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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.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis that catalyzes the conversion of glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglyceric acid (BPGA) in the presence of nicotinamide adenine dinucleotide (NAD+) and inorganic phosphate. Whereas animal cells contain only one isoform of GAPDH, plants contain several isoforms of GAPDH that are encoded by different types of genes (
Glyceraldehyde-3-phosphate dehydrogenase, however, is not only an enzyme, but also a moonlighting protein. In animal cells, the concept of moonlighting proteins was introduced after the discovery of proteins that were able to perform additional functions in respect to their originally assigned ones (
In contrast with the well established moonlighting properties of animal GAPDH, little is known on the multifunctional roles of plant cytoplasmic GAPDH, here referred as GAPC. Nonetheless, GAPC was identified as a potential target of diverse redox modifications and these modifications appear to be related to changes in subcellular localization in plant cells as well. Emerging evidence clearly show that, similar to animal GAPDH, plant cytoplasmic GAPDH may also perform alternative functions that are completely unrelated to its catalytic activity, strongly suggesting that plant GAPDH may also behave as a moonlighting protein.
The aim of this review is to provide an overview of what is known on plant cytoplasmic GAPDH, starting from its crystal structure and the structural features that determine its pronounced redox sensitivity. A comprehensive description of the molecular mechanisms underlying redox regulation of plant GAPCs will be provided, and a brief inventory of the redox-dependent multi-facetted properties of animal GAPDH will constitute a framework for the emerging role of oxidatively modified GAPDH in stress signaling pathways in plant cells.
All types of phosphorylating GAPDHs, both in plants and non-photosynthetic organisms, either involved in glycolysis or in the Calvin-Benson cycle, share a similar structure with variations on the theme. GAPDH is essentially a tetramer of identical or similar subunits, with the same overall organization in which the subunits are related to three symmetry axis. Subunits are identical in glycolytic GAPDHs (GAPC, GAPCp) and in A4-GAPDH of oxygenic phototrophs, and similar in the main photosynthetic isoform of land plants (A2B2-GAPDH). B-subunits share a high sequence homology with A-subunits but contain an extra sequence of 30 amino acids (C-terminal extension, CTE) that is essential for thioredoxin-based regulation and required for the association of A2B2-GAPDH isoforms into tetramers of tetramers (A8B8-GAPDH;
Several structures of GAPDH isoforms from animal, microbial and plant sources are available (
List of selected GAPDH isoforms from diverse organisms for which the three-dimensional structure has been solved.
Pdb code | Organism | Nomenclature | Metabolic pathway | Reference |
---|---|---|---|---|
1GD1 | GAPDH | Glycolysis/gluconeogenesis | ||
1GPD | GAPDH | Glycolysis/gluconeogenesis | ||
1ZNQ | GAPDH | Glycolysis/gluconeogenesis | ||
3E5R | GAPC | Glycolysis/gluconeogenesis | ||
1JN0 | A4-GAPDH | Calvin-Benson cycle | ||
2PKQ | A2B2-GAPDH | Calvin-Benson cycle | ||
2EUH | GAPN | Glycolysis |
This review is focused on GAPC-type GAPDH and a brief overview of the only known GAPC structure from plants is given. OsGAPC is a tetramer of four identical subunits related by three symmetry molecular axis (
A NAD+ molecule is bound to each enzyme subunit and anchored by several hydrogen bonds, either directly or through water molecules (
The structure of the active site of cytoplasmic OsGAPDH is very similar to that of other glycolytic and also chloroplastic GAPDHs. Sequence alignments show a highly conserved region between residues 150 and 161 (
The presence of basic residues as histidines, arginines or lysines located in a close proximity with a cysteine residue generally at a distance lower or equal to 6 Å, enhances the deprotonation of the thiol (-SH) to the nucleophilic thiolate (-S-), making the cysteine quite reactive to oxidative modifications. The positioning of the catalytic cysteine at the N-terminal side of an α-helix (α6), might further increase its reactivity due to the helix overall dipole moment (
With the aim of evaluating the reactivity of the catalytic cysteine as a function of its chemical environment, the p
In the OsGAPC structure, a second cysteine (Cys-158 corresponding to Cys-153 in
As already mentioned, plant cells contain a cytoplasmic non-phosphorylating NADP-GAPDH (GAPN), with low sequence homology with classical phosphorylating GAPDH (~20% with OsGAPC). The crystal structure of a GAPN from
Reactive oxygen and nitrogen species (ROS and RNS, respectively) play an important role in redox signaling mainly through a set of reversible post-translational modifications (PTMs) of cysteine thiols on proteins (
The reaction between ROS and RNS with protein thiols mainly involves cysteine residues with unique physico-chemical properties. These residues are considered reactive when their ionization constant (p
Since 2001, numerous proteomic approaches have been developed to identify plant proteins putatively regulated by TRXs via dithiol/disulfide interchange or undergoing glutathionylation or S-nitrosylation (
