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)

Leishmaniasis is a zoonotic disease caused by the species of the genus Leishmania, flagellated protozoa that multiply inside mammalian macrophages and are transmitted by the bite of the sandfly. The disease is widespread and due to the lack of fully effective treatment and vaccination the search for new drugs and immune targets is needed. Proteomics seems to be a suitable strategy because the annotated sequenced genome of L. major is available. Here, we present a high-resolution proteome for L. infantum promastigotes comprising of around 700 spots. Western blot with rabbit hyperimmune serum raised against L. infantum promastiogote extracts and further analysis by MALDI-TOF and MALDI-TOF/TOF MS allowed the identification of various relevant functional antigenic proteins. Major antigenic proteins were identified as propionil carboxilasa, ATPase beta subunit, transketolase, proteasome subunit, succinyl-diaminopimelate desuccinylase, a probable tubulin alpha chain, the full-size heat shock protein 70, and several proteins of unknown function. In addition, one enzyme from the ergosterol biosynthesis pathway (adrenodoxin reductase) and the structural paraflagellar rod protein 3 (PAR3) were found among non-antigenic proteins. This study corroborates the usefulness of proteomics in identifying new proteins with crucial biological functions in Leishmania parasites.
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PMID:Proteomic analysis of antigens from Leishmania infantum promastigotes. 1679 30

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target for cancer therapy. It operates as part of a multichaperone complex and is essential for the conformation, stability, and function of several key oncogenic client proteins such as mutant p53, ERBB2, B-RAF, C-RAF, and CDK4. The HSP90-based chaperone machine is driven by the hydrolysis of ATP and ADP/ATP nucleotide exchange. Many of the inhibitors of HSP90 interrupt the intrinsic ATPase activity, causing degradation of the client proteins via the ubiquitin-proteasome pathway. The first-in-class HSP90 inhibitor in clinical trials is the geldanamycin analog, 17-allylamino, 17-demethoxygeldanamycin (17-AAG). The results that have emerged from these trials have been encouraging, with stable disease observed in two melanoma patients. Pharmacodynamic endpoints, such as induction of HSP70 and downregulation of C-RAF and CDK4 in peripheral blood mononuclear cells and tumor biopsies from treated patients, provided evidence of HSP90 inhibition at well-tolerated doses. The toxicity of 17-AAG has been mild. Several preclinical studies have shown that 17-AAG may enhance the efficacy of a variety of chemotherapeutic agents. Phase II clinical trials in various cancers have been initiated as well as Phase I trials of combined therapy with 17-AAG. However, there are several limitations with 17-AAG such as solubility, stability, and hepatotoxicity. Thus, it is not surprising that new HSP90 agents are under development against this novel target for cancer therapy and several show promise.
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PMID:Inhibitors of the HSP90 molecular chaperone: current status. 1686 Jun 62

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

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid beta (Abeta) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Abeta aggregation. These observations led to the hypothesis that chaperones might interact with Abeta and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Abeta has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Abeta aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Abeta self-assembly and that these chaperones are effective at substoichiometric concentrations (approximately 1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Abeta is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.
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PMID:Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro. 1697 2

The vent shrimps, Mirocaris fortunata and Rimicaris exoculata, live in a highly fluctuating thermal environment and undergo frequent temperature bursts. As a first step in the investigation of the response to heat stress, this work aimed to characterize stress proteins in these two species. Complementary deoxyribonucleic acid (cDNA) clones encoding a 70-kDa heat shock protein (HSP) were isolated and characterized from M. fortunata and R. exoculata. The cDNA clones were of 2055 and 1941 base pairs in length, and contained a 2018-bp complete open reading frame (ORF) and a 1785-bp partial coding sequence, respectively. The amino acid sequences corresponding to these ORF are 645 residues in length for M. fortunata and 595 for R. exoculata, and were clearly characterized as members of the HSP70 family. The C-terminal extremity would identify R. exoculata sequence as a cytoplasm HSP70. The relationships between the crustacean HSP70 sequences were examined by two phylogenetic methods, i.e. Maximum Likelihood and Bayesian methods. The resulting trees suggested that M. fortunata sequence may correspond to constitutively expressed HSP70, named HSC70, whereas R. exoculata sequence may correspond to an inducible form of HSP70. The HSP70 sequences from the hydrothermal shrimps proved to be very similar to the other homologous shrimp sequences, except for the presence of an insertion of unknown function in the ATPase domain of R. exoculata sequence.
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PMID:First hsp70 from two hydrothermal vent shrimps, Mirocaris fortunata and Rimicaris exoculata: characterization and sequence analysis. 1709 61

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.
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PMID:Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members. 1714 52

