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)

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-epsilon interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
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PMID:In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins. 1173 92

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways. In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23. The role of these proteins in Hsp90-mediated assembly processes is poorly understood. It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro. Here we show, for the first time, that human Hsp90 has ATPase activity in vitro. The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli. Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C. Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold. This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity. In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity. We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90. In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate. Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further. In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis. Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding. We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo. We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur.
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PMID:Stimulation of the weak ATPase activity of human hsp90 by a client protein. 1181 47

Nearly 100 proteins are known to be regulated by hsp90. Most of these substrates or "client proteins" are involved in signal transduction, and they are brought into complex with hsp90 by a multiprotein hsp90/hsp70-based chaperone machinery. In addition to binding substrate proteins at the chaperone site(s), hsp90 binds cofactors at other sites that are part of the heterocomplex assembly machinery as well as immunophilins that connect assembled substrate*hsp90 complexes to protein-trafficking systems. In the 5 years since we last reviewed this subject, much has been learned about hsp90 structure, nucleotide-binding, and cochaperone interactions; the most important concept is that ATP hydrolysis by an intrinsic ATPase activity results in a conformational change in hsp90 that is required to induce conformational change in a substrate protein. The conformational change induced in steroid receptors is an opening of the steroid-binding cleft so that it can be accessed by steroid. We have now developed a minimal system of five purified proteins-hsp90, hsp70, Hop, hsp40, and p23- that assembles stable receptor*hsp90 heterocomplexes. An hsp90*Hop*hsp70*hsp40 complex opens the cleft in an ATP-dependent process to produce a receptor*hsp90 heterocomplex with hsp90 in its ATP-bound conformation, and p23 then interacts with the hsp90 to stabilize the complex. Stepwise assembly experiments have shown that hsp70 and hsp40 first interact with the receptor in an ATP-dependent reaction to produce a receptor*hsp70*hsp40 complex that is "primed" to be activated to the steroid-binding state in a second ATP-dependent step with hsp90, Hop, and p23. Successful use of the five-protein system with other substrates indicates that it can assemble signal protein*hsp90 heterocomplexes whether the substrate is a receptor, a protein kinase, or a transcription factor. This purified system should facilitate understanding of how eukaryotic hsp70 and hsp90 work together as essential components of a process that alters the conformations of substrate proteins to states that respond in signal transduction.
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PMID:Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. 1256 18

The 90-kD molecular chaperone hsp90 is the key component of a multiprotein chaperone complex that facilitates folding, stabilization, and functional modulation of a number of signaling proteins. The components of the animal chaperone complex include hsp90, hsp70, hsp40, Hop, and p23. The animal Hop functions to link hsp90 and hsp70, and it can also inhibit the ATPase activity of hsp90. We have demonstrated the presence of an hsp90 chaperone complex in plant cells, but not all components of the complex have been identified. Here, we report the isolation and characterization of soybean (Glycine max) GmHop-1, a soybean homolog of mammalian Hop. An analysis of soybean expressed sequence tags, combined with preexisting data in literature, suggested the presence of at least three related genes encoding Hop-like proteins in soybean. Transcripts corresponding to Hop-like proteins in soybean were detected under normal growth conditions, and their levels increased further in response to stress. A recombinant GmHop-1 bound hsp90 and its binding to hsp90 could be blocked by the tetratricopeptide repeat (TPR) domain of rat (Rattus norvegicus) protein phosphatase 5. Deletion of amino acids 325 to 395, adjacent to the TPR2A domain in GmHop-1, resulted in loss of hsp90 binding. In a minimal assembly system, GmHop-1 was able to stimulate mammalian steroid receptor folding. These data show that plant and animal Hop homologs are conserved in their general characteristics, and suggest that a Hop-like protein in plants is an important cochaperone of plant hsp90.
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PMID:Characterization of a plant homolog of hop, a cochaperone of hsp90. 1258 77

RAR1 and its interacting partner SGT1 play a central role in plant disease resistance triggered by a number of resistance (R) proteins. We identified cytosolic heat shock protein 90 (HSP90), a molecular chaperone, as another RAR1 interacting protein by yeast two-hybrid screening. RAR1 interacts with the N-terminal half of HSP90 that contains the ATPase domain. HSP90 also specifically interacts with SGT1 that contains a tetratricopeptide repeat motif and a domain with similarity to the cochaperone p23. In Arabidopsis, the HSP90 inhibitor geldanamycin reduces the hypersensitive response and abolishes resistance triggered by the R protein RPS2 against Pseudomonas syringae pv. tomato DC3000 (avrRpt2). One of four Arabidopsis cytosolic HSP90 isoforms, AtHSP90.1 is required for full RPS2 resistance and is rapidly induced upon pathogen challenge. We propose that RAR1 and SGT1 function closely with HSP90 in chaperoning roles that are essential for disease resistance.
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PMID:HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis. 1450 84

