ATPase Activity and ATP-dependent Conformational Change in the Co-chaperone HSP70/HSP90-organizing Protein (HOP)

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.     

Interaction of HSP90 to N-WASP leads to activation and protection from proteasome-dependent degradation

Neural Wiskott-Aldrich syndrome protein (N-WASP) regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. Here, we show that heat shock protein 90 (HSP90) regulates N-WASP-induced actin polymerization in cooperation with phosphorylation of N-WASP. HSP90 binds directly to N-WASP, but binding alone does not affect the rate of N-WASP/Arp2/3 complex-induced in vitro actin polymerization. An Src family tyrosine kinase, v-Src, phosphorylates and activates N-WASP. HSP90 increases the phosphorylation of N-WASP by v-Src, leading to enhanced N-WASP-dependent actin polymerization. In addition, HSP90 protects phosphorylated and activated N-WASP from proteasome-dependent degradation, resulting in amplification of N-WASP-dependent actin polymerization. Association between HSP90 and N-WASP is increased in proportion to activation of N-WASP by phosphorylation. HSP90 is colocalized and associated with active N-WASP at podosomes in 3Y1/v-Src cells and at growing neurites in PC12 cells, whose actin structures are clearly inhibited by blocking the binding of HSP90 to N-WASP. These findings suggest that HSP90 induces efficient activation of N-WASP downstream of phosphorylation signal by Src family kinases and is critical for N-WASP-dependent podosome formation and neurite extension.     

Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84).

A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion. HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.     

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

Background

The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.

Methods

PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.

Results

RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.

Conclusions

The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.     

The HSP90 complex of plants

Heat shock protein 90 (HSP90) is a highly conserved and essential molecular chaperone involved in maturation and activation of signaling proteins in eukaryotes. HSP90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis. HSP90 often functions together with co-chaperones that regulate the conformational cycle and/or load a substrate "client" protein onto HSP90. In plants, immune sensing NLR (nucleotide-binding domain and leucine-rich repeat containing) proteins are among the few known client proteins of HSP90. In the process of chaperoning NLR proteins, co-chaperones, RAR1 and SGT1 function together with HSP90. Recent structural and functional analyses indicate that RAR1 dynamically controls conformational changes of the HSP90 dimer, allowing SGT1 to bridge the interaction between NLR proteins and HSP90. Here, we discuss the regulation of NLR proteins by HSP90 upon interaction with RAR1 and SGT1, emphasizing the recent progress in our understanding of the structure and function of the complex. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Interaction of the PAS B domain with HSP90 accelerates hypoxia-inducible factor-1alpha stabilization.

Hypoxia-inducible factor (HIF) alpha subunits are induced under hypoxic conditions, when limited oxygen supply prevents prolyl hydroxylation-dependent binding of the ubiquitin ligase pVHL and subsequent proteasomal degradation. A short normoxic half-life of HIF-alpha and a very rapid hypoxic protein stabilization are crucial to the cellular adaptation to changing oxygen supply. However, the molecular requirements for the unusually rapid mechanisms of protein synthesis, folding and nuclear translocation are not well understood. We and others previously found that the chaperone heat-shock protein 90 (HSP90) can interact with HIF-1alpha in vitro. Here we show that HSP90 also interacts with HIF-2alpha and HIF-3alpha, suggesting a general involvement of HSP90 in HIF-alpha stabilization. The PAS B domain, common to all three alpha subunits, was required for HSP90 interaction. ARNT competed with HSP90 for binding to the PAS B domain since an excess of either component inhibited the activity of the other. HSP90 as well as the heterocomplex members HSP70 and p23, but not HSP40, were detected in immunoprecipitations of endogenous cellular HIF-1alpha. While HSP90 and HSP70 bound to HIF-1alpha predominantly under normoxic conditions, ARNT bound to HIF-1alpha primarily under hypoxic conditions, suggesting that ARNT displaced HSP90 from HIF-1alpha following nuclear translocation. Hypoxic accumulation of HIF-1alpha was delayed in a novel cell model deficient for HSP90beta as well as after treatment of wild-type cells with the HSP90 inhibitor geldanamycin, suggesting that HSP90 activity is involved in the rapid HIF-1alpha protein induction.     

低表达HSP90α和高表达HSP90β HepG2细胞株的建立

目的:建立HSP90α低表达和HSP90β高表达HepG2细胞株。方法:通过电转染方法将质粒pSilencerHSP90α和pSmycHSP90β转染入HepG2细胞中,应用Western-blotting和MTT法分别鉴定转染效果及绘制细胞生长曲线。结果:带有HSP90αsiRNA片段的质粒和带有HSP90β片段的质粒成功转入HepG2中,转染细胞与未转染细胞生长情况无差别。结论:电转染方法可以有效地将质粒转染入HepG2中去,转染细胞的生长情况将不会影响后续实验的结果。     

Localization of HSP90 in rat brain

• 1.1. In rats, HSP90 (90-kDa heat shock protein) was abundant in the brain compared with the liver and kidney. Immunohistochemical studies showed the presence of HSP90 in almost all neurons in the brain.
• 2.2. On immunoblotting using an anti-HSP90 antibody, HSP90 was present in a lower amount in the medulla oblongata and spinal cord.
• 3.3. In the pituitary gland, some of superficial cells of the anterior lobe adjacent to the middle lobe was specifically stained with anti-HSP90 antibody.

