Abundant Molecular Chaperone Research Articles

Hsp90 inhibitors as promising agents for radiotherapy

The 90-kD heat shock protein (Hsp90) is an abundant molecular chaperone catalyzing maturation and activation of client proteins. A number of the Hsp90 client proteins are components of cancer cell-associated signaling pathways that ensure unlimited growth of tumors and their resistance to chemotherapy and radiotherapy. Upon inhibition of the Hsp90 chaperone function, such client proteins are destabilized and degraded which disrupts multiple pathways essential for tumor cell survival; hence, pharmacological Hsp90 inhibitors could be applied in anticancer therapy. Several Hsp90-inhibiting compounds are currently tested in preclinical or phase I-III clinical trials as single anticancer agents or in combination with conventional drugs and radiation. The present review summarizes the data characterizing Hsp90 inhibitors as agents that sensitize human tumors to irradiation which may improve the outcome of radiotherapy. We also discuss molecular mechanisms of the Hsp90 inhibition-induced radiosensitization and its selectivity toward cancer cells.

Inhibition of Hsp90 Leads to Cell Cycle Arrest and Apoptosis in Human Malignant Pleural Mesothelioma

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone that mediates the maturation and stability of a variety of proteins associated with the promotion of cell growth and survival. Inhibition of Hsp90 function leads to proteasomal degradation of its mis-folded client proteins. Recently, Hsp90 has emerged as being of prime importance to the growth and survival of cancer cells and its inhibitors have already been used in phase I and II clinical trials. We investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM). We found that 17-AAG led to significant G1 or G2/M cell cycle arrest, inhibition of cell proliferation, and decrease of AKT, AKT1, and survivin expression in all human malignant pleural mesothelioma cell lines examined. We also observed significant apoptosis induction in all MM cell lines treated with 17-AAG. Furthermore, 17-AAG induced apoptosis in freshly cultured primary MM cells and caused signaling changes identical to those in 17-AAG treated MM cell lines. These results suggest that Hsp90 is strongly associated with the growth and survival of MM and that inhibition of Hsp90 may have therapeutic potential in the treatment of MM.

The Hsp90 Molecular Chaperone Modulates Multiple Telomerase Activities

The Hsp90 molecular chaperone is a highly abundant eukaryotic molecular chaperone. While it is understood that Hsp90 modulates a significant number of proteins, the mechanistic contributions made by Hsp90 to a client protein typically are not well understood. Here we investigate the yeast Hsp90 regulatory roles with telomerase. Telomerase lengthens chromosome termini by specifically associating with single-stranded telomeric DNA and appending nucleotides by reverse transcription. We have found that the yeast Hsp90 homolog Hsp82p promotes both telomerase DNA binding and nucleotide addition properties. By isolating telomerase from different allelic backgrounds we observed distinct defects. For example, in an hsp82 T101I strain telomerase displayed decreased nucleotide processivity, whereas both DNA binding and extension activities were lowered in a G170D background. The decline in telomerase DNA binding correlated with a loss of Hsp82p association. No matter the defect, telomerase activity was recovered upon Hsp82p addition. Importantly, telomere length and telomerase telomere occupancy was yeast Hsp90 dependent. Taken together, our results indicate that Hsp82p promotes telomerase DNA association and facilitates DNA extension once telomerase is engaged with the DNA.

2-color photobleaching experiments reveal distinct intracellular dynamics of two components of the Hsp90 complex

The abundant molecular chaperone Hsp90 functions in association with co-chaperones including p23 to promote the folding and maturation of a subset of cytosolic proteins. “Fluorescence recovery after photobleaching” (FRAP) experiments showed that the dynamics of p23 in live cells is dictated by Hsp90. Since Hsp90 is present in large excess over p23, the mobility of Hsp90 could conceivably be quite different. To facilitate the analysis and to allow a direct comparison with p23, we developed a 2-color FRAP technique. Two test proteins are expressed as fusion proteins with the two spectrally separable fluorescent proteins mCherry and enhanced green fluorescent protein (EGFP). The 2-color FRAP technique is powerful for the concomitant recording of two proteins located in the same area of a cell, two components of the same protein complex, or mutant and wild-type versions of the same protein under identical experimental conditions. 2-color FRAP of Hsp90 and p23 is virtually indistinguishable, consistent with the notion that they are both engaged in a multitude of large protein complexes. However, when Hsp90–p23 complexes are disrupted by the Hsp90 inhibitor geldanamycin, p23 moves by free diffusion while Hsp90 maintains its low mobility because it remains bound in remodeled multicomponent complexes.

Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies

Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies

Human and yeast Hsp110 chaperones exhibit functional differences

Hsp110 proteins constitute a heterogeneous family of abundant molecular chaperones, related to the Hsp70 proteins and exclusively found in the cytosol of eukaryotic organisms. Hsp110 family members are described as efficient holdases, preventing the aggregation and assisting the refolding of heat-denatured model substrates in the presence of Hsp70 chaperones and their co-chaperones. To gain more insights into the mode of action of this protein family we compared two homologues representing two subtypes of Hsp110 proteins, S. cerevisiae Sse1 and H. sapiens Apg-2, in their structural and functional properties in vitro. In contrast to previous publications both proteins exhibited intrinsic ATPase activities, which only in the case of Sse1 could be stimulated by the Hsp40 co-chaperone Sis1. Similar to Hsp70 proteins ATP binding and hydrolysis induced conformational rearrangements in both Hsp110 proteins as detected by tryptophane fluorescence. However, nucleotide induced changes in the proteolytic digestion pattern were detected only for Sse1. Sse1 and Apg-2 thus show significant differences in their biochemical properties, which may relate to differences in their functional roles in vivo.

Down-regulation of Hsp60 expression by RNAi impairs folding of medium-chain acyl-CoA dehydrogenase wild-type and disease-associated proteins

We have analyzed the role of the highly abundant molecular chaperone Hsp60 in the biogenesis of medium-chain acyl-CoA dehydrogenase (MCAD) using RNA interference (RNAi). MCAD is a mitochondrial enzyme involved in the fatty acid metabolism and previous studies in isolated rat mitochondria or prokaryotic expression systems have shown that Hsp60 and GroEL are involved in the folding of MCAD proteins. To elucidate the impact of Hsp60 levels for folding and assembly of MCAD proteins in intact mammalian cells, we report the design and in vivo synthesis of anti-human Hsp60 small-hairpin RNAs (shRNAs). Quantitative PCR analysis of transfected HEK-293 cells showed significant down-regulation of endogenous Hsp60 mRNA 48 h post-transfection and Western blot analysis confirmed the reduced levels of Hsp60 protein. Furthermore, expression of exogenous Myc-tagged Hsp60 was decreased in shRNA-transfected cells. Flow cytometry showed that shRNA-treatment only affects green fluorescent protein targeted to mitochondria, demonstrating that the shRNA effect is specific. In cells with reduced Hsp60 levels both the amounts of total MCAD proteins and folded MCAD were reduced for MCAD wild-type and the two disease-associated variants studied. A similar effect was observed in cells expressing mitochondrial short-chain acyl-CoA dehydrogenase. Thus, in intact human cells we demonstrate that Hsp60 is involved in the folding of MCAD variant proteins. The present system can be used to study the requirement of Hsp60 for folding of other mitochondrial proteins and to assess the role of Hsp60 for the severity of genetic defects involving these proteins.

Structures of the N-terminal and middle domains of E. coli Hsp90 and conformation changes upon ADP binding.

Hsp90 is an abundant molecular chaperone involved in many biological systems. We report here the crystal structures of the unliganded and ADP bound fragments containing the N-terminal and middle domains of HtpG, an E. coli Hsp90. These domains are not connected through a flexible linker, as often portrayed in models, but are intimately associated with one another. The individual HtpG domains have similar folding to those of DNA gyrase B but assemble differently, suggesting somewhat different mechanisms for the ATPase superfamily. ADP binds to a subpocket of a large site that is jointly formed by the N-terminal and middle domains and induces conformational changes of the N-terminal domain. We speculate that this large pocket serves as a putative site for binding of client proteins/cochaperones. Modeling shows that ATP is not exposed to the molecular surface, thus implying that ATP activation of hsp90 chaperone activities is accomplished via conformational changes.

