Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Partial inhibition of Na/K-ATPase by ouabain causes hypertrophic growth and regulates several early and late response genes, including that of Na/K-ATPase alpha3 subunit, in cultured neonatal rat cardiac myocytes. The aim of this work was to determine whether ouabain and other hypertrophic stimuli affect Na/K-ATPase beta1 subunit gene expression. When myocytes were exposed to non-toxic concentrations of ouabain, ouabain increased beta1 subunit mRNA in a dose- and time-dependent manner. Like the alpha3 gene, beta1 mRNA was also regulated by several other well-known hypertrophic stimuli including phenylephrine, a phorbol ester, endothelin-1, and insulin-like growth factor, suggesting involvement of growth signals in regulation of beta1 expression. Ouabain failed to increase beta1 subunit mRNA in the presence of actinomycin D. Using a luciferase reporter gene that is directed by the 5'-flanking region of the beta1 subunit gene, transient transfection assay showed that ouabain augmented the expression of luciferase. These data support the proposition that ouabain regulates the beta1 subunit through a transcriptional mechanism. The effect of ouabain on beta1 subunit induction, like that on alpha3 repression, was dependent on extracellular Ca2+ and on calmodulin. Inhibitions of PKC, Ras, and MEK, however, had different quantitive effects on ouabain-induced regulations of beta1 and alpha3 subunits. The findings show that partial inhibition of Na/K-ATPase activates multiple signaling pathways that regulate growth-related genes, including those of two subunit isoforms of Na/K-ATPase, in a gene-specific manner.
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PMID:Regulation of Na/K-ATPase beta1-subunit gene expression by ouabain and other hypertrophic stimuli in neonatal rat cardiac myocytes. 1120 57

We have previously identified five Na, K-ATPase beta1-mRNA species that are expressed in the rat heart, kidney, and brain. These mRNAs which are unequal in their abundance have an identical coding region but differ in the length of their 5'- and 3'-untranslated regions (UTRs). In this study we examined the possibility that the beta1-mRNA species exhibit differential translational efficiencies. We constructed expression plasmids encoding each of the five mRNAs and transcribed and translated them in vitro. Using rabbit reticulocyte system we determined the translation of the different mRNAs under conditions optimized for each beta1-cRNA and under an equivalent (standard) condition. The longest beta1-cRNA species (initiating at the first transcription start site and ending at the last [fifth] poly(A) site) exhibited the lowest relative translational efficiency averaging 0.2 +/- 0.05 units/mol of cRNA compared to the shortest beta1-cRNA species initiating at the first transcription start site and ending at the first poly(A) signal (with an assigned relative value of 1.0). These results suggested that the different translation rates of beta1-mRNAs may be due to their 3'-UTRs. To further define the role of beta1-3'-UTR, chimeric luciferase constructs containing different segments of the beta1-3'-UTR were transiently transfected into Clone 9 cells. Compared to the chimeric construct containing the shortest beta1-3'-UTR segment (ending at the first poly(A) site), the construct containing the full-length beta1-3'-UTR exhibited a luciferase expression of 0.23 +/- 0.04. To control for potential changes in the abundance of the expressed chimeric mRNAs which may lead to differences in luciferase expression, luciferase activity was normalized against chimeric luciferase-mRNA content measured in mixtures of cells stably transfected with the above constructs. The ratio of luciferase activity/chimeric luciferase-mRNA content in cells expressing the construct containing the entire beta1-3'-UTR region was 0.17 that in cells expressing chimeric luciferase mRNA containing beta1-3'-UTR up to the first poly(A) signal (P < 0.05). We conclude that the translational efficiency of the different beta1-mRNA species is negatively regulated by the 3'-UTR of the mRNA and that a regulating region appears to be localized between the second and fifth poly(A) signals of beta1-mRNA.
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PMID:Different Na, K-ATPase mRNA(beta1) species exhibit unique translational efficiencies. 1136 18

