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)

A model structure of the Hsc70/auxilin complex has been constructed to gain insight into interprotein substrate transfer and ATP hydrolysis induced conformational changes in the multidomain Hsc70 structure. The Hsc70/auxilin system, which is a member of the Hsp70/Hsp40 chaperone system family, uncoats clathrin-coated vesicles in an ATP hydrolysis-driven process. Incorporating previous results from NMR and mutant binding studies, the auxilin J-domain was docked into the Hsc70 ATPase domain lower cleft using rigid backbone/flexible side chain molecular dynamics, and the Hsc70 substrate binding domain was docked by a similar procedure. For comparison, J-domain and substrate binding domain docking sites were obtained by the rigid-body docking programs DOT and ZDOCK, filtered and ranked by the program ClusPro, and relaxed using the same rigid backbone/flexible side chain dynamics. The substrate binding domain sites were assessed in terms of conserved surface complementarity and feasibility in the context of substrate transfer, both for auxilin and another Hsp40 protein, Hsc20. This assessment favors placement of the substrate binding domain near D152 on the ATPase domain surface adjacent to the J-domain invariant HPD segment, with the Hsc70 interdomain linker in the lower cleft. Examining Hsc70 interdomain energetics, we propose that long-range electrostatic interactions, perhaps due to a difference in the pKa values of bound ATP and ADP, could play a major role in the structural change induced by ATP hydrolysis. Interdomain electrostatic interactions also appear to play a role in stimulation of ATPase activity due to J-domain binding and substrate binding by Hsc70.
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PMID:Experimentally biased model structure of the Hsc70/auxilin complex: substrate transfer and interdomain structural change. 1527 4

Immortalized human fibroblasts were used to investigate the putative interactions of the Hsp90 molecular chaperone with the wild-type p53 tumor suppressor protein. We show that geldanamycin or radicicol, specific inhibitors of Hsp90, diminish specific wild-type p53 binding to the p21 promoter sequence. Consequently, these inhibitors decrease p21 mRNA levels, which lead to a reduction in cellular p21/Waf1 protein, known to induce cell cycle arrest. In control experiments, we show that neither geldanamycin nor radicicol affect p53 mRNA levels. A minor decrease in p53 protein level following the treatment of human fibroblasts with the inhibitors suggests the potential involvement of Hsp90 in the stabilization of wild-type p53. To support our in vivo findings, we used a reconstituted system with highly purified recombinant proteins to examine the effects of Hsp90 on wild-type p53 binding to the p21 promoter sequence. The human recombinant Hsp90 alpha-isoform as well as bovine brain Hsp90 were purified to homogeneity. Both of these molecular chaperones displayed ATPase activity and the ability to refold heat-inactivated luciferase in a geldanamycin- and radicicol-sensitive manner, suggesting that post-translational modifications are not involved in the modulation of Hsp90alpha activity. We show that the incubation of recombinant p53 at 37 degrees C decreases the level of its wild-type conformation and strongly inhibits the in vitro binding of p53 to the p21 promoter sequence. Interestingly, Hsp90 in an ATP-dependent manner can positively modulate p53 DNA binding after incubation at physiological temperature of 37 degrees C. Other recombinant human chaperones from Hsp70 and Hsp40 families were not able to efficiently substitute Hsp90 in this reaction. Consistent with our in vivo results, geldanamycin can suppress Hsp90 ability to regulate in vitro p53 DNA binding to the promoter sequence. In summary, the results presented in this article state that chaperone activity of Hsp90 is important for the transcriptional activity of genotypically wild-type p53.
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PMID:Hsp90 chaperones wild-type p53 tumor suppressor protein. 1535 69

The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910-914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70/Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use.
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PMID:Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity. 1544 48

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.
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PMID:Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70. 1549 3

A proteomic approach was applied to liver cytosol from rats fed a diet consisting of high fat and ethanol to identify 4-hydroxy-2-nonenal (4-HNE)-modified proteins in vivo. Cytosolic Hsp72, the inducible variant of the Hsp70 heat shock protein family, was consistently among the proteins modified by 4-HNE. Despite 1.3-fold induction of Hsp72 in the livers of ethanol-fed animals, no increase in Hsp70-mediated luciferase refolding in isolated heptocytes was observed, suggesting inhibition of this process by 4-HNE. A 50% and 75% reduction in luciferase refolding efficiency was observed in rabbit reticulocyte lysate (RRL) supplemented with recombinant Hsp72 which had been modified in vitro with 10 and 100 microM 4-HNE, respectively. This observation was accompanied by a 25% and 50% decrease in substrate binding by the chaperone following the same treatment; however, no effect on complex formation between Hsp72 and its co-chaperone Hsp40 was observed. Trypsin digest and mass spectral analysis of Hsp72 treated with 10 and 100 microM 4-HNE consistently identified adduct formation at Cys267 in the ATPase domain of the chaperone. The role of this residue in the observed inhibition was demonstrated through the use of DnaK, a bacterial Hsp70 variant lacking Cys267. DnaK was resistant to 4-HNE inactivation. Additionally, Hsp72 was resistant to inactivation by the thiol-unreactive aldehyde malondialdehyde (MDA), further supporting a role for Cys in Hsp72 inhibition by 4-HNE. Finally, the affinity of Hsp72 for ATP was decreased 32% and 72% following treatment of the chaperone with 10 and 100 microM 4-HNE, respectively. In a model of chronic alcoholic liver injury, induction of Hsp72 was not accompanied by an increase in protein refolding ability. This is likely the result of 4-HNE modification of the Hsp72 ATPase domain.
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PMID:Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal. 1554 Sep 44

