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PubMed2003

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity.

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.

Adenosine Triphosphatases↗

PubMed2001

The measles virus (MV) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and MV infection upregulates chaperone expression.

The present study examines the coprecipitation of measles virus (MV) glycoproteins with host cell endoplasmic reticulum (ER) chaperone proteins. Both the haemagglutinin (H) and fusion (F) glycoproteins interacted with calnexin and GRP78, whereas interaction with calreticulin was only demonstrated for the H glycoprotein. The alpha-glucosidase inhibitor castanospermine reduced and delayed the association of F proteins with calnexin. We have previously shown that alpha-glucosidase activity is important for the functionality and antigenicity of the MV F glycoprotein and for release of MV particles from infected cells. Thus, interaction with calnexin appears vital for processing of nascent MV F protein into its functional conformation. In contrast to many other viral glycoproteins, a substantial proportion of the pulsed MV glycoproteins remained associated with ER chaperones for more than 2(1/2) h. Thus, the slow and incomplete migration of MV glycoproteins to the cell surface may result from their retention by ER chaperones, probably due to malfolding. MV infection upregulated the cellular expression of calreticulin and GRP78 and also increased their presence at the cell surface. The chaperone proteins are involved in a wide range of cellular processes, and their induction by MV may play a role for the pathogenesis of measles and its sequelae.

Calcium-Binding Proteins↗

PubMed1998

Multiple molecular chaperones interact with apolipoprotein B during its maturation. The network of endoplasmic reticulum-resident chaperones (ERp72, GRP94, calreticulin, and BiP) interacts with apolipoprotein b regardless of its lipidation state.

The present study was undertaken to identify and characterize molecular chaperones that assist in the folding of apolipoprotein (apo) B, a secretory protein that requires assembly with lipids (lipidation) for its secretion. Both HepG2 cells, normally secreting full-length apoB (apoB-100), and C127 cells transfected to secrete truncated forms of apoB, apoB-41, apoB-29, and apoB-17, respectively, were employed. C127 cells were used to determine whether chaperone binding is dependent on apoB lipidation as they secrete both unlipidated and lipidated apoB forms despite their lack of microsomal triglyceride transfer protein (MTP), which mediates lipidation of apoB in HepG2 cells. The endoplasmic reticulum (ER)-resident molecular chaperones GRP94, calreticulin, and ERp72 were co-immunoprecipitated with apoB-100 from HepG2 cell lysates following cross-linking of proteins in living cells. The same chaperones including BiP/GRP78 were also associated with all truncated forms of apoB. Sequential immunoprecipitation with antibodies to MTP and apoB revealed the presence of ternary complexes containing apoB-100, MTP, and ERp72. However, MTP is not obligatory for the binding of ERp72 as it was associated with all truncated forms of apoB in C127 cells that lack MTP. The interactions between apoB-100 and ERp72 or GRP94 persisted for at least 2 h following a 30-min pulse. Thus, BiP/GRP78, calreticulin, ERp72, and GRP94 may participate in critical steps in the folding of apoB before any substantial lipidation occurs. ERp72 and GRP94 may also mediate the folding of more advanced folding intermediates and/or target the misfolded underlipidated pool of apoB for degradation.

Animals↗

PubMed1998

Enzymes as chaperones and chaperones as enzymes.

Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into functional proteins in cells. Protein disulfide isomerase, a foldase, and ATP-dependent proteases, responsible for degradation of misfolded proteins in cells, both have intrinsic chaperone activities. Trigger factor and DnaJ, well known Escherichia coli chaperones, show peptidyl prolyl isomerase and protein disulfide isomerase activities respectively. It is suggested that the combination of chaperone and enzyme activities in one molecule is the result of evolution to increase molecular efficiency.

ATP-Dependent Proteases↗

PubMed2000

Heat shock cognate protein 70 chaperone-binding site in the co-chaperone murine stress-inducible protein 1 maps to within three consecutive tetratricopeptide repeat motifs.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone homologous with the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). The concomitant interaction of mSTI1 with hsp70 and hsp90 at its N- and C-termini respectively is mediated by the tetratricopeptide repeat (TPR) motifs in these regions. With the use of co-precipitation assays, we show here that the N-terminal TPR domain of mSTI1 without extensive flanking regions is both necessary and sufficient to mediate a specific interaction with hsc70. In contrast, other TPR-containing co-chaperones require TPR flanking regions for target substrate recognition, suggesting different mechanisms of TPR-mediated chaperone-co-chaperone interactions. Furthermore, the interaction between mSTI1 and hsc70 was analysed to ascertain the effect of replacing or deleting conserved amino acid residues and sequences within the three TPR motifs constituting the N-terminal TPR domain of full-length mSTI1. Replacement of a bulky hydrophobic residue in TPR1 disrupted the interaction of mSTI1 with hsc70. A highly conserved sequence in TPR2 was altered by deletion or single amino acid replacement. These derivatives retained a specific interaction with hsc70. These results are consistent with a model in which conserved residues within the N-terminal TPR region of mSTI1 contribute differentially to the interaction with hsc70, and in which TPR1 has a significant role in targeting mSTI1 to hsc70. The contribution of the TPR domain mutations and deletions are discussed with respect to their effect on target substrate interactions.

