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)

Spatial control of proteolysis is emerging as a common feature of regulatory networks in bacteria. In the spore-forming bacterium Bacillus subtilis, the peptidase ClpP can associate with any of three ATPases: ClpC, ClpE, and ClpX. Here, we report that ClpCP, ClpEP, and ClpXP localize in foci often near the poles of growing cells and that ClpP and the ATPase are each capable of polar localization independently of the other component. A region of ClpC containing an AAA domain was necessary and sufficient for polar localization. We also report that ClpCP and ClpXP proteases differentially localize to the forespore and mother cell compartments of the sporangium during spore formation. Moreover, model substrates for each protease created by appending recognition sequences for ClpCP or ClpXP to the green fluorescent protein were preferentially eliminated from the forespore or the mother cell, respectively. Biased accumulation of ClpCP in the forespore may contribute to the cell-specific activation of the transcription factor sigma(F) by preferential ClpCP-dependent degradation of the anti-sigma(F) factor SpoIIAB.
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PMID:Polar localization and compartmentalization of ClpP proteases during growth and sporulation in Bacillus subtilis. 1868 76

Clp is a barrel-shaped hetero-oligomeric ATP-dependent protease comprising a hexameric ATPase (ClpX or ClpA) that unfolds protein substrates and translocates them into the central chamber of the tetradecameric proteolytic component (ClpP) where they are degraded processively to short peptides. Chamber access is controlled by the N-terminal 20 residues (for Escherichia coli) in ClpP that prevent entry of large polypeptides in the absence of the ATPase subunits and ATP hydrolysis. Remarkably, removal of 10-17 residues from the mature N-terminus allows processive degradation of a large model unfolded substrate to short peptides without the ATPase subunit or ATP hydrolysis; removal of 14 residues is maximal for activation. Furthermore, since the product size distribution of Delta14-ClpP is identical to ClpAP and ClpXP, the ATPases do not play an essential role in determining this distribution. Comparison of the structures of Delta14-ClpP and Delta17-ClpP with other published structures shows R15 and S16 are labile and that residue 17 can adopt a range of rotomers to ensure protection of a hydrophobic pocket formed by I19, R24 and F49 and maintain a hydrophilic character of the pore.
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PMID:Turned on for degradation: ATPase-independent degradation by ClpP. 1903 48

A common feature of chaperone-proteases is architectural two-fold symmetry across the proteolytic cylinder. Here we investigate the role of symmetry for the function of ClpAP and ClpXP assemblies. We generated asymmetric ClpP particles in which the two rings differ in ClpA and ClpX binding capability and/or in proteolytic activity. Rapid-kinetic fluorescence measurements and steady-state experiments indicate that single 2:1 ClpAP or ClpXP complexes are as efficient in substrate degradation as two 1:1 ClpAP or ClpXP assemblies. This implies that the two chaperone components work independently. However, an asymmetric ClpP particle composed of one active and one inactive ring can stimulate ATPase activity of ClpA regardless of whether ClpA binds to the active ring or to the opposite side of ClpP, across the ring of inactivated protease. Thus, we propose that conformational transitions in ClpP are concerted and allosteric effects are transferred simultaneously to both associated chaperones, leading to synchronized activation.
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PMID:Optimal efficiency of ClpAP and ClpXP chaperone-proteases is achieved by architectural symmetry. 1936 79

The Clp protease ATPase subunit and chaperone ClpX is dispensable in some bacteria, but it is thought to be essential in others, including streptococci and lactococci. We confirm that clpX is essential in the Rx strain of Streptococcus pneumoniae but show that the requirement for clpX can be relieved by point mutations, frame shifts, or deletion of the gene spr1630, which is found in many isolates of S. pneumoniae. Homologs occur frequently in Staphylococcus aureus as well as in a few strains of Listeria monocytogenes, Lactobacillus johnsonii, and Lactobacillus rhamnosus. In each case, the spr1630 homolog is accompanied by a putative transcriptional regulator with an HTH DNA binding motif. In S. pneumoniae, the spr1630-spr1629 gene pair, accompanied by a RUP element, occurs as an island inserted between the trpA and cclA genes in 15 of 22 sequenced genomes.
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PMID:spr1630 is responsible for the lethality of clpX mutations in Streptococcus pneumoniae. 1946 54