In redox proteomic studies, plant glycolytic GAPDH (GAPC) has been identified as a potential target of TRX regulation (i.e., dithiol/disulfide interchange) and glutathionylation (
The identification of plant GAPC as a target of multiple redox modifications is consistent with the fact that GAPC contains a reactive catalytic cysteine (referred in the following as Cys-149) with a low p
Protein sulfenation consists of the formation of a sulfenic acid after the reaction between a reactive cysteine and an oxidant molecule such as H2O2 through a two-electron oxidation process. The sensitivity of GAPC toward H2O2 has been tested by several research groups and in all cases a strong inhibition of enzyme activity was observed, suggesting that the highly reactive catalytic Cys-149 was indeed the target of H2O2 oxidation (
Beyond sulfenation, a modification that is fully DTT-reversible, cysteine thiolates may be further oxidized to sulfinic and sulfonic acids (
Protein glutathionylation, consisting in the formation of a mixed-disulfide between a protein thiol and a molecule of GSH, can act as a mechanism of protection against irreversible oxidation of reactive cysteine thiols but has also a role in modulating protein functions (
Glutathionylation of GAPC
Removal of GSH from glutathionylated proteins (namely deglutathionylation) is, in most cases, a reaction that GSH cannot perform by itself with any significant efficiency. Glutathionylated GAPC (GAPC-SSG) and GRXs were recently used to investigate the molecular mechanisms of deglutathionylation reactions in a plant system (
Glutathionylated GAPC was also used to investigate the deglutathionylating activity of different plant TRXs. These enzymes are known to control the activity of a large number of proteins through dithiol/disulfide exchange reactions (
Protein S-nitrosylation, consisting in the formation of nitrosothiols (S-NO) by reaction of protein thiols with NO, can be triggered chemically by RNS such as NO or by trans-nitrosylation reactions mediated by small nitrosothiols (e.g., nitrosoglutathione, GSNO) or by other S-nitrosylated proteins (
The reduction of nitrosothiols on proteins, i.e., denitrosylation, entails two possible mechanisms, either dependent on reduced GSH or on the TRX system (NADPH, NADPH: TRX reductase and TRX;
Since cytoplasmic GAPDH is sensitive to oxidative stress due to its reactive catalytic cysteine, oxidative modification of this residue invariably inhibits enzyme activity and, in animal cells at least, also glycolysis (
Although mammalian GAPDH clearly retains its fundamental role as a glycolytic housekeeping enzyme in the cytoplasm, mounting evidence indicates that different redox PTMs can induce or regulate additional non-glycolytic functions of GAPDH. In this sense, GAPDH is considered as a moonlighting protein. Besides inhibiting enzyme activity, redox PTMs of GAPDH can induce or affect the interaction with other proteins or nucleic acids (
Animal GAPDH is involved in the signaling cascade that governs NO-dependent apoptosis. After apoptotic stimuli, GAPDH is found in the nucleus in a number of cell systems, including neurons (
Siah1-mediated GAPDH translocation to the nucleus is counteracted by GOSPEL (GAPDH’s competitor Siah1 protein enhances life;
Another mechanism counteracting the Siah1-mediated nuclear translocation of GAPDH is the formation of aggregates that immobilize GAPDH in the cytoplasm and may evolve into cytotoxic amyloid-like aggregates (
The interaction between SNO-GAPDH and Siah1 can be counteracted also in the nucleus. This mechanism of regulation of the SNO-GAPDH/Siah1-dependent pro-apoptotic cascade depends on the ability of SNO-GAPDH to function as a trans-nitrosylase, i.e., to transfer its nitroso-group to a receiving thiol on a different protein (
The trans-nitrosylating activity of SNO-GAPDH in the nucleus is not restricted to B23. Other nuclear proteins that are similarly trans-nitrosylated include deacetylases such as SIRT1 and HDAC (
GAPDH can bind to RNA and several examples of post-transcriptional regulation mechanisms involving GAPDH can be found in the literature. Binding of GAPDH to messenger RNA may have contrasting effects: in some cases GAPDH increases mRNA stability (e.g., CSF1), in others stimulates mRNA degradation (ET-1) or causes inhibition of translation (AT1R;
Post-transcriptional regulation of the vaso-constricting peptide endothelin (ET-1) is based on the ability of human GAPDH to bind AU-rich elements (ARE) of mRNA. AREs are common cis-regulatory elements of 3′-untranslated regions (UTR) of eukaryotic messenger RNAs and are important determinants of mRNA stability in mammals. Proteins binding to these elements can either increase or decrease mRNA stability and thus affect protein expression levels (
Thanks to its capability to interact with AREs via its Rossmann fold, GAPDH was shown to bind many other types of mRNA, and may well exert a more general role in post-transcriptional regulation. Although only in the case of ET-1, GAPDH-mRNA interaction was experimentally shown to be redox-regulated, it is plausible that this property may also apply to other GAPDH-mRNA combinations, as long as different mRNAs may bind to GAPDH in a similar fashion.
