Modulation of prion protein structural integrity by geldanamycin

H.-J. Ochel1,2, G. Gademann2, J. Trepel3 and L. Neckers3

2 Otto-von-Guericke-University, Medical Faculty, Clinic for Radiation Therapy, Radiobiological Laboratory, Leipziger Str. 44, 39120 Magdeburg, Germany
3 National Institutes of Health, National Cancer Institute, Medicine Branch, Tumor Cell Biology Section, 9610 Medical Center Drive Ste. 300, Rockville, MD 20850

Received on November 8, 2002; revised on May 5, 2003; accepted on May 7, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The cellular prion protein PrPc is of crucial importance for the development of neurodegenerative diseases called transmissible spongiform encephalopathies. We investigated if the function of members of the HSP90 family is required for the integrity of the normal, nonpathogenic prion protein called PrPc. Eukaryotic cells were treated with the structurally unrelated HSP90-inhibitors geldanamycin (GA) or radicicol (RC). In either case the cellular prion protein was induced and exhibited faster migrating bands on western blot analysis, whereas geldampicin (GE), an analog of GA known not to bind to HSP90, had no effect. Ongoing protein and messenger RNA synthesis during treatment were found to be necessary for the appearance of these bands. Cotreatment with tunicamycin abrogated any effect of HSP90 inhibitors on the cellular prion protein. Finally, enzymatic deglycosylation with peptide:N-glycosidase F of the normal prion protein as well as the variant induced by benzoquinone ansamycins resulted in very similar band patterns. These experiments indicate that either altered glycosylation, or a change in conformation, or both are involved in the induction of faster migrating bands by HSP90 inhibitors. Thus the inhibition of the function of members of the HSP90 family of molecular chaperones results in profound changes in the physicochemical properties of PrPc.

Key words: conformation / geldanamycin / glycosylation / Hsp90 / prion


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Transmissible spongiform encephalopathies (TSEs) occur as hereditary, sporadic, infectious, and iatrogenic diseases in various mammals including humans (Haywood, 1997Go; Prusiner et al., 1998Go). The etiology is still in dispute (Chesebro, 1998Go; Farquhar et al., 1998Go; Lasmezas et al., 1997Go; Mestel, 1996Go; Rohwer, 1984Go), but the prevailing theory assumes that a phosphatidylinositolglycan-anchored membrane protein called cellular prion protein (PrPc), a normal constituent of mammalian cells, plays a key role in the pathogenesis of this class of diseases (Basler et al., 1986Go; Oesch et al., 1985Go). Through unknown posttranslational mechanisms, PrPc is converted to PrPres, a protease-resistant and less soluble isoform believed to be the causative principle (Prusiner, 1982Go). This is supported by the finding that in hereditary TSEs, which constitute roughly 10% of all cases, mutations in the prion protein gene were found (Gabizon et al., 1996; Prusiner, 1991Go). In addition, knockout mice devoid of PrPc are resistant to infectious extracts purified from diseased animals (Büeler et al., 1993Go; Sailer et al., 1994Go) or to prions produced by infectious brain implants (Brandner et al., 1996Go). Enriched preparations of contagious prions regularly contain PrPres but few nucleic acids (Kellings et al., 1993Go). No mutations of the prion protein were found in the more prevalent sporadic cases. These are explained as either unrecognized infectious diseases or as the consequence of a hypothetical mutation in another protein.

In yeast the protein-linked epigenetic inheritance of [PSI+] was shown to share features of the postulated mode of replication of prions (Patino et al., 1996Go). Here, heat shock protein 104 (HSP104), which is unrelated to HSP90, was shown to be of significance for the perpetuation of [PSI+]. Some evidence for an involvement of molecular chaperones distinct from HSP90 in the pathogenesis of TSEs already exists (Kenward et al., 1994Go). Because frequently the conversion of proteins from one isoform to another is controlled by molecular chaperones, we asked if there is a role for the HSP90 family of molecular chaperones in the physiology of the normal prion protein. Identification of geldanamycin (GA) and radicicol (RC) as structurally unrelated small molecule inhibitors of HSP90 allows for the examination of HSP90 involvement in PrPc processing and stability.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Subconfluent PC3M prostate carcinoma cells were treated for 24 h with GA, geldampicin (GE), (1 and 4 µM) RC (1 µM) or with dimethyl sulfoxide (DMSO) (1 µl/ml) as solvent control. Both GA and RC bind to the same domain at the N-terminus of HSP90 and inhibit its function (Grenert et al., 1997Go). GE is a benzoquinone ansamycin analog of GA with poor affinity for HSP90 (Grenert et al., 1997Go). At 24 h, the cells treated with GA or RC exhibit typical, profound morphological changes. Although rounded but still adherent cells in control dishes occur only sparsely, they account for approximately 50% of the cells after treatment with HSP90 inhibitors. The cell bodies of still adherent cells are spindle-like, appear collapsed, and have curvilinear delineations (not shown).

