A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells

The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.     

Heat shock protein 90: The cancer chaperone

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.     

Overlapping sites of tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90

The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity.     

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.     

Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells

Summary Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 slowed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation attern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90α or-β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiological critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.     

The biochemical role of the heat shock protein 90 chaperone complex in establishing human telomerase activity

Telomerase is a ribonucleoprotein complex that synthesizes the G-rich DNA found at the 3'-ends of linear chromosomes. Human telomerase consists minimally of a catalytic protein (hTERT) and a template-containing RNA (hTR), although other proteins are involved in regulating telomerase activity in vivo. Several chaperone proteins, including hsp90 and p23, have demonstrable roles in establishing telomerase activity both in vitro and in vivo, and previous reports indicate that hsp90 and p23 are required for the reconstitution of telomerase activity from recombinant hTERT and hTR. Here we report that hTERT and hTR associate in the absence of a functional hsp90-p23 heterocomplex. We also report that hsp90 inhibitors geldanamycin and novobiocin inhibit recombinant telomerase even after telomerase is assembled. Inhibition by geldanamycin could be overcome by allowing telomerase to first bind its primer, suggesting a role for hsp90 in loading telomerase onto the telomere. Inhibition by novobiocin could not similarly be overcome but instead resulted in destabilization of the hTERT polypeptide. We propose that the hsp90-p23 complex fine tunes and stabilizes a functional telomerase structure, allowing primer loading and extension.     

Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells

Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-beta-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.     

热休克蛋白90在缺氧心肌细胞中表达的初步研究

观察热休克蛋白90(HSP90)在缺氧心肌细胞中的表达变化规律,并初步阐明HSP90在早期心肌缺氧损害中的作用。方法:原代培养SD大鼠乳鼠心肌细胞,随机分为正常对照组、缺氧组和加HSP90阻断剂格尔霉素(geldanamycin,GA)预处理后再缺氧组(GA 缺氧组),蛋白免疫印迹法(western blot)及间接免疫荧光法检测缺氧1、3、6、12、24h后心肌细胞中HSP90蛋白分布表达情况,并检测细胞上清中肌酸激酶同工酶(CK-MB)及乳酸脱氢酶(LDH)的含量变化。结果:心肌细胞缺氧3h后HSP90蛋白在胞浆内表达量升高,持续到12h达到峰值。与单纯缺氧组相比,GA 缺氧组中LDH,CK-MB含量在不同时相点均有显著升高。结论:缺氧早期即可引起心肌细胞胞浆中HSP90蛋白表达增强,HSP90可能在早期心肌缺氧损害中发挥其内源性抗损伤机制。     

The hexameric structures of human heat shock protein 90

Background

The human 90-kDa heat shock protein (HSP90) functions as a dimeric molecular chaperone. HSP90 identified on the cell surface has been found to play a crucial role in cancer invasion and metastasis, and has become a validated anti-cancer target for drug development. It has been shown to self-assemble into oligomers upon heat shock or divalent cations treatment, but the functional role of the oligomeric states in the chaperone cycle is not fully understood.

Principal Findings

Here we report the crystal structure of a truncated HSP90 that contains the middle segment and the carboxy-terminal domain, termed MC-HSP90. The structure reveals an architecture with triangular bipyramid geometry, in which the building block of the hexameric assembly is a dimer. In solution, MC-HSP90 exists in three major oligomer states, namely dimer, tetramer and hexamer, which were elucidated by size exclusion chromatography and analytical ultracentrifugation. The newly discovered HSP90 isoform HSP90N that lacks the N-terminal ATPase domain also exhibited similar oligomerization states as did MC-HSP90.

Conclusions

While lacking the ATPase domain, both MC-HSP90 and HSP90N can self-assemble into a hexameric structure, spontaneously. The crystal structure of MC-HSP90 reveals that, in addition to the C-terminal dimerization domain, the residue W320 in the M domain plays a critical role in its oligomerization. This study not only demonstrates how the human MC-HSP90 forms a hexamer, but also justifies the similar formation of HSP90N by using 3D modeling analysis.     

Chicken heat shock protein 90 is a component of the putative cellular receptor complex of infectious bursal disease virus

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The capsid protein VP2 of IBDV plays an important role in virus binding and cell recognition. VP2 forms a subviral particle (SVP) with immunogenicity similar to that of the IBDV capsid. In the present study, we first showed that SVP could inhibit IBDV infection to an IBDV-susceptible cell line, DF-1 cells, in a dose-dependent manner. Second, the localizations of the SVP on the surface of DF-1 cells were confirmed by fluorescence microscopy, and the specific binding of the SVP to DF-1 cells occurred in a dose-dependent manner. Furthermore, the attachment of SVP to DF-1 cells was inhibited by an SVP-induced neutralizing monoclonal antibody against IBDV but not by denatured-VP2-induced polyclonal antibodies. Third, the cellular factors in DF-1 cells involved in the attachment of SVP were purified by affinity chromatography using SVP bound on the immobilized Ni(2+) ions. A dominant factor was identified as being chicken heat shock protein 90 (Hsp90) (cHsp90) by mass spectrometry. Results of biotinylation experiments and indirect fluorescence assays indicated that cHsp90 is located on the surface of DF-1 cells. Virus overlay protein binding assays and far-Western assays also concluded that cHsp90 interacts with IBDV and SVP, respectively. Finally, both Hsp90 and anti-Hsp90 can inhibit the infection of DF-1 cells by IBDV. Taken together, for the first time, our results suggest that cHsp90 is part of the putative cellular receptor complex essential for IBDV entry into DF-1 cells.     

Dual modes of RNA-silencing suppression by Flock House virus protein B2

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.     

The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.     

Association of a cellular heat shock protein with simian virus 40 large T antigen in transformed cells.

The viral oncoprotein of simian virus 40, large T antigen (T-ag), is essential for viral replication and cellular transformation. To understand the mechanisms by which T-ag mediates its multifunctional properties, it is important to identify the cellular targets with which it interacts. A cellular protein of 73 kilodaltons (p73) which specifically associates with T-ag in simian virus 40-transformed BALB/c 3T3E cells has been identified. The binding of p73 to T-ag was demonstrated by coimmunoprecipitation analyses using polyclonal and monoclonal antibodies specific for T-ag. The interaction of p73 with T-ag was independent of T-ag complex formation with the cellular protein p53. Partial V8 protease cleavage maps for p73 and the cellular heat shock protein hsp70 were identical. Immunoblot analyses indicated that p73 complexed to T-ag was antigenically related to hsp70. T-ag deletion mutants were constructed that remove internal, amino-terminal, and carboxy-terminal sequences. These mutants mapped the p73 binding domain to the amino terminus of T-ag. The specific dissociation of p73 from the p73/T-ag complex was mediated by ATP; GTP, CTP, and UTP were also utilized as substrates. These characteristics suggest that p73 may be a member of the hsp70 family of heat shock proteins. The biologic significance of p73/T-ag complex formation has yet to be determined.     

Two-dimensional blue native/SDS-PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.