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J Buchner

Publications and source records attributed to J Buchner.

At least 37 records · Page 2Linked to original sources
PubMed1999

An unstructured C-terminal region of the Hsp90 co-chaperone p23 is important for its chaperone function.

p23 is a co-chaperone of the heat shock protein Hsp90. p23 binds to Hsp90 in its ATP-bound state and, on its own, interacts specifically with non-native proteins. In our attempt to correlate these functions to specific regions of p23 we have identified an unstructured region in p23 that maps to the C-terminal part of the protein sequence. This unstructured region is dispensible for interaction of p23 with Hsp90, since truncated p23 can still form complexes with Hsp90. In contrast, however, truncation of the C-terminal 30 amino acid residues of p23 affects the ability of p23 to bind non-native proteins and to prevent their non-specific aggregation. The isolated C-terminal region itself is not able to act as a chaperone nor is it possible to complement truncated p23 by addition of this peptide. These results imply that the binding site for Hsp90 is contained in the folded domain of p23 and that for efficient interaction of p23 with non-native proteins both the folded domain and the C-terminal unstructured region are required.

Adenosine Triphosphate↗
PubMed1999

Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.

The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein. Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.

Antibodies, Monoclonal↗
PubMed1999

BiP-binding sequences in HIV gp160. Implications for the binding specificity of bip.

BiP, a resident endoplasmic reticulum member of the HSP70 family of molecular chaperones, associates transiently with a wide variety of newly synthesized exocytotic proteins. In addition to immunoglobulin heavy and light chains, the first natural substrates identified for BiP, a number of viral polypeptides including the human immunodeficiency virus type 1 envelope glycoprotein gp160 interact with BiP during their passage through the endoplasmic reticulum. We have used a computer algorithm developed to predict BiP-binding sites within protein primary sequences to identify sites within gp160 that might mediate its association with BiP. Analysis of the ability of 22 synthetic heptapeptides corresponding to predicted binding sites to stimulate the ATPase activity of BiP or to compete with an unfolded polypeptide for binding to BiP indicated that about half of them are indeed recognized by the chaperone. All of the confirmed binding sites are localized within conserved regions of gp160, suggesting a conserved role for BiP in the folding of gp160. Information on the characteristics of confirmed BiP-binding peptides gained in this and previous studies has been utilized to improve the predictive power of the BiP Score algorithm and to investigate the differences in peptide binding specificities of HSP70 family members.

Adenosine Triphosphatases↗
PubMed1999

Analysis of GroE-assisted folding under nonpermissive conditions.

The molecular chaperones GroEL and GroES facilitate protein folding in an ATP-dependent manner under conditions where no spontaneous folding occurs. It has remained unknown whether GroE achieves this by a passive sequestration of protein inside the GroE cavity or by changing the folding pathway of a protein. Here we used citrate synthase, a well studied model substrate, to discriminate between these possibilities. We demonstrate that GroE maintains unfolding intermediates in a state that allows productive folding under nonpermissive conditions. During encapsulation of non-native protein inside GroEL.GroES complexes, a folding reaction takes place, generating association-competent monomeric intermediates that are no longer recognized by GroEL. Thus, GroE shifts folding intermediates to a productive folding pathway under heat shock conditions where even the native protein unfolds in the absence of GroE.

Chaperonin 10↗
PubMed1999

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.

Amino Acid Substitution↗
PubMed1999

Catalysis, commitment and encapsulation during GroE-mediated folding.

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspecific aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE. Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to define basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the efficient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.

ATP-Binding Cassette Transporters↗
PubMed1999

The dynamics of Hsp25 quaternary structure. Structure and function of different oligomeric species.

Small heat shock proteins (sHsps), including alpha-crystallin, represent a conserved and ubiquitous family of proteins. They form large oligomers, ranging in size from 140 to more than 800 kDa, which seem to be important for the interaction with non-native proteins as molecular chaperones. Here we analyzed the stability and oligomeric structure of murine Hsp25 and its correlation with function. Upon unfolding, the tertiary and quaternary structure of Hsp25 is rapidly lost, whereas the secondary structure remains remarkably stable. Unfolding is completely reversible, leading to native hexadecameric structures. These oligomers are in a concentration-dependent equilibrium with tetramers and dimers, indicating that tetramers assembled from dimers represent the basic building blocks of Hsp25 oligomers. At high temperatures, the Hsp25 complexes increase in molecular mass, consistent with the appearance of "heat shock granules" in vivo after heat treatment. This high molecular mass "heat shock form" of Hsp25 is in a slow equilibrium with hexadecameric Hsp25. Thus, it does not represent an off-pathway reaction. Interestingly, the heat shock form exhibits unchanged chaperone activity even after incubation at 80 degrees C. We conclude that Hsp25 is a dynamic tetramer of tetramers with a unique ability to refold and reassemble into its active quaternary structure after denaturation. So-called heat shock granules, which have been reported to appear in response to stress, seem to represent a novel functional species of Hsp25.

HSP27 Heat-Shock Proteins↗
PubMed1999

The charged region of Hsp90 modulates the function of the N-terminal domain.

Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.

Adenosine Triphosphate↗
PubMed1999

Hsp90 & Co. - a holding for folding.

