Association Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Mycobacterium tuberculosis glyceraldehyde-3-phosphate dehydrogenase plays a dual role-As an adhesin and as a receptor for plasmin(ogen).

The spread of infection is directly determined by the ability of a pathogen to invade and infect host tissues. The process involves adherence due to host-pathogen interactions and traversal into deeper tissues. Mycobacterium tuberculosis (Mtb) primarily infects the lung but is unique in its ability to infect almost any other organ of the human host including immune privileged sites such as the central nervous system (CNS). The extreme invasiveness of this bacterium is not fully understood. In the current study, we report that cell surface Mtb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a virulence factor by multiple mechanisms. Firstly, it serves as a dual receptor for both plasminogen (Plg) and plasmin (Plm). CRISPRi-mediated silencing of this essential enzyme confirmed its role in the recruitment of Plg/Plm. Our studies further demonstrate that soluble GAPDH can re-associate on Mtb bacilli to promote plasmin(ogen) recruitment. The direct association of plasmin(ogen) via cell surface GAPDH or by the re-association of soluble GAPDH enhanced bacterial adherence to and traversal across lung epithelial cells. Furthermore, the association of GAPDH with host extracellular matrix (ECM) proteins coupled with its ability to recruit plasmin(ogen) may endow cells with the ability of directed proteolytic activity vital for tissue invasion.

GAPDH binds Akt to facilitate cargo transport in the early secretory pathway

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes numerous post-translational modifications, which impart new function and influence intracellular location. For example, atypical PKC ι/λ phosphorylates GAPDH that locates to vesicular tubular clusters and is required for retrograde membrane trafficking in the early secretory pathway. GAPDH is also required in the endocytic pathway; substitution of Pro234 to Ser (Pro234Ser) rendered CHO cells defective in endocytosis. To determine if GAPDH (Pro234Ser) could inhibit endoplasmic reticulum to Golgi trafficking, we introduced the recombinant mutant enzyme into several biochemical and morphological transport assays. The mutant protein efficiently blocked vesicular stomatitis virus-G protein transport. Because GAPDH binds to microtubules (MTs), we evaluated MT binding and MT intracellular distribution in the presence of the mutant. Although these properties were not changed relative to wild-type, GAPDH (Pro234Ser) altered Golgi complex morphology. We determined that the GAPDH point mutation disrupted association between the enzyme and the serine/threonine kinase Akt. Interestingly Rab1, which functions in anterograde-directed trafficking, stimulates GAPDH-Akt association with membranes in a quantitative binding assay. In contrast, Rab2 does not stimulate GAPDH-Akt membrane binding but instead recruits GAPDH-aPKC. We propose a mechanism whereby the association of GAPDH with Akt or with aPKC serves as a switch to discriminate between anterograde directed cargo and recycling cargo retrieved back to the ER, respectively.

Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells.

BackgroundFor a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood.MethodsGAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses.ResultHere, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex.ConclusionPKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2172-x) contains supplementary material, which is available to authorized users.

High Glucose-induced Retinal Pericyte Apoptosis Depends on Association of GAPDH and Siah1

Diabetic retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive, and have limited effectiveness. Retinal pericyte death is an early pathologic feature of DR. Although it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this article we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5 mm normal glucose, 25 mm l- or d-glucose for 48 h (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to down-regulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1-directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this article can provide a foundation to identify novel targets for early treatment of DR.

Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Ciota (aPKCiota) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCiota in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCiota were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCiota. Because GAPDH binds to the carboxyl terminus of alpha-tubulin, we characterized the distribution of tyrosinated/detyrosinated alpha-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated alpha-tubulin; however, aPKCiota was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCiota-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.

Binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules.

The interaction of glyceraldehyde 3-phosphate dehydrogenase with microtubules has been studied by measurement of the amount of enzyme which co-assembles with in vitro reconstituted microtubules. The binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules is a saturable process; the maximum binding capacity is about 0.1 mole of enzyme bound per mole of assembled tubulin. Half saturation of microtubule binding sites is obtained at a concentration of glyceraldehyde 3-phosphate dehydrogenase of about 0.5 microM. Glyceraldehyde 3-phosphate dehydrogenase (between 0.1 and 2 microM) induces a concentration-dependent increase a) in the turbidity of the microtubule suspension without alteration of the net amount of polymer formed and b) in the amount of microtubule protein polymers after cold microtubule disassembly. There is a linear relationship between the intensity of the glyceraldehyde 3-phosphate dehydrogenase-induced effects and the amount of microtubule-bound enzyme. The specificity of the association of glyceraldehyde 3-phosphate dehydrogenase to microtubules has been documented by copolymerization experiments. Assembly-disassembly cycles of purified microtubules in the presence of a crude liver soluble fraction results in the selective extraction of a protein with an apparent molecular weight of 35,000 identified as the monomer of glyceraldehyde 3-phosphate dehydrogenase by peptide mapping and immunoblotting. In conclusion, microtubules possess a limited number of binding sites for glyceraldehyde 3-phosphate dehydrogenase. The binding of the glycolytic enzyme to microtubules shows a considerable specificity and is associated with alterations of assembly and disassembly characteristics of microtubules.