FKBP52

The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug FK506. FKBP52 exhibits peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is inhibited by the binding of FK506—properties that it shares with the smaller but better-studied immunophilin, FKBP12. Unlike FKBP12, however, FKBP52 does not mediate the immunosuppressive actions of FK506 and, due to its larger size, contains additional numerous functional domains. One such structure is a series of tetratricopeptide repeat (TPR) domains, which serve as binding sites for the ubiquitous and abundant molecular chaperone, Hsp90. It is this property as a TPR protein that best characterizes the known cellular roles of FKBP52. Here, we review the structural features of FKBP52 and relate them to the evolving and diverse functions of this protein. Although the most recognized role of FKBP52 is in regulation of steroid receptor signaling, other less well-known functions are also discussed.

Commonly used antibiotics induce expression of Hsp 27 and Hsp 60 and protect human lymphocytes from apoptosis

Heat shock proteins (Hsps) are abundant molecular chaperones participating in the cytoprotection. The kinetics of synthesis of Hsps closely correlates with the kinetics of development of resistance to cell death. In this study, we analysed the probable involvement of Hsp 27 and Hsp 60 in the protection of cells undergoing apoptosis. Human lymphocytes cultured in the presence of ampicillin or ceftriaxone produced Hsp 60 and Hsp 27, estimated by immunoblotting in a time-dependent manner and the increased levels of Hsp 60 and Hsp 27 correlated with enhanced resistance of the lymphocytes to apoptosis, as determined by flow cytometry. Cultures treated with ampicillin or ceftriaxone also exhibited smaller numbers of apoptotic cells than untreated cultures when exposed to the apoptosis-inducing agent staurosporine (1 mM). In contrast, cloramphenicol induced the production of only small amounts of Hsp 60, and no resistance apoptosis. Further studies are needed to clarify the potential role of Hsp 27 and Hsp 60 in the resistance of human cells to apoptosis and the effects of antibiotics on this phenomenon.

Commonly used antibiotics induce expression of Hsp 27 and Hsp 60 and protect human lymphocytes from apoptosis

Heat shock proteins (Hsps) are abundant molecular chaperones participating in the cytoprotection. The kinetics of synthesis of Hsps closely correlates with the kinetics of development of resistance to cell death. In this study, we analysed the probable involvement of Hsp 27 and Hsp 60 in the protection of cells undergoing apoptosis. Human lymphocytes cultured in the presence of ampicillin or ceftriaxone produced Hsp 60 and Hsp 27, estimated by immunoblotting in a time-dependent manner and the increased levels of Hsp 60 and Hsp 27 correlated with enhanced resistance of the lymphocytes to apoptosis, as determined by flow cytometry. Cultures treated with ampicillin or ceftriaxone also exhibited smaller numbers of apoptotic cells than untreated cultures when exposed to the apoptosis-inducing agent staurosporine (1 mM). In contrast, cloramphenicol induced the production of only small amounts of Hsp 60, and no resistance apoptosis. Further studies are needed to clarify the potential role of Hsp 27 and Hsp 60 in the resistance of human cells to apoptosis and the effects of antibiotics on this phenomenon.

Geldanamycin, a heat shock protein 90-binding agent, disrupts Stat5 activation in IL-2-stimulated cells.

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone in eukaryotic cells. Hsp90 plays a critical role in regulating signal transduction pathways that control cell proliferation since its chaperone function is restricted to a subset of proteins including some signal molecules. Improper function of these proteins can be induced by an anti-tumor agent geldanamycin (GA) which is the specific inhibitor of Hsp90. In this study, it was demonstrated that GA interferes with IL-2-stimulated proliferation of murine CTLL-2 cells. As to the signaling mechanisms underlying this inhibitory effect, we discovered GA disrupts the IL-2-stimulated activation and phosphorylation of the transcription factor Stat5, indicating the proper function of Hsp90 is indispensable for Stat5 activation. This conclusion is validated by the observation that Hsp90 interacts with Stat5 in the immunoprecipitation assay and GA interrupts their interaction. Furthermore, by constructing deletion mutants, we identified the c-terminal half of Stat5 coiled-coil region is responsible for binding with Hsp90.

Hsp90 Inhibition Accelerates Cell Lysis: ANTI-Hsp90 RIBOZYME REVEALS A COMPLEX MECHANISM OF Hsp90 INHIBITORS INVOLVING BOTH SUPEROXIDE- AND Hsp90-DEPENDENT EVENTS

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.

Hsp90 reaches new heights. Conference on the Hsp90 chaperone machine.

