The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.     

Role of heat shock protein Hsp90 in formation of protective reactions in acute toxic stress

The involvement of heat shock protein Hsp90 in pro-inflammatory response in male NMRI mice under conditions of acute toxic stress, caused by lipopolysaccharide from Gram negative bacteria, was studied using geldanamycin, a specific blocker of the activity of this protein. It is shown that the introduction of geldanamycin lowers total intoxication of the organism upon acute toxic stress caused by endotoxin. Thus, a decrease in cytokine TNF-α, IFN-γ, IL-1, and IL-10 concentrations in blood serum of the geldanamycin-treated animals with acute toxic stress was found along with normalization of functional activity of nitric oxide producing peritoneal macrophages. Studying expression of receptor protein Tlr-4 as well of proteins of two signal cascades, NF-κB and SAPK/JNK, has shown that mechanisms of the geldanamycin protective effect are realized at the level of inhibition of Tlr-4 receptor expression, which provides for endotoxin-to-cell binding, and due to lowering the endotoxin-stimulated activation of signal cascades NF-κB and SAPK/JNK. The results suggest Hsp90 might be a therapeutic target in diseases accompanied by acute toxic stress.     

The role of protein kinase SAPK/JNK in cell responses to low-intensity nonionizing radiation

The effect of low-intensity laser light (He-Ne, 0.2 mW/cm², 632.8 nm, exposure time 1 min) or centimeter waves (8.15–18 GHz, 1 μW/cm², exposure time 1 h) on Phospho-SAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes or cells incubated previously with geldanamycin, an inhibitor of Hsp90, were used in the experiments. Significant stimulation of Phospho-SAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models. It was proposed that activation of the SAPK/JNK signal pathway plays one of the central roles in cellular stress response to low-power nonionizing radiation.     

Effect of the Hsp90 modulators on the heat-shock response in eukaryotic cells

The possible role of the heat-shock protein 90 (Hsp90) complex on the heat-shock (HS) response in yeast using the Hsp90 inhibitors geldanamycin (GA) and 17-allylamino-17-demethoxygeldanamycin (AAG), and prednisolone and 17beta-estradiol as modulators was investigated. Following long- or short-term administration of the drugs, either alone or in combination, the response was determined as cell viability and growth after exposure to HS. Upon short-term preconditioning, both Hsp90 inhibitors conferred cycloheximide-dependent thermal resistance to the yeast cultures, while upon long-term treatment the induction of thermotolerance was confined only to AAG. Co-administration of prednisolone or 17beta-estradiol failed to significantly alter the response to Hsp90 inhibitors. However, since short-term incubation with prednisolone alone induced thermotolerance, increased the budding cell fraction and tended to reduce the adaptive response to GA, its effect on GA-induced thermotolerance is not yet explained. Generally, GA and AAG showed a comparable short-term action but a different long-term effect on the HS response in yeast; this response was not related to any regulation by prednisolone or 17beta-estradiol (while 17beta-estradiol was unable to modify the response, the action of prednisolone in both the stress response and the cell cycle was equivocal).     

The role of TLR4 receptor in the stress response of lymphocytes

In vitro effects of low-level electromagnetic waves (8.18 GHz, frequency swings within 1 s, intensity 1 microW/cm, exposure for 1 h) and low-energy laser light (He-Ne laser with 632.8 nm, 0.2 mW/cm, dose 1.2 x 10(-2) J/cm2) on the expression of receptor protein TLR4, which is known as a part of the system for microbal toxin recognition, were studied in mouse lymphocytes. In addition, TLR4 expression was examined in situations when stress responses to low-level nonionizing radiation were modified by the antibiotic geldanamycin, which suppresses the activity of the heat shock protein Hsp90. It was found that low-level microwaves significantly raised the amount of TLR4; in contrast, laser light decreased the expression of the receptor in lymphocytes. In cells pretreated with geldanamycin, the TLR4 expression in irradiated cells was reduced to minimum levels, much lower than control values. The results showed that TLR4, which is involved in specific binding of toxin from gram-negative bacteria, can regulate cell responses to signals of other origin, in particular to nonionizig radiation, including low-level microwaves and laser light.     

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.     

Changes in heat-shock protein synthesis and thermotolerance of Arabodopsis thaliana seedlings resulting from Hsp90 inhibition by geldanamycin

The influence of geldanamycin (GA), a specific inhibitor of heat-shock protein Hsp90, on the synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA under normal conditions induced synthesis of these stress proteins. Treatment of seeds with the Hsp90 inhibitor resulted in elevated constitutive levels of Hsp70 and Hsp90 in seedlings, as well as increased induction of their synthesis under heat shock. The GA effect increased with its concentration. Hsp up-regulation promoted thermotolerance of seedlings. The findings suggest autoregulation of heatshock protein synthesis and regulation of plant tolerance by Hsp90.     

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.     

