Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30-100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35-40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a K(D) of approximately 0.5 microM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule-membrane interactions by affecting the p22-microtubule, but not the p22-membrane, association.
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PMID:Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules. 1531 48

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.
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PMID:Identification of major proteins in maize egg cells. 1556 24

Although hypercholesterolemia is a well-known risk factor for atherosclerosis, little is known about the effect of hypercholesterolemia on cardiac contractile function. The objective of this study was to examine the effect of hypercholesterolemia on myocardial contractility. Fifteen New Zealand white rabbits were fed standard chow (control group) and another 15 were fed a cholesterol-enriched diet (HC group) for 12 weeks. The contractile response of ventricular muscle strips was measured in various extracellular calcium concentrations and at different pacing rates. The whole-cell calcium current recording, and mRNA and protein levels of cellular calcium-handling proteins were also analyzed. With 2 mM Ca2+ and stimulation at 3 Hz, the contractile force of HC strips was less than that of the controls (3.63 +/- 0.20 vs. 4.61 +/- 0.50 mN, p < 0.05). The time to peak tension was longer for HC strips (93.3 +/- 2.16 vs. 82.2 +/- 2.81 ms, p < 0.05). The peak L-type calcium inward current density was slightly higher in HC myocytes but did not reach statistical significance (-14.90 +/- 0.94 vs. -12.44 +/- 0.84 pA/pF, p = 0.15). The mRNA level of sarcoplasmic reticulum Ca2+-ATPase (SERCA), normalized to GAPDH, was significantly lower in the HC than that in the control group (2.85 +/- 0.14 vs. 7.67 +/- 0.67, p < 0.05), as was the ryanodine receptor (RyR; 0.42 +/- 0.06 vs. 0.71 +/- 0.13, p < 0.05). The mRNA of the Na+/Ca2+ exchanger (NCX) was statistically higher in the HC group (0.90 +/- 0.12 vs. 0.48 +/- 0.05, p < 0.05). Western blot experiments revealed that protein expression of SERCA in the HC strips decreased, but that of the NCX increased. The protein expression of the dihydropyridine receptor was similar between these two groups. We concluded that hypercholesterolemia results in suppression of the maximal contractile function and in a longer systolic contractile time course. These changes may partially be mediated through a decrease in SERCA and RyR but an increase in NCX expression.
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PMID:Effect of hypercholesterolemia on myocardial function in New Zealand white rabbits. 1559 80

NADH regulates the release of calcium from the endoplasmic reticulum by modulation of inositol 1,4,5-trisphosphate receptors (IP3R), accounting for the augmented calcium release of hypoxic cells. We report selective binding of IP3R to GAPDH, whose activity leads to the local generation of NADH to regulate intracellular calcium signaling. This interaction requires cysteines 992 and 995 of IP3R and C150 of GAPDH. Addition of native GAPDH and NAD+ to WT IP3R stimulates calcium release, whereas no stimulation occurs with C992S/995S IP3R that cannot bind GAPDH. Thus, the IP3R/GAPDH interaction likely enables cellular energy dynamics to impact calcium signaling.
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PMID:Inositol 1,4,5-trisphosphate receptor/GAPDH complex augments Ca2+ release via locally derived NADH. 1567 21

We report the isolation of a full length cDNA from cardiac muscle that encodes a approximately 73 kDa calcium/calmodulin (CaM) dependent kinase IIbeta isoform (CaMKIIbeta(C)) that was generated by alternative splicing of the CaMKIIbeta gene. Antipeptide antibodies raised to specific regions of the kinase identified a 73 kDa kinase polypeptide in cardiac SR. Anti-alpha kinase anchoring protein (alphaKAP) antibodies identified a 25 kDa polypeptide in cardiac SR and RT-PCR followed by sequence analysis confirmed the presence of a full length alphaKAP encoding transcript in myocardium. Protein interaction assays revealed that the 73 kDa CaMKIIbeta(C) binds GAPDH to modulate the production of NADH in a Ca2+/CaM dependent reaction. The presence of a CaMKIIbeta isoform that can target the SR presumably via its membrane anchor alphaKAP defines a previously unrecognized Ca2+/CaM regulatory system in myocardium.
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PMID:Alternative splicing generates a CaM kinase IIbeta isoform in myocardium that targets the sarcoplasmic reticulum through a putative alphaKAP and regulates GAPDH. 1579 70

Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o-phenyl-EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations.
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PMID:Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation. 1579 52