The first international meeting on the Hsp90 (heat‐shock protein 90) chaperone machine was held in Arolla, Switzerland, from 24 to 28 August 2002. Located in a beautiful isolated chalet, high in the Swiss Alps, this was a highly interactive and productive gathering, whose success owed much to its organizers, Didier Picard and Johannes Buchner. ![][1] Hsp90 (heat‐shock protein 90) is an abundant molecular chaperone, but its function seems to be restricted to the folding of proteins involved in cell signalling, such as transcription factors and protein kinases. This restricted set of ‘clients’ (see Fig. 1) makes Hsp90 an attractive target for cancer therapeutics. As an anti‐Hsp90 drug is now in clinical trials, the meeting was relevant to a broad range of scientists interested in its chaperone activity. However, even with this clinical relevance, a meeting devoted to just one chaperone might seem like too much of a good thing, unless one takes into account the myriad of co‐chaperones that regulate Hsp90. These co‐chaperones modulate the ATPase activity of Hsp90 and many of them have intrinsic chaperone activity of their own, providing some measure of specificity for different Hsp90 clients. There is also some crossover between different chaperone machineries, as some co‐chaperones interact with Hsp70 as well as Hsp90. Figure 1. Examples of roles of Hsp90 and its co‐chaperones in different cellular processes through their interactions with different client proteins. The conference was opened by D. Smith (Scottsdale, AZ, USA), who gave a historical perspective on the interactions of Hsp90 and its co‐chaperones with steroid receptors (reviewed by Pratt & Toft, 1997). His presentation began with a review of the work of Toft and Gorksi from the late 1960s, in which an oestrogen receptor was identified and found to exist in a large complex of proteins. Smith followed the identification and characterization of Hsp90 … [1]: /embed/graphic-1.gif

Oolemmal proteomics – identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane

BackgroundThe mature mouse egg contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins remain to be characterized, therefore in this study we have identified highly abundant egg proteins using a proteomic approach and found that several of these proteins also appear to localize to the egg surface. Characterization of such molecules will provide important insight into the cellular events of fertilization and early development.MethodsIn order to identify some of the more abundant egg proteins, whole egg extracts were resolved on coomassie-stained two-dimensional (2D) PAGE gels. Several highly abundant protein spots were cored and microsequenced by tandem mass spectrometry (TMS), and determined to be molecular chaperone proteins. Concurrent experiments were performed to identify oolemmal proteins using 2D avidin blotting. Proteins spots that appeared to be surface labeled by biotinylation were correlated with the initial coomassie-stained reference gel. Surprisingly, some of the surface labelled proteins corresponded to those abundant chaperone proteins previously identified. To confirm whether these molecules are accumulating at the oolemmal surface in eggs, we performed immunofluoresence on live, zona-free eggs using antibodies to HSP70, HSP90, GRP94, GRP78, calreticulin and calnexin.ResultsThe putative surface-labeled proteins identified by biotinylation included the molecular chaperones HSP70 (MW 70 KDa, pI 5.5), HSP90a (MW 85 KDa, pI 4.9), GRP94 (MW 92 KDa, pI 4.7), GRP78 (MW 72 KDa, pI 5.0), Oxygen regulated protein 150 (ORP150; MW 111 KDa, pI 5.1), Calreticulin (MW 48 KDa, pI 4.3), Calnexin (MW 65 KDa, pI 4.5), and Protein disulfide isomerase (PDI; MW 57 KDa, pI 4.8). Immunofluoresence results showed that antibodies to HSP90, GRP94, GRP78 and calreticulin were reactive with oolemmal proteins. We were unable to confirm surface localization of HSP70 or calnexin by this method.ConclusionsWe report here the identification of nine highly abundant molecular chaperones in the mouse egg proteome. In addition, we present preliminary data suggesting that these molecules localize to the oolemma of the mature mouse egg.

