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. 2016 Jul 19;12(8):2471-80.
doi: 10.1039/c6mb00175k.

A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism

Kusal T G Samarasinghe  1 , Dhanushka N P Munkanatta Godage  1 , Yani Zhou  2 , Fidelis T Ndombera  1 , Eranthie Weerapana  1 , Young-Hoon Ahn  1
Affiliations

A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism

Kusal T G Samarasinghe et al. Mol Biosyst. .

Abstract

Glucose metabolism and mitochondrial function are closely interconnected with cellular redox-homeostasis. Although glucose starvation, which mimics ischemic conditions or insufficient vascularization, is known to perturb redox-homeostasis, global and individual protein glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. In this report, we use our clickable glutathione approach, which forms clickable glutathione (azido-glutathione) by using a mutant of glutathione synthetase (GS M4), for detection and identification of protein glutathionylation in response to glucose starvation. We found that protein glutathionylation is readily induced in HEK293 cells in response to low glucose concentrations when mitochondrial reactive oxygen species (ROS) are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Proteomic and biochemical analysis identified over 1300 proteins, including SMYD2, PP2Cα, and catalase. We further showed that PP2Cα is glutathionylated at C314 in a C-terminal domain, and PP2Cα C314 glutathionylation disrupts the interaction with mGluR3, an important glutamate receptor associated with synaptic plasticity.

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Figures

Fig. 1
Fig. 1. A flow of clickable glutathione approach for detecting glutathionylation
Incubation of azido-Ala forms azido-glutathione (γGlu-Cys-azido-Ala) in situ in HEK293 cells stably expressing GS M4, which catalyzes azido-Ala in place of Gly. In response to stimulus, the subsequent click reaction allows for identifying glutathionylated proteins.
Fig. 2
Fig. 2. Protein glutathionylation is highly dependent on glucose metabolism in the presence of the electron transport chain blocker
After incubation of azido-Ala to HEK293/GS M4 cells for 20 h, cells were subjected to the following stimulus: (A) glucose starvation with antimycin A in a time-dependent manner, (B) glucose starvation with an increasing concentration of antimycin A, (C) decreasing glucose concentrations with antimycin A, and (D) increasing incubation time of antimycin A in a physiological glucose concentration (5 mM). After stimulus, cell lysates were subjected to click reaction with rhodamine-alkyne, and analyzed for fluorescence and Coomassie stain.
Fig. 3
Fig. 3. Glutathionylation is reversed by re-addition of glucose
After incubation of azido-Ala to HEK293/GS M4 cells for 20 h, glutathionylation was induced by glucose starvation with treatment of antimycin A for 2 h. Subsequently, (A) glucose (GL, 5 mM), pyruvate (Py, 5 mM) or (B) lactate (La, 10 mM) was individually added to the cells for the indicated time period. Cell lysates were subjected to click reaction with rhodamine-alkyne, and analyzed for fluorescence and Coomassie stain.
Fig. 4
Fig. 4. Proteomic analysis of glutathionylated proteins
(A) Approach for pull-down and mass analysis: after click reaction with biotin-alkyne, glutathionylated proteins enriched by streptavidin-agarose were eluted for silver staining or directly digested on-beads for LC-MS/MS analysis. (B) Analysis of enriched proteins in indicated conditions. (C) The number of glutathionylated proteins in indicated conditions by LC-MS/MS. ‘Positive’ and ‘negative’ indicate proteins enriched in lane 4 and 1, respectively, in Figure 4B. (D) DAVID gene ontology (GO) analysis of identified glutathionylated proteome. (E-F) Comparison of glutathionylated proteins in glucose starvation/antimycin A with previous proteomic data of glutathionylome (E) or sulfenylome (F) by treatment of exogenous H2O2.
Fig. 5
Fig. 5. Identification of glutathionylated proteins by pull-down and Western blotting
(A) After incubation of azido-Ala to HEK293/GS M4 cells for 20 h, cells were subjected to glucose starvation with antimycin A in a time-dependent manner. (B) After glucose starvation with treatment of antimycin A for 2 h, pyruvate was added to cells. After click reaction with biotin-alkyne, the individual proteins were probed by specific antibodies before and after pull-down by streptavidin-agarose.
Fig. 6
Fig. 6. The cysteine modification site and functional role of PP2Cα glutathionylation
(A) The structure (PDB:4RA2) and domains of PP2Cα that shows three cysteine residues, C204 and C314 that are exposed to solvent, and C238 that is close to the active site. Two manganese atoms (purple) and a phosphate (red) in active site are shown in space-filling model. (B) PP2Cα C314 is the major cysteine residue for glutathionylation. After inducing glutathionylation in HEK293/GS M4 cells transfected with HA-PP2Cα WT and mutants, cell lysates were subjected to click reaction with biotin-alkyne. HA-PP2Cα was probed before (bottom) and after (top) pull-down by streptavidin-agarose. (C) PP2Cα glutathionylation disrupts its interaction with mGluR3. With and without inducing glutathionylation, PP2Cα and mGluR3 were co-immunoprecipitated and probed by individual antibodies.
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