Amino Acid Motifs↗

PubMed1998

Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE.

The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM). However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ. substrate.DnaK.ADP.Pi complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.

Adenosine Triphosphate↗

PubMed1999

Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity.

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.

Adenosine Triphosphatases↗

PubMed1999

Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino- and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication.

The first discovery of an Hsp70 chaperone gene was the isolation of an Escherichia coli mutant, dnaK756, which rendered the cells resistant to lytic infection with bacteriophage lambda. The DnaK756 mutant protein has since been used to establish many of the cellular roles and biochemical properties of DnaK. DnaK756 has three glycine-to-aspartate substitutions at residues 32, 455, and 468, which were reported to result in defects in intrinsic and GrpE-stimulated ATPase activities, substrate binding, stability of the substrate-binding domain, interdomain communication, and, consequently, defects in chaperone activity. To dissect the effects of the different amino acid substitutions in DnaK756, we analyzed two DnaK variants carrying only the amino-terminal (residue 32) or the two carboxyl-terminal (residues 455 and 468) substitutions. The amino-terminal substitution interfered with the GrpE-stimulated ATPase activity. The carboxyl-terminal mutations (i) affected stability and function of the substrate-binding domain, (ii) caused a 10-fold elevated ATP hydrolysis rate, but (iii) did not severely affect domain coupling. Surprisingly, DnaK chaperone activity was more severely compromised by the amino-terminal than by the carboxyl-terminal amino acid substitutions both in vivo and in vitro. In the in vitro refolding of denatured firefly luciferase, the defect of the DnaK variant carrying the amino-terminal substitution results from its inability to release, upon GrpE-mediated nucleotide exchange, bound luciferase in a folding competent state. Our results indicate that the DnaK-DnaJ-GrpE chaperone system can tolerate suboptimal substrate binding, whereas the tight kinetic control of substrate dissociation by GrpE is essential.

Adenosine Diphosphate↗

PubMed2000

Folding pathway mediated by an intramolecular chaperone. The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked.

The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Upon completion of folding, subtilisin initiates IMC degradation to complete precursor maturation. Existing data suggest that the chaperone and inhibitory functions of the subtilisin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to disrupt three hydrogen bonds that stabilize the interface between the protease and its IMC domain. This mutation within subtilisin does not alter the folding kinetics but dramatically slows down autoprocessing of the IMC domain. Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-fold weaker than wild-type subtilisin. Although the IMC domain displays substantial loss of inhibitory function, its ability to chaperone E112A-subtilisin folding remains intact. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii) the Glu(112) residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin.

Catalysis↗

PubMed2003

Folding pathway mediated by an intramolecular chaperone. A functional peptide chaperone designed using sequence databases.

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

Algorithms↗

PubMed2006

The effect of aging on the chaperone concentrations in the hepatic, endoplasmic reticulum of male rats: the possible role of protein misfolding due to the loss of chaperones in the decline in physiological function seen with age.

The endoplasmic reticulum (ER) chaperones are highly conserved proteins that catalyze the posttranslational processing of all secretory and membrane proteins. Our studies suggest that chaperone declines are one of the two central defects in Alzheimer's disease. We propose that similar declines in other organ systems underlie the physiological deficits of aging. Rats were maintained in a colony from age 21 days to death. Animals were killed at regular intervals, and hepatic, ER chaperone contents were determined by immunoblotting. ERp55, ERp57, ERp72, BiP, and calnexin constitutive levels declined 30%-50% with age. Calreticulin was unaffected. BiP (also known as GRP78), ERp55, and ERp57 showed marked swings with peaks occurring in midwinter and midsummer. This cyclics declined 73% with age. Considering the role of the ER chaperones in membrane and secretory protein posttranslational processing, these data support the concept that their loss could lead to many of the physiological declines associated with aging.