Apoptosis of vascular smooth muscle cells (VSMC) plays an important role in remodeling the vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Apoptosis in VSMC can be inhibited by inversion of the intracellular [Na+]/[K+] ratio after the sustained blockage of the Na+,K+-ATPase by ouabain. Using two-dimensional gel electrophoresis followed by tandem mass spectroscopy, we compared proteomes of control VSMC and of those with ouabain-inhibited Na+,K+-ATPase and found that ouabain treatment led to overexpression of numerous soluble and membrane-bound proteins. Among proteins, which showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 superfamily and a marker for cellular mortal and immortal phenotypes. Further experiments showed that mortalin RNA and protein levels are induced in ouabain-treated VSMC, and that transient transfection of cells with mortalin cDNA inhibited serum deprivation-induced apoptosis via inactivation of the tumor suppressor gene, p53.
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PMID:Proteomic analysis of vascular smooth muscle cells treated with ouabain. 1717 93

A gene encoding a cytosolic heat shock protein 70 from pathogenic Naegleria fowleri (Nf-cHSP70) was identified. The Nf-cHSP70 was 2,062 bp in length with an open reading frame of 1,980 bp encoding 659 amino acid residues. The deduced amino acid sequence of the gene shared high sequence identities with HSP70s from other parasitic organisms and mammals. The characteristic domains, including N-terminal ATPase domain, calmodulin-binding domain, and EE(D)VD motif, found in HSP70s were also well conserved in this gene. The recombinant Nf-cHSP70 protein showed strong antigenicity against the sera from mice experimentally infected with N. fowleri. Immunofluorescence assay showed that Nf-cHSP70 localized in cytosol of the parasite. The results from semi-quantitative RT-PCR and Western blot analyses demonstrated the expression levels of gene transcripts, and its products were significantly increased at high temperature (42 degrees C). The definitive biological roles of Nf-cHSP70 are not clear, but it may protect the parasite under environmental changes especially high temperature.
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PMID:Molecular cloning and characterization of a cytosolic heat shock protein 70 from Naegleria fowleri. 1725 78

The endoplasmic reticulum HSP70 chaperone BiP/Kar2p is both the sensor for the unfolded protein response (UPR) in the yeast Saccharomyces cerevisiae and a target of transcriptional up-regulation by this signaling pathway. In this study, the molecular form of Kar2p that interacts with the Ire1p transmembrane receptor kinase to inhibit UPR signaling was shown to be the substrate-free, ATP-bound conformation. Oligosaccharide shielding experiments localized the binding site for Ire1p to the top of the back face of lobe IB of the Kar2p ATPase domain. The interaction between Kar2p and Ire1p is abolished by substitution of glutamic acid for glutamine 88, a residue on the surface of lobe IB that is likely to be shielded by ectopic oligosaccharide side-chains that also prevented the interaction between the two proteins. Glutamine 88 is conserved significantly throughout the HSP70 chaperone family and others have shown that the NMR resonances of the corresponding glutamine residue in Thermus thermophilus DnaK display chemical shift perturbations between the ATP-bound and ADP-bound states and in the presence of a substrate peptide. We conclude that glutamine 88 is part of or close to the Ire1p-binding site displayed on the ATP-bound conformation of Kar2p. Binding of an unfolded polypeptide to the substrate-binding domain of Kar2p could alter the positioning of glutamine 88 and other residues on lobe IB involved in binding Ire1p, releasing Ire1p for activation of UPR signaling.
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PMID:Lobe IB of the ATPase domain of Kar2p/BiP interacts with Ire1p to negatively regulate the unfolded protein response in Saccharomyces cerevisiae. 1727 61

Histone deacetylases (HDACs), histone acetyltransferases (HATs), and the molecular chaperone heat shock protein 90 (HSP90) are attractive anticancer drug targets. High-throughput screening plays a pivotal role in modern molecular mechanism-based drug discovery. Cell-based screens are particularly useful in that they identify compounds that are permeable and active against the selected target or pathway in a cellular context. We have previously developed time-resolved fluorescence cell immunosorbent assays (TRF-Cellisas) for compound screening and pharmacodynamic studies. These assays use a primary antibody to the single protein of interest and a matched secondary immunoglobulin labeled with an europium chelate (Eu). The availability of species-specific secondary antibodies labeled with different lanthanide chelates provides the potential for multiplexing this type of assay. The approach has been applied to the development of a 384-well duplexed cell-based screen to simultaneously detect compounds that induce the co-chaperone HSP70 as a molecular marker of potential inhibitors of HSP90 together with those that modulate cellular acetylation (i.e., potential inhibitors of histone deacetylase or histone acetyltransferase activity). The duplexed assay proved reliable in high-throughput format and approximately 64,000 compounds were screened. Following evaluation in secondary assays, 3 of 13 hits from the HSP70 arm were confirmed. Two of these directly inhibited the intrinsic ATPase activity of HSP90 whereas the third seems to have a different mechanism of action. In the acetylation arm, two compounds increased cellular acetylation, one of which inhibited histone deacetylase activity. A third compound decreased cellular histone acetylation, potentially through a novel mechanism of action.
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PMID:A duplexed phenotypic screen for the simultaneous detection of inhibitors of the molecular chaperone heat shock protein 90 and modulators of cellular acetylation. 1736 4


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