Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional) properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively modified GAPDH in stress signaling pathways in plants.

Keywords: S-nitrosylation; cysteine thiols; glycolytic glyceraldehyde-3-phosphate dehydrogenase; moonlighting protein; redox modifications.

FIGURE 1

Crystal structure of cytoplasmic GAPDH from rice. (A) Ribbon representation of the homotetramer of Oryza sativa (Os) GAPDH (PDB code 3E5R), each subunit is differently colored. The three symmetry molecular axes are represented (the x-axis is perpendicular to the plane). (B) Ribbon representation of the crystal structure of a single OsGAPDH subunit. The cofactor-binding domain is yellow, the catalytic domain blue and the S-loop gray. The NAD⁺, the sulfate ions occupying the Ps and Pi sites and the catalytic residues (Cys-154 and His-181) are represented as ball-and-sticks.

FIGURE 2

Partial sequence alignment of GAPDH isoforms from different organisms. The alignment is focused on two highly conserved regions of GAPDH, one belonging to the coenzyme-binding domain and one belonging to the catalytic domain. The first (N-terminal) region includes the strictly conserved sequence NGFGRIGR and other important residues for the binding of the pyridine nucleotide cofactor (Asp-35, Phe-37). The second region, located in the central part of GAPDH sequences, contains several residues involved in substrate binding including the catalytic cysteine (Cys-154), but also a second cysteine close to the active site (Cys-158) and His-181, essential for activating Cys-154. Residues are numbered according to the sequence of Oryza sativa (Os) cytoplasmic GAPDH (GAPC). Abbreviation and accession numbers: OsGAPC, Oryza sativa GAPC, Q0J8A4.1; AtGAPC1, Arabidopsis thaliana GAPC1, AEE74039.1; AtGAPC2, Arabidopsis thaliana GAPC1, AEE29016.1; AtGAPCp1, Arabidopsis thaliana GAPCp1, Q9SAJ6.1; AtGAPCp2, Arabidopsis thaliana GAPCp2, Q5E924.1; AtGAPA, Arabidopsis thaliana GAPA, AEE77191.1; AtGAPB, Arabidopsis thaliana GAPB; HsGAPDH, Homo sapiens GAPDH, P04406.3; BsGAPDH, Bacillus stearothermophilus GAPDH, PDB code 2DBV; HaGAPDH, Homarus americanus GAPDH, P00357; EcGAPDH, Escherichia coli GAPDH, ACI83895.1. Invariant residues are on a blue background while yellow background indicated residues with strongly similar properties. Catalytic Cys-154 (corresponding to Cys-149 and Cys-156 in BsGAPDH and AtGAPCs, respectively) and His-181 (corresponding to His-176 and His-183 in BsGAPDH and AtGAPCs, respectively) are highlighted by arrows. The sequences were aligned with the Clustal Omega program ().

FIGURE 3

The catalytic sites of rice cytoplasmic GAPDH and non-phosphorylating GAPDH from Streptococcus mutants. (A) Magnified representation of OsGAPC catalytic site (PDB code 3E5R). Important catalytic residues, the cofactor NAD⁺ and residue Cys-158 are shown as ball-and-sticks. The distances of the sulfur atom (SG) of catalytic Cys-154 from the basic residue His-181 (atom NE2), the sulfur atom of Cys-158 and the cofactor (atom C4N) are indicated by dashed lines. (B) Catalytic site representation of Streptococcus mutans non-phosphorylating GAPDH (SmGAPN; PDB code 2EUH). The catalytic residues Cys-302 and Glu-268, and the cofactor NADP⁺ are in ball-and-sticks representation.

FIGURE 4

GAPDH catalytic mechanism. In the glycolytic direction, the binding of the substrate glyceraldehyde-3-phosphate (G3P) to the holo-enzyme containing NAD⁺ (1) leads to the formation of a hemithioacetal intermediate (2). In the next redox step, hydride transfer from the covalently bound substrate to the coenzyme allows formation of NADH, that can leave the acyl-enzyme (3) and be substituted by a molecule of NAD. The NAD-acyl-enzyme (4) can finally accept a free inorganic phosphate to release the product 1,3-bisphosphoglycerate (BPGA).

