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At least 271 records · Page 15Linked to original sources
PubMed2005

Insights into hydrophobicity and the chaperone-like function of alphaA- and alphaB-crystallins: an isothermal titration calorimetric study.

Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.

Anilino Naphthalenesulfonates↗
PubMed2005

Cyanobacterial ClpC/HSP100 protein displays intrinsic chaperone activity.

HSP100 proteins are molecular chaperones that belong to the broader family of AAA+ proteins (ATPases associated with a variety of cellular activities) known to promote protein unfolding, disassembly of protein complexes and translocation of proteins across membranes. The ClpC form of HSP100 is an essential, highly conserved, constitutively expressed protein in cyanobacteria and plant chloroplasts, and yet little is known regarding its specific activity as a molecular chaperone. To address this point, ClpC from the cyanobacterium Synechococcus elongatus (SyClpC) was purified using an Escherichia coli-based overexpression system. Recombinant SyClpC showed basal ATPase activity, similar to that of other types of HSP100 protein in non-photosynthetic organisms but different to ClpC in Bacillus subtilis. SyClpC also displayed distinct intrinsic chaperone activity in vitro, first by preventing aggregation of unfolded polypeptides and second by resolubilizing and refolding aggregated proteins into their native structures. The refolding activity of SyClpC was enhanced 3-fold in the presence of the B. subtilis ClpC adaptor protein MecA. Overall, the distinctive ClpC protein in photosynthetic organisms indeed functions as an independent molecular chaperone, and it is so far unique among HSP100 proteins in having both "holding" and disaggregase chaperone activities without the need of other chaperones or adaptor proteins.

Adenosine Triphosphatases↗
PubMed2006

The Hsp70 chaperone Ssq1p is dispensable for iron-sulfur cluster formation on the scaffold protein Isu1p.

The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.

Amino Acid Motifs↗
PubMed2006

The C-terminal domain of Escherichia coli trigger factor represents the central module of its chaperone activity.

In bacteria, ribosome-bound Trigger Factor assists the folding of newly synthesized proteins. The N-terminal domain (N) of Trigger Factor mediates ribosome binding, whereas the middle domain (P) harbors peptidyl-prolyl isomerase activity. The function of the C-terminal domain (C) has remained enigmatic due to structural instability in isolation. Here, we have characterized a stabilized version of the C domain (C(S)), designed on the basis of the recently solved atomic structure of Trigger Factor. Strikingly, only the isolated C(S) domain or domain combinations thereof (NC(S), PC(S)) revealed substantial chaperone activity in vitro and in vivo. Furthermore, to disrupt the C domain without affecting the overall Trigger Factor structure, we generated a mutant (Delta53) by deletion of the C-terminal 53 amino acid residues. This truncation caused the complete loss of the chaperone activity of Trigger Factor in vitro and severely impaired its function in vivo. Therefore, we conclude that the chaperone activity of Trigger Factor critically depends on its C-terminal domain as the central structural chaperone module. Intriguingly, a structurally similar module is found in the periplasmic chaperone SurA and in MPN555, a protein of unknown function. We speculate that this conserved module can exist solely or in combination with additional domains to fulfill diverse chaperone functions in the cell.

Binding Sites↗
PubMed2006

Chaperone properties of mammalian mitochondrial translation elongation factor Tu.

The main function of the prokaryotic translation elongation factor Tu (EF-Tu) and its eukaryotic counterpart eEF1A is to deliver aminoacyl-tRNA to the A-site on the ribosome. In addition to this primary function, it has been reported that EF-Tu from various sources has chaperone activity. At present, little information is available about the chaperone activity of mitochondrial EF-Tu. In the present study, we have examined the chaperone function of mammalian mitochondrial EF-Tu (EF-Tumt). We demonstrate that recombinant EF-Tumt prevents thermal aggregation of proteins and enhances protein refolding in vitro and that this EF-Tumt chaperone activity proceeds in a GTP-independent manner. We also demonstrate that, under heat stress, the newly synthesized peptides from the mitochondrial ribosome specifically co-immunoprecipitate with EF-Tumt and are destabilized in EF-Tumt-overexpressing cells. We show that most of the EF-Tumt localizes on the mitochondrial inner membrane where most mitochondrial ribosomes are found. We discuss the possible role of EF-Tumt chaperone activity in protein quality control in mitochondria, with regard to the recently reported in vivo chaperone function of eEF1A.

Animals↗
PubMedDate not supplied

Chaperone-assisted folding of newly synthesized proteins in the cytosol.

