Binding Constants and Stoichiometries of Glyceraldehyde 3-Phosphate Dehydrogenase-Tubulin Complexes

doi:10.1006/abbi.1994.1385

Archives of Biochemistry and Biophysics

Volume 313, Issue 2, September 1994, Pages 253-260

https://doi.org/10.1006/abbi.1994.1385 Get rights and content

Abstract

The catalytic activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) decreased (almost linearly) as a function of increasing concentrations of tubulin; the total loss in activity was attained at a ratio of 1.2 to 1.8 tubulin dimer to GAPDH tetramer. Based on the inhibition data, a dissociation constant for the tubulin-GAPDH complex was calculated to be about 0.73 nM. The stoichiometry and the dissociation constants of the tubulin-GAPDH complex were found to be dependent upon the ionic strength of the assay media. Qualitatively similar results were obtained (i.e., inhibition and ionic strength effect) when the GAPDH-catalyzed reaction was measured in the presence of Sepharose-immobilized tubulin. The physical interaction between these two proteins, i.e., GAPDH and tubulin, was measured by the ability of one protein (immobilized on a Sepharose matrix) to copellet the other protein. By employing this copelleting technique, we measured the dissociation constant and stoichiometry of the immobilized tubulin-GAPDH complex to be about 6.4 nM and 0.91 tubulin dimer/GAPDH tetramer, respectively. The dissociation constant and stoichiometry thus obtained were found to be remarkably similar to those obtained by the tubulin-dependent GAPDH inhibition data. In contrast to these results, (soluble) tubulin had no effect on the catalytic activity of the immobilized GAPDH, albeit the soluble tubulin copelleted with the immobilized GAPDH. The dissociation constant and stoichiometry of immobilized GAPDH-tubulin complex were calculated to be 0.76 ± 0.13 μM and 3.23 ± 0.16 tubulin dimer/GAPDH tetramer, respectively. These data suggest that there are two classes of binding sites for tubulin on a tetrameric GAPDH; high-affinity and low-affinity sites. The enzyme is inhibited when tubulin binds at the high-affinity site while the catalytic function of the enzyme is unaffected when the tubulin binds at the low-affinity site. The latter site is suggested herein to be responsible for the crosslinking (bundling) of microtubules.

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Cited by (60)