N-terminal Residues Regulate the Catalytic Efficiency of the Hsp90 ATPase Cycle

Hsp90 is an abundant molecular chaperone involved in a variety of cellular processes ranging from signal transduction to viral replication. The function of Hsp90 has been shown to be dependent on its ability to hydrolyze ATP, and in vitro studies suggest that the dimeric nature of Hsp90 is critical for this activity. ATP binding occurs at the N-terminal domains of the Hsp90 dimer, whereas the main dimerization site resides in the very C-terminal domain. ATP hydrolysis is performed in a series of conformational changes. These include the association of the two N-terminal domains, which has been shown to stimulate the hydrolysis reaction. In this study, we set out to identify regions in the N-terminal domain that are important for this interaction. We show that N-terminal deletion variants of Hsp90 are severely impaired in their ability to hydrolyze ATP. However, nucleotide binding of these constructs is similar to that of the wild type protein. Heterodimers of the Hsp90 deletion mutants with wild type protein showed that the first 24 amino acids play a crucial role during the ATPase reaction, because their deletion abolishes the trans-activation between the two N-terminal domains. We propose that the turnover rate of Hsp90 is decisively controlled by intermolecular interactions between the N-terminal domains.

Exogenous Expression of Heat Shock Protein 90kDa Retards the Cell Cycle and Impairs the Heat Shock Response

The 90-kDa heat shock protein, HSP90, is an abundant molecular chaperone which functions in cellular homeostasis in prokaryotes and eukaryotes. It is well known that HSP90 plays a critical and indispensable role in regulating cell growth through modulations of various signal transduction pathways, but its roles in cell cycle control are not so well known. We transferred human HSP90 (wild-type or mutated types) expression vectors into NIH-3T3 cells in order to study certain functions of HSP90 in the cell cycle and cell growth under physiological conditions. We found that the exogenous expression of HSP90 (wild-type) induced a decrease in cell growth via retardation of the G1/S transition. The inhibition of cell growth was caused by reduced expressions of cyclin D3 and cyclin A mRNA and protein. On the other hand, no stable transfectants with the three types of mutated HSP90 were obtained. Unexpectedly, exogenous HSP90 expression impaired the heat shock response by inhibiting both heat shock transcription factor 1(HSF1) activation and transportation of HSF1 into the nucleus. The HSF1 function was disrupted by the direct association between HSF1 and exogenous HSP90, which was present as a monomer. These results reveal important roles of HSP90 in cell cycle control and in the stress response of nontransformed cells.

Hsp104 interacts with Hsp90 cochaperones in respiring yeast.

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.

Three-dimensional structure topology of the calreticulin P-domain based on NMR assignment

Calreticulin (CRT) is an abundant molecular chaperone of the endoplasmic reticulum. Its central, proline-rich P-domain, comprising residues 189–288, contains three copies of each of two repeat sequences (types 1 and 2), which are arranged in a characteristic ‘111222’ pattern. Here we show that the three-dimensional structure of CRT(189–288) contains a single hairpin fold formed by the entire polypeptide chain. The loop at the bottom of the hairpin consists of residues 227–247, and is closed by an anti-parallel β-sheet of residues 224–226 and 248–250. Two additional β-sheets contain residues 207–209 and 262–264, and 190–192 and 276–278. The 17-residue spacing of the β-strands in the N-terminal part of the hairpin and the 14-residue spacing in the C-terminal part reflect the length of the type 1 and type 2 sequence repeats. As a consequence of this topology the peptide segments separating the β-strands in the N-terminal part of the hairpin are likely to form bulges to accommodate the extra residues. These results are based on nearly complete sequence-specific NMR assignments for CRT(189–288), which were obtained using standard NMR techniques with the 13C/ 15N-labeled protein, and collection of nuclear Overhauser enhancement upper distance constraints.

Reactive Cysteines of the 90-kDa Heat Shock Protein, Hsp90

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of the eukaryotic cytoplasm. Its cysteine groups participate in the interactions of Hsp90 with the heme-regulated eIF-2α kinase and molybdate, a stabilizer of Hsp90–protein complexes. In our present studies we investigated the reactivity of the sulfhydryl groups of Hsp90. Our data indicate that Hsp90 as well as two Hsp90 peptides containing Cys-521 and Cys-589/590 are able to reduce cytochrome c. The effect of Hsp90 can be blocked by sulfhydryl reagents including arsenite and cadmium, which indicates the involvement of the vicinal cysteines Cys589/590 in the reduction of cytochrome c. Hsp90 neither reduces the disulfide bonds of insulin nor possesses a NADPH:quinone oxidoreductase activity. Oxidizing conditions impair the chaperone activity of Hsp90 toward citrate synthase. The high and specific reactivity of Hsp90 cysteine groups toward cytochrome c may indicate a role of this chaperone in modulation of the redox status of the cytosol in resting and apoptotic cells.