After resolution of total cell lysates by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), western blot analysis for the prion protein with 3F4 as the detection antibody showed the appearance of faster migrating bands in the case of treatment with GA or RC but not for GE (Figure 1). Occasionally, the uppermost, slowest migrating PrPc bands appeared less intense after treatment with GA or RC. In a separate experiment these results were confirmed for GA-treated cells using the polyclonal anti-PrPc antibody N-12 (not shown). A low level of faster migrating bands was detectable in DMSO-treated cells only after overexposure of the membranes. Thus the treatment with HSP90 reactive compounds induced changes in PrPc migration, whereas solvent-only treated cells exhibited no PrPc alterations.



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Fig. 1. Treatment with HSP90 inhibitors induces electrophoretically faster migrating prion protein bands. Immunoblot analysis for PrPc from PC3M cells left untreated or treated with 1 µl/ml DMSO, 1 µM GE, 4 µM GE, 1 µM GA, 4 µM GA, or 1 µM RC for 24 h. Faster migrating prion protein bands appear only in cells treated with the HSP90 inhibitors GA and RC but not in those left untreated or treated with solvent DMSO or non-HSP90-binding GA-analog GE. Bar: molecular weight marker.

 
After 24 h of treatment the appearance of faster migrating PrPc bands could be seen for GA and for RC at a dose of 500 nM, and for GA at 250 nM faint faster migrating bands were already visible (Figure 2). Higher doses of up to 8 µM did not cause any further changes (not shown).




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Fig. 2. Dose-dependence of HSP90 inhibitors on PrPc electrophoretic mobility. Western blot analysis of PrPc from PC3M cells treated with HSP90-inhibitors. (Top) PC3M cells treated for 24 h with solvent DMSO or with increasing concentrations of GA as indicated. (Bottom) PC3M cells treated for 24 h with 1 µl/ml DMSO or with increasing concentrations of RC as indicated. Bar, molecular weight marker.

 
To investigate whether these observations could be extended to a different prostate carcinoma cell line, we did the same dose-dependence analysis with DU145 cells. After treatment for 24 h with GA at 1–8 µM, a pattern strikingly similar to the one described for PC3M cells was found (Figure 3).



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Fig. 3. Immunoblot analysis PrPc from DU145 cells treated with GA. DU145 cells were treated for 24 h with increasing concentrations of solvent DMSO or GA. Faster migrating prion protein bands similar to those seen in PC3M cells are detectable. Bar, molecular weight marker.

 
Several regulatory proteins, including mutant P53 (Blagosklonny et al., 1995Go), Raf-1 (Schulte et al., 1995Go), and focal adhesion kinase (FAK) (Ochel et al., 1999Go), along with such transmembrane receptors as ErbB2 (Miller et al., 1994Go), are destabilized by treatment with GA after less than 24 h. No such effect on the prion protein was observed 24 h after drug addition (Figure 4). Instead, the overall signal for PrPc was stronger in treatment as opposed to control lanes. In this experiment, a distinct alteration in PrPc migration was detectable as soon as 6 h after start of treatment. The lower panel in Figure 4 shows the detection of actin as a loading control.



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Fig. 4. Time-dependence of PrPc electrophoretic mobility. Immunoblot study of PrPc from PC3M cells treated with 1 µM GA for increasing time intervals as indicated. Faster migrating PrPc bands are detectable at 6 h after start of treatment and remain stable for the duration of the experiment. The lower panel shows the immunoblot detection of actin as a loading control.

 
Next we addressed the question of whether ongoing protein and/or mRNA synthesis are necessary for the observed effects. PC3M prostate carcinoma cells were treated with 4 µM GA alone or in combination with 100 µM cycloheximide or 1 µg/ml actinomycin D for up to 16 h. In the case of cotreatment with cycloheximide, no faster migrating bands appeared (Figure 5), and only a faint signal could be observed with actinomycin D cotreatment (Figure 5). Therefore we reasoned that the faster migrating bands are newly synthesized but alternatively processed variants of PrPc.