The way in which a newly synthesized polypeptide chain folds into its unique three-dimensional structure remains one of the fundamental questions in molecular biology. Protein folding in the cell is a problematic process and, in many cases, requires the assistance of a network of molecular chaperones to support productive protein foldingin vivo. During protein biosynthesis, ribosome-associated chaperones guide the folding of the nascent polypeptide emerging from the ribosomal tunnel. In this review we summarize the basic principles of the protein-folding process and the involved chaperones, and focus on the role of ribosome-associated chaperones. Our discussion emphasizes the bacterial Trigger Factor, which is the best studied chaperone of this type. Recent advances have determined the atomic structure of the Trigger Factor, providing new, exciting insights into the role of ribosome-associated chaperones in co-translational protein folding.

Animals↗
PubMed2003

Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function?

Small heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow, off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsps and clusterin accompanies a range of diseases that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils, e.g., Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases. The interaction of sHsps and clusterin with fibril-forming species is discussed along with their ability to prevent fibril formation.

Animals↗
PubMed2003

Polyglutamine diseases and molecular chaperones.

The polyglutamine diseases, a group of diseases currently thought to consist of nine inherited neurodegenerative diseases, are caused by the expansion of unstable CAG trinucleotide repeats that code for polyglutamine tracts in the responsible genes. These diseases are now recognized as being of a type with conformationally abnormal or amyloid-related proteins, and thus are called 'conformational diseases'. Recently, many studies using cell cultures and model organisms have suggested that the two major machineries for protein quality control (the molecular chaperone and the protein degradation machineries) play important roles in the pathogenesis of the polyglutamine diseases. Interestingly, molecular chaperones have been shown to behave in totally different ways in these studies, namely in suppressing as well as enhancing neurodegeneration or cell death. These apparently opposite actions of molecular chaperones suggest that a certain balance between the activities of molecular chaperones and the expression level of polyglutamine is an important determinant of the pathogenesis. In this review, we summarize recent findings on such ambiguous effects of molecular chaperones on polyglutamine diseases, and discuss possible mechanisms by which molecular chaperones, especially VCP, are involved in the pathogenesis.

Humans↗
PubMed2003

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins.

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.

Adenosine Triphosphate↗
PubMed2004

Chaperone-assisted translocation.

We investigate the translocation of a stiff polymer through a nanopore in a membrane, in the presence of binding particles (chaperones) that bind reversibly to the polymer on both sides of the membrane. A bound chaperone covers one (univalent binding) or many (multivalent binding) binding sites. Assuming that the diffusion of the chaperones is fast compared to the rate of translocation we describe the process by a one-dimensional master equation. We expand previous models by a detailed study of the effective force in the master equation, which is obtained by the appropriate statistical mechanical average over the chaperone states. The dependence of the force on the degree of valency (the number of binding sites occupied by a chaperone) is studied in detail. We obtain finite size corrections (to the thermodynamical expression for the force), which, for univalent binding, can be expressed analytically. We finally investigate the mean velocity for translocation as a function of chaperone binding strength and size. For both univalent and multivalent binding simple results are obtained for the case of a sufficiently long translocating polymer.

Binding Sites↗
PubMed1997

Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp70 was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp70 and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp70 and, perhaps, the hsp90 families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.

Anti-Bacterial Agents↗
PubMed2005

BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP.

Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.

Amino Acid Sequence↗
PubMed2000

Role of the heat shock response and molecular chaperones in oncogenesis and cell death.

Exposure of cells to conditions of environmental stress-including heat shock, oxidative stress, heavy metals, or pathologic conditions, such as ischemia and reperfusion, inflammation, tissue damage, infection, and mutant proteins associated with genetic diseases-results in the inducible expression of heat shock proteins that function as molecular chaperones or proteases. Molecular chaperones are a class of proteins that interact with diverse protein substrates to assist in their folding, with a critical role during cell stress to prevent the appearance of folding intermediates that lead to misfolded or otherwise damaged molecules. Consequently, heat shock proteins assist in the recovery from stress either by repairing damaged proteins (protein refolding) or by degrading them, thus restoring protein homeostasis and promoting cell survival. The events of cell stress and cell death are linked, such that molecular chaperones induced in response to stress appear to function at key regulatory points in the control of apoptosis. On the basis of these observations-and on the role of molecular chaperones in the regulation of steroid aporeceptors, kinases, caspases, and other protein remodeling events involved in chromosome replication and changes in cell structure-it is not surprising that the heat shock response and molecular chaperones have been implicated in the control of cell growth. In this review, we address some of the molecular and cellular events initiated by cell stress-the interrelationships between stress signaling, cell death, and oncogenesis-and chaperones as potential targets for cancer diagnosis and treatment.

