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

The oligomeric AAA+ chaperone Hsp104 is essential for thermotolerance development and prion propagation in yeast. Thermotolerance relies on the ability of Hsp104 to cooperate with the Hsp70 chaperone system in the reactivation of heat-aggregated proteins. Prion propagation requires the Hsp104-dependent fragmentation of prion fibrils to create infectious seeds. It remained elusive whether both processes rely on common or different activities of Hsp104. Specifically, protein reactivation has been suggested to require a substrate threading activity of Hsp104 whereas fibril fragmentation may be mediated by a crowbar activity. Here we engineered an Hsp104 variant, HAP, which cooperates with the bacterial peptidase ClpP to form a novel proteolytic system. HAP threads aggregated model substrates as well as the yeast prion Sup35 through its central pore into associated ClpP. HAP variants that harbour a reduced threading activity were affected in both protein disaggregation and prion propagation, demonstrating that substrate threading represents the common mechanism for the processing of both substrate classes.
Mol Microbiol 2008 Apr
PMID:Substrate threading through the central pore of the Hsp104 chaperone as a common mechanism for protein disaggregation and prion propagation. 1831 64

ClpX, an archetypal proteolytic AAA+ unfoldase, must engage the ssrA tags of appropriate substrates prior to ATP-dependent unfolding and translocation of the denatured polypeptide into ClpP for degradation. Here, specificity-transplant and disulfide-crosslinking experiments reveal that the ssrA tag interacts with different loops that form the top, middle, and lower portions of the central channel of the ClpX hexamer. Our results support a two-step binding mechanism, in which the top loop serves as a specificity filter and the remaining loops form a binding site for the peptide tag relatively deep within the pore. Crosslinking experiments suggest a staggered arrangement of pore loops in the hexamer and nucleotide-dependent changes in pore-loop conformations. This mechanism of initial tag binding would allow ATP-dependent conformational changes in one or more pore loops to drive peptide translocation, force unfolding, and mediate threading of the denatured protein through the ClpX pore.
Mol Cell 2008 Feb 29
PMID:Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates. 1831 82

ClpP and its ATPase compartment, ClpX or ClpA, remove misfolded proteins in cells and are of utmost importance in protein quality control. The ring hexamers of ClpA or ClpX recognize, unfold, and translocate target substrates into the degradation chamber of the double-ring tetradecamer of ClpP. The overall reaction scheme catalyzed by ClpXP or ClpAP has been proposed; however, the molecular mechanisms associated with substrate recognition and degradation have not yet been clarified in detail. To investigate these mechanisms, we determined the crystal structures of ClpP from Helicobacter pylori in complex with product peptides bound to the active site as well as in the apo state. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, thus providing structural explanations for the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. The structures also suggest that substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP.
J Mol Biol 2008 Jun 13
PMID:The structural basis for the activation and peptide recognition of bacterial ClpP. 1846 23

ATP-dependent protein degradation in bacteria is carried out by barrel-shaped proteases architecturally related to the proteasome. In Escherichia coli, ClpP interacts with two alternative ATPases, ClpA or ClpX, to form active protease complexes. ClpAP and ClpXP show different but overlapping substrate specificities. ClpXP is considered the primary recipient of ssrA-tagged substrates while ClpAP in complex with ClpS processes N-end rule substrates. Notably, in its free form, but not in complex with ClpS, ClpAP also degrades ssrA-tagged substrates and its own chaperone component, ClpA. To reveal the mechanism of ClpAP-mediated ClpA degradation, termed autodegradation, and its possible role in regulating ClpAP levels, we dissected ClpA to show that the flexible C-terminus of the second AAA module serves as the degradation signal. We demonstrate that ClpA becomes largely resistant to autodegradation in the absence of its C-terminus and, conversely, transfer of the last 11 residues of ClpA to the C-terminus of green fluorescent protein (GFP) renders GFP a substrate of ClpAP. This autodegradation tag bears similarity to the ssrA-tag in its degradation behavior, displaying similar catalytic turnover rates when coupled to GFP but a twofold lower apparent affinity constant compared to ssrA-tagged GFP. We show that, in analogy to the prevention of ssrA-mediated recognition, the adaptor ClpS inhibits autodegradation by a specificity switch as opposed to direct masking of the degradation signal. Our results demonstrate that in the presence of ssrA-tagged substrates, ClpA autodegradation will be competitively reduced. This simple mechanism allows for dynamic reallocation of free ClpAP versus ClpAPS in response to the presence of ssrA-tagged substrates.
J Mol Biol 2008 Dec 12
PMID:An intrinsic degradation tag on the ClpA C-terminus regulates the balance of ClpAP complexes with different substrate specificity. 1883 67