Hsp90 is an abundant molecular chaperone that is involved in the folding of a defined set of signalling molecules including steroid-hormone receptors and kinases. Recent in vitro experiments suggest that Hsp90 contains two different binding sites for non-native proteins, which allow it to combine the properties of a promiscuous chaperone with those of a dedicated folding-helper protein. Significant progress has been made in analysing co-chaperones, which form defined, substrate-dependent complexes with Hsp90 in vivo. Structural studies have identified the ATP-binding site in the N-terminal domain of Hsp90, which can be blocked by high-affinity inhibitors. Although a detailed understanding of the mechanism of Hsp90 action is still lacking, recent advances suggest that the protein is the centre of a dynamic, multifunctional and multicomponent chaperone machinery that extends the limits of protein folding in the cell.

Antibiotics, Antineoplastic↗
PubMed1999

Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae.

The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co-chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90-depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v-Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo.

Cell Division↗
PubMed1998

GroEL traps dimeric and monomeric unfolding intermediates of citrate synthase.

The prokaryotic molecular chaperone GroE is increasingly expressed under heat shock conditions. GroE protects cells by preventing the irreversible aggregation of thermally unfolding proteins. Here, the interaction of GroE with thermally unfolding citrate synthase (CS) was dissected into several steps that occur before irreversible aggregation, and the conformational states of the unfolding protein recognized by GroEL were determined. The kinetic analysis of CS unfolding revealed the formation of inactive dimeric and monomeric intermediates. GroEL binds both intermediates without affecting the unfolding pathway. Furthermore, the dimeric intermediates are not protected against dissociation in the presence of GroEL. Monomeric CS is stably associated with GroEL, thus preventing further irreversible unfolding steps and subsequent aggregation. During refolding, monomeric CS is encapsulated inside the cavity of GroEL. GroES complexes. Taken together our results suggest that for protection of cells against heat stress both the ability of GroEL to interact with a large variety of nonnative conformations of proteins and the active, GroES-dependent refolding of highly unfolded species are important.

Binding Sites↗
PubMed1998

Folding and association of beta-Galactosidase.

beta-D-Galactosidase from Escherichia coli is one of the largest tetrameric enzymes known at present. Although its physiological importance, the regulation of its synthesis, its enzymatic properties and its structure are well established, little is known about the stability and the folding pathway of this enzyme. Here we show that the overall folding mechanism of chemically denatured beta-galactosidase consists of three stages: (i) formation of elements of secondary structure; (ii) collapse to subdomains and structured monomers; (iii) association to the native quaternary structure via dimeric intermediates. The first rate-limiting step is the association of structured monomers to form dimers in a bi-molecular reaction, with a rate constant of 4.3x10(3) M-1 s-1 at 20 degreesC. The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5x10(-3) s-1 at 20 degreesC. Tetramers form from these dimers in a fast reaction. By determining a similar mechanism for alpha-complementation of beta-galactosidase fragments it could be confirmed that beta-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association.

Dimerization↗
PubMed1998

The Hsp90 complex--a super-chaperone machine as a novel drug target.

Cells respond to sudden changes in the environmental temperature with increased synthesis of a distinct number of heat shock proteins (Hsps). Analysis of the function of these proteins in recent years has shown that all the major classes of conserved Hsps are molecular chaperones involved in assisting cellular protein folding and preventing irreversible side-reactions, such as unspecific aggregation. In addition to their function under stress conditions, molecular chaperones also play a critical role under physiological conditions. Hsp90 is one of the most abundant chaperones in the cytosol of eukaryotic cells. It is part of the cell's powerful network of chaperones to fight the deleterious consequences of protein unfolding caused by nonphysiological conditions. In the absence of stress, however, Hsp90 is an obligate component of fundamental cellular processes such as hormone signaling and cell cycle control. In this context, several key regulatory proteins, such as steroid receptors, cell cycle kinases, and p53, have been identified as substrates of Hsp90. Recently, Hsp90 was shown to be the unique target for geldanamycin, a potent new anti-tumor drug that blocks cell proliferation. Interestingly, under physiological conditions, Hsp90 seems to perform its chaperone function in a complex with a set of partner proteins, suggesting that the Hsp90 complex is a multi-chaperone machine specialized in guiding the maturation of conformationally labile proteins. The regulation of key signaling molecules of the cell by the Hsp90 machinery is a stimulating new concept emerging from these studies, and Hsp90 has become a promising new drug target.

Chaperonins↗
PubMed1998

Stabilization of proteins and peptides in diagnostic immunological assays by the molecular chaperone Hsp25.

Diagnostic assays for proteins devoid of enzymatic activity are becoming increasingly important. Antibodies generated against the respective proteins are used for their detection in enzyme-linked immunosorbent assay or patient sera are used to monitor disease-related antibodies against recombinantly produced antigens. A problem frequently encountered with these assays is that the proteins or fragments thereof used as standards have a limited shelf life. A similar problem arises when activities of labile enzymes are used for diagnostic detection. Here, we present a novel approach to 'stabilizing' enzymatic activity and antigenicity of proteins used for immunogenic detection by molecular chaperones. We have exploited the ability of molecular chaperones to keep proteins in their active conformation to overcome the biotechnological problems encountered in protein-based diagnostics of heart attack, stroke, and viral infections such as hepatitis C. We show that Hsp25, a member of the family of small heat shock proteins, known to act as a molecular chaperone in protein folding reactions, can stably bind labile standard proteins. Complex formation does not interfere with immunogenic detection and, importantly, antigenic as well as enzymatic activity remains constant for weeks. This strategy seems to be applicable to a wide range of assays involving unstable proteins, including the generation of vaccines.

Amino Acid Sequence↗
PubMed1998

The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network.

The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.

Adenosine Triphosphate↗
PubMed1998

Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence.

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.

Adenosine Triphosphate↗