Animals↗
PubMed2006

The triage of damaged proteins: degradation by the ubiquitin-proteasome pathway or repair by molecular chaperones.

Accumulation of damaged proteins is causally related to many age-related diseases. The ubiquitin-proteasome pathway (UPP) plays a role in selective degradation of damaged proteins, whereas molecular chaperones, such as heat shock proteins, are involved in refolding denatured proteins. This work demonstrates for the first time that the UPP and molecular chaperones work in a competitive manner and that the fates of denatured proteins are determined by the relative activities of the UPP and molecular chaperones. Enhanced UPP activity suppresses the refolding of denatured proteins whereas elevated chaperone activity inhibits the degradation of denatured proteins. CHIP, a co-chaperone with E3 activity, plays a pivotal role in determining the fates of the damaged proteins. The delicate balance between UPP-mediated degradation and refolding of denatured proteins is governed by relative levels of CHIP and other molecular chaperones. Isopeptidases, the enzymes that reverse the actions of CHIP, also play an important role in determining the fate of denatured proteins.

Animals↗
PubMed2005

Genetic disorders involving molecular-chaperone genes: a perspective.

Molecular chaperones are important for maintaining a functional set of proteins in all cellular compartments. Together with protein degradation machineries (e.g., the ubiquitin-proteasome system), chaperones form the core of the cellular protein-quality control mechanism. Chaperones are proteins, and as such, they can be affected by mutations. At least 15 disorders have been identified that are associated with mutations in genes encoding chaperones, or molecules with features suggesting that they function as chaperones. These chaperonopathies and a few other candidates are presented in this article. In most cases, the mechanisms by which the defective genes contribute to the observed phenotypes are still uncharacterized. However, the reported observations definitely point to the possibility that abnormal chaperones participate in pathogenesis. The available data open novel perspectives and should encourage searches for new genetic chaperonopathies, as well as further analyses of the disorders discussed in this article, including detection of new cases.

Animals↗
PubMed2005

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector.

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1' was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1', suggesting that ShcS1 is a TTS chaperone for HopS1' and that amino acids 1 to 118 of HopS1' are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1' :: AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1' and HopO1-1, that share only 16% amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

Amino Acid Sequence↗
PubMed2000

Structural changes in alpha-synuclein affect its chaperone-like activity in vitro.

Alpha-synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of alpha-synuclein has not yet been established. We here present biochemical evidence that recombinant alpha-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, alpha-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and alpha-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of alpha-synuclein, which is believed to reorganize the molecular surface of alpha-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, alpha-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, alpha-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that alpha-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that alpha-synuclein could act as a molecular chaperone, and that the conformational change of the alpha-synuclein could explain the aggregation kinetics of alpha-synuclein, which may be related to the abolishment of the chaperonic-like activity.

Albumins↗
PubMed1996

Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome.

Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclear-encoded mitochondrial proteins and of proteins encoded by mitochondrial DNA. Although the regulation of heat-shock genes has been the subject of considerable investigation, regulation of the genes encoding mitochondrial chaperones is not well defined. We have found that stress applied specifically to the mitochondria of mammalian cells is capable of eliciting an organelle-specific, molecular chaperone response. Using the loss of mitochondrial DNA as a means of producing a specific mitochondrial stress, we show by Western-blot analysis that mtDNA-less (rho 0) rat hepatoma cells show an increase in the steady-state levels of chaperonin 60 (cpn 60) and chaperonin 10 (cpn 10). Nuclear transcription assays show that the upregulation of these chaperones is due to transcriptional activation. There was no effect on the inducible cytosolic Hsp 70, Hsp 72, nor on mtHsp 70 in rho 0 cells, leading us to concluded that stress applied selectively to mitochondria elicits a specific molecular chaperone response. Heat stress was able to provide an additional induction of cpn 60 and cpn 10 above that obtained for the rho 0 state alone, indicating that these genes have separate regulatory elements for the specific mitochondrial and general stress responses. Since the mitochondrial-specific chaperones are encoded by nuclear DNA, there must be a mechanism for molecular communication between the mitochondrion and nucleus and this system can address how stress is communicated between these organelles.

Animals↗