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PubMed1990

The ClpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5.

The genes that encode the subunits of the Clp protease of Escherichia coli, clpA and clpP, appear to be regulated differently from each other. The clpA gene does not seem to be under heat shock control (Y. S. Katayama, S. Gottesman, J. Pumphrey, S. Rudikoff, W. P. Clark, and M. R. Maurizi, J. Biol. Chem. 263:15226-15236, 1988). In contrast, the level of ClpP protein was increased in rpoH+ cells but not in null rpoH cells after an upshift in temperature from 17 to 43 degrees C. The level of ClpP protein in a null dnaK strain was also elevated relative to the level of ClpP protein in an otherwise isogenic dnaK+ strain. In two-dimensional gels, the ClpP protein was located in the position of the previously unidentified heat shock protein F21.5. No protein spot corresponding to F21.5 was present in two-dimensional gels of a null clpP strain. The clpP gene, therefore, appears to be a heat shock gene, expressed in a sigma 32-dependent manner and negatively regulated by DnaK; the product of clpP is the previously unidentified heat shock protein F21.5.

ATP-Dependent Proteases↗
PubMed1990

Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli.

The ATP-dependent Clp protease of Escherichia coli contains two dissimilar components: the Clp A regulatory polypeptide, with two ATP binding sites and intrinsic ATPase activity, and the Clp P subunit, which contains the proteolytic active site. The DNA sequence of the clpP gene predicts a protein of 207 amino acids (Mr 21,679), which is in close agreement with the size determined by sodium dodecyl sulfate-gel electrophoresis of purified Clp P. Clp P has a native Mr of approximately 240,000, and electron micrographs of the protein show superimposed disk-like structures with a central cavity, similar in appearance to purified proteasomes from eukaryotic cells. Clp P is synthesized with a 14-amino acid leader which is rapidly cleaved in vivo to yield the 193-amino acid protein which has activity in vitro. The clpP gene is at 10 min on the E. coli map, close to that for the ATP-dependent Lon protease of E. coli and far from the gene for clpA. Primer extension experiments indicate that transcription initiates immediately upstream of the coding region for Clp P, with a major transcription start at 120 bases in front of the start of translation. Insertion mutations in clpP have been isolated and transferred to the chromosome; strains devoid of Clp P are viable in the presence or absence of Lon protease. Mutations in clpP stabilize the same Clp A-beta-galactosidase fusion protein specifically stabilized by clpA mutations, providing the first genetic evidence that Clp A and Clp P act together in vivo.

ATP-Dependent Proteases↗
PubMed2023

Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis.

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated β-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds.

Humans↗
PubMed1991

Expression of ClpB, an analog of the ATP-dependent protease regulatory subunit in Escherichia coli, is controlled by a heat shock sigma factor (sigma 32).

Escherichia coli K-12 produces at least two ATP-dependent proteases, Lon (La) and Clp (Ti), the latter consisting of a regulatory subunit (ClpA) and a proteolytic subunit (ClpP). The gene clpB encoding an analog of ClpA had been found at 57 min on the E. coli chromosome. Cloning and examination of novel heat shock promoters led us to identify a major clpB promoter specifically controlled by a heat shock sigma factor, sigma 32 (the rpoH [= htpR] gene product). beta-Galactosidase synthesis from a PclpB-lacZ operon fusion was transiently induced upon temperature shift from 30 to 42 degrees C, and the induction depended on the rpoH function. Chromosomal clpB transcripts also increased upon temperature upshift and were totally absent in the rpoH deletion strain. In the in vitro transcription experiments, the clpB promoter was specifically recognized and transcribed by RNA polymerase-sigma 32. Nucleotide sequencing and determination of mRNA start sites permitted us to identify a major heat shock promoter located upstream of the clpB coding sequence. The results clearly indicate that clpB expression is under direct control of sigma 32. Since ClpP was recently shown to be a sigma 32-dependent heat shock protein, the present finding suggests the possibility that a potential ATP-dependent protease, ClpB-ClpP complex, plays an important role against thermal stress in E. coli.

