Molecular Chaperone Machinery Research Articles

Understanding ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution

Molecular switching and ligand-based modulation of the 90-kDa heat-shock protein (Hsp90) chaperone activity may ultimately facilitate conformational coupling to the ATPase cycle along with activation and recruitment of the broad range of client proteins. We present an atomic resolution analysis of the Hsp90 N-terminal domain (NTD) binding energy landscape by simulating protein dynamics with a range of binding partners. We show that the activity of the molecular chaperone may be linked to (i) local folding-unfolding transitions and conformational switching of the "active site lid" upon binding and (ii) differences in the underlying protein dynamics as a function of the binding partner. This study suggests that structural plasticity of the Hsp90 NTD can be exploited by the molecular chaperone machinery to modulate enhanced structural rigidity during ATP binding and increased protein flexibility as a consequence of the inhibitor binding. The present study agrees with the experimental structural data and provides a plausible molecular model for understanding mechanisms of modulation of molecular chaperone activities by binding partners.

Cloning and Characterization of the HSP70 Gene, and Its Expression in Response to Diapauses and Thermal Stress in the Onion Maggot, Delia antiqua

The cytosolic members of the HSP70 family of proteins play key roles in the molecular chaperone machinery of the cell. In the study we cloned and sequenced the fulllength cDNA of Delia antiqua HSP70 gene, which is 2461 bp long and encodes 643 a.a. with a calculated molecular mass of 70,787 Da. We investigated gene copies of cytosolic HSP70 members of 4 insect species with complete genome available, and found that they are quite variable with species. In order to characterize this protein we carried out an alignment and a phylogenetic analysis with 41 complete protein sequences from insects. The analysis divided the cytosolic members of the family into two classes, HSP70 and HSC70, distinguishable on the basis of 15 residues. HSP70 class members were slightly shorter in length and smaller in molecular mass relative to the HSC70 class members, and the conservative and functional regions in these sequences were documented. Mainly, we investigated the expression of Delia antiqua HSP70 gene, in response to diapauses and thermal stresses. Both summer and winter diapauses elevated HSP70 transcript levels. Cold-stress led to increased HSP70 expression levels in summer- and winter-diapausing pupae, but heat-stress elevated the levels only in the winter-diapausing pupae. In all cases, the expression levels, after being elevated, gradually decreased with time. HSP70 expression was low in non-diapausing pupae but was up-regulated following cold- and heatstresses. Heat-stress gradually increased the mRNA level with time whereas cold-stress gradually decreased levels after an initial increase.

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90's diverse "clientele." Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

The mechanism of GroEL/GroES folding/refolding of protein substrates revisited

The thermodynamics and kinetics of zinc-cytochrome c (ZnCyt c) interactions with Escherichia coli molecular chaperone GroEL (Chaperonin 60; Cpn60) are described. Zinc(II)-porphyrin represents a flexible fluorescent probe for thermodynamic complex formation between GroEL and ZnCyt c, as well as for stopped-flow fluorescence kinetic experiments. Data suggests that GroEL and GroEL/GroES-assisted refolding of unfolded ZnCyt c takes place by a mechanism that is quite close to the Anfinsen Cage hypothesis for molecular chaperone activity. However, even in the presence of ATP, GroEL/GroES-assisted refolding of ZnCyt c takes place at approximately half the rate of refolding of ZnCyt c alone. On the other hand, there is little evidence for refolding behaviour consistent with the Iterative Annealing hypothesis. This includes a complete lack of GroEL or GroEL/GroES-assisted enhancement of refolding rate constant k(2) associated with the unfolding of a putative misfolded state I (Zn) on the pathway to the native state. Reviewing our data in the light of data from other laboratories, we observe that all forward rate enhancements or reductions could be accounted for in terms of thermodynamic coupling (adjusting positions of refolding equilibria) due to binding interactions between GroEL and unfolded protein substrates, driven by thermodynamic considerations. Therefore, we propose that passive kinetic partitioning should be considered the core mechanism of the GroEL/GroES molecular chaperone machinery, wherein the core function is to bind unfolded protein substrates leading to a blockade of aggregation pathways and to increases in molecular flux through productive folding pathway(s).

