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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding and internalization of a protein substrate by E. coli ClpXP was investigated by electron microscopy. In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface. When substrate lambda O protein was added, extra density attached to this surface. Upon addition of ATP, this density disappeared as lambda O was degraded. When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX. We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.
Mol Cell 2000 Dec
PMID:Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP. 1116 24

A new method to analyze the similarity between multiply aligned protein motifs (blocks) was developed. It identifies sets of consistently aligned blocks. These are found to be protein regions of similar function and structure that appear in different contexts. For example, the Rossmann fold ligand-binding region is found similar to TIM barrel and methylase regions, various protein families are predicted to have a TIM-barrel fold and the structural relation between the ClpP protease and crotonase folds is identified from their sequence. Besides identifying local structure features, sequence similarity across short sequence-regions (less than 20 amino acid regions) also predicts structure similarity of whole domains (folds) a few hundred amino acid residues long. Most of these relations could not be identified by other advanced sequence-to-sequence or sequence-to-multiple alignments comparisons. We describe the method (termed CYRCA), present examples of our findings, and discuss their implications.
J Mol Biol 2001 Mar 30
PMID:Consistency analysis of similarity between multiple alignments: prediction of protein function and fold structure from analysis of local sequence motifs. 1127 12

The proteolytic machinery of plant organelles is largely unknown, although indications so far point to several proteases of bacterial origin. In this study an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX, a molecular chaperone and regulatory subunit of the ATP-dependent, serine-type Clp protease. Computer analysis of the predicted plant ClpX revealed a putative mitochondrial transit peptide at the N-terminus, as well as overall sequence similarity to other eukaryotic ClpX homologues. Specific polyclonal antibodies were made to the Arabidopsis ClpX protein and used to confirm its localization in plant mitochondria. In addition to ClpX, a ClpP protein located in mitochondria was also identified from the numerous ClpP isomers in Arabidopsis. Localization of this nuclear-encoded protein, termed ClpP2, was determined first by its close sequence similarity to mitochondrial ClpP human, and later experimentally using ClpP2-specific antibodies with isolated plant organellar fractions. In Arabidopsis, transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions, with no significant variation in mRNA level (i.e. 2-fold) for each gene between samples. Using beta-casein as a substrate, plant mitochondria were found to possess an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. The recent discovery of homologous mitochondrial ClpX and ClpP proteins in mammals suggests that this type of protease may be common to multicellular eukaryotes.
Plant Mol Biol 2001 Mar
PMID:Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease. 1135 64

Gene trees of Plasmodium species have been reported for the nuclear encoded genes (e.g. the Small Subunit rRNA) and a mitochondrial encoded gene, cytochrome b. Here, we have analyzed a plastid gene coding for caseinolytic protease ClpC, whose structure, function and evolutionary history have been studied in various organisms. This protein possesses a 220-250 amino acid long AAA domain (ATPases associated with a variety of cellular activities) that belongs to the Walker super family of ATPases and GTPases. We have sequenced the AAA motif of this gene, encoding the protein from nine different species of Plasmodium infecting rodents, birds, monkeys, and humans. The codon usage and GC content of each gene were nearly identical in contrast to the widely varying nucleotide composition of genomic DNAs. Phylogenetic trees derived from both DNA and inferred protein sequences have consistent topologies. We have used the ClpC sequence to analyze the phylogenetic relationship among Plasmodium species and compared it with those derived from mitochondrial and genomic sequences. The results corroborate well with the trees constructed using the mitochondrially encoded cytochrome b. However, an important element distinguishes the trees: the placement of Plasmodium elongatum near the base of the plastid tree, indicating an ancient lineage of parasites in birds that branches from the tree prior to other lineages of avian malaria and the human parasite, P. falciparum.
Mol Biochem Parasitol 2001 Apr 25
PMID:A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species. 1135 17

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.
Mol Microbiol 2001 Jul
PMID:Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis. 1145 3

Mutations in clpP and clpX have pleiotropic effects on growth and developmentally regulated gene expression in Bacillus subtilis. ClpP and ClpX are needed for expression of comK, encoding the competence transcription factor required for the expression of genes within the competence regulon. ClpP, in combination with the ATPase ClpC, degrades the inhibitor of ComK, MecA. Proteolysis of MecA is stimulated by a small protein, ComS, which interacts with MecA. Suppressor mutations (cxs) were isolated that bypass the requirement for clpX for comK expression. These were found also to overcome the defect in comK expression conferred by a clpP mutation. These mutations were identified as missense mutations (cxs-5, -7 and -12) and a nonsense (UAG) codon substitution (cxs-10) in the yjbD coding sequence in a locus linked to mecA. That a yjbD disruption confers the cxs phenotype, together with its complementation by an ectopically expressed copy of yjbD, indicated that the suppressor alleles bear recessive, loss-of-function mutations of yjbD. ClpP- and ClpX-independent comK expression rendered by inactivation of yjbD was still medium-dependent and required ComS. MecA levels in a clpP-yjbD mutant were lower that those of clpP mutant cells and ComK protein concentration in the clpP mutant was restored to wild-type levels by the yjbD mutation. Consequently, the yjbD mutation bypasses the defect in competence development conferred by clpP and clpX. YjbD protein is barely detectable in wild-type cells, but is present in large amounts in the clpP mutant cells. The results suggest that the role of ClpP in competence development is to degrade YjbD protein so that ComS can productively interact with the MecA-ClpC-ComK complex. Alternatively, the result could suggest that YjbD has a negative effect on regulated proteolysis and that MecA is degraded independently of ClpP when YjbD is absent.
Mol Microbiol 2001 Oct
PMID:Loss-of-function mutations in yjbD result in ClpX- and ClpP-independent competence development of Bacillus subtilis. 1170 62

MecA targets the competence transcription factor ComK to ClpC. As a consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. In vivo, this mutation had no effect on thermal resistance, but had a clpC null phenotype with respect to competence. Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10-fold but was not eliminated. In vivo, the K551Q mutation showed a partial defect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain. Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC.
Mol Microbiol 2001 Nov
PMID:Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response. 1172 37

ATP-dependent proteases of the ClpP type are widespread in eubacteria. These proteolytic complexes are composed of a proteolytic subunit and an ATPase subunit. They are involved in the degradation of denatured proteins, but also play a role in specific regulatory pathways. In Streptomyces lividans strains which lack the proteolytic subunit ClpP1, cell cycle progression has been shown to be blocked at early stages of growth. In this study, we examined the role of the ATPase subunit ClpX, a possible partner of the products of the clpP1 operon. A clpX mutant was obtained and it was shown that its growth was impaired only on acidic medium. Thus, the clpX phenotype differs from the clpP1 phenotype, indicating that these two components have only partially overlapping roles. We also analyzed the expression of clpX. Although clpX expression is increased under heat-shock conditions in many bacteria, we found that this is not the case in S. lividans.
Mol Genet Genomics 2003 Feb
PMID:The ATPase ClpX is conditionally involved in the morphological differentiation of Streptomyces lividans. 1258 31

ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been identified. Using a tagged and inactive variant of ClpP, substrates of E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.
Mol Cell 2003 Mar
PMID:Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. 1266 50

Clp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature-tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus. Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325-4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall-associated proteins. In the mutant cells, the amount of alpha-haemolysin (hla) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum-sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.
Mol Microbiol 2003 Jun
PMID:Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence. 1279 Nov 39


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