During the development of transformability (competence), Bacillus subtilis synthesizes a set of proteins that mediate both the uptake of DNA at the cell poles and the recombination of this DNA with the resident chromosome. Most, if not all, of these Com proteins localize to the poles of the cell, where they associate with one another, and are then seen to delocalize as transformability declines. In this study, we use fluorescence microscopy to analyse the localization and delocalization processes. We show that localization most likely occurs by a diffusion-capture mechanism, not requiring metabolic energy, whereas delocalization is prevented in the presence of sodium azide. The kinetics of localization suggest that this process requires the synthesis of a critical protein or set of proteins, which are needed to anchor the Com protein complex to the poles. We further show that the protein kinase proteins McsA and McsB are needed for delocalization, as are ClpP and either of the AAA(+) (ATPases associated with a variety of cellular activities) proteins ClpC or ClpE. Of these proteins, at least McsB, ClpC and ClpP localize to the cell poles of competent cells. Our evidence strongly suggests that delocalization depends on the degradation of the postulated anchor protein(s) by the McsA-McsB-(ClpC or ClpE)-ClpP protease in an ATP-dependent process that involves the autophosphorylation of McsB. The extent of cell-pole association at any given time reflects the relative rates of localization and delocalization. The kinetics of this dynamic process differs for individual Com proteins, with the DNA-binding proteins SsbB and DprA exhibiting less net localization.
Mol Microbiol 2009 Apr
PMID:McsA and B mediate the delocalization of competence proteins from the cell poles of Bacillus subtilis. 1922 26

The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli: Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway.
Mol Microbiol 2009 Apr
PMID:ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway. 1931 33

The AAA(+) (ATPases associated with a variety of cellular activities) superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility and conformational exchange on the microsecond-to-millisecond timescale. The electrostatic surface of N-ClpCR differs substantially from the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC.
J Mol Biol 2009 Apr 03
PMID:Structural and motional contributions of the Bacillus subtilis ClpC N-domain to adaptor protein interactions. 1936 34

The yeast AAA(+) chaperone Hsp104 is essential for the development of thermotolerance and for the inheritance of prions. Recently, Hsp104, together with the actin cytoskeleton, has been implicated in the asymmetric distribution of carbonylated proteins. Here, we investigated the interplay between Hsp104 and actin by using a dominant-negative variant of Hsp104 (HAP/ClpP) that degrades substrate proteins instead of remodeling them. Coexpression of HAP/ClpP causes defects in morphology and the actin cytoskeleton. Taking a candidate approach, we identified Spa2, a member of the polarisome complex, as an Hsp104 substrate. Furthermore, we provided genetic evidence that links Spa2 and Hsp104 to Hof1, a member of the cytokinesis machinery. Spa2 and Hof1 knockout cells are affected in the asymmetric distribution of damaged proteins, suggesting that Hsp104, Spa2, and Hof1 are members of a network controlling the inheritance of carbonylated proteins.
Mol Cell Biol 2009 Jul
PMID:The yeast AAA+ chaperone Hsp104 is part of a network that links the actin cytoskeleton with the inheritance of damaged proteins. 1939 83

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.
Mol Cell Proteomics 2009 Aug
PMID:Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism. 1942 72

Haemosporidian parasites of birds and mammals reproduce asexually inside nucleated and nonnucleated host erythrocytes, respectively. Because of these different parasite environments and because bird parasites are paraphyletic, we evaluated whether patterns of parasite molecular evolution differ between host groups. We compared two mitochondrial (mt) genes and one apicoplast gene across mammal Plasmodium, bird Plasmodium, and bird Parahaemoproteus. Using a molecular phylogenetic approach, we show that the parasite mt cytochrome b (cyt b), mt cytochrome oxidase I (COI), and the apicoplast caseinolytic protease C (ClpC) exhibit similar levels of sequence divergence, yet each gene tree presents a strikingly different pattern of internal versus terminal branch lengths. In cyt b, the ratio of nonsynonymous (NS)-to-synonymous substitutions (d(N)/d(S)) is markedly elevated along the internal branch linking mammalian and avian parasites despite the sister relationship between mammal and bird Plasmodium. This is not the case for either COI or ClpC. When NS substitutions are excluded from the parasite cyt b alignment, the resulting phylogenetic tree resembles that of COI (both with and without NS substitutions). The high d(N)/d(S) ratio in the cyt b branch separating avian and mammalian parasites and a mammal-parasite codon bias suggest that adaptive evolution has distinguished mammal and bird parasites.
Mol Biol Evol 2010 Mar
PMID:Comparative gene evolution in haemosporidian (apicomplexa) parasites of birds and mammals. 1993 37


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