     

HSP90 modulates actin dynamics: Inhibition of HSP90 leads to decreased cell motility and impairs invasion

HSP90, a major molecular chaperone, plays an essential role in the maintenance of several signaling molecules. Inhibition of HSP90 by inhibitors such as 17-allylamino-demethoxy-geldanamycin (17AAG) is known to induce apoptosis in various cancer cells by decreasing the activation or expression of pro-survival molecules such as protein kinase B (Akt). While we did not observe either decrease in expression or activation of pro-survival signaling molecules in human breast cancer cells upon inhibiting HSP90 with 17AAG, we did observe a decrease in cell motility of transformed cells, and cell motility and invasion of cancer cells. We found a significant decrease in the number of filopodia and lamellipodia, and in the F-actin bundles upon HSP90 inhibition. Our results show no change in the active forms or total levels of FAK and Pax, or in the activation of Rac-1 and Cdc-42; however increased levels of HSP90, HSP90α and HSP70 were observed upon HSP90 inhibition. Co-immuno-precipitation of HSP90 reveals interaction of HSP90 with G-actin, which increases upon HSP90 inhibition. FRET results show a significant decrease in interaction between actin monomers, leading to decreased actin polymerization upon HSP90 inhibition. We observed a decrease in the invasion of human breast cancer cells in the matrigel assay upon HSP90 inhibition. Over-expression of αB-crystallin, known to be involved in actin dynamics, did not abrogate the effect of HSP90 inhibition. Our work provides the molecular mechanism by which HSP90 inhibition delays cell migration and should be useful in developing cancer treatment strategies with known anti-cancer drugs such as cisplatin in combination with HSP90 inhibitors.     

Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90

The 90-kDa heat shock protein, HSP90, of the mouse has two isoforms, alpha and beta, which are electrophoretically separable. We have investigated the native forms of HSP90 molecules under physiological conditions and determined their isoform compositions. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that HSP90 purified from mouse lymphoma L5178Y cells consists of approximately 40% alpha and 60% beta isoforms. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the purified HSP90 exists predominantly as a dimer, but a considerable amount of monomer was also detected. Western blotting using polyclonal anti-mouse HSP90 antibodies revealed that the native forms of HSP90 in the crude L5178Y cell lysates are also dimer and monomer. The nondenaturing polyacrylamide gel electrophoresis resolved the dimeric forms into two separate bands that were identified as alpha/alpha and beta/beta homodimers by two methods: sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. In addition, the results showed that the monomeric form consists mainly of the beta isoform. Both the alpha and beta isoforms were shown to bind equally to actin filaments.     

Functional interaction of HSP90 with steroid receptors

Hsp90 (Heat Shock Protein 90) is a component of the inactive and metastable hetero-oligomeric structure of steroid receptors. Recent data on Hsp90 structure and function as a stress protein and dedicated molecular chaperone are here reviewed with a particular focus on Hsp90 chaperone cycle interfering with steroid receptor action. The dual role of Hsp90 as a positive and negative modulator of steroid receptor function is considered along the activation-desactivation process of the receptors. It is proposed that Hsp90 chaperone machinery assists the receptor during its synthesis thus avoiding collapse and facilitating an open structure able to bind ligand efficiently. Moreover, it is suggested that Hsp90 may help the folding of the hydrophobic core of the receptor around the ligand and finally Hsp90 may chaperone the receptor after the dissociation of the ligand.     

Apoptosis Versus Cell Differentiation: Role of Heat Shock Proteins HSP90, HSP70 and HSP27

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs     

Mitochondrial HSP90s and tumor cell metabolism

The control of protein homeostasis, or proteostasis, has been traditionally viewed through the lenses of a general housekeeping function that all cells need, regardless of pathway specification or link to defined cellular responses. A more updated perspective considers proteostasis as an essential adaptive mechanism, taking place in specialized subcellular organelles, and maintaining the functionality of defined cellular networks. Fresh experimental evidence now identifies heat shock protein 90 (HSP90) chaperones as pivotal regulators of proteostasis in mitochondria, selectively in tumor cells. This function connects to a global network of cellular compensation, linking autophagy, endoplasmic reticulum (ER) stress and metabolic reprogramming in a single adaptive continuum, and offers prime opportunities for novel cancer therapeutics.