Disturbances in neuronal calcium homeostasis have been implicated in a variety of neuropathological conditions, including cerebral ischemia, hypoglycemia, and epilepsy, and possibly constitute part of the cell death process associated with chronic neurodegenerative disorders. We investigated if endoplasmic reticulum (ER) calcium stores participate in neuronal death triggered by moderate glycolysis inhibition induced by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, in cultured hippocampal neurons. Results show that exposure to iodoacetate leads to a slow partial decrease in cell survival, which is significantly prevented in the absence of Ca(2+) or in the presence of the calcium chelator BAPTA-AM. Treatment with caffeine and a low (1 microM) concentration of ryanodine, which activates the ryanodine receptor (RyR), exacerbates neuronal death, whereas dantrolene and 25 microM ryanodine, which antagonizes RyR, prevents damage. Xestospongin C (XeC), an antagonist of the inositol-3-phosphate (IP(3)) receptor (IP(3)R) also prevents neuronal damage. Inhibitors of the ER calcium ATPase (sarcoendoplasmic reticulum Ca(2+) ATPase; SERCA) have no effect. The decrease in ATP levels induced by iodoacetate is potentiated by caffeine and prevented by dantrolene. Although only a slight increase in glutamate extracellular levels is observed 3.5 hr after iodoacetate exposure, the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, MK-801, efficiently prevents neuronal damage. Taken together, the data suggest that neuronal death induced during moderate glycolysis inhibition involves calcium influx through NMDA receptors and calcium release from intracellular ER stores. These results might be relevant to the understanding the mechanisms involved in neuronal damage related to aging and chronic neurodegenerative diseases, which have been associated with decreased glucose metabolism.
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PMID:Disruption of endoplasmic reticulum calcium stores is involved in neuronal death induced by glycolysis inhibition in cultured hippocampal neurons. 1617 70

Programmed cell death, also called apoptosis, participates not only in normal physiologic processes such as development of the immune system, but also in many diseases. A loss of normal cell death may occur in cancer, and excessive cell death is found in a variety of neurodegenerative conditions. We describe 3 distinct pathways that regulate cell death. First, bilirubin, often thought to be a toxic end product of heme metabolism, serves as a physiologic cytoprotectant that may attenuate multiple forms of morbidity. In a second pathway, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mediates a novel cell death cascade. Cytotoxic stimuli, via nitric oxide generation, lead to the binding of GAPDH to the protein Siah1, translocation of GAPDH-Siah1 to the nucleus, and ultimately cell death. Third, cytochrome c, released from mitochondria early in apoptosis, synergizes with inositol-1,4,5-triphosphate (IP3) to elicit massive cellular calcium release, resulting in cell death. These pathways may regulate cell survival in a variety of pathologic states and represent fertile targets for novel therapies.
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PMID:Messenger molecules and cell death: therapeutic implications. 1639 Dec 20

It is known that the extracellular matrix (ECM) is able to signal to cells and thereby direct or modulate the transcription of certain mRNAs. This signaling plays an important role in tumor invasion and metastasis, wound healing, remodeling of the ECM and cell differentiation. There are several mechanisms whereby the ECM signals cells to change their metabolism: (1) receptor molecules binding to specific domains in the ECM, (2) direct phagocytosis of the ECM molecules or domains into the cell, (3) structural changes of the ECM domains. We report the effect of an ECM containing either mutant or normal Fbn1 on the transcription levels of several collagen mRNAs. Tsk/Tsk, Tsk/+ and +/+ mouse embryonic fibroblast cell lines were used. Tsk/Tsk cells produce only mutated fibrillin-1 which arises from mRNA containing an in-frame duplication of exons 17-40. To test the effect of the ECM containing mutant Fbn1, cells of the Tsk/Tsk, Tsk/+ and the wild-type (+/+) genotype were each grown on an ECM produced by either Tsk/Tsk, Tsk/+ cells or by wild-type cells (+/+). The embryonic cells were genotyped by Northern analyses for Fbn1 and grown to confluence. The cultures were then harvested and the cells removed, leaving the matrix in the flasks. Matrices produced from Tsk/Tsk, Tsk/+ and from +/+ cells were reseeded with Tsk/Tsk cells, Tsk/+ cells or +/+ cells. The cells were plated at a confluent concentration and incubated on the matrices for 48 h, after which total RNA was harvested and cDNA generated. Real-time PCR using cDNA or Northern analyses using RNA were performed for Fbn1 and Types I, III and V collagens. The PCR and Northern results were normalized using beta-actin and GAPDH, respectively. The Northern analyses showed that the steady state levels of mRNA for Col1a1 were depressed in both Tsk/Tsk and +/+ cells when grown on the matrix produced by Tsk/Tsk cells. Real-time PCR was then performed with primers specific for Col1a2, Col3a1, Col5a1 and Col5a2. The results showed that cells with the Tsk/Tsk, Tsk/+, and +/+ genotype all had lower steady-state levels of the above 4 collagen mRNAs when grown on the matrix produced by homozygous Tsk/Tsk cells or the matrix produced by heterozygous Tsk/+ cells compared with those grown on a matrix produced by +/+ cells. We hypothesize that the mutated Fbn1 molecules with many additional EGF-calcium binding regions and TGF-beta binding domains may (1) change the homeostasis of the ECM by binding additional growth factors and/or (2) present a radically different ECM 3-dimensional architecture. Either or both of these changes could signal the cell to produce less collagen.
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PMID:Extracellular matrix containing mutated fibrillin-1 (Fbn1) down regulates Col1a1, Col1a2, Col3a1, Col5a1, and Col5a2 mRNA levels in Tsk/+ and Tsk/Tsk embryonic fibroblasts. 1658 19