FIGURE 5

Molecular mechanisms of plant GAPC glutathionylation and deglutathionylation. (A) Oxidation and glutathionylation of plant GAPC. Plant GAPC can undergo primary oxidation to sulfenic acid (GAPC-SOH) in the presence of H₂O₂ that can subsequently react with GSH leading to the glutathionylated form (GAPC-SSG) and protecting the enzyme from irreversible oxidation (GAPC-SO_nH). In principle, GAPC glutathionylation may also be performed by GSSG with release of GSH, but this reaction does not occur under physiological GSH/GSSG ratios (dashed arrow). GSSG can be formed by the reaction of GSH with ROS. (B) GRX- and TRX-dependent deglutathionylation mechanism of plant GAPC. The most reactive cysteine of both GRX and TRX (the N-terminal active-site cysteine) performs the nucleophilic attack on the glutathione-mixed disulfide on GAPC, resulting in the release of the reduced GAPC and the formation of a glutathionylated GRX intermediate (right side) or an oxidized TRX (left side). Subsequently, the mixed disulfide on GRX is reduced by a GSH molecule to form GSSG and reduced GRX and the oxidized TRX is reduced by NADPH-TRX reductase (NTR) in the presence of NADPH.

FIGURE 6

Molecular mechanisms of plant GAPC nitrosylation and denitrosylation. (A) Nitrosylation of plant GAPC. Plant GAPC undergoes reversible S-nitrosylation in the presence of NO donors or trans-nitrosylation by GSNO with concomitant release of GSH. GSNO can be generated by the reaction of GSH with RNS. (B) Denitrosylation mechanism of plant GAPC. Denitrosylation of plant nitrosylated GAPC (GAPC1-SNO) is not catalyzed by plant cytoplasmic TRXs (dashed arrow), but it is efficiently catalyzed by GSH with formation of GSNO.

FIGURE 7

Schematic representation of glycolysis showing the NADPH-producing systems in a situation of oxidative modification of GAPC. Under stress conditions, GAPC might undergo different type of oxidative modifications (GAPC-S_ox: sulfenation, S-OH; glutathionylation, S-SG; or nitrosylation, S-NO) with important effects on cytoplasmic primary metabolism. Indeed, inhibition of GAPC activity and the consequent down-regulation of glycolysis pathway would promote entry of glucose equivalents into the OPP pathway leading to the generation of NADPH (red arrows). Although inhibition of GAPC would down-regulate the glycolytic pathway, plant cells also contain a non-phosphorylating GAPDH (GAPN) that can by-pass the GAPC-catalyzed reaction providing an alternative source of NADPH for the antioxidant enzymes (blue arrows). Glutathione reductase and thioredoxin reductases (GR and NTR, respectively) are major antioxidants enzymes in the cytoplasm of plant cells. Glutathione reductase, using NAPDH as electron donor, can keep the glutathione pool reduced providing the reductant (GSH) for the efficient reduction of nitrosylated GAPC or the deglutathionylation via cytoplasmic glutaredoxins (GRXs). Alternatively, GAPC may be also deglutathionylated by a GSH-independent system involving NADPH, NTR and cytoplasmic thioredoxins (TRXs). Overall, redirection of primary metabolism in stressed plant cells would allow reinforcing the antioxidant systems and creating the conditions for recovery (e.g., reduction/reactivation of redox-modified proteins such as GAPC). 3PGA, 3-phosphoglycerate; BPGA, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phospate.

FIGURE 8

GAPDH and apoptosis in animal cells. Interactions and processes that have been observed in different studies and cell types are here summarized in a single hypothetical animal cell. Different apoptotic stimuli may induce NO biosynthesis and the figure shows how apoptosis in animal cells may be triggered by the initial nitrosylation of GAPDH, and counteracted by several processes. For a detailed explanation, see text (Animal GAPDH: redox-dependent non-glycolytic (moonlighting) functions). GOSPEL, GAPDH’s competitor Siah1 protein enhances life; N-CoR, nuclear co-repressor; Siah1, seven in absentia homologue 1.

FIGURE 9

Metabolic and non-metabolic functions of GAPC in plant cells under normal and different stress conditions. Functional roles of GAPC that have been observed in different studies and cell types are here summarized in a single hypothetical plant cell. Under normal conditions, GAPC fulfills metabolic function by participating to glycolysis (left side of the figure) while it can play non-metabolic functions under specific stress conditions (e.g., drought, salinity and cold stress, treatments in the presence of cadmium (Cd), hydrogen peroxide (H₂O₂) and nitrosoglutathione (GSNO; right side of the figure). Overall, these conditions are supposed to induce different type of redox modifications of GAPC (GAPC-S_ox) and stimulate interaction with several partner proteins or to induce protein expression, accumulation of inactive protein and relocalization to the nucleus. Additional functions of GAPC are not clearly related to either specific stress conditions or redox modification or sub-cellular localization of the GAPC protein and are indicated by dashed arrows. For a detailed explanation, see text (Plant Glycolytic GAPDH: Metabolic and Non-Metabolic Functions). NtOSAK, Nicotiana tabacum osmotic stress activated kinase; PA, phosphatidic acid; PLD-δ, phospholipase D-δ; VDAC, voltage-dependent anion channels.