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Fig. 5. (Top) Inhibition of protein synthesis abrogates the induction of electrophoretically faster migrating PrPc-bands by GA. Western blot analysis for PrPc. PC3M cells were left untreated or were treated with 4 µM GA alone or in combination with 100 µM cycloheximide for increasing time intervals as indicated. Bar, molecular weight marker. (Bottom) Inhibition of mRNA synthesis inhibits the induction of electrophoretically faster migrating PrPc-bands by GA. Western blot analysis for PrPc. PC3M cells were left untreated or were treated with 4 µM GA alone or in combination with 1 µg/ml actinomycin D for increasing time intervals. Bar, molecular weight marker.

 
The prion protein is highly glycosylated and is usually seen as a smear on western blots (Endo et al., 1989Go). To gain more insight into the nature of PrPc-alterations induced by HSP90-active drugs, we manipulated the glycosylation status of the protein with tunicamycin (TU), an inhibitor of N-glycosylation (Duksin and Mahoney, 1982Go). The presence of TU in the medium effectively abrogated any difference between samples treated with GA or DMSO (Figure 6). This supports the notion that GA-induced changes in PrPc mobility most probably are the result of accumulation of hypoglycosylated or incompletely glycosylated variants of this protein. An alternative explanation postulates that the faster migrating, GA-induced bands represent fully glycosylated prion proteins with variant conformations, which prevent complete denaturation by SDS–PAGE.



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Fig. 6. Deglycosylated PrPc is unaffected by GA treatment. Western blot analysis for PrPc. PC3M cells were left untreated or treated with 4 µl/ml DMSO, 4 µM GA, 4 µl/ml DMSO + 5 µg/ml TU, or 4 µM GA + 5 µg/ml TU for 24 h. In the presence of TU no difference was detectable between PrPc bands from solvent-only- or GA-treated cells. Bar, molecular weight marker.

 
A more direct approach to show that altered glycosylation plays a role in the observed electrophoretic mobility changes of the prion protein allows for the induction of these abberant bands followed by enzymatic deglycosylation. Subconfluent PC3M cells were lysed, and boiled lysates and fetuin as a positive control were digested for 4 h with peptide:N-glycosidase F (PNGase F), which removes N-linked oligosaccharides (Fan and Lee, 1997Go; Plummer et al., 1984Go). The PNGase F digest of fetuin caused a complete band shift as evidenced by Coomassie blue staining of the membrane, indicating deglycosylation of the protein (not shown). As demonstrated in Figure 7, digestion of the prion protein from solvent only treated or from GA-treated cells with PNGase F results in very similar band patterns.



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Fig. 7. PNGase F digest abrogates differences in PrPc electrophoretic mobility. PC3M cells were scraped into phosphate-containing buffer and lysed by aspiration through a small needle. Precleared, boiled lysates were digested with PNGase F. After enzymatic deglycosylation no major differences in band appearance are discernible anymore.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Several lines of evidence indicate that PrPc plays a central role in the pathogenesis of TSEs. A protease-resistant isoform, enriched in beta sheets, is a major component of the infectious principle that is found in TSEs like bovine spongiform encephalopathy (BSE), scrapie, or kuru (Prusiner, 1991Go). Renewed interest in this class of diseases stems from the recent discovery of a new variant of Creutzfeld-Jakob disease (Will et al., 1996Go) that may reflect human infection by the BSE agent through the food chain. Evidence supporting this view stems from in vitro conversion assays (Raymond et al., 1997Go), glycosylation-dependent electrophoretic migration patterns of the prion protein in cases of BSE and new variant Creutzfeld-Jakob disease (Collinge et al., 1996Go), and the comparative analysis of survival times and lesion profiles after experimental transmission of BSE, sporadic Creutzfeld-Jacob disease, and new variant Creutzfeld-Jacob disease to mice (Bruce et al., 1997Go).