Understanding the mode of action of Hsp90 requires that molecular detail of its interactions with client proteins and co-chaperones are known. The structure determination of the N-terminal domain of Hsp90/Hsp90beta, proof that it is an ATPase, that this activity is regulated and the identification of co-chaperones that facilitate Hsp90 function were landmarks towards understanding conformational changes in Hsp90 brought about by ATP, co-chaperones and client proteins. Sti1 and Cdc37/p50, which associate with early Hsp90 complexes, were shown to be inhibitors of Hsp90 ATPase activity and therefore promote its 'open' state, whereas Sba1/p23, which associates with mature complexes, inhibits ATPase activity and stabilises the 'closed' state. The isolation and characterisation of Aha1, the only known strong activator of Hsp90 ATPase activity, which promotes the 'closed' state of Hsp90, will also be of major importance in understanding Hsp90 function. The structure determination of the middle region of Hsp90 has shed further light on the complex ATP-cycle of Hsp90, identifying a catalytic loop, with key residues that are essential for ATP hydrolysis. These studies, together with biochemical ones, suggest that ATP hydrolysis, is dependent on a complex rate-limiting step, involving N-terminal dimerization and association of the middle region, and therefore the catalytic loop, of Hsp90 with the N-terminal domains. The structure of the middle region of Hsp90 will also accelerate our understanding of client protein interactions since this region is implicated in their recognition and in particular their active-site openings.
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PMID:Structure and functional relationships of Hsp90. 1452 83

The 90-kDa heat shock protein (Hsp90) is an abundant chaperone that regulates a diverse set of intracellular signaling proteins. Drugs that inhibit Hsp90 activity have been useful in the identification of novel Hsp90-dependent signaling pathways. One class of inhibitory compounds disrupts Hsp90-dependent processes by binding to the N-terminal ATPase/p23-binding domain of Hsp90, whereas a second inhibitor class binds within the C-terminal domain. We used signaling by aryl hydrocarbon receptor (AhR), an Hsp90-dependent transcription factor, as a functional probe to study the effects of Hsp90 inhibitors in yeast strains with deletion mutations of individual Hsp90 and p23 cochaperone genes. The more abundant and constitutively expressed Hsp90 isoform, Hsc82, functioned best in supporting AhR signaling. Deletion of the more inducible isoform, Hsp82, had no effect on signaling. AhR complexes containing Hsc82 were preferentially sensitive to the effects of low concentrations of the N-terminal inhibitors radicicol and herbimycin A. However, both Hsp90 isoforms were equally sensitive to the AhR-specific effects of novobiocin, which binds to the C terminus. Hsp90 inhibitors had no preferential effects on AhR signaling in strains that lacked p23, suggesting that the inhibitors exert their effects through a p23-independent mechanism. In contrast, overexpression of p23 buffered the effects of radicicol and herbimycin A, but not novobiocin, on AhR signaling. The data collectively suggest preferential use or function of the Hsc82 isoprotein in AhR signaling and provide new insight into the effects of three structurally unrelated Hsp90 inhibitors.
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PMID:Pharmacological and genetic analysis of 90-kDa heat shock isoprotein-aryl hydrocarbon receptor complexes. 1464 86

Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by the binding of xenobiotic and endogenous ligands. AhR interacts with heat shock protein (Hsp) 90 complexes and can be used as a functional substrate to detect chaperone-dependent processes. Yeast Hsp90 (hsp82) mutants that variably affected AhR signaling were identified using reporter gene assays. Some mutated alleles resided in the p23/adenosine triphosphate (ATP)-binding pocket of Hsp90, so the relationship between the cochaperone Sba1 (yeast p23) and adenosine triphosphatase (ATPase) activity was investigated. Deletion of the p23 gene in the hsp82G170D mutant background had a greater effect on AhR signaling than the individual mutations, suggesting that these 2 mutations have separate actions on AhR signaling. In contrast, p23 overexpression suppressed temperature sensitivity and AhR signaling defects in the hsp82G170D mutant strain, suggesting that there is a relationship between these 2 proteins. The mutated hsp82G170D protein lacked detectable ATPase activity and p23 binding in vitro, which may relate to the weakened AhR signaling observed in mutant cells. Sba1 (p23) suppressed Hsp82 ATPase activity in vitro. These studies implicate the p23 protein and the G170 region of Hsp90 as being important, but not essential, for AhR signaling. Our results are consistent with a model in which p23 inhibits Hsp90 ATPase activity, thereby stabilizing ATP-Hsp90-client protein complexes.
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PMID:Cooperation of heat shock protein 90 and p23 in aryl hydrocarbon receptor signaling. 1527 73

The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.
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PMID:The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle. 1536 69

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.
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PMID:Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle. 1546 38


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