Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2

The benzoquinone ansamycin geldanamycin (GA) stimulates proteasome-mediated degradation of plasma membrane-associated ErbB2, a receptor tyrosine kinase. Drug sensitivity is mediated by ErbB2's kinase domain and occurs subsequent to the disruption of Hsp90 interaction with this domain. Full-length ErbB2 is efficiently processed via the endoplasmic reticulum (ER) and Golgi network, so that at steady state most of the detectable protein is plasma membrane associated. However, previous studies have also demonstrated the GA sensitivity of newly synthesized ErbB2, normally a minor component of the total cellular pool of the kinase. Drug sensitivity of nascent ErbB2 is distinguished by 2 characteristics--protein instability and inability to traverse the ER. As nascent ErbB2 can associate with both cytoplasmic Hsp90 and its ER luminal homolog Grp 94, also a GA-binding protein, the purpose of this study was to examine the relative contributions of the cytoplasmic and ER luminal domains of ErbB2 to the GA sensitivity of the nascent kinase. By studying the drug sensitivity of ErbB2/DK, a construct lacking ErbB2's cytoplasmic kinase domain, and by examining the activity of a GA derivative that preferentially binds Hsp90, we conclude that both the stability and the maturation of nascent ErbB2 are regulated by its cytoplasmic, Hsp90-interacting domain.     

Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.     

Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [³⁵S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.     

Surface charge and hydrophobicity determine ErbB2 binding to the Hsp90 chaperone complex

The molecular chaperone Hsp90 modulates the function of specific cell signaling proteins. Although targeting Hsp90 with the antibiotic inhibitor geldanamycin (GA) may be a promising approach for cancer treatment, little is known about the determinants of Hsp90 interaction with its client proteins. Here we identify a loop within the N lobe of the kinase domain of ErbB2 that determines Hsp90 binding. The amino acid sequence of the loop determines the electrostatic and hydrophobic character of the protein's surface, which in turn govern interaction with Hsp90. A point mutation within the loop that alters ErbB2 surface properties disrupts Hsp90 association and confers GA resistance. Notably, the immature ErbB2 point mutant remains sensitive to GA, suggesting that mature and nascent client kinases may use distinct motifs to interact with the Hsp90 chaperone complex.     

Benzoquinoid ansamycins (herbimycin A and geldanamycin) interfere with the maturation of growth factor receptor tyrosine kinases

Benzoquinoid ansamycins, such as herbimycin A (HA) and geldanamycin (GA), are antibiotics that exhibit anti-tumor effects. These compounds have been shown to result in the intracellular depletion of important growth signaling molecules. Recently, GA has been shown to bind tightly to Hsp90, thereby implicating Hsp90 as a possible chaperone for those signaling molecules adversely affected by the benzoquinoid ansamycins. Here we have investigated the effects of HA and GA on the synthesis, maturation and stability of different protein tyrosine kinases. Exposing cells to either compound blocked normal maturation of the epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, and pp60(v-src). We show that only the nascent forms of the EGF and PDGF receptors are degraded under these conditions. Once the newly synthesized receptors had been translocated into the endoplasmic reticulum membrane, addition of the drugs no longer affected their stability. For the cytoplasmic tyrosine kinase, pp60(v-src), both the nascent as well as the mature forms of the protein were degraded in cells treated with the drugs. We discuss these observations as they pertain to the possible role of Hsp90 as a substrate-specific molecular chaperone, perhaps involved in the maturation and/or stability of proteins important for growth control.     

Analysis of Conformational Determinants Underlying HSP90-Kinase Interaction

The role of HSP90 in stabilization of oncogenic tyrosine kinases made it an attractive therapeutic target for treating cancer but the molecular basis underlying the interaction between the HSP90 chaperone and client kinases is not elucidated yet. Using kinase inhibitors we show that the inactive conformation of ERBB2 does not interact with HSP90 chaperone and is thus not amenable to degradation upon HSP90 inhibitor treatment, while active ERBB2 kinase conformation promotes interaction with the HSP90 machinery and thus is degraded upon HSP90 inhibitor treatment. Interestingly, the kinase-chaperone interaction is disrupted in case of BCR-ABL and FLT3-ITD when bound to inhibitors irrespective of whether they block the kinase in an active or inactive conformation and thus our results indicate that the stability of the active kinase conformation varies between different kinases.     

G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

Akt shows variable sensitivity to an Hsp90 inhibitor depending on cell context

Hsp90 inhibitors are currently in clinical trials for cancer therapy based on their ability to promote proteasomal degradation of oncogenic protein kinases and nuclear receptors. Results from recent studies suggest that cancer cells are more sensitive to these inhibitors than cells from healthy tissues. We analyzed an immortalized cell line Ba/F3 for sensitivity to the Hsp90 inhibitor geldanamycin in the absence and presence of the oncogenic tyrosine fusion kinase NPM-ALK expressed from a retroviral vector. Our results showed that NPM-ALK expression makes Akt and Cdk4 more resistant to degradation in the presence of geldanamycin, and there was a slightly reduced amount of apoptosis. The mechanism underlying the effect of NPM-ALK on Akt stability was probed by comparison of the turnover of the kinase after translation inhibition and geldanamycin treatment. We observed that Akt was degraded more rapidly in the presence of GA than upon translation inhibition without NPM-ALK expression. This suggests that NPM-ALK protects the mature kinase. Furthermore, Akt failed to bind to the Cdc37 chaperone in cells expressing NPM-ALK, which also correlates with increased Akt stability.     

