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PubMed1991

Chemical synthesis of 10 kDa chaperonin. Biological activity suggests chaperonins do not require other molecular chaperones.

Molecular chaperones are required for the correct folding and assembly of certain other polypeptides. It is not known whether molecular chaperones themselves require other chaperones to become functional. A 97-amino acid chaperone, the chaperonin 10 protein was chemically synthesised so that during synthesis and purification there was no contact of the chaperone with any other protein. The purified, synthetic chaperonin 10 protein formed oligomeric structures spontaneously and was biologically active as a chaperonin. This is the first description of a chemically synthesised chaperonin, and suggests that no other chaperones are required for correct folding, polymerisation and biological activity of this chaperone.

Bacterial Proteins↗
PubMed1989

Pediatricians' use of chaperones when performing gynecologic examinations on adolescent females.

This study assesses the influence of the interest and skill in adolescent health care on a pediatrician's use of a chaperone during the pelvic examination. A national sample of 558 pediatricians selected at random and 384 members of the American Academy of Pediatrics' Section on Adolescent Health (SAH) completed questionnaires assessing their perceived skills in and practice of adolescent medicine. Some 90.4% of the non-SAH males and 31.0% non-SAH females always used a chaperone compared to 69.4% of SAH males and 12.5% of SAH females (p less than or equal to 0.05). The actual frequency of chaperone use was less among SAH members than non-SAH pediatricians (p less than or equal to 0.013) and less among females in both groups (p less than or equal to 0.0001). Among male physicians, the variables found to have the strongest association with a less frequent use of chaperones included the frequency that oral contraceptives were prescribed, perceived skill in providing contraceptive counseling, percent of adolescents in the pediatrician's practice, and the frequency with which confidential services were provided. These correlations were stronger among SAH members than non-SAH pediatricians. Among female SAH members, not using a chaperone was associated with a higher perceived skill in managing sexually transmitted diseases, the frequency with which they performed pelvic examinations, and the percent of adolescents in their practice. Among non-SAH female physicians, the perceived skill to perform a pelvic examination and provide contraceptive counseling were associated with increased chaperone use. These data suggest that many factors other than gender and medicolegal influences are associated with the physician's decision concerning the use of a chaperone.

Adolescent↗
PubMed2005

Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways.

The mechanisms by which molecular chaperones assist quality control of cytosolic proteins are poorly understood. Analysis of the chaperone requirements for degradation of misfolded variants of a cytosolic protein, the VHL tumor suppressor, reveals that distinct chaperone pathways mediate its folding and quality control. While both folding and degradation of VHL require Hsp70, the chaperonin TRiC is essential for folding but is dispensable for degradation. Conversely, the chaperone Hsp90 neither participates in VHL folding nor is required to maintain misfolded VHL solubility but is essential for its degradation. The cochaperone HOP/Sti1p also participates in VHL quality control and may direct the triage decision by bridging the Hsp70-Hsp90 interaction. Our finding that a distinct chaperone complex is uniquely required for quality control provides evidence for active and specific chaperone participation in triage decisions and suggests that a hierarchy of chaperone interactions can control the alternate fates of a cytosolic protein.

Cells, Cultured↗
PubMed2003

Attitudes towards pelvic examination and chaperones: a questionnaire survey of patients and providers.

Some women find pelvic examinations distressing. Some doctors use this as an excuse not to do them. Most professional organizations recommend the presence of a chaperone for every pelvic examination. In family-planning clinics, usually staffed predominantly by women, the provision of a chaperone for every pelvic examination is a burden to the efficient running of clinics and may not be necessary. Views about pelvic examinations and chaperones were sought from 1000 women attending a family-planning clinic and 98 health-care professionals. The majority of women (59%) do not mind being examined, and when the examiner is female most women do not particularly want a chaperone. One-third of women (34%) actively object to a chaperone. Health professionals are not good at predicting women's feelings and expectations about pelvic examinations. Young women (under 25) and nulligravid women are more likely to find pelvic examinations distressing but not more likely to want a chaperone present. Chaperones should be offered to, but not inflicted upon, women undergoing pelvic examination. Younger women need a particularly sensitive approach. Providers should not be deterred from doing pelvic examinations if they are clinically indicated in the mistaken belief that women will object.