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein
2020, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
The changes in the spatial structure of hGAPDH in response to S-glutathionylation may be involved in regulation of numerous processes. There are many reports on the ability of GAPDH to interact with nucleic acids [42–45], heme [46], proteins such as transferrin [47,48], tubulin [49], alpha-synuclein [50], Siah1 and GOSPEL [51,52] and on the possible role of these interactions in the regulation of various cellular processes (transcription, iron uptake, apoptosis, etc.). The interaction of GAPDH with Siah1 (an E3-ubiquitinligase) is of particular interest in connection with the induction of apoptosis [51].
We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H₂O₂ and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated.
Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH.
S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S.
S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1.
The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.
Oxidatively modified glyceraldehyde-3-phosphate dehydrogenase in neurodegenerative processes and the role of low molecular weight compounds in counteracting its aggregation and nuclear translocation
2018, Ageing Research Reviews
Citation Excerpt :
It has also been shown to facilitate membrane fusion (Glaser and Gross, 1995; Rodacka, 2013) and exhibits Ca2+ dependent fusogenic activity (Hessler et al., 1998; Tristan et al., 2011). The cytosolic form of dehydrogenase also participates in microtubule formation and actin polymerisation by binding tubulin and actin, respectively (Durrieu et al., 1987; Muronetz et al., 1994). However this function is displayed only by glycolytically inactive monomeric molecules of GAPDH.
A number of independent studies have shown the contribution of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the pathogenesis of several neurodegenerative disorders. Indeed, GAPDH aggregates have been found in many post-mortem samples of brains of patients diagnosed with Alzheimer’s and Parkinson disease. Currently, it is accepted that GAPDH-mediated cell death pathways in the neurodegenerative processes are associated with apoptosis caused by GAPDH nuclear translocation and excessive aggregation under oxidative stress conditions. Also the role of GAPDH in neurodegenerative diseases is linked to it directly binding to specific amyloidogenic proteins and petides such as β-amyloid precursor protein, β-amyloid peptide and tau protein in Alzheimer’s disease, huntingtin in Huntington’s disease and α-synuclein in Parkinson disease. One of the latest studies indicated that GAPDH aggregates significantly accelerate amyloidogenesis of the β-amyloid peptide, which implies that aggregates of GAPDH may act as a specific aggregation “seed” in vitro.
Previous detailed studies revealed that the active-site cysteine (Cys152) of GAPDH plays an essential role in the oxidative stress-induced aggregation of GAPDH associated with cell death. Furthermore, oxidative modification of this cysteine residue initiates the translocation of the enzyme to the nucleus, subsequently leading to apoptosis. The crystallographic structure of GAPDH shows that the Cys152 residue is located close to the surface of the molecule in a hydrophilic environment, which means that it can react with low molecular weight compounds such as hydroxynonenal or piceatannol. Therefore, it is highly possible that GAPDH may serve as a target for small molecule compounds with the potential to slow down or prevent the progression of neurodegenerative disorders.
Recently appearing new evidence has highlighted the significance of low molecular weight compounds in counteracting the oxidation of GAPDH and consequently its aggregation and other unfavourable pathological processes. Hence, this review aims to present all recent findings concerning molecules that are able to interact with GAPDH and counteract its aggregation and translocation to the nucleus.
Glyceraldehyde-3-phosphate dehydrogenase: Aggregation mechanisms and impact on amyloid neurodegenerative diseases
2017, International Journal of Biological Macromolecules
Citation Excerpt :
The most contradicting is the information on the complex between GAPDH and lactate dehydrogenase [106–109], however, such a possibility cannot be excluded. GAPDH also binds to structural proteins (actin, tubulin, and troponin) and plays an important role in functioning systems formed by these proteins [39,110–115]. The preferable form for the binding to structural proteins is presumably GAPDH dimer, whose stability is supported by the interaction between GAPDH and a structural protein.
The review analyses data on specific features of aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and possible role of this enzyme in the development of neurodegenerative diseases. Different post-translational modifications of the enzyme are considered: oxidation, nitrosylation, and S-glutathionylation of the active site sulfhydryl groups, as well as phosphorylation, glycation and homocysteinylation of other amino acid residues. Modification of the sulfhydryl groups of the enzyme inhibits the enzymatic activity of GAPDH, resulting in slowdown of glycolysis, and may lead to the dissociation of the cofactor NAD from the active site of the enzyme. The resulting apo-GAPDH (without NAD) is less stable and prone to dissociation, denaturation, and subsequent aggregation. These processes could play a crucial role in the translocation of GAPDH subunits from the cytoplasm into the nucleus, which is linked to the induction of apoptosis. Phosphorylation and glycation of GAPDH are presumably involved in the regulation of protein-protein interactions and intracellular localization of the enzyme. Besides, glycation by dicarbonyl compounds and aldehydes may directly inhibit glycolysis. Homocysteinylation of GAPDH may stabilize aggregates of the enzyme by additional disulfide bonding. All types of post-translational modifications affect aggregation of GAPDH. A special attention is given to the role of chaperones in the amyloidogenic transformation of proteins and to confirmation of the hypothesis on blocking of the chaperones by misfolded protein forms. The denatured GAPDH forms were shown to interact directly with amyloidogenic proteins (alpha-synuclein and amyloid-beta peptide) and to play a crucial role in blocking of chaperone system.
A dimer interface mutation in glyceraldehyde-3-phosphate dehydrogenase regulates its binding to AU-rich RNA
2015, Journal of Biological Chemistry
Citation Excerpt :
Each 335-amino acid polypeptide contains two domains: an NAD+ binding/Rossmann-fold domain (residues 1–150 and 317–335) and a catalytic domain (residues 151–316). GAPDH is a multifunctional enzyme implicated in a variety of cellular processes including nucleic acid binding (13, 15–19), DNA replication and repair (20), nuclear tRNA transport (18), apoptosis (21, 22), microtubule bundling (23), membrane fusion (24), neurodegenerative disorders (25, 26), and heme incorporation (27). The mechanism by which GAPDH switches among these various functions in the cell is unknown but may depend on the cell status and GAPDH posttranslational modifications (for reviews, see Refs. 28 and 29).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD⁺ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.The glycolytic enzyme GAPDH is a non-canonical RNA-binding protein.
A dimer interface mutation impairs the two-step binding of GAPDH to AU-rich elements from TNF-α mRNA and leads to RNA structural changes.
Dimer and tetramer interface residues are important for GAPDH-RNA binding.
We propose a novel model for GAPDH binding to AU-rich RNA via the oligomeric interfaces.
Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: Comparison with influence of poly(sulfoanions)
2013, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
However, the bound polysulfoanions could induce pronounced denaturation of the protein [3], whereas the poly(carboxylic) acids did not suffer this disadvantage and, in some cases, were able to exhibit a chaperone activity [4]. The results of above model studies could be relevant to living systems since a lot of proteins contain charged areas on the globule surface [5,6]. Moreover, many proteins undergo posttranslational modifications that change their functions [7,8].
It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.
Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.
The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.
Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.
Direct Binding of Glyceraldehyde 3-Phosphate Dehydrogenase to Telomeric DNA Protects Telomeres against Chemotherapy-Induced Rapid Degradation
2009, Journal of Molecular Biology
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel-shift assays, we show that recombinant GAPDH binds directly with high affinity (K_d = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats, and that nucleotides T1, G5, and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA:GAPDH), and GAPDH appears to form a high-molecular-weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active-site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth-inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition; this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.

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Regular ArticleBinding Constants and Stoichiometries of Glyceraldehyde 3-Phosphate Dehydrogenase-Tubulin Complexes

Abstract

Regular Article
Binding Constants and Stoichiometries of Glyceraldehyde 3-Phosphate Dehydrogenase-Tubulin Complexes