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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.
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PMID:Heat shock response of Archaeoglobus fulgidus. 1610 46

Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D(3) binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (K(d), approximately 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.
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PMID:Adenosine 5'-triphosphate-dependent vitamin D sterol binding to heat shock protein-70 chaperones. 1614 93

We have examined the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioreductive metabolism of 17-allylamino-demethoxygeldanamycin (17-AAG). High-performance liquid chromatography (HPLC) analysis of the metabolism of 17-AAG by recombinant human NQO1 revealed the formation of a more polar metabolite 17-AAGH2. The formation of 17-AAGH2 was NQO1 dependent, and its formation could be inhibited by the addition of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based (suicide) inhibitor of NQO1. The reduction of 17-AAG to the corresponding hydroquinone 17-AAGH2 was confirmed by tandem liquid chromatography-mass spectrometry. 17-AAGH2 was relatively stable and only slowly underwent autooxidation back to 17-AAG over a period of hours. To examine the role of NQO1 in 17-AAG metabolism in cells, we used an isogenic pair of human breast cancer cell lines differing only in NQO1 levels. MDA468 cells lack NQO1 due to a genetic polymorphism, and MDA468/NQ16 cells are a stably transfected clone that express high levels of NQO1 protein. HPLC analysis of 17-AAG metabolism using cell sonicates and intact cells showed that 17-AAGH2 was formed by MDA468/NQ16 cells, and formation of 17-AAGH2 could be inhibited by ES936. No 17-AAGH2 was detected in sonicates or intact MDA468 cells. Following a 4-hour treatment with 17-AAG, the MDA468/NQ16 cells were 12-fold more sensitive to growth inhibition compared with MDA468 cells. More importantly, the increased sensitivity of MDA468/NQ16 cells to 17-AAG could be abolished if the cells were pretreated with ES936. Cellular markers of heat shock protein (Hsp) 90 inhibition, Hsp70 induction, and Raf-1 degradation were measured by immunoblot analysis. Marked Hsp70 induction and Raf-1 degradation was observed in MDA468/NQ16 cells but not in MDA468 cells. Similarly, downstream Raf-1 signaling molecules mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase and ERK also showed decreased levels of phosphorylation in MDA468/NQ16 cells but not in MDA468 cells. The ability of 17-AAG and 17-AAGH2 to inhibit purified yeast and human Hsp90 ATPase activity was examined. Maximal 17-AAG-induced ATPase inhibition was observed in the presence of NQO1 and could be abrogated by ES936, showing that 17-AAGH2 was a more potent Hsp90 inhibitor compared with 17-AAG. Molecular modeling studies also showed that due to increased hydrogen bonding between the hydroquinone and the Hsp90 protein, 17-AAGH2 was bound more tightly to the ATP-binding site in both yeast and human Hsp90 models. In conclusion, these studies have shown that reduction of 17-AAG by NQO1 generates 17-AAGH2, a relatively stable hydroquinone that exhibits superior Hsp90 inhibition.
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PMID:Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition. 1626 26

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651bp in length, having a 5' untranslated region (UTR) of 96bp, a 3' UTR of 575bp, and an open reading frame (ORF) of 1980bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8h and lasted to 16h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop.
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PMID:The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress. 1653 Apr 26

Previously, we demonstrated gender differences in Na-K-ATPase (NKA) expression and function after renal ischemia-reperfusion (I/R) injury (Sex differences in the alterations of Na(+), K(+)-ATPase following ischemia-reperfusion injury in the rat kidney. J Physiol 555: 471-480, 2004). Postischemic membrane destruction causes inhibition of NKA, whereas heat shock protein (HSP) 72 helps to preserve it. We tested the sex differences in postischemic expression of HSP72 and colocalization with NKA. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 (T2), 16 (T16), and 24 h (T24) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls. Postischemic blood urea nitrogen (BUN), serum creatinine, and renal histology were analyzed. HSP72 mRNA expression was detected by RT-PCR, protein levels by Western blot analysis. Fluorescent immunohistochemistry was performed to evaluate the localization of HSP72 and NKA alpha(1)-subunit. Postischemic BUN and creatinine were higher, and renal histology showed more rapid progression in M vs. F (P < 0.05). HSP72 mRNA expression was higher in F vs. M in control and in all I/R groups (P < 0.05). Similar changes were observed in HSP72 protein levels (F vs. M, P < 0.05, control, T2, T16, T24, respectively). Immunohistochemical localization of HSP72 and NKA alpha(1) was similar in control F and M. In postischemic F kidneys, the majority of NKA alpha(1) and HSP72 was colocalized on the basolateral membrane of tubular cells, whereas in M prominent staining was observed in the cytosol and apical domain. This study indicates that in female kidneys the higher basal and postischemic levels of HSP72 and different colocalization with NKA might contribute to the gender differences in renal I/R injury.
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PMID:Sex differences in heat shock protein 72 expression and localization in rats following renal ischemia-reperfusion injury. 1660 51

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.
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PMID:Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. 1662 88