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer-mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin-induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.
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PMID:Localization and function in endoplasmic reticulum stress tolerance of ERdj3, a new member of Hsp40 family protein. 1554 63

The essential Hsp40, Sis1, is a J-protein cochaperone for the Ssa class of Hsp70's of Saccharomyces cerevisiae. Sis1 is required for the maintenance of the prion [RNQ(+)], as Sis1 lacking its 55-amino-acid glycine-rich region (G/F) does not maintain [RNQ(+)]. We report that overexpression of Sis1DeltaG/F in an otherwise wild-type strain had a negative effect on both cell growth and [RNQ(+)] maintenance, while overexpression of wild-type Sis1 did not. Overexpression of the related Hsp40 Ydj1 lacking its G/F region did not cause inhibition of growth, indicating that this dominant effect of Sis1DeltaG/F is not a characteristic shared by all Hsp40's. Analysis of small deletions within the SIS1 G/F region indicated that the observed dominant effects were caused by the absence of sequences known to be important for Sis1's unique cellular functions. These inhibitory effects of Sis1DeltaG/F were obviated by alterations in the N-terminal J-domain of Sis1 that affect interaction with Ssa's ATPase domain. In addition, a genetic screen designed to isolate additional mutations that relieved these inhibitory effects identified two residues in Sis1's carboxy-terminal domain. These alterations disrupted the interaction of Sis1 with the 10-kD carboxy-terminal regulatory domain of Ssa1, indicating that Sis1 has a bipartite interaction with Ssa in vivo.
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PMID:In vivo bipartite interaction between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae. 1568 71

Hsp110 proteins constitute a heterogeneous family of abundant molecular chaperones, related to the Hsp70 proteins and exclusively found in the cytosol of eukaryotic organisms. Hsp110 family members are described as efficient holdases, preventing the aggregation and assisting the refolding of heat-denatured model substrates in the presence of Hsp70 chaperones and their co-chaperones. To gain more insights into the mode of action of this protein family we compared two homologues representing two subtypes of Hsp110 proteins, S. cerevisiae Sse1 and H. sapiens Apg-2, in their structural and functional properties in vitro. In contrast to previous publications both proteins exhibited intrinsic ATPase activities, which only in the case of Sse1 could be stimulated by the Hsp40 co-chaperone Sis1. Similar to Hsp70 proteins ATP binding and hydrolysis induced conformational rearrangements in both Hsp110 proteins as detected by tryptophane fluorescence. However, nucleotide induced changes in the proteolytic digestion pattern were detected only for Sse1. Sse1 and Apg-2 thus show significant differences in their biochemical properties, which may relate to differences in their functional roles in vivo.
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PMID:Human and yeast Hsp110 chaperones exhibit functional differences. 1636 15

To perform effectively as a molecular chaperone, DnaK (Hsp70) necessitates the assistance of its DnaJ (Hsp40) co-chaperone partner, which efficiently stimulates its intrinsically weak ATPase activity and facilitates its interaction with polypeptide substrates. In this study, we address the function of the conserved glycine- and phenylalanine-rich (G/F-rich) region of the Escherichia coli DnaJ in the DnaK chaperone cycle. We show that the G/F-rich region is critical for DnaJ co-chaperone functions in vivo and that despite a significant degree of sequence conservation among the G/F-rich regions of Hsp40 homologs from bacteria, yeast, or humans, functional complementation in the context of the E. coli DnaJ is limited. Furthermore, we found that the deletion of the whole G/F-rich region is mirrored by mutations in the conserved Asp-Ile/Val-Phe (DIF) motif contained in this region. Further genetic and biochemical analyses revealed that this amino acid triplet plays a critical role in regulation of the DnaK chaperone cycle, possibly by modulating a crucial step subsequent to DnaK-mediated ATP hydrolysis.
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PMID:The role of the DIF motif of the DnaJ (Hsp40) co-chaperone in the regulation of the DnaK (Hsp70) chaperone cycle. 1653 11

The heat shock cognate protein hsc70 has been implicated as a postattachment cell receptor for rotaviruses. Here we show that hsc70 interacts specifically with rotaviruses through its peptide-binding domain, since a recombinant full-length hsc70 protein and its peptide-binding domain, but not its ATPase domain, bound triple-layered particles in a solid-phase assay, and known ligands of hsc70 competed this binding. The peptide ligands of hsc70 were also shown to block rotavirus infectivity when added to cells before virus infection, suggesting that hsc70 on the surface of MA104 cells also interacts with the virus through its peptide-binding domain and that this interaction is important for virus entry. When purified infectious virus was incubated with soluble hsc70 in the presence of the cochaperone hsp40 and ATP and then pelleted through a sucrose cushion, the recovered virus had lost 60% of its infectivity, even though hsc70 was not detected in the pellet fraction. The hsc70-treated virus showed slightly different reactivities with monoclonal antibodies and was more susceptible to heat and basic pHs than the untreated virus, suggesting that hsc70 induces a subtle conformational change in the virus that results in a reduction of its infectivity. The relevance of the ATPase activity of hsc70 for reducing virus infectivity was demonstrated by the finding that in the presence of a nonhydrolyzable analogue of ATP, virus infectivity was not affected, and a mutant protein lacking ATPase activity failed to reduce virus infection. Altogether, these results suggest that during cell infection, the interaction of the virus with hsc70 on the surface of MA104 cells results in a conformational change of virus particles that facilitates their entry into the cell cytoplasm.
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PMID:The peptide-binding and ATPase domains of recombinant hsc70 are required to interact with rotavirus and reduce its infectivity. 1653 99


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