Role of molecular chaperones in neurodegenerative disorders

Many major neurodegenerative diseases, including Amyotrophic Lateral Sclerosis, Alzheimer's disease, Parkinson's disease, Huntington Disease and other polyglutamine expansion disorders, are associated with degeneration and death of specific neuronal populations due to accumulation of certain abnormal polypeptides. These misfolded species aggregate and form inclusion bodies and their neurotoxicity is associated with the aggregation. To handle a build-up of abnormal proteins cells employ a complicated machinery of molecular chaperones and various proteolytic systems. Chaperones facilitate refolding or degradation of misfolded polypeptides, prevent protein aggregation and play a role in formation of aggresome, a centrosome-associated body to which small cytoplasmic aggregates are transported. The ubiquitin-proteasome proteolytic system is critical for reducing the levels of soluble abnormal proteins, while autophagy plays the major role in clearing of cells from protein aggregates. Accumulation of the aggregation prone proteins activates signal transduction pathways that control cell death, including JNK pathway that controls viability of a cell in various models of Parkinson's and Huntington's diseases. The major chaperone Hsp72 can interfere with this signalling pathway, thus promoting survival. A very important consequence of a build-up and aggregation of misfolded proteins is impairment of the ubiquitin-proteasome degradation system and suppression of the heat shock response. Such an inhibition of the major cell defense systems may play a critical role in neurodegeneration. Here, it is suggested that these changes may reflect a senescence-like programme initiated by the aggregated abnormal polypeptides. Pathways that control the fate of misfolded proteins, for example molecular chaperones or proteolytic systems, may become interesting novel targets for therapy of neurodegenerative disorders.

The Hsp70 Ssz1 modulates the function of the ribosome-associated J-protein Zuo1

J-proteins are obligate partners of Hsp70s, forming a ubiquitous class of molecular chaperone machinery. The ribosome-associated Hsp70 of yeast Ssb binds nascent polypeptides as they exit the ribosome. Here we report that the ribosome-associated J-protein Zuo1 is the partner of Ssb. However, Zuo1 efficiently stimulates the ATPase activity of Ssb only when in complex with another Hsp70, Ssz1. Ssz1 binds ATP, but none of the 11 different amino acid substitutions in the ATP-binding cleft affected Ssz1 function in vivo, suggesting that neither nucleotide binding nor hydrolysis is required. We propose that Ssz1's predominant function in the cell is to facilitate Zuo1's ability to function as a J-protein partner of Ssb on the ribosome, serving as an example of an Hsp70 family member that has evolved to carry out functions distinct from that of a chaperone.

Characterization of ST13 Protein Expression in Human Colorectal Cancer Tissues

Objective: To characterize the expression of ST13 protein in human tissues for investigation of the function of colorectal cancer related gene ST13.Methods: ST13 ORF was cloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinity chromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinant protein. Immunoblot and immunohistochemical staining were employed to analyze ST13 protein expression in human tissues. Results:The expression and purification of the recombinant ST13 protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterial culture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from 104 to 105 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed that the apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility was approximately 50000, which was about 10000 larger than the 41324 calculated, but the glycosylation of the protein was excluded. Computer modeling revealed the protein to be a hydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributed in cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in the staining intensity of the protein were observed between normal and cancer tissues as well as among different normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with an apparent Mr of 50000. The protein is expressed in colorectal and other epithelial tissues. The expression level of the protein is down-regulated in colorectal cancer and varies among different normal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicates that ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibility that ST13 protein is involved in the development of colorectal cancer through Hsp70 molecular chaperone machinery.

Chaperoning prions: the cellular machinery for propagating an infectious protein?

Newly made polypeptide chains require the help of molecular chaperones not only to rapidly reach their final three-dimensional forms, but also to unfold and then correctly refold them back to their biologically active form should they misfold. Most prions are an unusual type of protein that can exist in one of two stable conformations, one of which leads to formation of an infectious alternatively folded form. Studies in Baker's yeast (Saccharomyces cerevisiae) have revealed that prions can exploit the molecular chaperone machinery in the cell in order to ensure stable propagation of the infectious, aggregation-prone form. The disaggregation of yeast prion aggregates by molecular chaperones generates forms of the prion protein that can seed the protein polymerisation that underlies the prion propagation cycle. In this article, we review what we have learnt about the role of molecular chaperones in yeast prion propagation, describe a model that can explain the role of various classes of molecular chaperones and their co-chaperones, and speculate on the possible involvement of chaperones in the propagation of mammalian prions.

Tetratricopeptide repeat cochaperones in steroid receptor complexes

The molecular chaperone machinery contains multiple protein components that have 1 or more structural domains composed of tetratricopeptide repeat (TPR) motifs. Many other proteins of separate or unknown function also have TPR domains, so this motif is not exclusive to molecular chaperones. A general function of TPR domains is to bind other polypeptides, but this otherwise prosaic function has been exploited in an assortment of ways that link chaperones and other protein systems into cooperative networks. Among the best-characterized TPR proteins are several cochaperones that participate in assembly and regulation of steroid receptor complexes. Steroid receptors, members of the nuclear receptor subfamily, are hormone-dependent transcription factors that regulate many vertebrate pathways of homeostasis, growth, differentiation, reproduction, and pathology and, as such, have been of great interest to biologists and clinicians. Moreover, the steroid receptors are among the first recognized native clients for chaperones and have been widely studied models for complex chaperone interactions. To provide a coherent, representative minireview of TPR protein function, the scope of this article has been narrowed down primarily to functions of steroid receptor-associated TPR cochaperones.

Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity.

The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by proteins with expanded polyglutamine (polyQ) repeats. However, the mechanistic relationship between polyQ aggregation and its toxic effects on neurons remains unclear. Two main hypotheses have been put forward for how polyQ expansions may cause cellular dysfunction. In one model neurotoxicity results from the ability of polyQ-expanded proteins to recruit other important cellular proteins with polyQ stretches into the aggregates. In the other model, aggregating polyQ proteins partially inhibit the ubiquitin-proteasome system for protein degradation. These two mechanisms are not exclusive but may act in combination. In general, protein misfolding and aggregation are prevented by the machinery of molecular chaperones. Some chaperones such as the members of the Hsp70 family also modulate polyQ aggregation and suppress its toxicity. These recent findings suggest that an imbalance between the neuronal chaperone capacity and the production of potentially dangerous polyQ proteins may trigger the onset of polyQ disease.

Induction of heat shock protein 40 and GrpE mRNAs following transient focal cerebral ischemia in the rat

Cerebral ischemia is associated with the induction of several heat shock proteins (HSPs), but the effects on HSP40 and GrpE are less clear. The present study investigated the induction of Hsp40 and GrpE mRNAs following 30 min of middle cerebral artery occlusion in the rat model. Reverse transcription–polymerase chain reaction (PCR) and in situ hybridization analyses showed significant induction of both mRNAs in the ischemic cortex. These results demonstrate the synergic induction of HSP70 molecular chaperone machinery in cerebral ischemia.

Two Distinct Regions of the Immunophilin-like Protein XAP2 Regulate Dioxin Receptor Function and Interaction with hsp90

The dioxin (aryl hydrocarbon) receptor is a ligand inducible transcription factor, which mediates cellular responses to a variety of xenobiotic compounds such as dioxins. In the absence of ligand the receptor is associated with the molecular chaperone hsp90 and the tetratricopeptide repeat (TPR-) containing immunophilin-like protein XAP2. XAP2 has been implicated in regulation of the intracellular localization of the dioxin receptor and protection of the receptor against degradation. In this study a series of XAP2 mutants has been generated in order to identify the structural motif(s) mediating interaction with the dioxin receptor-hsp90 complex and modulation of receptor function. Immunoprecipitation experiments demonstrated that the C-terminal part of XAP2, including the TPR motifs and the region outside the TPR motifs, was required to directly contact hsp90. The N-terminal part of XAP2 was required for the stability of the ternary dioxin receptor-hsp90-XAP2 complex. In addition, the integrity of the N-terminal region of XAP2 was essential for XAP2 to regulate the intracellular localization of the dioxin receptor. In conclusion, these data demonstrate that two distinct regions of XAP2 modulate dioxin receptor function and interaction with hps90, illustrating the complexity in regulation of dioxin receptor signaling by the hsp90 molecular chaperone machinery.

Hyperthermic stress stimulates the association of both constitutive and inducible isoforms of 70 kDa heat shock protein with rat liver glucocorticoid receptor

Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 °C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive—Hsc70 and inducible—Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24 h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70.

The Immunophilin-like Protein XAP2 Regulates Ubiquitination and Subcellular Localization of the Dioxin Receptor

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent transcription factor that induces expression of a number of genes encoding drug metabolizing enzymes. The nonactivated form of the dioxin receptor is associated with heat shock protein (hsp) 90, the co-chaperone p23, and the immunophilin-like protein XAP2. Whereas hsp90 has a role in maintenance of the high-affinity ligand binding conformation of the dioxin receptor complex, and p23 stabilizes receptor-hsp90 interaction, the exact role of XAP2 is largely unknown. Here we show that XAP2 protected the ligand-free form of receptor against ubiquitination, resulting in increased dioxin receptor protein levels. Upon exposure to ligand, nuclear translocation of the dioxin receptor was markedly delayed by XAP2, indicating an additional role of XAP2 in regulation of the subcellular localization of the receptor by a mechanism of cytoplasmic retention. In order to mediate these effects, XAP2 required stable association with the hsp90-p23 molecular chaperone complex. The association of XAP2 as well as p23 with the dioxin receptor was determined by the functional state of hsp90. These data indicate a novel mode of regulation of dioxin receptor signaling by the hsp90-dependent molecular chaperone machinery.

Phosphorylation by CK2 and MAPK enhances calnexin association with ribosomes.

Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of calnexin. Analysis by two-dimensional phosphopeptide maps revealed that calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK-1 led to increased association of calnexin with membrane-bound ribosomes. In vivo, calnexin-associated ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-associated calnexin over unstimulated cells. Taken together with studies revealing calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of calnexin leads to a potential increase in glycoprotein folding close to the translocon.

Cns1 is an essential protein associated with the hsp90 chaperone complex in Saccharomyces cerevisiae that can restore cyclophilin 40-dependent functions in cpr7Delta cells.

Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60(v-src). In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Delta slow-growth phenotype. The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Delta growth defect. Overexpression of CNS1 in cpr7Delta cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

Requirement for Hsp90 and a CyP-40-type Cyclophilin in Negative Regulation of the Heat Shock Response

The heat shock response is a highly conserved mechanism that allows cells to withstand a variety of stress conditions. Activation of this response is characterized by increased synthesis of heat shock proteins (HSPs), which protect cellular proteins from stress-induced denaturation. Heat shock transcription factors (HSFs) are required for increased expression of HSPs during stress conditions and can be found in complexes containing components of the Hsp90 molecular chaperone machinery, raising the possibility that Hsp90 is involved in regulation of the heat shock response. To test this, we have assessed the effects of mutations that impair activity of the Hsp90 machinery on heat shock related events in Saccharomyces cerevisiae. Mutations that either reduce the level of Hsp90 protein or eliminate Cpr7, a CyP-40-type cyclophilin required for full Hsp90 function, resulted in increased HSF-dependent activities. Genetic tests also revealed that Hsp90 and Cpr7 function synergistically to repress gene expression from HSF-dependent promoters. Conditional loss of Hsp90 activity resulted in both increased HSF-dependent gene expression and acquisition of a thermotolerant phenotype. Our results reveal that Hsp90 and Cpr7 are required for negative regulation of the heat shock response under both stress and nonstress conditions and establish a specific endogenous role for the Hsp90 machinery in S. cerevisiae.

Sequence motifs shared between chaperone components participating in the assembly of progesterone receptor complexes.

Steroid receptors typically exist in a heteromeric complex with Hsp90 and other components of the molecular chaperone machinery. Assembly of functional receptor complexes follows an ordered pathway involving at least eight chaperone components, some only participating in early assembly stages that are prerequisite for formation of mature complexes. The mechanisms directing the order of assembly steps and the nature of transitional interactions between assembly steps are largely unknown, but likely are encoded in the primary sequence and functional domains of the participating chaperones. Several common sequence motifs are shared between participants that may be key in ordering the steroid receptor assembly pathway.

Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein.

A 48-kDa protein (p48) that transiently associates with progesterone receptor during cell-free assembly in rabbit reticulocyte lysate was isolated by two-dimensional gel separation. Tryptic peptide sequences were generated and used to develop an antipeptide antiserum recognizing p48 by Western immunostaining, and this antiserum was used to monitor purification of native p48 from reticulocyte lysate. Eight mouse monoclonal antibodies capable of immunoprecipitating vertebrate p48 were generated. These monoclonal antibodies served as probes to clone ten p48 cDNAs from a HeLa cDNA expression library. One of the cloned cDNAs was sequenced in its entirety and codes for a 369-amino acid protein (calculated Mr = 41,324). Northern blot analysis of RNA from multiple human tissues suggest that p48 may be ubiquitously expressed. Expression of human p48 cDNA in vitro yielded a product that comigrated with rabbit p48 by SDS-PAGE and associated with progesterone receptor in a similar manner. Immunoprecipitation of p48 complexes revealed a common association of p48 with hsp70 and, to a lesser extent, with hsp90 and p60. Thus, it appears that p48 is a novel component of the cytoplasmic molecular chaperone machinery.

A pathway of multi-chaperone interactions common to diverse regulatory proteins: estrogen receptor, Fes tyrosine kinase, heat shock transcription factor Hsf1, and the aryl hydrocarbon receptor.

A variety of regulatory proteins, including different classes of transcription factors and protein kinases, have been identified in complexes with Hsp90. On careful examination of unactivated progesterone receptor complexes, eight different protein participants have been identified, and each can be considered a component of the cytoplasmic molecular chaperone machinery. These proteins are Hsp90, Hsp70, Hip, p60, p23, FKBP51, FKBP52 and Cyp40. Studies in a cell-free assembly system have helped to define a highly ordered, dynamic pathway for assembly of progesterone receptor complexes. In the present study, target proteins other than progesterone receptor were used in this cell-free system to assemble complexes in vitro and to compare the composition of resulting complexes. Targets used were human estrogen receptor, human Fes protein-tyrosine kinase, human heat shock transcription factor Hsf1, and human aryl hydrocarbon receptor. The striking similarity of resulting target complexes with previously characterized progesterone receptor complexes suggest that each of these targets undergoes a common assembly pathway involving multiple chaperone components in addition to Hsp90.