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)

Atypical protein kinase C iota/lambda (PKCiota/lambda) is essential for protein transport in the early secretory pathway. The small GTPase Rab2 selectively recruits the kinase to vesicular tubular clusters (VTCs) where PKCiota/lambda phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). VTCs are composed of small vesicles and tubules and serve as transport intermediates that shuttle cargo from the endoplasmic reticulum to the Golgi complex. These structures are the first site of segregation of the anterograde and retrograde pathways. When Rab2 binds to a VTC subcompartment, the subsequent recruitment of PKCiota/lambda and soluble components, including COPI (coatomer and ADP-ribosylation factor), results in the release of retrograde-directed vesicles. Because Rab2 stimulates PKCiota/lambda membrane association in a dose-dependent manner, we investigated whether the two proteins physically interact. Using a combination of in vivo and in vitro assays, we found that Rab2 interacts directly with PKCiota/lambda and that this interaction occurs through the Rab2 amino terminus (residues 1-19) and the PKCiota/lambda regulatory domain. A mutant lacking the PKCiota/lambda binding domain (Rab2N'Delta19) was functionally characterized. In contrast to Rab2, Rab2N'Delta19 failed to recruit PKCiota/lambda to normal rat kidney microsomes in a quantitative binding assay. To determine whether Rab2 modulates the ability of PKCiota/lambda to phosphorylate GAPDH, an in vitro kinase assay was supplemented with Rab2 or Rab2N'Delta19. Rab2 inhibited PKCiota/lambda-dependent GAPDH phosphorylation, whereas no effect was observed when the assay was performed with the aminoterminal truncation mutant. These results suggest that a downstream effector recruited to the VTC stimulates PKCiota/lambda-mediated GAPDH phosphorylation by alleviating the inhibition imposed by Rab2-PKCiota/lambda interaction.
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PMID:Rab2 interacts directly with atypical protein kinase C (aPKC) iota/lambda and inhibits aPKCiota/lambda-dependent glyceraldehyde-3-phosphate dehydrogenase phosphorylation. 1457 Aug 76

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.
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PMID:Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity. 1548 21

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

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.
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PMID:Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells. 1597 15

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

The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and aPKCiota/lambda and GAPDH interact. Based on the reports demonstrating aPKCiota-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCiota/lambda and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCiota/lambda on the VTC. To determine the consequence of aPKCiota/lambda tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCiota/lambda. Since Rab2 requires aPKCiota/lambda for the downstream recruitment of beta-coat protein (beta-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of beta-COP. Like aPKCiota/lambda, the membranes contained a negligible amount of beta-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCiota/lambda facilitates aPKCiota/lambda association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of beta-COP and release of Rab2-mediated retrograde-directed vesicles.
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PMID:Src-dependent aprotein kinase C iota/lambda (aPKCiota/lambda) tyrosine phosphorylation is required for aPKCiota/lambda association with Rab2 and glyceraldehyde-3-phosphate dehydrogenase on pre-golgi intermediates. 1645 74

The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway. Rab2 associates with vesicular tubular clusters (VTCs) located between the endoplasmic reticulum (ER) and the Golgi complex. VTCs function as transport intermediates and sort anterograde-directed cargo from recycling proteins. Rab2 selectively recruits atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate (GAPDH) to VTCs where aPKCiota/lambda phosphorylates GAPDH. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and this interaction ultimately results in COPI recruitment and the release of retrograde-directed vesicles. This chapter describes a protocol to purify recombinant Rab2 from Rab2 cDNA transformed bacteria and methods to assess recombinant Rab2 biological activity. Additionally, in vivo and in vitro assays are outlined that are employed to demonstrate Rab2 interaction with the downstream effectors aPKCiota/lambda and GAPDH.
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PMID:Rab2 purification and interaction with protein kinase C iota/lambda and glyceraldehyde-3-phosphate dehydrogenase. 1647 4