We have previously evaluated the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioreductive metabolism of 17-(allylamino)-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone, a more potent 90-kDa heat shock protein (Hsp90) inhibitor. Here, we report an extensive study with a series of benzoquinone ansamycins, which includes gel-danamycin, 17-(amino)-17-demethoxygeldanamycin, and 17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin. The reduction of these benzoquinone ansamycins by recombinant human NQO1 to the corresponding hydroquinone ansamycins was monitored by high-performance liquid chromatography (HPLC) and confirmed by liquid chromatography/mass spectrometry. Inhibition of purified yeast Hsp90 ATPase activity was augmented in the presence of NQO1 and abrogated by 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl-]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1, showing that the hydroquinone ansamycins were more potent Hsp90 inhibitors than their parent quinones. An isogenic pair of human breast cancer cell lines, MDA468 and MDA468/NQ16, differing in expression of NQO1, was used, and HPLC analysis showed that hydroquinone ansamycins were formed by the MDA468/NQ16 cells, which could be prevented by ES936 pretreatment. The MDA468/NQ16 cells were more sensitive to growth inhibition after treatment with the benzoquinone ansamycins compared with the MDA468 cells; this increased sensitivity could be reduced by ES936 pretreatment. The increased duration of benzoquinone ansamycin exposure showed increased potency and -fold inhibition in MDA468/NQ16 cells relative to the parental MDA468 cells. Computational-based molecular modeling studies displayed additional contacts between yeast Hsp90 and the hydroquinone ansamycins, which translated to greater interaction energies compared with the corresponding benzoquinone ansamycins. In conclusion, these studies show that the reduction of this series of benzoquinone ansamycins by NQO1 generates the corresponding hydroquinone ansamycins, which exhibit enhanced Hsp90 inhibition.
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PMID:The bioreduction of a series of benzoquinone ansamycins by NAD(P)H:quinone oxidoreductase 1 to more potent heat shock protein 90 inhibitors, the hydroquinone ansamycins. 1682 87

Striated muscle activity is always accompanied by oxidative stress (OxStress): the more intense muscle work and/or its duration, the more a redox imbalance may be attained. In spite of cardiac muscle functioning continuously, it is well known that the heart does not suffer from OxStress-induced damage over a broad physiological range. Although the expression of antioxidant enzymes may be of importance in defending heart muscle against OxStress, a series of combined antioxidant therapeutic approaches have proved to be mostly ineffective in avoiding cellular injury. Hence, additional mechanisms may be involved in heart cytoprotection other than antioxidant enzyme activities. The strong cardiotoxic effect of doxorubicin-induced cancer chemotherapy shed light on the possible role for multidrug resistance-associated proteins (MRP) in this context. Muscle activity-induced 'physiological' OxStress enhances the production of glutathione disulfide (GSSG) thus increasing the ratio of GSSG to glutathione (GSH) content inside the cells, which, in turn, leads to redox imbalance. Since MRP1 gene product (a GS-X pump ATPase) is a physiological GSSG transporter, adult Wistar rats were tested for MRP1 expression and activity in the heart and skeletal muscle (gastrocnemius), in as much as the latter is known to be extremely sensitive to muscle activity-induced OxS. MRP1 expression was completely absent in skeletal muscle. In contrast, the heart showed an exercise training-dependent induction of MRP1 protein expression which was further augmented (2.4-fold) as trained rats were challenged with a session of acute exercise. On the other hand, inducible expression of the 70-kDa heat shock protein (HSP70), a universal marker of cellular stress, was completely absent in the heart of sedentary and acutely exercised rats, whereas skeletal muscle showed a conspicuous exercise-dependent HSP70 expression, which decreased by 45% with exercise training. This effect was paralleled by a 58% decrease in GSH content in skeletal muscle which was even higher (an 80%-fall) after training thus leading to a marked redox imbalance ([GSSG]/[GSH] raised up to 38-fold). In the heart, GSH contents and [GSSG]/[GSH] ratio remained virtually unchanged even after exercise challenges, while GS-X pump activity was found to be 20% higher in the heart related to skeletal muscle. These findings suggest that an intrinsic higher capacity to express the MRP1/GS-X pump may dictate the redox status in the heart muscle thus protecting myocardium by preventing GSSG accumulation in cardiomyocytes as compared to skeletal muscle fibres.
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PMID:MRP1/GS-X pump ATPase expression: is this the explanation for the cytoprotection of the heart against oxidative stress-induced redox imbalance in comparison to skeletal muscle cells? 1686 18

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.
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PMID:Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein. 1687 3

In animal cells, arsenite has been reported to cause sulfhydryl depletion, generate reactive oxygen species and increase the level of large ubiquitin-protein conjugates. Plant viability tests and DNA laddering experiments have shown that Lemna minor remains viable after exposure to 50 microM NaAsO(2) for periods of at least 6 h. However, protein metabolism is affected in two major ways: the synthesis of an array of stress proteins, which confer thermotolerance; and an increase in the amount of large ubiquitin-protein conjugates, particularly evident after 2-3 h of stress, indicative of a role for the ubiquitin/proteasome pathway. This outcome is primarily attributed to an increased availability of protein substrates during arsenite treatment for three main reasons: an increase in protein carbonyl content after 1-2 h of stress; moderate increments in the transcript levels of the sequences coding for the ubiquitin pathway components chosen as markers (polyubiquitin, E1 and E2, and the beta subunit and the ATPase subunits of the 26S proteasome); the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. This study is the first report on the involvement of the ubiquitin/proteasome pathway in response to arsenite in plants. In addition, it addresses the simultaneous expression of selected genes encoding the various components of the pathway. The results suggest that in plants, unlike in animals, the response to a relatively low level of arsenite does not induce apoptotic cell death. As a whole, the response to arsenite apparently involves a conjugation of salvage and proteolytic machineries, including heat shock protein synthesis and the ubiquitin/proteasome pathway.
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PMID:Exposure of Lemna minor to arsenite: expression levels of the components and intermediates of the ubiquitin/proteasome pathway. 1692 64


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