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation have been the subject of much study. Using an in vitro experimental system we have identified mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation, and how these responses differ in cells isolated from diseased cartilage. We have previously shown that mechanical stimulation of normal articular chondrocytes leads to a cell membrane hyperpolarisation. Within 1 hour following mechanical stimulation there is an increase in aggrecan mRNA levels. These responses are mediated via alpha5beta1 integrins, the neuropeptides substance P and NMDA, and the cytokine interleukin-4. In OA chondrocytes mechanical stimulation leads to cell membrane depolarisation, but no change in aggrecan mRNA at 1 hour. The depolarisation response is mediated via alpha5beta1 integrins, substance P and interleukin-4, but the cells show an altered response to NMDA. Having identified that the NMDA receptor is present in human articular cartilage and may play an important role in a chondroprotective mechanotransduction pathway, we were interested in whether other components associated with NMDA signalling may be involved in the chondrocyte mechanotransduction pathways. One such component is calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII mediates many cellular responses to elevated Ca2+ in a wide variety of cells and tissues. It is involved in the regulation of ion channels, cytoskeletal dynamics, gene transcription, neurotransmitter synthesis, insulin secretion, and cell division. CaMKII also shows a broad substrate specificity and is abundant in brain tissue, indicating that this kinase may play a number of roles in the functioning of the central nervous system. This kinase has been studied extensively in brain, but there is only a limited understanding of CaMKII in other tissues. CAMKII has four subunit isoforms (alpha,beta,gamma,delta). The alpha- and beta-isoforms have narrow distributions restricted mainly to neuronal tissues, but the gamma- and delta-isoforms are ubiquitously expressed within neuronal and non-neuronal tissues. The aim of this study was to investigate the expression of CaMKII in normal and OA cartilage and chondrocytes, and whether this enzyme is involved in the response of chondrocytes to cyclical mechanical stimuli. Reverse transcriptase-polymerase chain reaction (RT-PCR), using primers specific for the different CaMKII isoforms, was carried out to assess which isoforms are expressed in human articular chondrocytes. To assess whether CaMKII is expressed in human articular chondrocytes at the protein level, cultured chondrocytes were extracted and analysed by Western blotting using a pan-CaMKII antibody. Immunohistochemistry was carried out to investigate whether CaMKII is expressed by human articular chondrocytes in vivo. Frozen sections of normal, OA and ankle cartilage were incubated for one hour with CaMKII antibody and visualised using ABC and DAB. To assess the role of CaMKII in the mechanotransduction responses of normal and OA chondrocytes, human normal and OA articular chondrocytes were mechanically stimulated at 0.33 Hz, or by addition of recombinant IL-4 for 20 minutes. Cell responses to these stimuli, in the absence or presence of an inhibitor of CaMKII were assessed by measuring changes in cell membrane potential or changes in relative levels of aggrecan mRNA compared with the housekeeping gene GAPDH. Normal, OA, and ankle chondrocytes expressed the gamma and delta isoforms of CaMKII mRNA, but not the alpha and beta isoforms as demonstrated by RT-PCR. Western blotting showed a band at approximately 60 kDa consistent with the expression of CaMKII. Immunohistochemistry revealed the positive staining in the middle and deep zones, but not the superficial zone, of normal, OA, and ankle cartilage. The presence of a CaMKII inhibitor inhibits the membrane hyperpolarisation response and upregulation of aggrecan mRNA in normal chondrocytes following mechanical stimulation, but has no effect on the hyperpolarisation response to recombinant IL4. The depolarisation response of OA chondrocytes to mechanical stimulation is unaffected by the presence of the CaMKII inhibitor. The CaMKII isoforms gamma and delta are expressed in both normal and OA chondrocytes, both in vitro and in vivo, but are only involved in the response of normal chondrocytes to mechanical stimulation. This response is upstream of the effect of IL4. These findings are consistent with previous findings for the NMDA receptor, and suggest that dysregulation of NMDA-CaMKII signalling may be important in onset and progression of osteoarthritis.
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PMID:Calcium/calmodulin-dependent protein kinase II in human articular chondrocytes. 1691 96


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