ATP-Dependent Proteases↗
PubMed1991

ClpB is the Escherichia coli heat shock protein F84.1.

ClpB is thought to be involved in proteolysis because of its sequence similarity to the ClpA subunit of the ClpA-ClpP protease. It has recently been shown that ClpP is a heat shock protein. Here we show that ClpB is the Escherichia coli heat shock protein F84.1. The F84.1 protein was overproduced in strains containing the clpB gene on a plasmid and was absent from two-dimensional gels from a clpB null mutation. Besides possessing a slower growth rate at 44 degrees C, the null mutant strain had a higher rate of death at 50 degrees C. We used reverse transcription of in vivo mRNA to show that the clpB gene was expressed from a sigma 32-specific promoter consensus sequence at both 37 and 42 degrees C. We noted that the clpB+ gene also caused the appearance of a second protein spot, F68.5, on two-dimensional gels. This spot was approximately 147 amino acids smaller than F84.1 and most probably is the result of a second translational start on the clpB mRNA. F68.5 can be observed on many published two-dimensional gels of heat-induced E. coli proteins, but the original catalog of 17 heat shock proteins did not include this spot.

Base Sequence↗
PubMed1992

The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase.

The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClpP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM. ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.

Adenosine Triphosphatases↗
PubMed1990

Clp P represents a unique family of serine proteases.

The amino acid sequence of Clp P, the proteolytic subunit of the ATP-dependent Clp protease of Escherichia coli, closely resembles a protein encoded by chloroplast DNA, which is well conserved between chloroplasts of different plant species. The homology extends over almost the full length of the sequences of both proteins and consists of approximately 46% identical and approximately 70% similar amino acids. Antibodies against E. coli Clp P cross-reacted with proteins with Mr of 20,000-30,000 in bacteria, lower eukaryotes, plants, and animal cells. Since the regulatory subunit of Clp protease, Clp A, also has a homolog in plants, as well as in other bacteria and in lower eukaryotes, it is likely that ATP-dependent proteolysis in chloroplasts is catalyzed in part by a Clp-like protease and that both components of Clp-like proteases are widespread in living cells. We have identified Ser-111 as the active site serine in E. coli Clp P modified by diisopropyl fluorophosphate. Mutational alteration of Ser-111 or His-136 eliminates proteolytic activity of Clp P. Both residues are found in highly conserved regions of the protein. The sequences around the active site residues suggest that Clp P represents a unique class of serine protease. Amino-terminal processing of cloned Clp P mutated at either Ser-111 or His-136 occurs efficiently when wild-type clpP is present in the chromosome but is blocked in clpP- hosts. Processing of Clp P appears, therefore, to involve an intermolecular autocatalytic cleavage reaction. Since processing of Clp P occurs in clpA- cells, the autoprocessing activity of Clp P is independent of Clp A.

ATP-Dependent Proteases↗
PubMed2026

Exercise elicits mitonuclear protein imbalance and UPRmt in the liver of mice with obesity.

Mitochondrial dysfunction plays a critical role in the development of metabolic dysfunction-associated steatotic liver disease (MASLD). It has been proposed that mitochondrial unfolded-protein response (UPRmt) activation improves mitochondrial function in the liver. Growing evidence demonstrates that physical exercise effectively prevents and treats MASLD. However, the effects of exercise on UPRmt activation in the liver are unknown. Thus, we investigated the impact of aerobic training on the mechanisms involved in mitochondrial quality control in the liver in a mouse model of obesity. Liver transcript data from a genetic reference panel of BXD isogenic mice revealed a negative correlation between UPRmt-related genes and hepatic triacylglycerol content. In addition, the liver UPRmt markers were strongly associated with several mitochondrial-related genes in the hepatic tissue of BXD mice and humans. Notably, 4 weeks of aerobic exercise strongly impacted the liver metabolism, preventing intrahepatic lipid accumulation in HFD-fed mice. Physical exercise boosted the NAD-biosynthesis pathway, elicited the mitonuclear protein imbalance, stimulated the protein content of UPRmt-markers, including CLpP, Lonp1, and Yme1L1, and improved the mitochondrial proteostasis and function in the liver in HFD-fed mice. Thus, our findings link the mitonuclear protein imbalance and UPRmt activation in the liver to mitochondrial proteostasis and MASLD prevention in response to physical exercise.