Molecular insights into the maturation of phosphodiesterase 6 by the specialized chaperone complex of HSP90 with AIPL1

Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state.     

A role of Rab7 in stabilizing EGFR-Her2 and in sustaining Akt survival signal

Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA‐mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage‐independent growth of cancer cells in soft agar. Anoikis (matrix‐detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked‐down. More robust reduction of EGFR and Her2 levels was observed when knocked‐down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50–100 nm)‐induced apoptosis of the Rab7 knocked‐down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome‐mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors. J. Cell. Physiol. 227: 2788–2797, 2012. © 2011 Wiley Periodicals, Inc.     

Signaling through ERBB receptors: multiple layers of diversity and control

The four known ERBB receptors in humans are involved in a broad range of cellular responses, and their deregulation is a significant aspect in a large number of disease states. However, their mechanism of action and modes of control are still poorly understood. This is largely due to the fact that the control of ERBB activity is a multilayered process with significant differences between the various ERBB members. In contrast to other receptor tyrosine kinases, the kinase domain of EGFR (ERBB1) does not require phosphorylation for activation. Consequently, the overall activation state of the receptor is controlled by constant balancing of activity favoring and activity suppressing actions within the receptor molecule. Influences of the membrane microenvironment and context dependent interactions with varying sets of signaling partners are superimposed on this system of intramolecular checks and balances. We will discuss current models of the control of ERBB signaling with an emphasis on the multilayered nature of activation control and aspects that give rise to diversity between ERBB receptors.     

Heat shock protein 90 and tyrosine kinase regulate eNOS NO* generation but not NO* bioactivity

An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-*) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO* release from stimulated endothelial cell cultures and increased O(2)(-*) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-*) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.     

A critical role for HSP90 in cancer cell invasion involves interaction with the extracellular domain of HER-2

HSP90 is a ubiquitously expressed molecular chaperone that controls the folding, assembly, intracellular disposition, and proteolytic turnover of many proteins, most of which are involved in signal transduction processes. Recently, a surface form of HSP90 has been identified and associated with cell migration events. In this paper, we explore the interaction of surface HSP90 with HER-2, a receptor-like glycoprotein and member of the ErbB family of receptor tyrosine kinases that play central roles in cellular proliferation, differentiation, and migration as well as in cancer progress. The involvement of HSP90 in the regulation of HER-2 has been attributed so far to receptor stabilization via interaction with its cytoplasmic kinase domain. Here we present evidence, using glutathione S-transferase pull-down and transfection assays, for a novel interaction between surface HSP90 and the extracellular domain of HER-2. Specific disruption of this interaction using mAb 4C5, a function-blocking monoclonal antibody against HSP90, inhibits cell invasion accompanied by altered actin dynamics in human breast cancer cells under ligand stimulation conditions with heregulin. Additionally, disruption of surface HSP90/HER-2 interaction leads to inhibition of heregulin-induced HER-2-HER-3 heterodimer formation, reduced HER-2 phosphorylation, and impaired downstream kinase signaling. Interestingly, this disruption does not affect HER-2 internalization. Our data suggest that surface HSP90 is involved in heregulin-induced HER-2 activation and signaling, leading to cytoskeletal rearrangement, essential for cell invasion.     

Intracellular retention and degradation of the epidermal growth factor receptor, two distinct processes mediated by benzoquinone ansamycins

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an oncogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185(c-)(erbB-2). Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.     

Systematic identification of the HSP90 candidate regulated proteome

HSP90 is a central player in the folding and maturation of many proteins. More than two hundred HSP90 clients have been identified by classical biochemical techniques including important signaling proteins with high relevance to human cancer pathways. HSP90 inhibition has thus become an attractive therapeutic concept and multiple molecules are currently in clinical trials. It is therefore of fundamental biological and medical importance to identify, ideally, all HSP90 clients and HSP90 regulated proteins. To this end, we have taken a global and a chemical proteomic approach in geldanamycin treated cancer cell lines using stable isotope labeling with amino acids in cell culture and quantitative mass spectrometry. We identified >6200 proteins in four different human cell lines and ~1600 proteins showed significant regulation upon drug treatment. Gene ontology and pathway/network analysis revealed common and cell-type specific regulatory effects with strong connections to unfolded protein binding and protein kinase activity. Of the 288 identified protein kinases, 98 were geldanamycin treatment including >50 kinases not formerly known to be regulated by HSP90. Protein turn-over measurements using pulsed stable isotope labeling with amino acids in cell culture showed that protein down-regulation by HSP90 inhibition correlates with protein half-life in many cases. Protein kinases show significantly shorter half lives than other proteins highlighting both challenges and opportunities for HSP90 inhibition in cancer therapy. The proteomic responses of the HSP90 drugs geldanamycin and PU-H71 were highly similar suggesting that both drugs work by similar molecular mechanisms. Using HSP90 immunoprecipitation, we validated several kinases (AXL, DDR1, TRIO) and other signaling proteins (BIRC6, ISG15, FLII), as novel clients of HSP90. Taken together, our study broadly defines the cellular proteome response to HSP90 inhibition and provides a rich resource for further investigation relevant for the treatment of cancer.     

Inhibition of HSP90 in Trypanosoma cruzi induces a stress response but no stage differentiation

The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite Leishmania donovani. We investigated the role of HSP90 in the related parasite Trypanosoma cruzi by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G₁ phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in T. cruzi and serves as a feedback inhibitor in the cellular stress response. In contrast to L. donovani cells, however, T. cruzi cells treated with GA do not begin to differentiate into relevant life cycle stages.     

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.