liquid chromatography coupled to tandem mass spectrometry detection. We have identified 94 putative targets, involved in many processes, including oxidative stress response [peroxiredoxins (Prxs), ascorbate peroxidase, catalase], nitrogen, sulfur, and carbon metabolisms (methionine synthase, alanine aminotransferase, phosphoglycerate kinase), translation (elongation factors E and Tu), or protein folding (heat shock protein 70). Some of these proteins were previously found to interact with Trx or to be glutathiolated in other organisms, but others could be more specific partners of Grx. To substantiate further these data, Grx was shown to support catalysis of the stroma beta-type carbonic anhydrase and Prx IIF of Arabidopsis thaliana, but not of poplar 2-Cys Prx. Overall, these data suggest that the interaction could occur randomly either with exposed cysteinyl disulfide bonds formed within or between target proteins or with mixed disulfides between a protein thiol and glutathione.

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

“…7 and Fig. S8D) may be linked to a functional backup by this system, as PrxQ is a target candidate of the glutathione/ glutaredoxin system (60). Other factors such as the variation and/or severity of environmental stresses possibly affect the manner of chloroplast redox regulation.…”

The Mitochondrial Type II Peroxiredoxin F Is Essential for Redox Homeostasis and Root Growth of Arabidopsis thaliana under Stress

“…⌬28-AtPrxII F was cloned as described previously (35). For cDNA amplification of Atgrx-CxxS10, the following primers were used to obtain a product that lacks the N-terminal 7-amino acid signal peptide: grxcxxs10-f, 5Ј-gcgtcgcaaagagcggtggt-3Ј, and grx-cxxs10-r, 5Ј-taaagccacaaagcaccagcct-3Ј.…”

Prediction of thioredoxin and glutaredoxin target proteins by identifying reversibly oxidized cysteinyl residues

“…According to the redox potential of each player in the network, electrons are transferred through specific donor-acceptor pairs [4][5][6][7]. Besides experimental strategies such as affinity chromatography and gel based methods [10][11][12][13], Marino and Gladyshev have adapted an integrative methodology in bioinformatics to detect thiol oxidoreductases and their catalytic redox-active cysteine residues [14]. Thioredoxins and glutaredoxins regulate various categories of proteins, such as proteins involved in photorespiration, citric acid cycleassociated reactions, lipid metabolism, electron transport, etc.…”

“…Thioredoxins and glutaredoxins regulate various categories of proteins, such as proteins involved in photorespiration, citric acid cycleassociated reactions, lipid metabolism, electron transport, etc. [11,15]. Due to the functional diversity of the target proteins, simple sequence analysis usually fails in predicting novel target proteins.…”

“…The lack of database and experimental information thus becomes an obstacle in the network construction process. With the advance of proteomics techniques, chromatography coupled with gel-based or mass spectrometry can experimentally identify thioredoxin/glutaredoxin target proteins or proteins that undergo thiol-disulfide transitions [10][11][12][13]. Due to technical limitation, further experiments are needed to eliminate the false positive proteins.…”