Attitude↗
PubMed1997

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

BACKGROUND: The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. RESULTS: The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. CONCLUSIONS: The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.

Adenosine Diphosphate↗
PubMed2006

Chaperones in ENT out-patients; the patients' perspective.

Chaperones are used in order to avoid misunderstandings that can lead to false accusations. Consultations and examinations may be a minefield especially if the doctors are accused of sexual misconduct. The allegations may have irreparable effects on their reputation and may lead to removal from the medical register and to criminal charges being brought. We present the results of a questionnaire from 85 patients, exploring patient experience in ENT out-patients particularly with respect to examination of the ear under the microscope. This procedure necessitates close contact between the patient and doctor for several minutes and there may be inadvertent contact with intimate areas on the patient. Up to 30 per cent of patients expressed a preference for a chaperone during the ENT consultation. There was noticeably a greater proportion of men than women patients stating a preference for a chaperone during the consultation. Interestingly, there was a strong positive correlation between the presence of friends/relatives and the preference for a chaperone. Patients should be given the freedom to choose for or against the presence of a chaperone in clinic consultations. The most commonly predicted scenario requiring a chaperone is with the male doctor and female patient. This study shows that based on patient feedback, this scenario, although common is not the exclusive area in which chaperones should be used.

Female↗
PubMedDate not supplied

Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins.

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.

Gene Expression Regulation, Fungal↗
PubMed2004

Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays.

Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.

Animals↗
PubMed2003

Chaperone-mediated in vitro assembly of Polyomavirus capsids.

The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for approximately 70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperone-mediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly.

Adenosine Triphosphate↗
PubMed2002

Distinct roles of the N-terminal-binding domain and the C-terminal-solubilizing domain of alpha-synuclein, a molecular chaperone.

alpha-Synuclein, an acidic neuronal protein of 140 amino acids, is extremely heat-resistant and is natively unfolded. Recent studies have demonstrated that alpha-synuclein has chaperone activity both in vitro and in vivo, and that this activity is lost upon removing its C-terminal acidic tail. However, the detailed mechanism of the chaperone action of alpha-synuclein remains unknown. In this study, we investigated the molecular mechanism of the chaperone action of alpha-synuclein by analyzing the roles of its N-terminal and C-terminal domains. The N-terminal domain (residues 1-95) was found to bind to substrate proteins to form high molecular weight complexes, whereas the C-terminal acidic tail (residues 96-140) appears to be primarily involved in solubilizing the high molecular weight complexes. Because the substrate-binding domain and the solubilizing domain for chaperone function are well separated in alpha-synuclein, the N-terminal-binding domain can be substituted by other proteins or peptides. Interestingly, the resultant engineered chaperone proteins appeared to display differential efficiency and specificity in terms of the chaperone function, which depended upon the nature of the binding domain. This finding implies that the C-terminal acidic tail of alpha-synuclein can be fused with other proteins or peptides to engineer synthetic chaperones for specific purposes.

Animals↗
PubMed2006

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p.

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Role of chaperones in the biogenesis and maintenance of the mitochondrion.

All cells depend on correctly folded proteins for optimal function. A central question in cellular biology is how such folded structures are formed and maintained, a process that is now recognized to rely heavily on a group of proteins called molecular chaperones. Molecular chaperones constitute distinct families of proteins that are ubiquitous and highly conserved from bacteria to humans. They appear to bind nonnative conformations of most, if not all, proteins, thereby preventing their aggregation and subsequent inactivation. The chaperones not only protect newly synthesized proteins during transport and folding, but also serve to maintain the cell in a healthy state during exposure to a multitude of stress conditions. Accordingly, chaperones are expressed constitutively, but their synthesis is further enhanced during stress conditions. Detailed insights into the role of molecular chaperones have come from studies of mitochondrial protein biogenesis, a process in which chaperones act as unfoldases, pulling devices, and foldases. In this review we summarize these developments and further discuss the potential role of chaperones in mitochondrial DNA metabolism and human mitochondrial disease states.

