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)

Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro. The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication. Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.
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PMID:Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins. 787 26

In Escherichia coli, the molecular chaperones (DnaK, DnaJ, and GrpE) are essential for the rapid degradation of certain proteins. To see if chaperones are involved more generally in proteolysis, we studied the degradation of a short-lived fusion protein, CRAG, which associates with DnaK and GroEL in vivo. Its rapid degradation requires ATP and ClpP, the proteolytic subunit of protease Ti (Clp). However, this process is not reduced in strains lacking the complementary ATPase subunit, ClpA, or its homologs, ClpB and ClpX. At 37 degrees C, but not at 42 degrees C, protease La also contributes partially to CRAG degradation. Nevertheless, CRAG is not degraded in cell-free extracts or upon incubation with ClpP or protease La. We tested whether the chaperones associated with CRAG might be involved in its degradation. CRAG breakdown was accelerated 2-3-fold in strains with high levels of heat-shock proteins (hsps), i.e. in those that overproduce the hsp transcription factor (sigma 32) or carry a dnaK deletion. A similar stimulation of proteolysis was observed in cells overproducing GroEL or both GroEL and GroES; in these cells, more CRAG was associated with GroEL than in the wild type. In a temperature-sensitive groEL44 mutant at the nonpermissive temperature, CRAG breakdown was accelerated, and more CRAG was found complexed with GroEL. However, in a temperature-sensitive groES mutant, CRAG was completely stable at the nonpermissive temperature and accumulated bound to GroEL. These findings indicate that the association of CRAG with GroEL is a rate-limiting step in CRAG degradation, which also requires a subsequent action of GroES. We propose that if the hsp60/hsp10 chaperonins fail to catalyze the proper folding of a protein, they can facilitate its rapid degradation.
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PMID:Rapid degradation of an abnormal protein in Escherichia coli involves the chaperones GroEL and GroES. 791 44

A DnaK homolog (T.DnaK) has been purified as a stable complex with a DnaJ homolog (T.DnaJ) from a thermophilic bacterium, Thermus thermophilus. This complex has an approximate molecular size of 300 kDa and appears to contain three copies of each of T.DnaK and T.DnaJ molecules. Consistently, trigonal ring structures with a diameter (trigonal apex-to-apex) of about 11 nm were observed with electron microscopy. The complex has no endogenously bound AT(D)P and is stable in the presence of Mg-AT(D)P. It possesses a weak ATPase activity and retains about 3 mol of ADP/mole of the complex when incubated with Mg-ATP. This complex is able to interact with the reduced carboxymethylated alpha-lactalbumin which we used as a model unfolded protein.
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PMID:Isolation of the stable hexameric DnaK.DnaJ complex from Thermus thermophilus. 792 50

A temperature-sensitive mutant of DnaK, the principal Escherichia coli member of the 70 kDa heat shock protein family, has been isolated. The mutation, dnaK25, lies in the putative ATP binding pocket of DnaK. It consists of a C to T transition that changes the highly conserved proline 143 to serine. Mutant strains do not support the propagation of bacteriophage lambda or of plasmids that require DnaA for replication. They are also defective in the utilization of mannose and sorbitol. ATPase activity of the mutant protein is reduced 20-fold relative to wild-type, while autophosphorylation is unaffected. DnaK25 has a fourfold faster rate of nucleotide exchange than wild-type DnaK; nucleotide exchange by both proteins is markedly increased by GrpE. The DnaK25 ATPase is still stimulated by DnaJ and GrpE and by peptide substrates. However, the affinity of most peptides tested for stimulating the DnaK25 ATPase is reduced significantly. These results indicate that a mutation in the N-terminal nucleotide binding domain can alter substrate interactions with the C-terminal substrate binding site. Nucleotide exchange by both wild-type DnaK and DnaK25 proceeds at a much faster rate than ATP hydrolysis, and therefore cannot be the rate limiting step of ATP hydrolysis under the conditions used in these experiments. Consistent with this, peptides, which stimulate ATP hydrolysis, have no effect on nucleotide exchange. Peptides thus appear to stimulate the ATPase by acting at another step, such as increasing the rate of phosphate bond cleavage.
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PMID:Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity. 793 96