Hsp70s assist the folding of proteins in an ATP-dependent manner. DnaK, the Hsp70 of Escherichia coli, acts in concert with its co-chaperones DnaJ and GrpE. Amino acid substitutions (D388R and L391S/L392G) in the linker region between the ATPase and substrate-binding domain did not affect the functional domain coupling and oligomerization of DnaK. The intrinsic ATPase activity was enhanced up to 10-fold. However, the ATPase activity of DnaK L391S/L392G, if stimulated by DnaJ plus protein substrate, was five times lower than that of wild-type DnaK and DnaK D388R. This defect correlated with the complete loss of chaperone action in luciferase refolding. Apparently, the conserved leucine residues in the linker mediate the synergistic effects of DnaJ and protein substrate on ATPase activity, a function which might be essential for chaperone action.
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PMID:Mutations in the interdomain linker region of DnaK abolish the chaperone action of the DnaK/DnaJ/GrpE system. 1137 62

We have identified a cDNA encoding the mouse homologue of the yeast V-ATPase 21-kDa subunit c" (Vma16p). The encoded protein contains 205 amino acid residues with five putative membrane spanning segments and shows 48% identity and 64% similarity to the yeast protein. Despite this homology, however, the mouse cDNA does not complement the phenotype of a yeast strain in which the VMA16 gene has been disrupted. Northern blot analysis demonstrated that the 21-kDa subunit is expressed in most tissues examined and showed an expression pattern almost identical to that of the 16-kDa proteolipid subunit (subunit c). The presence of multiple mRNA species suggests the existence of alternatively spliced forms of the 21-kDa subunit which, from Southern blot analysis, are derived from a single gene. Promoter analysis using the luciferase reporter gene revealed that a region 186 bases upstream of the initiation site is sufficient to show a low level of transcriptional activity but that transcription is significantly enhanced by inclusion of the region -186 to -706. The 21-kDa protein was Myc-tagged and the 16-kDa protein was HA-tagged and the tagged proteins were co-expressed in COS-1 cells in order to study their intracellular localization by immunofluorescence microscopy. Both proteins showed significant punctate and perinuclear staining and were predominantly co-localized throughout the cell, consistent with their presence in the same V(0) complexes. Selective permeabilization of cells with digitonin (to permeabilize the plasma membrane) or Triton X-100 (to permeabilize both intracellular and plasma membranes) followed by immunofluorescence microscopy revealed that the carboxyl terminus of the 21-kDa subunit is exposed on the cytoplasmic side of the membrane whereas the carboxyl terminus of the 16-kDa subunit is located on the lumenal side of the membrane.
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PMID:Expression and localization of the mouse homologue of the yeast V-ATPase 21-kDa Subunit c" (Vma16p). 1144 Oct 17

The cytosol of mammalian cells contains several Hsp70 chaperones and an arsenal of cochaperones, including the anti-apoptotic Bag-1M protein, which regulate the activities of Hsp70s by controlling their ATPase cycles. To elucidate the regulatory function of Bag-1M, we determined its influence on nucleotide exchange, substrate release, ATPase rate, and chaperone activity of the housekeeping Hsc70 and stress-inducible Hsp70 homologs of humans. Bag-1M and a C-terminal fragment of it are potent nucleotide exchange factors as they stimulated the ADP dissociation rate of Hsc70 and Hsp70 up to 900-fold. The N-terminal domain of Bag-1M decreased the affinity of Bag-1M for Hsc70/Hsp70 by 4-fold, indicating a modulating role of the N terminus in Bag-1M action as nucleotide exchange factor. Bag-1M inhibited Hsc70/Hsp70-dependent refolding of luciferase in the absence of P(i). Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of P(i), Bag-1M activates the chaperone action of Hsc70/Hsp70 in luciferase refolding. Bag-1M accelerated ATP-triggered substrate release by Hsc70/Hsp70. We propose that Bag-1M acts as substrate discharging factor for Hsc70 and Hsp70.
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PMID:Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor. 1144 Oct 21

In the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion-like, self-propagating conformation giving rise to the non-Mendelian [PSI+] determinant. The replication of [PSI+] prion seeds can be readily blocked by growth in the presence of low concentrations of guanidine hydrochloride (GdnHCl), leading to the generation of prion-free [psi-] cells. Here, we provide evidence that GdnHCl blocks seed replication in vivo by inactivation of the molecular chaperone Hsp104. Although growth in the presence of GdnHCl causes a modest increase in HSP104 expression (20-90%), this is not sufficient to explain prion curing. Rather, we show that GdnHCl inhibits two different Hsp104-dependent cellular processes, namely the acquisition of thermotolerance and the refolding of thermally denatured luciferase. The inhibitory effects of GdnHCl protein refolding are partially suppressed by elevating the endogenous cellular levels of Hsp104 using a constitutive promoter. The kinetics of GdnHCl-induced [PSI+] curing could be mimicked by co-expression of an ATPase-negative dominant HSP104 mutant in an otherwise wild-type [PSI+] strain. We suggest that GdnHCl inactivates the ATPase activity of Hsp104, leading to a block in the replication of [PSI+] seeds.
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PMID:The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation. 1144 34

Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.
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PMID:Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization. 1160 58

A -1027 bp to + 108 bp region of Na-K-ATPase alpha3 gene promoter has been searched for the presence of thyroid response elements (TRE). Computer analysis of this sequence using a consensus TRE sequence revealed the presence of four putative TRE rich regions referred to as regions I (-636 to -457 bp), II (-218 to -106 bp), III (-106 to -6 bp) and IV (-6 to +108 bp). Cotransfection of the luciferase linked full length construct as well as constructs progressively devoid of the TRE rich regions in Cos1 cells revealed that regions I and III are positively regulated by T3 whereas there are some sequences in region II which can suppress the positive regulatory effect of region III but not of region I. TRE IV seems to have no functional role. EMSA of the three functional TRE rich regions (I, II and III) showed strong and specific interaction with thyroid hormone receptor (TR) cloned and expressed in baculovirus. The overall results suggest the regulation of Na-K-ATPase alpha3 gene by T3 is complex involving several thyroidal regulatory elements.
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PMID:Identification of thyroid regulatory elements in the Na-K-ATPase alpha3 gene promoter. 1171 May 59

Extracellular signal-regulated protein kinases (ERKs) are important in many cellular functions. We and others have previously reported that prolonged exposure of gastric parietal cells to epidermal growth factor (EGF) enhanced gastric acid secretion stimulated by secretagogues via ERKs. In this study, we examined whether ERKs regulated H(+),K(+)-ATPase alpha-subunit gene expression using a gastric cancer cell line, AGS. EGF induced ERK activity time- and dose-dependently with a maximal effect observed at 10 min and 10 nM, respectively. The MEK inhibitors, U0126 and PD-98059, dose-dependently inhibited the ERK activity stimulated by EGF. To test H(+),K(+)-ATPase alpha-subunit gene expression, we transfected AGS cells with a plasmid containing a canine H(+),K(+)-ATPase alpha-subunit gene promoter fused to a luciferase reporter gene. EGF induced luciferase activity in transfected cells; this effect was inhibited by the MEK inhibitors, suggesting that EGF-induced gene expression involved the ERK pathway. When AGS cells were transfected with the reporter plasmids in conjunction with an expression vector encoding constitutively active MEK1, luciferase activity was strongly enhanced; this effect was attenuated by the MEK inhibitors or by an additional cotransfection of dominant negative MEK1. Taken together, our results led us to conclude that the ERK pathway may mediate H(+),K(+)-ATPase alpha-subunit gene expression, contributing to gastric acid secretion in parietal cells.
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PMID:Extracellular signal-regulated protein kinases mediate H(+),K(+)-ATPase alpha-subunit gene expression. 1181 3

The ability of two high-affinity Hsc70-binding peptides [FYQLALT (peptide-Phi) and NIVRKKK (peptide-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both peptide-Phi and peptide-K inhibited chaperone-dependent renaturation of luciferase in RRL. Peptide-Phi, but not peptide-K, blocked Hsp90/Hsc70-dependent transformation of the heme-regulated eIF2 alpha kinase (HRI) into an active, heme-regulatable kinase. In contrast, peptide-K, but not peptide-Phi, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. Furthermore, HDJ2 (Human DnaJ homologue 2), but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Mechanistically, peptide-K inhibited, while peptide-Phi enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. The data presented support the hypotheses that peptide-Phi acts to inhibit Hsc70 function by binding to the hydrophobic peptide-binding cleft of Hsc70, while peptide-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. Overall, the data indicate that peptide-Phi and peptide-K have differential effects on Hsc70 functions under quasi-physiological conditions in RRL, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.
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PMID:Differential inhibition of Hsc70 activities by two Hsc70-binding peptides. 1188 92


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