Animals↗
PubMed1999

Chaperone-mediated protein folding.

The folding of most newly synthesized proteins in the cell requires the interaction of a variety of protein cofactors known as molecular chaperones. These molecules recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding. There are several families of chaperones; those most involved in protein folding are the 40-kDa heat shock protein (HSP40; DnaJ), 60-kDa heat shock protein (HSP60; GroEL), and 70-kDa heat shock protein (HSP70; DnaK) families. The availability of high-resolution structures has facilitated a more detailed understanding of the complex chaperone machinery and mechanisms, including the ATP-dependent reaction cycles of the GroEL and HSP70 chaperones. For both of these chaperones, the binding of ATP triggers a critical conformational change leading to release of the bound substrate protein. Whereas the main role of the HSP70/HSP40 chaperone system is to minimize aggregation of newly synthesized proteins, the HSP60 chaperones also facilitate the actual folding process by providing a secluded environment for individual folding molecules and may also promote the unfolding and refolding of misfolded intermediates.

Chaperonins↗
PubMed1992

Chaperoning practices of Ohio family physicians.

BACKGROUND: The medical literature contains no consistent recommendations regarding chaperon practices during physical examination of patients. The objective of this study was to determine the chaperon practices of Ohio family physicians. METHODS: A questionnaire was mailed to all 1,786 active members of the Ohio Academy of Family Physicians. The questionnaire solicited information about the physicians' chaperon practices during physical examinations of the rectum, sexual and genital organs, and the female chest. RESULTS: The response rate was 74.6%. During female genital examinations, chaperons were used by 79.4% of male and 31.9% of female family physicians. Only 1.4% of male and 14.4% of female physicians used chaperons during male genital examinations. Chaperon practices were influenced by the gender of the physician and patient, the type of examination, and the availability of an assistant. CONCLUSIONS: No standard of care exists regarding chaperon practices among Ohio family physicians.

Age Factors↗
PubMed1993

alpha-Crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract.

This study reports the first demonstration of a marked reduction in alpha-crystallin chaperone activity in an experimental model of cataract, and the study implicates activation of the cysteine protease calpain II (EC 3.4.22.17) as the in vivo protease responsible for decreased chaperone activity. Chaperone activity of normal alpha-crystallin from lenses of young rats was assayed by measuring attenuation of heat-induced aggregation and scattering of beta L-crystallin. alpha-Crystallin from the nucleus of lenses with selenite cataract showed specific selective proteolysis, and chaperone activity was diminished. Proteolysis of alpha-crystallin from selenite cataract lenses was mimicked by incubating normal alpha-crystallin with calpain II, and this also resulted in loss of chaperone activity. Two-dimensional gel electrophoresis and peptide mapping were used to identify four partially degraded alpha A- and alpha B-crystallin polypeptides following incubation of normal alpha-crystallin with calpain. Similar partially degraded alpha A and alpha B polypeptides were found in selenite cataract. Previous experiments indicated that alpha-crystallin chaperone activity decreases because of removal of the COOH terminus. Our experiments support this observation and suggest that calpain proteolysis of alpha-crystallin at the COOH terminus may result in a loss of chaperone activity in selenite cataract.

Amino Acid Sequence↗
PubMed1998

Chemical chaperones enhance superantigen and conventional antigen presentation by HLA-DM-deficient as well as HLA-DM-sufficient antigen-presenting cells and enhance IgG2a production in vivo.

Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide loading onto MHC II proteins and also stabilizes empty MHC II molecules prior to their acquisition of antigenic peptides. APC that lack HLA-DM exhibit quantitative defects in protein Ag as well as superantigen presentation. Here we show that both the superantigen and protein presentation defect in MHC II-transfected, HLA-DM-deficient T2 cells can be partially overcome by treating the APC with the chemical chaperones glycerol, DMSO, or trimethylamine oxide. These chemical chaperones also enhance superantigen and conventional Ag presentation by wild-type APC. In vivo, glycerol was found to act as an adjuvant and resulted in enhanced IgG2a production to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). In vitro, the enhancement of Ag presentation by chemical chaperones was found to take place at the level of the APC and took several hours to develop. Subcellular fractionation experiments show that HLA-DM enhances presentation of peptides by dense endosome fractions whereas chemical chaperones enhance presentation by light membrane fractions (early endosome or plasma membrane). The mechanism by which these chemical chaperones augment Ag presentation is not defined, but flow cytometric analysis suggests that the enhancement may be due to a subtle effect on the stability of several different proteins at the cell surface.