Microtubules bind glyceraldehyde 3-phosphate dehydrogenase and modulate its enzyme activity and quaternary structure

Glyceraldehyde 3-phosphate dehydrogenase, a tetramer of 140,000 Da, interacts with in vitro reconstituted microtubules. It results in a partial inhibition of the activity of the microtubule-bound enzyme. After cold depolymerization of the microtubule-glyceraldehyde 3-phosphate dehydrogenase complexes, a fraction of the enzyme is recovered in an active form in the disassembly supernatant; the other fraction devoid of activity, identified by polyacrylamide gel electrophoresis, remains associated with the undepolymerizable microtubule protein pellet. The inactivation of the microtubule-bound enzyme is related to the concentration of microtubule protein. Higher the concentration of microtubule protein, lower the fraction of inactivated enzyme; consequently, glyceraldehyde 3-phosphate dehydrogenase is able to copolymerize quantitatively with microtubule protein through one assembly-disassembly cycle, provided that the concentration of microtubule protein is high. Monomeric glyceraldehyde 3-phosphate dehydrogenase (molecular weight: 35,000) devoid of enzyme activity, prepared by reversible dissociation of the tetrameric enzyme, also binds to microtubules and is quantitatively recovered in the undepolymerizable microtubule protein fraction after cold treatment. These results indicate that (i) interacting with microtubules, glyceraldehyde 3-phosphate dehydrogenase partly dissociates into inactive monomers, (ii) this process is regulated by the concentration of assembled microtubule protein, and (iii) active and inactive glyceraldehyde 3-phosphate dehydrogenase bound to microtubules have different fate at the step of microtubule disassembly. These data suggest that an association of glyceraldehyde 3-phosphate dehydrogenase to microtubules could play a role in modulating the activity of the glycolytic enzyme in intact cells.

Association of glyceraldehyde 3-phosphate dehydrogenase with the membrane of the intact human erythrocyte.

Intact human erythrocytes were exposed to low concentrations of glutaraldehyde. After washing and subsequent lysis of the cells, glyceraldehyde 3-phosphate dehydrogenase activity is found to be associated with a membrane fraction and cannot be eluted by salt treatment. Lactate dehydrogenase activity is associated with a supernatant fraction under the same conditions. Preincubation of the intact cells under conditions designed to increase internal NADH concentrations, leads to a lower membrane-associated activity of glyceraldehyde 3-phosphate dehydrogenase after lysis.

Specificity in the Association of Glyceraldehyde 3-Phosphate Dehydrogenase with Isolated Human Erythrocyte Membranes

Abstract We have characterized the binding of glyceraldehyde 3phosphate dehydrogenase to isolated human erythrocyte membranes in an effort to establish whether this in vitro association has physiologic significance. At pH 8 and µ = 0.015 m, approximately 2 x 106 glyceraldehyde-3-P dehydrogenase molecules bound reversibly and homogeneously to the inner (but not the outer) membrane surface with an association constant of 1 x 107 m-1. This binding capacity exceeded by roughly 10-fold the cell's glyceraldehyde-3-P dehydrogenase content. Binding diminished with increasing ionic strength and pH, suggesting a strong electrostatic component in the association. Millimolar amounts of certain metabolites also released bound enzyme from the membrane. In contrast, an indifferent strongly basic protein, cytochrome c, lacked specificity in its association with the erythrocyte membrane. That is, binding was relatively weak, occurred at both membrane surfaces, and may have been limited in extent by close packing (i.e. monolayer formation) rather than by a fixed number of binding sites. Cytochrome c showed much stronger association with carboxymethylcellulose than did glyceraldehyde-3-P dehydrogenase, but was more readily eluted from the membrane by electrolytes. Cytochrome c binding to membranes was not altered by any metabolite tested. We hypothesize that a fraction of erythrocyte glyceraldehyde-3-P dehydrogenase may be specifically bound to the inner surface of the membrane in vivo in a fashion which is responsive to metabolic and ionic transients.

Association of Glyceraldehyde 3-Phosphate Dehydrogenase with the Human Erythrocyte Membrane: EFFECT OF DETERGENTS, TRYPSIN, AND ADENOSINE TRIPHOSPHATE

Abstract Glyceraldehyde 3-phosphate dehydrogenase is one of the major protein components associated with the erythrocyte membrane. This has been shown by activity studies of specific membrane extracts and by the specificity of iodoacetate inactivation and labeling of the enzyme. Studies on the association of the enzyme with the membrane are complicated by the tendency of the ghosts to seal under certain conditions, thus rendering the enzyme inaccessible to its substrates. This crypticity of the enzyme activity can be eliminated by detergent treatment. Sodium dodecyl sulfate is preferred, since at low concentrations it causes only minor decreases in the enzyme activity, while Triton X-100 gives more extensive inactivation at all concentrations tested. Trypsin digestion can release glyceraldehyde 3-phosphate dehydrogenase from the membrane, but it also causes a fraction of the ghosts to seal. The fraction which reseals is somewhat variable between different membrane preparations and is related to the prior treatments of the membranes. Under standard hypotonic hemolysis conditions (10 mm Tris), 60 to 80% of the glyceraldehyde 3-phosphate dehydrogenase remains associated with the membrane. Aldolase exhibits similar behavior, while lactic dehydrogenase and hemoglobin are almost completely released. In the presence of ATP (1 mm) the amount of glyceraldehyde 3-phosphate dehydrogenase associated with the membrane is reduced to 5 to 20%. EDTA, Ca++, Mg++, and 2,3-diphosphoglycerate show little effect at concentrations of 1 mm, although release of the enzyme is promoted by these agents at higher concentrations. At 0°, solubilized glyceraldehyde 3-phosphate dehydrogenase is inactivated by ATP, a process that can be prevented if the enzyme is preincubated with NAD or if the incubation with ATP is performed at 25°. The ATP-promoted release of glyceraldehyde 3-phosphate dehydrogenase during hemolysis is not altered by preincubation with NAD or by raising the temperature to 25°. Therefore, the two phenomena (enzyme inactivation and release from the membrane) appear to be unrelated. It is postulated that the effect of ATP on enzyme release is due to a direct action of ATP on the membrane.