Effects of 50 Hz sinusoidal magnetic fields on Hsp27, Hsp70, Hsp90 expression in porcine aortic endothelial cells (PAEC)

The aim of the present study was to investigate the influence of 50 Hz sinusoidal magnetic field on Hsp27, Hsp70, and Hsp90 expression in a model of primary culture of porcine aortic endothelial cells (PAEC). We took into consideration the Hsp profile in terms of mRNA expression, protein expression and protein localization inside the cells. The choice of the cell system was motivated by the involvement of the endothelial cells in the onset of many diseases; moreover, only few reports describe the effects of extremely low frequency magnetic fields (ELF-MFs) on such cells. ELF-MF exposure induced an increase in the mRNA levels of the three proteins, which was statistically significant for Hsp70. On the contrary, we did not observe any influence on Hsp27, Hsp70, and Hsp90 protein levels. Analysis in situ by immunofluorescence revealed that ELF-MF exposure affected the cellular distribution of Hsp27; in particular a partial relocalization in the nucleus was observed.     

Hsp90 isoforms: functions, expression and clinical importance

The 90 kDa heat shock protein, Hsp90, is a main functional component of an important cytoplasmic chaperone complex, and it is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. Identification of Hsp90 as a molecular target of various anticancer drugs highlighted its importance from the clinical point of view. Here we summarize the current knowledge of various Hsp90 isoforms regarding their genomic location, molecular evolution, functional differences, differential induction after various environmental stresses and in pathological conditions as well as the growing importance of discriminating between Hsp90 isoforms in clinical practice.     

A small molecule that preferentially binds the closed conformation of Hsp90

Described is the synthesis of three different fluorescein-tagged derivatives of a macrocycle, and their binding affinity to heat shock protein 90 (Hsp90). Using fluorescence polarization anisotropy, we report the binding affinity of these fluorescein-labeled compounds to Hsp90 in its open state and ATP-dependent closed state. We show that the compounds demonstrate a conformation-dependent preference for binding to the closed state.     

Post-translational modifications of Hsp90 and their contributions to chaperone regulation

Molecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport, and degradation of proteins as well as in facilitating cell signaling. Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the varied roles of known post-translational modifications of cytosolic and nuclear Hsp90 (phosphorylation, acetylation, S-nitrosylation, oxidation and ubiquitination) in fine-tuning chaperone function in eukaryotes. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

FAT10 protein binds to polyglutamine proteins and modulates their solubility

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.     

Microtubule-associated protein 4 binds to actin filaments and modulates their properties

We previously reported that an isoform of microtubule-associated protein 4 (MAP4) is localized to the distal area of developing neurites, where microtubules are relatively scarce, raising the possibility that MAP4 interacts with another major cytoskeletal component, actin filaments. In the present study, we examined the in vitro interaction between MAP4 and actin filaments, using bacterially expressed MAP4 and its truncated fragments. Sedimentation assays revealed that MAP4 and its microtubule-binding domain fragments bind to actin filaments under physiological conditions. The apparent dissociation constant and the binding stoichiometry of the fragments to actin were about 0.1 μm and 1 : 3 (MAP4/actin), respectively. Molecular dissection studies revealed that the actin-binding site on MAP4 is situated at the C-terminal part of the proline-rich region, where the microtubule-binding site is also located. Electron microscopy revealed that the MAP4-bound actin filaments become straighter and longer and that the number of actin bundles increases with greater concentrations of added MAP4 fragment, indicating that MAP4 binding alters the properties of the actin filaments. A multiple sequence alignment of the proline-rich regions of MAP4 and tau revealed two putative actin-binding consensus sequences.     

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L

A stress treatment of 32 degrees C for at least 8h was able to change the gametophytic program of the microspore, switching it to embryogenesis in Brassica napus, an interesting model for studying this process in vitro. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores, not sensitive to induction, followed their original gametophytic development. In this work the distribution and ultrastructural localization of two heat-shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins are rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution patterns of these proteins, these changes being assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and noninduced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores, suggesting a possible additional role for Hsp70 in the switch to embryogenesis. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction. The differential appearance and distribution of the two proteins and their association at specific stages have been determined between the two systems coexisting in the same culture: embryogenic development (induced cells) and development of gametes (noninduced cells).     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Aha1 binds to the middle domain of Hsp90, contributes to client protein activation,and stimulates the ATPase activity of the molecular chaperone

The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of Hsp90 5-fold. To establish their cellular role further, we deleted the genes encoding Aha1 and Hch1 in S. cerevisiae. In vivo experiments demonstrated that Aha1 and Hch1 contributed to efficient activation of the heterologous Hsp90 client protein v-Src. Moreover, Aha1 and Hch1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90.     

Chaperone Hsp27 modulates AUF1 proteolysis and AU-rich element-mediated mRNA degradation

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues--Ser(15), Ser(78), and Ser(82)-by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2-Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization.     

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.     

Hsp90 binds microtubules and is involved in the reorganization of the microtubular network in angiosperms

Microtubules (MTs) are essential for many processes in plant cells. MT-associated proteins (MAPs) influence MT polymerization dynamics and enable them to perform their functions. The molecular chaperone Hsp90 has been shown to associate with MTs in animal and plant cells. However, the role of Hsp90-MT binding in plants has not yet been investigated. Here, we show that Hsp90 associates with cortical MTs in tobacco cells and decorates MTs in the phragmoplast. Further, we show that tobacco Hsp90_MT binds directly to polymerized MTs in vitro. The inhibition of Hsp90 by geldanamycin (GDA) severely impairs MT re-assembly after cold-induced de-polymerization. Our results indicate that the plant Hsp90 interaction with MTs plays a key role in cellular events, where MT re-organization is needed.     

Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90beta,a property that can be exploited in screens for new Hsp90 chaperone inhibitors

Heat shock protein 90 (Hsp90) is essential for activation of many of the most important regulatory proteins of eukaryotic cells. It is an extremely conserved protein, such that heterologous expressions of either human Hsp90beta or Caenorhabditis elegans Hsp90 will provide the essential Hsp90 function in yeast. The ability of these metazoan Hsp90s to provide this Hsp90 function to yeast cells requires Sti, a Hsp90 system cochaperone. Yeast that is expressing human Hsp90beta in place of the normal native yeast Hsp90 is selectively hypersensitised to Hsp90 inhibitor drugs. Hsp90 drugs are promising anticancer agents, their administration simultaneously destabilizing a number of the proteins critical to multistep carcinogenesis. Though one of these drugs (17-allylaminogeldanamycin, 17-AAG) is now progressing to Phase 2 clinical trials, there is a pressing need to identify selective Hsp90 inhibitors that are more soluble than 17-AAG. High-throughput screening for chemical agents that exert greater inhibitory effects against yeast expressing the human Hsp90beta relative to yeast expressing its native Hsp90 should therefore facilitate the search for new Hsp90 inhibitors.     

TRAP-1, the mitochondrial Hsp90

Protein folding quality control does not occur randomly in cells, but requires the action of specialized molecular chaperones compartmentalized in subcellular microenvironments and organelles. Fresh experimental evidence has now linked a mitochondrial-specific Heat Shock Protein-90 (Hsp90) homolog, Tumor Necrosis Factor Receptor-Associated Protein-1 (TRAP-1) to pleiotropic signaling circuitries of organelle integrity and cellular homeostasis. TRAP-1-directed compartmentalized protein folding is broadly exploited in cancer and neurodegenerative diseases, presenting new opportunities for therapeutic intervention in humans. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).