The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70-amino-acid J domain. Escherichia coli DnaJ interacts with the 70-kDa heat-shock protein (DnaK), in vitro, to stimulate the 70-kDa heat-shock protein ATPase activity and modify substrate binding. The conservation of the interaction of DnaJ-like proteins with the 70-kDa heat-shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E. coli DnaJ. Human neurone-specific DnaJ-like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine-rich region in common with E. coli DnaJ, although the overall amino acid identity is less than 23%. We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70-kDa heat-shock protein (hsc70). The weak intrinsic ATPase activity of the constitutive 70-kDa heat-shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b. This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected. HSJ1 proteins appear to regulate the affinity of the 70-kDa constitutive heat-shock protein for the permanently unfolded substrate, carboxymethylated alpha-lactalbumin. A recent report [Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J. & Fink, A. L. (1993) Nature 365, 664-666] has suggested that substrate release by 70-kDa heat-shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding. In the presence of ATP, HSJ1 proteins reduce 70-kDa constitutive heat-shock protein/carboxymethylated alpha-lactalbumin complex formation both in the presence and absence of K+. This suggests that HSJ1 proteins induce a conformational change in the 70-kDa constitutive heat-shock protein that can mimic the effect mediated by K+ and therefore modulate 70-kDa heat-shock protein substrate release by another mechanism rather than merely stimulating the 70-kDa heat-shock protein ATPase activity. As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70-kDa heat-shock-protein substrate binding will be common to all proteins that contain a J domain.
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PMID:Regulation of 70-kDa heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJ1a and HSJ1b. 795 63

The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK). Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites. We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone. ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein. RepA is activated by the conversion of dimers to monomers. We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis. In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase.
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PMID:A molecular chaperone, ClpA, functions like DnaK and DnaJ. 799 9

The Escherichia coli heat shock proteins DnaK and DnaJ function cooperatively as molecular chaperones. Central to their biochemical functions is the ability of DnaJ to interact with DnaK and to stimulate its ATPase activity. Here, we report the genetic isolation of dnaJ12, which has a nonsense mutation at codon 109, yet was able to support lambda growth at 30 degrees C. The 12-kDa DnaJ12 protein was purified to homogeneity and shown to be active in an in vitro lambda-DNA replication system and to be capable of stimulating DnaK's ATPase activity, specifically at the step of ATP hydrolysis. The previously well studied and characterized dnaJ259 mutation was also cloned and sequenced, revealing a single His-->Gln amino acid change at codon 33. The purified DnaJ259 protein was inactive in an in vitro lambda-DNA replication system and was unable to stimulate DnaK's ATPase activity. Consistent with this, an NH2-terminal deletion of the first 34 amino acids or an Asp insertion at residue 35 of DnaJ resulted in a protein that completely lacked DnaJ activity. Collectively, these results demonstrate that the highly conserved NH2-terminal region of DnaJ, the so-called J region, is necessary and sufficient for stimulating both DnaK's ATPase activity and lambda-DNA replication. These results may be applicable to other eukaryotic proteins that contain this conserved J domain as proteins that interact and stimulate the hydrolysis of ATP by their cognate HSP70 proteins.
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PMID:The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. 810 26

In Saccharomyces cerevisiae Ydj1p, a DnaJ homolog, is localized to the cytosol with the Ssa and Ssb Hsp70 proteins. Ydj1p helps facilitate polypeptide translocation across mitochondrial and endoplasmic reticulum membranes (Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992) Cell 71, 1143-1155) and can directly interact with Ssa1p to regulate chaperone activity (Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927-20931). In this study, the role of Ydj1p in modulating ATP-dependent reactions catalyzed by Ssa and Ssb Hsp70 proteins has been examined using purified components and compared with that of other Hsp70 homologs BiP and DnaK. Ssa1p, Ssa2p, and Ssb1/2p all formed stable complexes with the mitochondrial presequence peptide, F1 beta(1-51). ATP alone had only modest effects on polypeptide complex formation with Ssa1p and Ssa2p, but prevented the majority of polypeptide binding to BiP and DnaK. ATP by itself also reduced polypeptide binding to Ssb1/2p to a level that was intermediate between that observed for the Ssa Hsp70 proteins tested and BiP and DnaK. ATP hydrolysis by Ssa1p, Ssa2p, and Ssb1/2p occurred at similar rates. Ydj1p was a potent modulator of the both the ATPase and polypeptide binding activities of Ssa1p and Ssa2p. In contrast, Ydj1p had little effect on the ATPase and polypeptide binding activity of Ssb1/2p. Therefore the chaperone-related activities of Ssa and Ssb Hsp70 proteins exhibit significant differences in sensitivity to ATP and YDJ1p. These data indicate that regulation of Hsp70 activity by DnaJ homologs can be specific. The specificity of interactions between Ydj1p and the Ssa and Ssb Hsp70 proteins observed could contribute in determining the functional specificity of these chaperones in the cytosol. In related experiments, F1 beta(1-51) was found to reduce the extent to which Ydj1p stimulated Ssa1p ATPase activity. This effect correlated with the formation of F1 beta(1-51).Ssa1p complexes. We propose that intramolecular communication between the polypeptide binding, ATPase and DnaJ regulatory domains on Ssa1p plays a role in the regulation of chaperone activity.
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PMID:Differential regulation of Hsp70 subfamilies by the eukaryotic DnaJ homologue YDJ1. 814 72

DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.
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PMID:Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes. 853 Apr 9

The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein. Like all J homologues, it shares homology with the highly conserved NH2-terminal "J-domain" of DnaJ. A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures. It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides. Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions. Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity. Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding. This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release. The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34.
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PMID:A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding. 862 99


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