Animals↗
PubMed1992

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.

ATP-Dependent Proteases↗
PubMed2026

Plastid genome evolution and phylogenomics with broad taxon sampling: insights into intrafamilial classification of Hamamelidaceae.

Hamamelidaceae, within the order Saxifragales, comprises 27 genera and approximately 120 species. The family has a pantropical and temperate distribution across the Americas, Asia, Africa, and Australia. Previous molecular investigations, constrained by limited taxon sampling and inadequate genetic markers, supported a five-subfamily classification system. However, these studies predominantly focused on Asian taxa, resulting in poor resolution of the evolutionary relationships among American, African, and Australian genera. To address these sampling gaps, we employed near-complete generic sampling (26 of 27 genera) to investigate plastome architecture, structural variation, and phylogenetic relationships. We newly sequenced and assembled 15 plastid genomes representing geographically and taxonomically underrepresented genera and analyzed them alongside 59 publicly available plastomes retrieved from GenBank. Plastid genomes exhibited conserved quadripartite architecture with sizes ranging from 158, 076 bp to 160, 814 bp, minimal structural variation, consistent GC content (37.7-38.2%), and identical gene order. Inverted repeat (IR) regions had limited size variation (26, 211-26, 429 bp). Simple sequence repeat (SSR) distribution (2, 219 loci) showed no clear correlation with the genus-level phylogenetic relationships. We identified ten hypervariable regions, including coding sequences (accD, ycf1, clpP, ndhF, and rpl22) and intergenic spacers (rpl33-rps18, the trnG-UCC intron, trnH-GUG-psbA, accD-psaI, and petA-psbJ), as promising candidate regions for future applications in species delimitation and phylogenetic studies. Phylogenetic analyses revealed largely congruent topologies across datasets and methods, providing improved resolution and strong support for most subfamilial and tribal relationships compared with previous studies. This study highlights the utility of plastid genome data for resolving deep-level phylogenetic relationships within Hamamelidaceae. The genome architecture reflects the high conservation of plastid genomes, while the identified mutation hotspots represent potential resources for future taxonomic and phylogenetic studies. Our results support the existing subfamily classification while improving geographical coverage and generic representation, providing a robust framework for future taxonomic and evolutionary studies of this globally distributed and taxonomically complex family.

Hamamelidaceae↗
PubMed2017

The Influence of the Toxin/Antitoxin mazEF on Growth and Survival of Listeria monocytogenes under Stress.

A major factor in the resilience of Listeria monocytogenes is the alternative sigma factor B (σB). Type II Toxin/Antitoxin (TA) systems are also known to have a role in the bacterial stress response upon activation via the ClpP or Lon proteases. Directly upstream of the σB operon in L. monocytogenes is the TA system mazEF, which can cleave mRNA at UACMU sites. In this study, we showed that the mazEF TA locus does not affect the level of persister formation during treatment with antibiotics in lethal doses, but exerts different effects according to the sub-inhibitory stress added. Growth of a ΔmazEF mutant was enhanced relative to the wildtype in the presence of sub-inhibitory norfloxacin and at 42 °C, but was decreased when challenged with ampicillin and gentamicin. In contrast to studies in Staphylococcus aureus, we found that the mazEF locus did not affect transcription of genes within the σB operon, but MazEF effected the expression of the σB-dependent genes opuCA and lmo0880, with a 0.22 and 0.05 fold change, respectively, compared to the wildtype under sub-inhibitory norfloxacin conditions. How exactly this system operates remains an open question, however, our data indicates it is not analogous to the system of S. aureus, suggesting a novel mode of action for MazEF in L. monocytogenes.

Anti-Bacterial Agents↗
PubMed1988

The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component.

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.

ATP-Dependent Proteases↗