Adjuvants, Immunologic↗
PubMed2006

Multi-chaperone complexes regulate the folding of interferon-gamma in the endoplasmic reticulum.

The quality control mechanisms directing the folding of cytokines in the endoplasmic reticulum (ER) are poorly understood. We have investigated ER chaperone usage by the cytokine interferon-gamma (IFN-gamma). ATP-depletion or inhibition of N-glycosylation was found to cause IFN-gamma to accumulate into detergent-insoluble aggregates in the ER. Six chaperones, GRP94, GRP78, ERp72, PDI, CaBP1/P5 and CRT were found to associate with IFN-gamma during its steady state folding. Interaction of the five first chaperones with IFN-gamma was regulated co-ordinately by ATP. These chaperones were recently reported to be part of a multi-chaperone complex involved in the folding of complex, multi-subunit proteins. Our data suggest that also proteins with a relatively simple quaternary structure such as cytokines may fold in association with this complex. In addition, we identified calreticulin as the major chaperone interacting with IFN-gamma, and the related class II cytokine interleukin-10, during heat-shock in vivo. IFN-gamma was maintained in a folding-competent form by calreticulin during heat-shock and released during subsequent recovery at 37 degrees C. This interaction was observed in both recombinant (CHO-F11) and natural producer cells (Jurkat, NK-92MI) of IFN-gamma. Since cytokines such as IFN-gamma and IL-10 are frequently produced in the course of inflammatory conditions associated with fever, the thermo-protective effect of calreticulin may constitute a previously unrecognized component of the cellular cytokine production machinery, of likely relevance in sustaining cytokine folding and secretion in pathophysiological conditions.

Adenosine Triphosphate↗
PubMed2006

Celecoxib upregulates endoplasmic reticulum chaperones that inhibit celecoxib-induced apoptosis in human gastric cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in cancer cells and this effect is involved in their antitumor activity. We recently demonstrated that NSAIDs upregulate GRP78, an endoplasmic reticulum (ER) chaperone, in gastric mucosal cells in primary culture. In the present study, induction of ER chaperones by NSAIDs and the effect of those chaperones on NSAID-induced apoptosis were examined in human gastric carcinoma cells. Celecoxib, an NSAID, upregulated ER chaperones (GRP78 and its cochaperones ERdj3 and ERdj4) but also C/EBP homologous transcription factor (CHOP), a transcription factor involved in apoptosis. Celecoxib also upregulated GRP78 in xenograft tumors, accompanying with the suppression of tumor growth in nude mice. Celecoxib caused phosphorylation of eukaryotic translation initiation factor 2 kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha) and production of activating transcription factor (ATF)4 mRNA. Suppression of ATF4 expression by small interfering RNA (siRNA) partially inhibited the celecoxib-dependent upregulation of GRP78. Celecoxib increased the intracellular Ca2+ concentration, while 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, an intracellular Ca2+ chelator, inhibited the upregulation of GRP78 and ATF4. These results suggest that the Ca2+-dependent activation of the PERK-eIF2alpha-ATF4 pathway is involved in the upregulation of ER chaperones by celecoxib. Overexpression of GRP78 partially suppressed the apoptosis and induction of CHOP in the presence of celecoxib and this suppression was stimulated by coexpression of either ERdj3 or ERdj4. On the other hand, suppression of GRP78 expression by siRNA drastically stimulated cellular apoptosis and production of CHOP in the presence of celecoxib. These results show that upregulation of ER chaperones by celecoxib protects cancer cells from celecoxib-induced apoptosis, thus may decrease the potential antitumor activity of celecoxib.

Activating Transcription Factor 4↗