Phylogenetic Relationships of the Glycolytic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, from Parabasalid Flagellates

Over 90% of the open reading frame of gap genes for glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was obtained with PCR from five species of Parabasala. With gap1 from Trichomonas vaginalis obtained earlier, the data include two sequences each for three species. All sequences were colinear with T. vaginalis gap1 and shared with it as a synapomorphy a 10- to 11-residue insertion not found in any other gap and an S-loop with characteristic features of eubacterial GAPDH. All residues known to be highly conserved in this enzyme were present. The parabasalid sequences formed a robust monophyletic group in phylogenetic reconstructions with distance-based, maximum-parsimony, and maximum-likelihood methods. The two genes of the amphibian commensal, Trichomitus batrachorum, shared a common ancestor with the rest, which separate into two well-supported lineages. T. vaginalis and Tetratrichomonas gallinarum (both representatives of Trichomonadinae) formed one, while Monocercomonas sp. and Tritrichomonas foetus formed the other. These data agreed with and/or were close to published reconstructions based on other macromolecules. They did not support the ancestral position of Monocercomonas sp. proposed on the basis of morphological characteristics but confirmed an early emergence of Trichomitus batrachorum. The sequence pairs obtained from three species indicated either gene duplications subsequent to the divergence of the corresponding lineages or a strong gene conversion later in these lineages. The parabasalid clade was a robust part of the eubacterial radiation of GAPDH and showed no relationships to the clade that contained all other eukaryotic gap genes. The data clearly reveal that the members of this lineage use in their glycolytic pathway a GAPDH species with properties and an evolutionary history that are unique among all eukaryotes studied so far. Received: 28 April 1997 / Accepted: 14 October 1997     

Rheb and mTOR regulate neuronal polarity through Rap1B

The development of polarized hippocampal neurons with a single axon and multiple dendrites depends on the activity of phosphoinositide 3-kinase (PI3K) and the GTPase Rap1B. Here we show that PI3K regulates axon specification and elongation through the GTPase Rheb and its target mammalian target of rapamycin (mTOR). Overexpression of Rheb induces the formation of multiple axons, whereas its suppression by RNA interference blocks axon specification. mTOR is a central regulator of translation that phosphorylates eIF4E-binding proteins like 4E-BP1. Axon formation was suppressed by inhibition of mTOR and expression of mTOR-insensitive 4E-BP1 mutants. Inhibition of PI3K or mTOR reduced the level of Rap1B, which acts downstream of Rheb and mTOR. The ubiquitin E3 ligase Smurf2 mediates the restriction of Rap1B by initiating its degradation. Suppression of Smruf2 by RNA interference is able to compensate the loss of Rheb. These results indicate that the mTOR pathway is required to counteract the Smurf2-initiated degradation of Rap1B during the establishment of neuronal polarity.     

The Evolution of Duplicate Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Drosophila

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea mays L. Leaves

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10(-5)m and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions.     

Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N -ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.     

Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.     

Tail-to-Head Arrangement of a Partial Chicken Glyceraldehyde-3-Phosphate Dehydrogenase Processed Pseudogene

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5′ region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization. Received: 15 November 1996 / Accepted: 1 April 1997     

The Phylogeny of Glyceraldehyde-3-Phosphate Dehydrogenase Indicates Lateral Gene Transfer from Cryptomonads to Dinoflagellates

Sequence analysis of two nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes isolated from the dinoflagellate Gonyaulax polyedra distinguishes them as cytosolic and chloroplastic forms of the enzyme. Distance analysis of the cytosolic sequence shows the Gonyaulax gene branching early within the cytosolic clade, consistent with other analyses. However, the plastid sequence forms a monophyletic group with the plastid isoforms of cryptomonads, within an otherwise cytosolic clade, distinct from all other plastid GAPDHs. This is attributed to lateral gene transfer from an ancestral cryptomonad to a dinoflagellate, providing the first example of genetic exchange accompanying symbiotic associations between the two, which are common in present day cells. Received: 29 April 1998 / Accepted: 7 July 1998     

A Dimer Interface Mutation in Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Its Binding to AU-rich RNA

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.     

Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart

Mitochondrial proteins have been shown to be common targets of S-nitrosylation (SNO), but the existence of a mitochondrial source of nitric oxide remains controversial. SNO is a nitric oxide-dependent thiol modification that can regulate protein function. Interestingly, trans-S-nitrosylation represents a potential pathway for the import of SNO into the mitochondria. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown to act as a nuclear trans-S-nitrosylase, has also been shown to enter mitochondria. However, the function of GAPDH in the mitochondria remains unknown. Therefore, we propose the hypothesis that S-nitrosylated GAPDH (SNO-GAPDH) interacts with mitochondrial proteins as a trans-S-nitrosylase. In accordance with this hypothesis, SNO-GAPDH should be detected in mitochondrial fractions, interact with mitochondrial proteins, and increase mitochondrial SNO levels. Our results demonstrate a four-fold increase in GAPDH levels in the mitochondrial fraction of mouse hearts subjected to ischemic preconditioning, which increases SNO-GAPDH levels. Co-immunoprecipitation studies performed in mouse hearts perfused with the S-nitrosylating agent S-nitrosoglutathione (GSNO), suggest that SNO promotes the interaction of GAPDH with mitochondrial protein targets. The addition of purified SNO-GAPDH to isolated mouse heart mitochondria demonstrated the ability of SNO-GAPDH to enter the mitochondrial matrix, and to increase SNO for many mitochondrial proteins. Further, the overexpression of GAPDH in HepG2 cells increased SNO for a number of different mitochondrial proteins, including heat shock protein 60, voltage-dependent anion channel 1, and acetyl-CoA acetyltransferase, thus supporting the role of GAPDH as a potential mitochondrial trans-S-nitrosylase. In further support of this hypothesis, many of the mitochondrial SNO proteins identified with GAPDH overexpression were no longer detected with GAPDH knock-down or mutation. Therefore, our results suggest that SNO-GAPDH can act as a mitochondrial trans-S-nitrosylase, thereby conferring the transfer of SNO from the cytosol to the mitochondria.     

Glyceraldehyde-3-Phosphate Dehydrogenases from Archaea: Objects for Studying Protein Thermoadaptation

Functional and structural properties of the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic archaeum Methanobacterium bryantii and from the hyperthermophilic archaea Methan-othermus fervidus, Pyrococcus woesei and Thermoproteus tenax were compared to characterize the thermophilic phenotype. Site dierected mutagenesis with the M. fervidus enzyme were performed to analyse the structural background of the thermoadaptive features of the archaeal glyceraldehyde-3-phosphate dehydrogenase.     

Cotransport of Glyceraldehyde-3-Phosphate Dehydrogenase and Actin in Axons of Chicken Motoneurons

1.To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [³⁵S]methionine and, 1–20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I–Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography.2.In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).3.The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods.4.Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3–5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity.5.Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

植物中磷酸甘油醛-3-磷酸脱氢酶（GAPDH）在氧化胁迫下的生理功能

文章介绍植物中持家蛋白磷酸甘油醛-3-磷酸脱氢酶（GAPDH）在氧化胁迫下抑制活性氧生成、诱发磷酸化过程从而激活MAPK信号级联反应、诱导聚合体形成、参与谷胱甘肽修饰和控制电子转运中的生理功能研究进展。