Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hamster gene encoding the 78-kDa glucose-regulated protein (Grp78) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. After induction with isopropyl beta-D-thiogalactopyranoside, the recombinant Grp78 was purified to homogeneity by affinity column chromatography of the fusion protein followed by thrombin cleavage. The purified recombinant protein was compared with liver Grp78 for its ability to interact with ATP. Like liver Grp78, the recombinant protein contained a weak ATPase activity and a Ca(2+)-stimulated autophosphorylation activity. However, unlike liver Grp78, in which the autophosphorylation reaction is stimulated less than 50% by CaCl2, the reaction with the recombinant Grp78 was stimulated about 15-fold in the presence of Ca2+. Although the liver protein showed at least four isoforms after two-dimensional gel electrophoresis, the recombinant Grp78 had one major species corresponding to the most basic form seen in liver. Both the liver Grp78 and the recombinant protein existed primarily as monomers and dimers. A small amount of oligomers was also present in the liver Grp78. When either protein was incubated with ATP, there was a conversion of the higher molecular weight species to the monomeric form.
 ...
PMID:Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation. 153 51

The calcium ionophore A23187 has been shown to induce the expression of a set of glucose-regulated protein (GRP) genes through the depletion of Ca2+ from the intracellular Ca2+ stores. Here we demonstrate that thapsigargin, which inhibits specifically the endoplasmic reticulum Ca(2+)-ATPase and causes a discharge of the intracellular Ca2+ store, is able to induce the transcription of two grp genes (grp78/BiP and grp94) with kinetics and magnitude similar to that of A23187. The induction of the grp genes by both reagents requires several hours of sustained treatment, in contrast to the rapid induction previously described for c-jun and c-fos. The transactivation of the rat grp78 promoter by A23187 is mediated through sequences spanning -154 to -130 and -99 to -90. Further, simultaneous mutation of two 10-base pair regions, spanning -139 to -130 and -99 to -90, severely reduced the A23187 response. The induction by thapsigargin is also partially mediated through these same promoter elements, without the involvement of the TRE and CRE-like elements of the grp78 promoter. The Ca2+ response elements are further defined by their ability to confer Ca2+ stress inducibility to a heterologous promoter. We show that subdomains of the grp78 promoter are capable of conferring the Ca2+ stress response. In particular, two copies of a 50-base pair region spanning -159 to -110, when cloned in either orientation, can confer a 5- and 9-fold induction by A23187 and thapsigargin, respectively. Our results lend support to the hypothesis that the induction of grp78 by A23187 and thapsigargin following ER Ca2+ discharge acts through a novel pathway in which a Ca2+ signal is transduced through redundant elements containing CCAAT box-like motifs flanked by GC-rich regions.
 ...
PMID:Transactivation of the grp78 promoter by Ca2+ depletion. A comparative analysis with A23187 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. 850 25

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.
 ...
PMID:Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65. 851 33

We have cloned a cDNA encoding the human 150 kDa oxygen-regulated protein (ORP150) from hypoxia-treated astrocytoma U373 cDNA library. The deduced amino acid sequence of 999 residues contains a signal peptide and an ER retention-like signal at the N- and C-termini, respectively. It has a striking sequence similarity (91% identity) with Chinese hamster 170 kDa glucose-regulated protein (GRP170). The N-terminal half of ORP150 exhibits significant similarity to the ATPase domain of HSP70 family proteins with well-conserved ATP binding motifs. Northern blot analysis revealed that induction of ORP150 in U373 cells was not limited to hypoxia but also observed by 2-deoxyglucose or tunicamycin treatment. Furthermore, tissue specificity of expression of ORP150 was quite similar to that of GRP78. These findings suggest that ORP150 participates in quality control of proteins in the ER in response to diverse environmental stresses.
 ...
PMID:Cloning and expression of cDNA encoding the human 150 kDa oxygen-regulated protein, ORP150. 902 69

High hydrostatic pressure (HP) has recently been shown to increase cellular heat shock protein 70 (Hsp70) level in a specific way that does not involve transcriptional activation of the gene, but rather the stabilisation of the mRNA for Hsp70. In this study, we investigated whether there are other observable changes caused by HP stress, and compared them with those induced by certain other forms of stressors. A chondrocytic cell line T/C28a4 was exposed to 30 MPa continuous HP, heat shock at 43 degrees C, and increased cytosolic calcium concentration by the addition of sarco-endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin (25 nM) or calcium ionophore A23187 (1 microM) in the cultures. The protein synthesis was studied by in vitro metabolic labelling followed by one- and two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry was utilized to confirm the identity of the protein spots on two-dimensional gels. Continuous 30 MPa HP increased remarkably the relative labelling of Hsp70. Labelling of Hsp90 was also increased by 15-20%, although no clear change was evident at the protein level in Western blots. Elevated intracellular Ca(2+) concentration induced by thapsigargin and calcium ionophore A23187 increased mainly the synthesis of glucose-regulated protein 78 (Grp78/BiP), whereas Hsp70 and Hsp90 were decreased by the treatment. Heat shock was the strongest inducer of Hsp70 and Hsp90. This study further confirmed the induction of Hsp70 in chondrocytic cells exposed to high HP, but it also showed that calcium-mediated responses are unlikely to cause the stress response observed in the hydrostatically pressurized cells.
 ...
PMID:Differential regulation of stress proteins by high hydrostatic pressure, heat shock, and unbalanced calcium homeostasis in chondrocytic cells. 1099 52

Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer. We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF complex. We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA. Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling. Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1. The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair.
 ...
PMID:Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex. 1172 52

Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominant-negative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.
 ...
PMID:Uncoating ATPase Hsc70 is recruited by invariant chain and controls the size of endocytic compartments. 1181 72

Metallothionein (MT) is a small sulfhydryl-rich protein whose levels are elevated by various inducers of organelle stresses, such as nuclear stress (cisplatin), mitochondrial stress (antimycin A, 2,4-dinitrophenol) and lysosomal stress (paraquat). Although abnormal folding of protein in the endoplasmic reticulum (ER) causes ER stress, induction of MT synthesis by ER stress has never been investigated. In this study, we examined the induction of MT by an inducer of ER stress, tunicamycin (Tun), which induces ER stress by inhibiting N-linked glycosylation of protein in the ER. Administration of Tun (0.5-1.5 mg/kg, sc) increased hepatic MT levels in C57BL/6J mice (3.1-fold). The maximal increase in hepatic MT was observed 48-96 h after the administration of Tun (1.0 mg/kg). Expressions of MT-I, II and glucose-regulated protein 78 (Bip/GRP78), which is a molecular chaperone induced by ER stress, mRNA were also detected by administration of Tun. Thapsigargin (Thap), a generator of ER stress by inhibiting ER Ca(2+)-ATPase, also increased both hepatic MT levels and expression of MT-I and -II mRNA. The level of expression of Bip/GRP78 mRNA induced by Tun administration in MT-null mice was greater than that in wild-type mice. Taken together, these findings suggest that inhibitors of ER are potent inducers of MT.
 ...
PMID:Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice. 1501 97

Nitric oxide (NO)-induced neurotoxicities are involved in the pathogenesis of several neurodegenerative disorders featured by misfolded proteins. However, the details remain to be investigated. In the present work, we focus on the study of some endoplasmic reticulum-related events in S-nitrosoglutathione (GSNO)-induced neurotoxicity in cerebellar granule cells (CGCs) and we demonstrated that: (1) GSNO caused sustained elevation of intracellular calcium; (2) This calcium elevation resulted partially from the depletion of endoplasmic reticulum (ER) calcium stores; (3) There was ER stress which was indicated by the incomplete splicing of X-box binding protein (XBP-1) mRNA by 8-polysialyltransferase (Pst1); (4) GSNO upregulated the expression of the proapoptotic growth arrest and DNA damage-inducible gene (Gadd153) and caused the depletion of intracellular glutathione (GSH) pools. At the same time, GSNO downregulated the expression of the antiapoptotic gene Sarco/endoplasmic reticulum calcium-ATPase (SERCA2b) in parallel with the downregulation of the antiapoptotic ER chaperones-glucose-regulated protein genes (Grp78 and Grp94). These effects indicate that ER is one of the NO targets in GSNO-induced neurotoxicity in cerebellar granule cells besides mitochondria.
 ...
PMID:The endoplasmic reticulum-related events in S-nitrosoglutathione-induced neurotoxicity in cerebellar granule cells. 1522 63

Nitric oxide (NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen. This action of NO has both physiological and pathophysiological consequences. Here we report that endogenously generated NO, which disrupts the respiratory chain, may cause changes in mitochondrial calcium flux. This induces cleavage of the endoplasmic reticulum (ER) stress-regulated transcription factor p90 ATF6 into an active p50 form. Cleavage depends on a calcium-dependent serine protease through a regulated intramembrane proteolysis (RIP) process. p50 ATF6 then translocates to the nucleus to upregulate expression of the ER-resident molecular chaperone, glucose-regulated protein 78 (Grp78). The increase in Grp78 provides significant cytoprotection against toxic agents, including thapsigargin, a selective ER calcium-ATPase inhibitor. Cytoprotection is abolished after treatment with cyclosporin A (CsA), which disrupts mitochondrial calcium signalling, or with the calcium chelator BAPTA-AM. The NO-mediated ER stress response is diminished in rho(0) cells devoid of mitochondrial DNA, consistent with our evidence that NO-dependent mitochondrial disruption is coupled to the ER stress response.
 ...
PMID:Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response. 1550 20


1 2 3 4 Next >>