Mirko Zaffagnini, Christophe H. Marchand, Marco Malferrari, Samuel Murail, Sara Bonacchi, Damiano Genovese, Marco Montalti, Giovanni Venturoli, Giuseppe Falini, Marc Baaden, Stéphane D. Lemaire, Simona Fermani, and Paolo Trost

PNAS December 17, 2019 116 (51) 26057-26065

Significance

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant enzyme whose activity depends on a reactive catalytic cysteine. Here, we determined the effect of 2 cysteine-based redox modifications, namely oxidation and S-glutathionylation, on the functionality and structural stability of GAPDH of Arabidopsis thaliana. Hydrogen peroxide causes the irreversible oxidation of the catalytic cysteine without altering the GAPDH structure. Conversely, S-glutathionylation, consisting of the formation of a glutathionyl-mixed disulfide with its catalytic cysteine, reversibly inactivates GAPDH and protects the enzyme from irreversible oxidation. The persistence, however, of the glutathionylated state alters the native folding of GAPDH, causing the irreversible collapse into insoluble oligomeric aggregates whose growth, but not breakdown, is under the control of physiological reductases such as thioredoxins and glutaredoxins.

Abstract

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

See https://www.pnas.org/content/116/51/26057

Figure 1: Oxidative treatments alter AtGAPC1 stability, inducing globular aggregation. (A) Turbidity analyses monitored at Abs₄₀₅ of AtGAPC1 (5 μM) incubated with 0.125 mM H₂O₂ in the absence (gray closed circles) or presence of 0.625 mM GSH (black closed circles). In the control experiment, change in turbidity was measured following incubation of AtGAPC1 in buffer alone (open circles). The turbidity of control and H₂O₂-treated AtGAPC1 showed no variation over 210 min, while H₂O₂/GSH-treated AtGAPC1 has a lag phase (15 to 20 min; Inset) followed by a rapid increase reaching a plateau after 2 h incubation. Data represent mean ± SDs (n = 3 experiments with technical duplicates). When not visible, SDs are within the symbols. (B) Dynamic light scattering (DLS) measurement of AtGAPC1 incubated for 90 min in the presence of buffer alone (white bar), 0.125 mM H₂O₂ (light gray bar), or 0.125 mM H₂O₂ supplemented with 0.625 mM GSH (dark gray bar). No appreciable variation of protein diameter (d_H) was observed for control and H₂O₂-treated AtGAPC1, while H₂O₂/GSH-treated AtGAPC1 formed aggregates with a diameter of ∼2 μm. This value might be underestimated due to technical limitations of the DLS instrument linked to the polydispersity index of samples. Data represent mean ± SDs (n = 3 experiments with technical duplicates; **P < 0.01). Representative TEM (C) and SEM (D) images of aggregated AtGAPC1 obtained after 90 min incubation in the presence of 0.125 mM H₂O₂ and 0.625 mM GSH. (Scale bars: 5 μm and 0.5 μm.) (E) Relative content in secondary structures of native AtGAPC1 determined by FTIR analysis. FTIR spectra were acquired after H₂O-to-D₂O substitution achieved through exhaustive concentrating/diluting steps. (F) Relative content in secondary structures of aggregated AtGAPC1 determined by FTIR analysis. The protein sample was incubated for 90 min in the presence of 0.125 mM H₂O₂ and 0.625 mM GSH, and, after treatment, the sample was centrifuged and the pellet resuspended in D₂O. In E and F, the percentages of secondary structures derived from the crystallographic 3D structure of native AtGAPC1 are also indicated as dashed lines.