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 cDNA representing the plastid-encoded homolog of the prokaryotic ATP-dependent protease ClpP was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. ClpP and a previously isolated cDNA designated ClpC, encoding an ATPase related to proteins encoded by the ClpA/B gene family, were expressed in Escherichia coli. Antibodies directed against these recombinant proteins recognized proteins in a wide variety of organisms. N-terminal analysis of the Clp protein isolated from crude leaf extracts showed that the N-terminal methionine is absent from ClpP and that the transit peptide is cleaved from ClpC. A combination of chloroplast subfractionation and immunolocalization showed that in Arabidopsis, ClpP and ClpC localize to the stroma of the plastid. Immunoblot analyses indicated that ClpP and ClpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E. coli ClpP and ClpA. ClpP, immunopurified from tobacco extracts, hydrolyzed N-succinyl-Leu-Tyr-amidomethylcoumarin, a substrate of E. coli ClpP. Purified recombinant ClpC facilitated the degradation of 3H-methylcasein by E. coli ClpP in an ATP-dependent fashion. This demonstrates that ClpC is a functional homolog of E. coli ClpA and not of ClpB or ClpX. These data represent the only in vitro demonstration of the activity of a specific ATP-dependent chloroplast protease reported to date.
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PMID:The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease. 758 Feb 59

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.
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PMID:The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. 774 94

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

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.
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PMID:A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. 802 80

The ATP-dependent Clp protease of Escherichia coli consists of two subunits, the ClpP subunit, which has the proteolytic active site, and ClpA, which possesses ATPase activity and activates the proteolytic activity of ClpP in vitro. Recently, Zylicz and co-workers (Wojtkowiak, D., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 22609-22617) identified another E. coli protein that activated ATP-dependent degradation of lambda O protein in the presence of ClpP. The amino-terminal sequence of this protein corresponds to the translated amino-terminal sequence of a gene that we have named clpX. clpX encodes a protein with M(r) 46,300, similar to that observed for the protein purified by Wojtkowiak et al. clpX is an operon with clpP; both genes are cotranscribed in a single heat-inducible 2200-base mRNA, with clpP the promoter proximal gene. The sequence of ClpX includes a single consensus ATP-binding site motif and has limited homology to regions of ClpA and other members of the ClpA/B/C family. A third group of proteins, ClpY, closely related to ClpX, has been identified by sequence homology. Mutations in either clpX or clpP abolish degradation of the highly unstable lambda O protein in vivo. clpX mutants are not defective in degradation of previously identified ClpA/ClpP substrates such as a ClpA-beta-galactosidase fusion protein. It appears that selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits.
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PMID:ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities. 822 70

In Escherichia coli, starvation (stationary-phase)-mediated differentiation involves 50 or more genes and is triggered by an increase in cellular sigma s levels. Western immunoblot analysis showed that in mutants lacking the protease ClpP or its cognate ATPase-containing subunit ClpX, sigma s levels of exponential-phase cells increased to those of stationary-phase wild-type cells. Lack of other potential partners of ClpP, i.e., ClpA or ClpB, or of Lon protease had no effect. In ClpXP-proficient cells, the stability of sigma s increased markedly in stationary-phase compared with exponential-phase cells, but in ClpP-deficient cells, sigma s became virtually completely stable in both phases. There was no decrease in ClpXP levels in stationary-phase wild-type cells. Thus, sigma s probably becomes more resistant to this protease in stationary phase. The reported sigma s-stabilizing effect of the hns mutation also was not due to decreased protease levels. Studies with translational fusions containing different lengths of sigma s coding region suggest that amino acid residues 173 to 188 of this sigma factor may directly or indirectly serve as at least part of the target for ClpXP protease.
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PMID:Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease. 855 Apr 68

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.
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PMID:HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. 865 Jan 74

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.
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PMID:Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli. 866 28

ClpQ (HslV) is a homolog of the beta-subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HslU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit Mr 19,000) and ClpY (subunit Mr 49,000) were purified separately as oligomeric proteins with molecular weights of approximately 220,000 and approximately 350,000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP-dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six-fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry-matched ClpYQ system.
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PMID:Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY. 897 22

The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter. BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter. The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.
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PMID:Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance. 964 46


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