Activation Of Hsp90 Research Articles

Asymmetric Activation of the Hsp90 Dimer by Its Cochaperone Aha1

The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.

Polypeptide from Chlamys farreri inhibits UVB-induced apoptosis of HaCaT cells via iNOS/NO and HSP90

Polypeptide from Chlamys farreri (PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri, and was found to be an effective antioxidant in our recent studies. In this study, we investigated the effect of PCF on ultraviolet B (UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved. Pretreatment with the inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis, indicating that iNOS and NO play important roles in apoptosis. On the other hand, the inhibition of UVB-induced apoptosis in the immortalized keratinocyte (HaCaT) cells by PCF was estimated using a DNA ladder. PCF treatment inhibited UVB-induced iNOS activation, as determined by RT-PCR, NO production, as determined by ESR, and up-regulated heat shock protein (HSP) 90 activation, as determined by Western blotting. Our results indicate that iNOS and NO are involved in UVB-induced apoptosis of HaCaT cells and the protective effect of PCF against UVB irradiation is exerted by suppressing the expression of iNOS, followed by inhibition of NO release and enhanced activation of HSP90.

Silencing of HSP90 Cochaperone AHA1 Expression Decreases Client Protein Activation and Increases Cellular Sensitivity to the HSP90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin

AHA1 (activator of HSP90 ATPase) is a cochaperone of the ATP-dependent molecular chaperone, HSP90, which is involved in the maturation, stabilization/degradation, and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of cells with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) results in the degradation of client proteins via the ubiquitin-proteasome pathway. As AHA1 increases the ATPase activity of HSP90, we hypothesized that modulation of AHA1 expression could influence the activity of client proteins and/or the cellular response to 17-AAG. We show that the basal expression of AHA1 is different across a panel of human cancer cell lines, and that treatment with 17-AAG resulted in sustained AHA1 up-regulation. Increasing the expression of AHA1 did not affect the sensitivity to 17-AAG, but did increase C-RAF activity and the levels of phosphorylated MEK1/2 and ERK1/2 without affecting total levels of these proteins or of client proteins C-RAF, ERBB2, or CDK4. Conversely, small interfering RNA-selective knockdown of >80% of AHA1 expression decreased C-RAF activity and reduced the levels of MEK1/2 and ERK1/2 phosphorylation. Moreover, the AHA1 knockdown resulted in a significant (P < 0.05) increase in sensitivity to 17-AAG, due in part to a 2- to 3-fold increase in apoptosis. These results show that the reduction of AHA1 levels could decrease the phosphorylation of key signal transduction proteins, and for the first time, separate the activation and stabilization functions of HSP90. Furthermore, AHA1 knockdown could sensitize cancer cells to 17-AAG. We conclude that modulation of AHA1 might be a potential therapeutic strategy to increase sensitivity to HSP90 inhibitors.

Activation of Multiple Signal Transduction Pathways as a Prognostic Marker in Acute Myelogenous Leukemia.

Deregulation of signal transduction pathways (STPs) including JAK/STAT, RAS/Raf/MEK/ERK and PI3K/AKT may promote leukemogenesis by conferring cells proliferation and survival advantages in acute myelogenous leukemia (AML). The activation of these pathways had an adverse prognosis in AML and development of targeted therapies seems to be promising. Heat-shock proteins (HSP) are involved in the conformational maturation of a number of signaling proteins, and HSPs expression in AML is associated with other adverse prognostic factors (Bcl2, MRP). The aim of this work was to study STPs expression in AML, and there correlation to other adverse prognostic factors and complete remission rate. Sixty five patients with primary AML were analyzed by flow cytometry for constitutive ERK, PI3K and AKT activation, HSP90, Bcl2 and P170 expression. FAB subtypes were M0=3, M1=19, M2=16, M4=10, M5=16, M6=1. All patients received an induction treatment, 44 patients reached complete remission. Cytogenetics was available in 63 cases (Intermediate = 36, favorable = 3, unfavorable= 24). We performed a 3 color flow cytometry protocol using CD45 and CD34 to identify blast cells, and used a third antibody to the following transduction proteins (ERK, pERK, AKT, pAKT, PI3K) or intracytoplasmic proteins (Bcl2, HSP90, p170). Spontaneous growth of leukemic progenitor cells (CFU-L) was investigated in 44 samples. In AML, activated proteins were found in CD34+ cells. ERK and PI3K/AKT were frequently co-activated. Flow cytometry results showed that levels of STPs were significantly higher (p<0,05) in patients who did not reach complete remission, with a shorter survival: PI3K (12% of positive cells for CR patients vs 68% for non CR patients), ERK (17% vs 69%), pERK (15% vs 77%), AKT (24% vs 76%) and pAKT (10% vs 79%). All patients who reached complete remission had a percentage of positive cells < 20% (except 2 cases for AKT). Interestingly, the levels did not differ significantly according to cytogenetics, and were only marginally higher in patients with high WBC, which suggests an independent prognostic value. By contrast, we found a good correlation between activation of signaling pathways and HSP90, Bcl2 and p170 expression (p<0,05). We observed a spontaneous growth in 30 cases (defined by more than 10 colonies/105cells), with a good correlation with HSP90, Bcl2, p170 and CD34 expression. By contrast, there was no correlation between spontaneous growth and activation of STPs, suggesting that spontaneous growth of leukemic cells which is also a prognostic factor is not directly related to activation of STPs. These results indicate that the activation of multiple STPs is common in AML and is associated with poor prognosis, and that flow cytometry technique can be used for rapid detection of AML patients who are resistant to conventional chemotherapy. This is of particular interest because of development of targeted therapies for signaling transduction pathways, chaperones and anti-apoptotic proteins.

Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase-Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase-Akt cytoprotective pathway.

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.

ClC-2 channels get new partners. Focus on “Association between Hsp90 and the ClC-2 chloride channel upregulates channel function”

clc-2 is a chloride channel that belongs to the ClC family of anion channels and transporters. ClC-2 is activated by hyperpolarization, leading to strict inward rectification, and is further activated by intracellular chloride and extracellular acidification. Work with recombinant ClC-2 in different

The nuclear factor interleukin-6 (NF-IL6) and signal transducer and activator of transcription-3 (STAT-3) signalling pathways co-operate to mediate the activation of the hsp90beta gene by interleukin-6 but have opposite effects on its inducibility by heat shock.

The levels of the 90 kDa heat-shock protein (hsp90) and the activity of the hsp90beta gene promoter are increased in response to treatment by interleukin (IL)-6. The hsp90beta gene promoter contains binding sites for the transcription factors nuclear factor IL-6 (NF-IL6) and signal transducer and activator of transcription 3 (STAT-3), which are activated respectively by the mitogen-activated-protein-kinase and Jak-kinase pathways following IL-6 treatment. Both these factors can activate the hsp90 promoter and have a strong synergistic effect on its activity, which appears to be critical for IL-6-mediated activation of the promoter. Interestingly, the two factors interact differently with the heat-shock factor (HSF) and a heat-shock stress. Thus STAT-3 reduces the stimulatory effect of heat shock whereas NF-IL6 enhances it. When applied together, heat shock and IL-6 produce only weak activation of the hsp90 promoter compared with either stimulus alone, indicating that the inhibitory effect of STAT-3 on HSF predominates under these conditions. In contrast, IL-1, which activates only the NF-IL6 pathway, synergizes with heat shock to produce strong activation of hsp90. These effects are discussed in terms of the multiple stimuli that may regulate the hsp90 promoter in unstressed cells and their interaction with its stress-mediated activation.