Heat shock protein 90 regulates the stability of MEKK3 in HEK293 cells

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the conformational maturation and function of certain signaling proteins. Hsp90 inhibitors cause the inactivation, destabilization and eventual degradation of Hsp90 client proteins through occupying the ATP/ADP binding pocket of Hsp90. In the present study, we found that Hsp90 interacted with MEKK3 in HEK293 cells. Hsp90 inhibitors reduced the level of endogenous MEKK3 in time- and dose-dependent manners, and this decrease was reversed by Hsp90 overexpression. In addition, Hsp90 RNAi destabilized MEKK3. A selective inhibitor of Hsp90, geldanamycin (GA), shortened MEKK3 half-life, and induced ubiquitination and proteasomal degradation of MEKK3. These results strongly suggested that Hsp90 could work as the molecular chaperone of MEKK3.     

Plasmodial heat shock proteins: targets for chemotherapy

Heat shock proteins act as molecular chaperones, facilitating protein folding in cells of living organisms. Their role is particularly important in parasites because environmental changes associated with their life cycles place a strain on protein homoeostasis. Not surprisingly, some heat shock proteins are essential for the survival of the most virulent malaria parasite, Plasmodium falciparum . This justifies the need for a greater understanding of the specific roles and regulation of malarial heat shock proteins. Furthermore, heat shock proteins play a major role during invasion of the host by the parasite and mediate in malaria pathogenesis. The identification and development of inhibitor compounds of heat shock proteins has recently attracted attention. This is important, given the fact that traditional antimalarial drugs are increasingly failing, as a consequence of parasite increasing drug resistance. Heat shock protein 90 (Hsp90), Hsp70/Hsp40 partnerships and small heat shock proteins are major malaria drug targets. This review examines the structural and functional features of these proteins that render them ideal drug targets and the challenges of targeting these proteins towards malaria drug design. The major antimalarial compounds that have been used to inhibit heat shock proteins include the antibiotic, geldanamycin, deoxyspergualin and pyrimidinones. The proposed mechanisms of action of these molecules and the pathways they inhibit are discussed.     

Stress protein flux during recovery from simulated ischemia: induced heat shock protein 70 confers cytoprotection by suppressing JNK activation and inhibiting apoptotic cell death

Multiple stress proteins are recruited in response to stress in living cells. There are limited reports in the literature analyzing multiple stress protein shifts and their functional consequences on stress response. Using two-dimensional electrophoresis we have analyzed shifts in stress protein profiles in response to energy deprivation as a model of ischemic injury to kidneys. A group of chaperones and stress-induced mitogen activated protein (MAP) kinases were analyzed. In addition to examining stress protein induction and phosphorylation we have also examined the mechanism of cytoprotection by heat shock protein 70 (Hsp70). Our results show that, of the different stress proteins examined, only binding protein (BiP) and Hsp70 were significantly induced upon energy deprivation. Other stress proteins, including Hsp27, calnexin, Hsp90 and ERp57 showed alterations in their phosphorylation profiles. Three different MAP kinases, namely p38, extracellular signal regulated kisase and c-jun N-terminal kinase (JNK) were activated in response to energy deprivation. While JNK activation was linked to apoptosis, activated-p38 was involved in phosphorylation of Hsp27. Study of inhibitors of Hsp70 induction or pre-induction of Hsp70 indicated that induced Hsp70 was involved in the suppression of JNK activation thereby inhibiting apoptotic cell death. Our results provide important insights into the flux in stress protein profiles in response to simulated ischemia and highlight the antiapoptotic, cytoprotective mechanism of Hsp70 action.     

Protein quality control of DYRK family protein kinases by the Hsp90-Cdc37 molecular chaperone

The DYRK (Dual-specificity tYrosine-phosphorylation Regulated protein Kinase) family consists of five related protein kinases (DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4). DYRKs show homology to Drosophila Minibrain, and DYRK1A in human chromosome 21 is responsible for various neuronal disorders including human Down syndrome. Here we report identification of cellular proteins that associate with specific members of DYRKs. Cellular proteins with molecular masses of 90, 70, and 50-kDa associated with DYRK1B and DYRK4. These proteins were identified as molecular chaperones Hsp90, Hsp70, and Cdc37, respectively. Microscopic analysis of GFP-DYRKs showed that DYRK1A and DYRK1B were nuclear, while DYRK2, DYRK3, and DYRK4 were mostly cytoplasmic in COS7 cells. Overexpression of DYRK1B induced nuclear re-localization of these chaperones with DYRK1B. Treatment of cells with specific Hsp90 inhibitors, geldanamycin and 17-AAG, abolished the association of Hsp90 and Cdc37 with DYRK1B and DYRK4, but not of Hsp70. Inhibition of Hsp90 chaperone activity affected intracellular dynamics of DYRK1B and DYRK4. DYRK1B and DYRK4 underwent rapid formation of cytoplasmic punctate dots after the geldanamycin treatment, suggesting that the chaperone function of Hsp90 is required for prevention of protein aggregation of the target kinases. Prolonged inhibition of Hsp90 by geldanamycin, 17-AAG, or ganetespib, decreased cellular levels of DYRK1B and DYRK4. Finally, DYRK1B and DYRK4 were ubiquitinated in cells, and ubiquitinated DYRK1B and DYRK4 further increased by Hsp90 inhibition with geldanamycin. Taken together, these results indicate that Hsp90 and Cdc37 discriminate specific members of the DYRK kinase family and play an important role in quality control of these client kinases in cells.