Aging↗

PubMed2006

Chaperone and anti-chaperone: two-faced synuclein as stimulator of synaptic evolution.

Previous studies have shown that beta-synuclein (beta-syn), the homologue of alpha-syn, inhibited alpha-syn aggregation and stabilized Akt cell survival signaling molecule, suggesting that beta-syn was protective against alpha-syn-related neurodegenerative disorders, such as Parkinson's disease and diffuse Lewy body disease. However, emerging evidence argues that the situation may be not so simple. Two missense mutations of beta-syn were identified in familial and sporadic diffuse Lewy body disease, and wild type beta-syn was induced to form fibril structures in vitro, while, alpha-syn was shown to be protective against neurodegeneration caused by deletion of cysteine-string protein-alpha, the presynaptic cochaperone to Hsc70 in mice. Collectively, alpha- and beta-syn are both, but in varying degrees, featured with two opposite properties, namely normal chaperone and anti-chaperone. By reviewing recent progress in syn biology with a particular focus on beta-syn, this manuscript refers to the intriguing possibility that the dual syn proteins might have acquired a driving force for synaptic evolution. Hypothetically, the anti-chaperone syn may provoke stress-induced diverse responses, whereas, the chaperone syn may provide buffering for them, allowing accumulation of nonlethal phenotypic variations in synapses. Consequently, dual syn proteins may cope with forthcoming stresses in the brain by stimulating adaptive evolution. In this context, failure to regulate this process due to various causes, such as gene mutations and environmental risk factors, may result in imperfect adaptability against stresses, leading to neurodegenerative disorders.

Animals↗

PubMed1996

Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.

Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple chaperone components that maintain substrates in an intermediate folded state.

Amino Acid Isomerases↗

PubMed2005

Chaperon and adjuvant activity of hsp70: different natural killer requirement for cross-priming of chaperoned and bystander antigens.

Heat shock proteins (HSP) convey both chaperoned propeptide and danger signal to dendritic cells (DC). However, few studies have compared the two activities. Using a murine inducible hsp70 secreted by cells distinct from those providing the tumor antigens, we showed that hsp70 exerts efficacious adjuvant effects toward DC cross-priming. Hsp70 induces DC maturation and phagocytosis of cellular debris both in vitro and in vivo, which are conducive to CTL response to chaperoned and nonchaperoned antigens. Whereas the ability of hsp70 to induce cross-presentation of chaperoned peptides is natural killer (NK) independent, the adjuvant activity requires NK cells at the site of DC-hsp70 interaction to induce CTL response and therapeutic effect against lung metastases. However, although bystander activity provides equal CTL induction, the best therapeutic efficacy rests on cell vaccine secreting hsp70 that combines chaperoned antigen and danger signal within the same cell.

Adenocarcinoma↗

PubMed1994

Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.

Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit "anti-chaperone" activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).

Animals↗

PubMed2004

The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle.

The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.

Adenosine Triphosphatases↗

PubMed1998

NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction.

The solution structure of the 21 kDa substrate-binding domain of the Escherichia coli Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision of 1.00 A (backbone of the beta-domain) from 1075 experimental restraints obtained from multinuclear, multidimensional NMR experiments. The domain is observed to bind to its own C-terminus and offers a preview of the interaction of this chaperone with other proteins. The bound protein region is tightly held at a single amino acid position (a leucyl residue) that is buried in a deep pocket lined with conserved hydrophobic residues. A second hydrophobic binding site was identified using paramagnetically labeled peptides. It is located in a region close to the N-terminus of the domain and may constitute the allosteric region that links substrate-binding affinity with nucleotide binding in the Hsp70 chaperones.

Amino Acid Sequence↗

PubMed2004

Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture.

The 17-kDa protein (Skp) of Escherichia coli is a homotrimeric periplasmic chaperone for newly synthesized outer-membrane proteins. Here we present its X-ray structure at a resolution of 2.35 A. Three hairpin-shaped alpha-helical extensions reach out by approximately 60 A from a trimerization domain, which is composed of three intersubunit beta-sheets that wind around a central axis. The alpha-helical extensions approach each other at their distal turns, resulting in a fold that resembles a 'three-pronged grasping forceps'. The overall shape of Skp is reminiscent of the cytosolic chaperone prefoldin, although it is based on a radically different topology. The peculiar architecture, with apparent plasticity of the prongs and distinct electrostatic and hydrophobic surface properties, supports the recently proposed biochemical mechanism of this chaperone: formation of a Skp(3)-Omp complex protects the outer membrane protein from aggregation during passage through the bacterial periplasm.

Amino Acid Sequence↗