GCUNC45 is the first Hsp90 co-chaperone to show alpha/beta isoform specificity

Hsp90 is an essential molecular chaperone required for the normal functioning of many key regulatory proteins in eukaryotic cells. Vertebrates have two closely related isoforms of cytosolic Hsp90 (Hsp90alpha and Hsp90beta). However, specific functions for each isoform are largely unknown, and no Hsp90 co-chaperone has been reported to distinguish between the two isoforms. In this study, we show that the Hsp90 co-chaperone GCUNC45 bound preferentially to the beta isoform of Hsp90 in vitro. GCUNC45 efficiently blocked the progression of progesterone receptor chaperoning in an in vitro functional system when Hsp90beta was used, but did so with much less efficacy when Hsp90alpha was used. Knockdown experiments in HeLa cells showed that GCUNC45 is required for the normal cellular distribution of Hsp90beta, but not Hsp90alpha. This is the first example of a co-chaperone with isoform selectivity, and this approach may open novel avenues to understanding the functional differences between Hsp90 isoforms.     

The role of membrane-bound heat shock Hsp90 proteins in the migration of tumor cells in vitro and the involvement of cell surface heparan sulfate proteoglycans in protein binding to the plasma membrane

The heat-shock protein Hsp90, which is found in the extracellular space and on the cell membrane, plays an important role in cell motility, migration, invasion, and metastasis of tumor cells. Currently, the functional role and molecular mechanisms of Hsp90 binding to the plasma membrane are not clear. Using isoform-specific antibodies against Hsp90, Hsp90α, and Hsp90β, we showed that the membrane-bound Hsp90α and Hsp90β proteins play a significant role in the migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Impairment of sulfonation of cellular heparan sulfates, cleavage of cellular heparan sulfates by heparinase I/III, as well as the treatment of cells with heparin, lead to a dramatic reduction in the expression levels of the Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. These results demonstrate that two isoforms of membrane-bound Hsp90 are involved in tumor-cell migration in vitro. As well, the cell surface heparan sulfate proteoglycans are shown to play a pivotal role in the “anchoring” of Hsp90α and Hsp90β to the plasma membrane.     

Importance of the C-terminal domain of Harc for binding to Hsp70 and Hop as well as its response to heat shock

Hsp90 is a molecular chaperone that acts in concert with Hsp70 to mediate the folding of many important regulatory proteins (e.g., protein kinases) into functional conformations. The chaperone activity of Hsp90 is primarily regulated by its cochaperones. For example, the Hsp90 cochaperone Cdc37 recruits Hsp90 to protein kinases as well as inhibiting its ATPase activity to promote the binding of Hsp90 to protein kinases. Harc is a structurally related Hsp90 cochaperone with a three-domain structure in which the middle domain binds Hsp90. In contrast to Cdc37 though, Harc also binds to Hsp70 and Hop (Hsp70/Hsp90 organizing protein). Here we demonstrate that deletion of the C-terminal domain of Harc abolished the binding of Hsp70 and Hop and reduced the affinity of Hsp90 binding to Harc. Significantly, the C-terminal domain of Harc bound Hsp70, but it did not bind Hop or Hsp90. Size exclusion chromatography of cell lysates revealed that Hop only formed a complex with Harc in the presence of Hsp90 and Hsp70, consistent with a model in which the interaction of Hop with Harc is mediated via the binding of Hop to Harc-bound Hsp90 and Hsp70. Notably, heat shock resulted in a marked decrease in the solubility of Harc, a response that was further augmented by the deletion of the C-terminal domain of Harc. This latter finding is especially interesting given that bioinformatics analysis indicated that cells may express splice variants of Harc that encode C-terminally truncated Harc isoforms. Together, these findings indicate that the C-terminal domain of Harc is a key determinant of its cochaperone functions.     

A comparison of Hsp90alpha and Hsp90beta interactions with cochaperones and substrates.

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90alpha and Hsp90beta, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90alpha was recovered after heat shock. The data demonstrate that Hsp90alpha and Hsp90beta exhibit similar interactions with cochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.     

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.     

Hsp90 phosphorylation,Wee1 and the cell cycle

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1? yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.     

Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients.     

A yeast-based assay reveals a functional defect of the Q488H polymorphism in human Hsp90alpha

It has been argued that the molecular chaperone Hsp90 guards the organism against genetic variations by stabilizing variant Hsp90 substrate proteins. However, little is known about polymorphisms affecting its own functions. We have followed up on a recent study describing two polymorphisms that alter the amino acid sequences of the two Hsp90 isoforms Hsp90alpha and Hsp90beta. Hsp90 is essential for cell proliferation in the budding yeast Saccharomyces cerevisiae, but the human proteins can replace the endogenous ones. In this growth assay, the variant V656M of Hsp90beta was indistinguishable from wild-type. In contrast, the Hsp90alpha variant Q488H, which carries an alteration of a very highly conserved residue, was severely defective for growth compared to wild-type Hsp90alpha. Hence, the characteristics of this yeast-based system-simplicity, rapidity, low cost-make it ideal for phenotype screening of polymorphisms in HSP90 and possibly many other human genes.     

Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-beta 1 (TGF beta), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGF beta is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGF beta can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGF beta on the hsp's. Analysis of the induction of hsp's using one- and two-dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGF beta, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an approximately twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGF beta, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGF beta, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGF beta.     

Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach

The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins ("cochaperones") that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages. To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast. These "bait" fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD). High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome. Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress. Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 "prey" fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the "relaxed" conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90. A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques.     

The middle domain of Hsp90 acts as a discriminator between different types of client proteins

The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.     

Multiple Hsp70 Isoforms in the Eukaryotic Cytosol: Mere Redundancy or Functional Specificity?

Hsp70 molecular chaperones play a variety of functions in every organism, cell type and organelle, and their activities have been implicated in a number of human pathologies, ranging from cancer to neurodegenerative diseases. The functions, regulations and structure of Hsp70s were intensively studied for about three decades, yet much still remains to be learned about these essential folding enzymes. Genome sequencing efforts revealed that most genomes contain multiple members of the Hsp70 family, some of which co-exist in the same cellular compartment. For example, the human cytosol and nucleus contain six highly homologous Hsp70 proteins while the yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized Hsp70s. The reasons and significance of the requirement for multiple Hsp70s is still a subject of debate. It has been postulated for a long time that these Hsp70 isoforms are functionally redundant and differ only by their spatio-temporal expression patterns. However, several studies in yeast and higher eukaryotic organisms challenged this widely accepted idea by demonstrating functional specificity among Hsp70 isoforms. Another element of complexity is brought about by specific cofactors, such as Hsp40s or nucleotide exchange factors that modulate the activity of Hsp70s and their binding to client proteins. Hence, a dynamic network of chaperone/co-chaperone interactions has evolved in each organism to efficiently take advantage of the multiple cellular roles Hsp70s can play. We summarize here our current knowledge of the functions and regulations of these molecular chaperones, and shed light on the known functional specificities among isoforms.     

Unc45 activates Hsp90-dependent folding of the myosin motor domain

Myosin folding and assembly in striated muscle are mediated by the general chaperones Hsc70 and Hsp90 and involve a myosin-specific co-chaperone related to the Caenorhabditis elegans gene unc-45. Two unc-45 genes are found in vertebrates, a general cell isoform, unc45a, and a striated muscle-specific isoform, unc45b. We have investigated the role of both isoforms of mouse Unc45 in myosin folding using an in vitro synthesis and folding assay. A smooth muscle myosin motor domain (MD) fused to green fluorescent protein (GFP) (MD::GFP) was used as substrate, and folding was measured by native gel electrophoresis and functional assays. In the absence of Unc45, the MD::GFP chimera folds poorly. Addition of either Unc45a or Unc45b dramatically enhances the folding in a reaction that is dependent on Hsp90 ATPase activity. Unc45a is more effective than Unc45b with a higher apparent affinity and greater extent of folding. The Unc45-Hsp90 chaperone complex acts late in the folding pathway and promotes motor domain maturation after release from the ribosome. Unc45a behaves kinetically as an activator of the folding reaction by stimulating the rate of the Hsp90-dependent folding by >20-fold with an apparent K(act) of 33 nm. This analysis of vertebrate Unc45 isoforms clearly demonstrates a direct role for Unc45 in Hsp90-mediated myosin motor domain folding and highlights major differences between the isoforms in substrate specificity and mechanism.     

Heat shock protein expression in relation to reproductive cycle in land snails: Implications for survival

Land snails are subject to daily and seasonal variations in temperature and in water availability and use heat shock proteins (HSPs) as part of their survival strategy. We tested whether the reproductive cycle of land snails affects the endogenous levels of HSPs, and their involvement in the reproductive process. We examined HSP levels in the foot tissue of two Sphincterochila species, S. cariosa and S. zonata, before and after laying eggs, and analyzed the albumen gland (reproductive organ) of both species and eggs of S. cariosa for the presence and quantity of various HSPs. Our study shows reduction in the expression level of Hsp70 isoforms and Hsp90 in S. zonata foot and of Hsp74 in S. cariosa foot during the period preceding egg laying compared to the post-reproductive stage. Hsp70 isoforms and Hsp25 were highly expressed in both large albumen glands and in freshly laid eggs of S. cariosa, whereas large albumen glands of S. zonata expressed mainly Hsp70 isoforms. We conclude that a trade-off between survival and fertility is responsible for the expression level of HSPs in the foot tissue of Sphincterochila snails. Our study shows that HSPs are involved in the reproductive process. We propose that parental provision of HSPs may be part of a "be prepared" strategy of Sphincterochila snails, and that HSPs may play important roles in the survival strategy of land snails during the early life stages. Our observations also highlight the importance of the reproductive status in study of whole organisms, especially when assessing the HSP response to stress.