The methylotrophic yeast Ogataea minuta IFO 10746 was selected as a suitable strain for producing human-compatible glycoproteins by means of analyses of its cell-wall mannoproteins. First, the OmURA3 gene encoding an orotidine-5'-phosphate decarboxylase was cloned and disrupted to generate a host strain with a uracil auxotrophic marker. Second, both the promoters and the terminators from the OmAOX1 gene encoding an alcohol oxidase for an inducible promoter, or those from the OmTDH1 gene encoding a glyceraldehyde-3-phosphate dehydrogenase for a constitutive promoter, were isolated to construct an expression vector system for heterologous genes. Next, the OmOCH1 gene encoding a starting enzyme with alpha-1,6-mannosyltransferase activity to form a backbone of the N-linked outer sugar chain peculiar to yeast was disrupted, and an alpha-1,2-mannosidase gene from Aspergillus saitoi with an endoplasmic reticulum retention signal (HDEL) under the control of the OmAOX1 promoter was introduced to convert the sugar chain to Man5GlcNAc2 in O. minuta. As a result, we succeeded in breeding a new methylotrophic yeast, O. minuta, producing a Man5GlcNAc2-high-mannose-type sugar chain as a prototype of a human-compatible sugar chain. We also elucidate here the usefulness of the strategy for producing human-compatible sugar chains in yeast.
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PMID:Production of Man5GlcNAc2-type sugar chain by the methylotrophic yeast Ogataea minuta. 1704 55

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset Motor Neuron Disease (MND), characterized by motor neurons death in the cortex, brainstem and spinal cord. Ten loci linked to Familial ALS have been mapped. ALS8 is caused by a substitution of a proline by a serine in the Vesicle-Associated Membrane Protein-Associated protein-B/C (VAP-B/C). VAP-B belongs to a highly conserved family of proteins implicated in Endoplasmic Reticulum-Golgi and intra-Golgi transport and microtubules stabilization. Previous studies demonstrated that the P56S mutation disrupts the subcellular localization of VAP-B and that this position would be essential for Unfolded Protein Response (UPR) induced by VAP-B. In the present work we expressed and purified recombinant wild-type and P56S mutant VAP-B-MSP domain for the analysis of its interactions with other cellular proteins. Our findings suggest that the P56S mutation may lead to a less stable interaction of this endoplasmic reticulum protein with at least two other proteins: tubulin and GAPDH. These two proteins have been previously related to other forms of neurodegenerative diseases and are potential key points to understand ALS8 pathogenesis and other forms of MND. Understanding the role of these protein interactions may help the treatment of this devastating disease in the future.
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PMID:A mutation in human VAP-B--MSP domain, present in ALS patients, affects the interaction with other cellular proteins. 1754 May 79

In bladder outlet obstruction (BOO), mechanical stress and ischemia/hypoxia are implicated in structural and functional alterations of the urinary bladder. Because mechanical stress and hypoxia may trigger endoplasmic reticulum (ER) stress, we examined involvement of ER stress in the damage of the bladder caused by BOO. An experimental model of BOO was established in rats by complete ligature of the urethra for 24 h, and bladders were subjected to northern blot analysis and assessment of apoptosis. Isolated urinary bladders and bladder-derived smooth muscle cells (BSMCs) were also exposed to mechanical strain and hypoxia and used for analyses. To examine involvement of ER stress in the damage of the bladder, the effects of a chemical chaperone 4-phenylbutyrate (4-PBA) were evaluated in vitro and in vivo. Outlet obstruction for 24 h induced expression of ER stress markers, GRP78 and CCAAT/enhancer-binding protein-homologous protein (CHOP), in the bladder. It was associated with induction of markers for mechanical stress (cyclooxygenases 2) and hypoxia (vascular endothelial growth factor and glyceraldehyde-3-phosphate dehydrogenase). When isolated bladders and BSMCs were subjected to mechanical strain, induction of GRP78 and CHOP was not observed. In contrast, when BSMCs were exposed to hypoxic stress caused by CoCl2 or thenoyltrifluoroacetone (TTFA), substantial upregulation of GRP78 and CHOP was observed, suggesting involvement of hypoxia in the induction of ER stress. In the bladder subjected to BOO, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells increased in the epithelial cells and BSMCs. Similarly, treatment with TTFA or CoCl2 induced apoptosis of BSMCs, and 4-PBA significantly attenuated ER stress and apoptosis triggered by these agents. Furthermore, in vivo administration with 4-PBA significantly reduced apoptosis in the bladder subjected to BOO. These results suggested that outlet obstruction caused ER stress via hypoxic stress in the bladder and that hypoxia-triggered ER stress may be involved in the induction of apoptosis in BOO.
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PMID:Involvement of hypoxia-triggered endoplasmic reticulum stress in outlet obstruction-induced apoptosis in the urinary bladder. 1834 81


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