Compared to mammalian cells in which a single gene codes for GAPDH, Arabidopsis contains two cytoplasmic isozymes, GAPC1 (At3g04120) and GAPC2 (At1g13440), 98% identical in amino acid sequence, and both similarly contributing to the total NAD-specific GAPDH activity of the cytoplasm (
In spite of its role in a fundamental metabolic pathway such as glycolysis, reduction of GAPC protein levels in transgenic plants generally leads to relatively mild phenotypes under normal growth conditions, in agreement with the notion that plants contain possible alternatives to cytoplasmic GAPC, e.g., non-phosphorylating GAPN and plastidic GAPDHs (A4- and A2B2-GAPDH and GAPCp).
Partial suppression by antisense technology of cytoplasmic GAPC was obtained in transgenic potato (
Quite unexpectedly, a strong metabolic-related phenotype is displayed by GAPCp double mutants in Arabidopsis. These plants are dwarf and suffer of arrested root development and sterility. In spite of the little contribution, in quantitative terms, of GAPCp to the total glycolytic activity of plant tissues, GAPCp (together with phosphoglycerate kinase) is essential in providing 3-phosphoglyceric acid (3-PGA) precursors for serine biosynthesis in plastids through the phosphorylating pathway (
Besides the multitude of examples in mammalian cells, emerging evidence for non-metabolic functions of glycolytic GAPDHs (GAPCs) are also coming from research carried out in plants (
Arabidopsis mutants in which both GAPC isoforms were knocked out are impaired in ABA-induced stomatal closure, and consequently suffer of a higher transpirational water loss than wild type plants under drought stress (
Similar to mammalian cells, also in plant cells GAPDH was detected both in the cytoplasm and in the nucleus (
In a recent study (
Cadmium-induced nuclear translocation of GAPC1 in Arabidopsis reminds the nuclear translocation of mammalian GAPDH upon apoptotic stimuli. However, in this latter case translocation is triggered by the S-nitrosylation of GAPDH catalytic cysteine and inhibited by its mutation to serine [
In BY-2 tobacco cells, cytoplasmic GAPDH isoforms NtGAPCa and NtGAPCb were found to interact, both in the cytoplasm and the nucleus, with an osmotic stress activated kinase (NtOSAK;
Whatever the mechanism required for nuclear localization, a clear role of plant GAPDHs in the nucleus is still missing. Preliminary evidence that GAPC may bind to a DNA sequence coding for NADP-malate dehydrogenase (
The capability of GAPDH to bind nucleic acids does also explains its recently discovered role in regulating virus replication in plants. Indeed GAPC interacts with the replicase complex of different phytopathogenic viruses with a single-stranded, positive-sense RNA genome. The effect of GAPC binding consists in either repressing or stimulating virus replication. In
Moonlighting properties of GAPC, possibly shared with other glycolytic enzymes, are also suggested by the specific and dynamic binding to sub-cellular structures such as membranes and the cytoskeleton. A portion of all glycolytic enzymes, including GAPC, is associated to the cytoplasmic face of the outer membrane of plant mitochondria (
Plant glycolytic GAPDH can also interact with the cytoskeleton and promote actin bundling, again a function of GAPDH that is stimulated by oxidizing conditions (
Glyceraldehyde-3-phosphate dehydrogenase is an abundant protein of animal and plant cells, whose catalytic role in glycolysis is based on a highly reactive catalytic cysteine. GAPDH is also a moonlighting, multifunctional protein with non-glycolytic roles that often involve oxidative modifications of the catalytic cysteine. Plant cytoplasmic GAPDH (GAPC) proved an excellent tool to study the different types of redox PTMs that may affect a plant protein (
Every function of GAPDH that depends, or is regulated by a redox modification that blocks its catalytic activity is by definition alternative to its role in glycolysis. However, alternative functions of GAPC, signaling functions in particular, may well involve only a small fraction of the large pool of GAPC in plant cells. Moreover, plant cells contain metabolic alternatives to keep the glycolytic flux operative under conditions in which GAPC is oxidatively inactivated. Overall, GAPC appears to have suitable properties to behave as an oxidative stress sensor in plant cells and this function may not be in contrast with its classical glycolytic role.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by PRIN 2008 and 2009 (to Mirko Zaffagnini and Paolo Trost), by ANR Grant 12-BSV5-0019 REDPRO2 (to Stéphane D. Lemaire), by LABEX DYNAMO ANR-LABX11-0011 (to Stéphane D. Lemaire), by the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) through the grant FIRB 2010 RBFR10S1LJ_001 (to Alex Costa), by FARB2012 University of Bologna (to Mirko Zaffagnini and Simona Fermani) and by “Research in Paris” fellowship from Ville de Paris (to Mirko Zaffagnini). No conflict of interest declared. All authors participated in planning and writing the paper.