Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.

Glyceraldehyde-3-phosphate dehydrogenase tetramer dissociation and amyloid fibril formation induced by negatively charged membranes.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme related with Huntington's, Parkinson's and Alzheimer's diseases. The ability of negatively charged membranes to induce a rapid formation of GAPDH amyloid fibrils has been demonstrated, but the mechanisms by which GAPDH reaches the fibrillar state remains unclear. In this report, we describe the structural changes undergone by GAPDH at physiological pH and temperature conditions right from its interaction with acidic membranes until the amyloid fibril is formed. According to our results, the GAPDH-membrane binding induces a beta-structuring process along with a loss of quaternary structure in the enzyme. In this way, experimental evidences on the initial steps of GAPDH amyloid fibrils formation pathway are provided.

Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) is an anchorless, multifunctional protein displayed on the surface of several fungi and Gram-positive pathogens, which contributes to their adhesion and virulence. To date a role for extracellular GAPDH in the pathogenesis of Gram-negative bacteria has not been described. The aim of this study was to analyze the extracellular localization of GAPDH in enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains and to examine its interaction with host components that could be related to the infection mechanism. Recombinant E. coli GAPDH was purified and polyclonal antibodies were obtained. Western blotting and immunoelectron microscopy showed that GAPDH is located on the bacterial surface and released to the culture medium of EHEC and EPEC strains. GAPDH export in these Gram-negative pathogens depends on the external medium, is not mediated by vesicles and leads to an extracellular active enzyme. Non-pathogenic E. coli strains do not secrete GAPDH. Two-dimensional electrophoresis analysis showed that in E. coli GAPDH is present at least in two major forms with different isoelectric points. Of these forms, the more basic is secreted. Purified GAPDH was found to bind human plasminogen and fibrinogen in Far-Western blot and ELISA-based assays. In addition, GAPDH remained associated with colonic Caco-2 epithelial cells after adhesion of EHEC or EPEC. These observations indicate that exported GAPDH may act as a virulence factor which could contribute to EHEC and EPEC pathogenesis. This is the first description of an extracellular localization for this enzyme, with a function other than its glycolytic role in Gram-negative pathogens.

Striking conformational change suspected within the phosphoribulokinase dimer induced by interaction with GAPDH.

A multitechnique approach was used to study the [glyceraldehyde-3-phosphate dehydrogenase](2 x 4)-[phosphoribulokinase](2 x 2) multienzymatic complex of the alga Chlamydomonas reinhardtii. On the one hand, each component of the complex was compared with known atomic structures of related enzymes or of similar enzymes originating from different organisms. On the other hand, the overall low resolution architecture of the whole complex was studied using cryoelectron microscopy and image processing techniques. The dimers of phosphoribulokinase are suspected to undergo a dramatic change in activity during a cycle of binding and detaching from tetramers of glyceraldehyde-3-phosphate dehydrogenase. This is likely supported by strong structural differences between the modeled phosphoribulokinase dimers and the counterpart in the three-dimensional reconstruction volume of the whole complex obtained from cryoelectron microscope images.

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis.

Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.

A helix-turn-strand structural motif common in alpha-beta proteins.

By exhaustive structural comparisons, we have found that about one-third of the alpha-helix-turn-beta-strand polypeptides in alpha-beta barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the alpha-helix and the beta-strand is somewhat constrained, and second, the beta-strand contains a hydrophobic patch that fits into a hydrophobic pocket on the alpha-helix. The geometry of the turn does not seem to be a major determinant of the alpha-beta unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn-for instance, when the turns between the alpha-helix and the beta-strands are very long. It also occurs much less frequently in flat-sheet alpha-beta proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which alpha-beta barrels are constructed.