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)

Deoxyspergualin (DSG), a spermidinyl, alpha-hydroxyglycyl, 7-guanidinoheptanoyl peptidomimetic, shows immunosuppressive activity. In confirmation of a recent report that immobilized methoxyDSG selectively retains the heat shock protein Hsc70, we report here quantitative binding of DSG and analogs to both Hsc70 and the 90-kDa heat shock protein Hsp90. We have utilized affinity capillary electrophoresis to obtain Kd values for DSG and analogs, and stimulation of the ATPase activity of Hsc70 to obtain Km values for DSG, that are comparable and corroborative. Kd values are 4 microM for DSG binding to Hsc70 and 5 microM for DSG binding to Hsp90. Two active analogs, methoxy- and glycylDSG, bind with similar affinities. Glyoxylylspermidine and des(aminopropyl)DSG, two inactive metabolites, have much reduced affinity for Hsc70 and Hsp90. These data validate binding of these novel immunosuppressant agents to these molecular chaperones, at concentrations in the range of pharmacologically active doses, and indicate that further characterization of Hsc70 and/or Hsp90 as potential targets for DSG is warranted.
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PMID:Quantitation of the interaction of the immunosuppressant deoxyspergualin and analogs with Hsc70 and Hsp90. 811 17

Decreased intracellular levels of FtsH, a membrane-bound ATPase, led to retardation of growth and protein export, as well as to an abnormal translocation of alkaline phosphatase that had been attached to a cytoplasmic domain of a multispanning membrane protein, SecY. The last phenotype is designated Std (stop transfer defective). In this study, we examined the effects of overproduction of some molecular chaperones on the phenotypes of ftsH mutants. The growth retardation was partially suppressed by overproduction of GroEL/GroES (Hsp60/Hsp10) or HtpG (Hsp90), although these chaperones could not totally substitute for FtsH. Overproduction of HtpG specifically alleviated the Std phenotype, while that of GroEL/GroES alleviated the protein export defect of ftsH mutants. These results suggest that FtsH functions can be somehow compensated for when the cellular concentrations of some molecular chaperones increase.
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PMID:Suppression of ftsH mutant phenotypes by overproduction of molecular chaperones. 857 50

Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques. Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90.
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PMID:Assessment of the ATP binding properties of Hsp90. 862 58

The homo-oligomeric Hip protein cooperates with the 70-kDa heat shock cognate Hsc70 in the folding of newly synthesized polypeptide chains and in the conformational regulation of signaling molecules known to interact with Hsc70 and Hsp90. In order to further assess the role of Hip during protein biogenesis, a structure-function analysis of the Hip protein was initiated. By employing the yeast two-hybrid system, the Hsc70-binding site of Hip was mapped to a domain comprising multiple tetratricopeptide repeats and flanking charged alpha-helices. Affinity chromatography confirmed direct interaction of isolated Hip fragments and protein fusions bearing this region with the ATPase domain of Hsc70 in an ATP- and salt-dependent manner. Contact of Hip with the ATPase domain appears to be mediated primarily by the positively charged alpha-helix following the tetratricopeptide repeats. Furthermore, a domain required for homo-oligomerization was identified at the extreme amino terminus of Hip.
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PMID:Characterization of functional domains of the eukaryotic co-chaperone Hip. 899 28

Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion. Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with half-maximal binding at 400 microM ATP was detected. The functional significance of this weak interaction remains enigmatic.
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PMID:ATP-binding properties of human Hsp90. 922 28

The BAG-1 protein appears to inhibit cell death by binding to Bcl-2, the Raf-1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG-1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG-1 is a regulator of the Hsc70 chaperone. BAG-1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady-state ATP hydrolysis activity approximately 40-fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG-1 accelerates the release of ADP from Hsc70. Thus, BAG-1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70-interacting protein Hip, which stabilizes the ADP-bound state. Intriguingly, BAG-1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti-apoptotic function of BAG-1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.
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PMID:GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1. 932

Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90. We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase. We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate. Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system. Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90. Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1. In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90. Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70. Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23. Conversely, once p23 is bound to hsp90, Hop binding is diminished. These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.
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PMID:Hop modulates Hsp70/Hsp90 interactions in protein folding. 945 98

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.
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PMID:Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence. 946 43

The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70's cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.
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PMID:The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. 952 74

Chaperones are a functionally related group of proteins assisting protein folding in the cell under physiological and stress conditions. They share the ability to recognize and bind nonnative proteins thus preventing unspecific aggregation. The underlying functional principles of the different chaperone classes are beginning to be understood. A landmark feature of molecular chaperones is the involvement of energy-dependent reactions in the folding process. Nucleotide binding to ATP-dependent chaperones (e.g. GroEL, Hsp70, Hsp90) leads to sometimes large conformational changes in the chaperone which allow to shift between high- and low-affinity states for substrate proteins. Interestingly, the ATPase activity which is the key determinant for functional cycles is tightly regulated by a set of co-chaperones. While for ATP-dependent chaperones binding sites for nucleotide and protein are found in one protein, in the case of ATP-independent chaperones (e. g. sHsps, SecB) the energy-dependent step is performed by another chaperone (Hsp70, SecA). Therefore, the ATP-independent chaperones can be regarded as efficient 'holding' components. Cooperation of different chaperone machineries creates a synergistic network of folding helpers in the cell, which allows to maintain protein homeostasis under conditions nonpermissive for spontaneous folding.
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PMID:How chaperones fold proteins. 956 19


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