Chaperone-proteases are responsible for the processive breakdown of proteins in eukaryotic, archaeal and bacterial cells. They are composed of a cylinder-shaped protease lined on the interior with proteolytic sites and of ATPase rings that bind to the apical sides of the protease to control substrate entry. We present a real-time FRET-based method for probing the reaction cycle of chaperone-proteases, which consists of substrate unfolding, translocation into the protease and degradation. Using this system we show that the two alternative bacterial ClpAP and ClpXP complexes share the same mechanism: after initial tag recognition, fast unfolding of substrate occurs coinciding with threading through the chaperone. Subsequent slow substrate translocation into the protease chamber leads to formation of a transient compact substrate intermediate presumably close to the chaperone-protease interface. Our data for ClpX and ClpA support the mechanical unfolding mode of action proposed for these chaperones. The general applicability of the designed FRET system is demonstrated here using in addition an archaeal PAN-proteasome complex as model for the more complex eukaryotic proteasome.
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PMID:Studying chaperone-proteases using a real-time approach based on FRET. 1959 40

The Escherichia coli ATP-dependent protease, ClpAP, is composed of the hexameric ATPase/protein-unfoldase, ClpA, and the tetradecameric proteolytic component, ClpP. ClpP proteolytically degrades folded proteins only when associated with the motor protein ClpA or ClpX, both of which use ATP binding and/or hydrolysis to unfold and translocate proteins into the tetradecameric serine protease ClpP. In addition to ClpA's role in regulating the proteolytic activity of ClpP, ClpA catalyzes protein unfolding of proteins that display target sequences to "remodel" them, in vivo, for regulatory roles beyond proteolytic degradation. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA first requires nucleoside triphosphate binding to assemble into an oligomeric form with protein substrate binding activity. In addition to this nucleotide driven assembly activity, ClpA self-associates in the absence of nucleoside triphosphate binding. An examination of the energetics of the nucleotide driven assembly process cannot be performed without a thermodynamic model of the self-assembly process in the absence of nucleotide cofactor. Here we report an examination of the self-association properties of the E. coli ClpA protein unfoldase through the application of analytical ultracentrifugation and light scattering techniques, including sedimentation velocity, sedimentation equilibrium, and dynamic light scattering approaches. In contrast to published results, application of these approaches reveals that ClpA exists in a monomer-tetramer equilibrium (300 mM NaCl, 10 mM MgCl(2), and 25 mM HEPES, pH 7.5 at 25 degrees C). The implications of these results for the E. coli ClpA self-association and ligand linked association activities are discussed.
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PMID:The Escherichia coli ClpA molecular chaperone self-assembles into tetramers. 1965 Jun 43

ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.
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PMID:Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. 1991 67

ClpP is a serine protease whose active sites are sequestered in a cavity enclosed between two heptameric rings of subunits. The ability of ClpP to process folded protein substrates depends on its being partnered by an AAA+ ATPase/unfoldase, ClpA or ClpX. In active complexes, substrates are unfolded and fed along an axial channel to the degradation chamber inside ClpP. We have used cryoelectron microscopy at approximately 11-A resolution to investigate the three-dimensional structure of ClpP complexed with either one or two end-mounted ClpA hexamers. In the absence of ClpA, the apical region of ClpP is sealed; however, it opens on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops (residues 1-17) of ClpP, which tend to be poorly visible in crystal structures, indicative of conformational variability. Nevertheless, we were able to model the closed-to-open transition that accompanies ClpA binding in terms of movements of these loops; in particular, "up" conformations of the loops correlate with the open state. The main part of ClpP, the barrel formed by 14 copies of residues 18-193, is essentially unchanged by the interaction with ClpA. Using difference mapping, we localized the binding site for ClpA to a peripheral pocket between adjacent ClpP subunits. Based on these observations, we propose that access to the ClpP degradation chamber is controlled allosterically by hinged movements of its N-terminal loops, which the symmetry-mismatched binding of ClpA suffices to induce.
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PMID:Binding of the ClpA unfoldase opens the axial gate of ClpP peptidase. 2023 30

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.
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PMID:ClpX contributes to innate defense peptide resistance and virulence phenotypes of Bacillus anthracis. 2037 6

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.
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PMID:Validation of the essential ClpP protease in Mycobacterium tuberculosis as a novel drug target. 2212 55


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