The general interest to explore chaperones in the context of TSEs stems from the fact that a change in conformation, that is, the transition of PrPc to PrPres, seems to be a crucial pathogenetic step. This is reminiscent of the main task of chaperones, that is, the surveillance of protein folding and conformation. The heat-shock response was found to be different in scrapie-infected as opposed to uninfected neuroblastoma cells (Tatzelt et al., 1995Go), but so far differences were described only for HSP28 and HSP72, which are unrelated to HSP90. Because PrPc may be internalized and later detected in lysosomes, a prominent role for HSP70, which is an abundant constituent of lysosomes, in the pathogenesis of TSEs was proposed (Laszlo et al., 1992Go; Mayer et al., 1992Go). A direct interaction with PrPc could be demonstrated for HSP60 in a yeast two-hybrid screen and was verified by immunoprecipitation of pertinent fusion proteins (Edenhofer et al., 1996Go). A concentration-dependent role for HSP104 for the maintenance of the prion-like state [PSI+] in yeast was also reported (DebBurman et al., 1997Go). Taken together, these findings suggest that multiple chaperones may interact directly or indirectly with PrPc. Despite extensive efforts to prove the existence of a complex containing HSP90 as well as PrPc, so far no such interaction was demonstrable in our model system (not shown).

PrPSc (the protease-resistant, less soluble isoform of the prion protein found in scrapie) is a sialoglycoprotein (Bolton et al., 1985Go; Haraguchi et al., 1989Go), and the glycosylation of PrPc differs from the glycosylation pattern of PrPSc (Rudd et al., 1999Go). It is conceivable that alterations of glycosylation may contribute to the development of epigenetic inheritance and may occur under conditions of stress, where less chaperoning activity is available because HSPs are forced to perform alternative functions. Various groups have demonstrated in different model systems that the inhibition of PrPc glycosylation contributes to the acquisition of hallmarks of PrPres, that is, detergent insolubility and proteinase K resistance (Lehmann and Harris, 1997Go; Ma and Lindquist, 1999Go). In the cell lines used in our experiments, HSP90 inhibitors did not induce proteinase K resistance or decreased solublity of PrPc (not shown).

The data presented here are the first to implicate HSP90-family member function in the structural and conformational integrity of the prion protein. After treatment of eukaryotic cells with structurally unrelated HSP90 inhibitors, faster migrating PrPc variants are consistently detectable. The joint interpretation of the experiments on the role of transcription and translation for the induction of faster migrating bands is that they probably represent newly synthesized protein. Two alternative, not mutually exclusive, explanations are possible. The faster migrating fraction of the prion protein smear is either due to hypoglycosylation or indicates variant conformations of PrPc. It is our opinion that the latter interpretation is less likely, because only few proteins are not denatured by SDS–PAGE. Still, it cannot be ruled out completely. The small difference in band patterns after PNGase F digest in Figure 7 might also depend on posttranslational modifcations different from glycosylation. However, this would be a low-level occurence and is not supported by other data.

The reaction of PrPc to treatment with HSP90 inhibitors is profoundly different from the response of all other hitherto studied cellular proteins. GA induces the rapid destabilization of several signal transducers like ErbB2 (Chavany et al., 1996Go), mutant P53 (Blagosklonny et al., 1996Go), Raf-1 (Schulte et al., 1995Go), and FAK (Ochel et al., 1999Go), and the list of proteins for which stability is dependent on the continuous presence of functional HSP90 is still growing (for review, see Richter and Buchner, 2001Go). In contrast, with the obvious exception of heat-shock proteins, the prion protein is the only known protein that is induced by benzoquinone ansamycins.

A future task will be to characterize further the mechanism that causes the effects of HSP90 inhibitors on PrPc. Maybe an enzyme that is part of the oligosaccharide-synthesis apparatus depends on functional HSP90. Conceivably, the pharmakological knockout of HSP90 and the subsequent loss of function of the putative enzyme does not permit proper oligosaccharide maturation of the prion protein. The induction of PrPc may be a compensatory reaction on the presence of immaturely glycosylated variants, although alternative interpretations implicating protein conformation are possible.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Cell lines and tissue culture
PC3M and DU145 prostate carcinoma cells were maintained in RPMI (PC3M) or Dulbecco's modified essential medium (DU145) at 37°C in a humidified atmosphere containing 5% CO2 and were allowed to adhere to culture dishes for at least 16 h prior to initiation of experiments. All media contained 20 mM glutamine, 10% fetal bovine serum, and 10 mM HEPES (Biofluids, Rockville, MD).

Materials
GA and GE were obtained from the Developmental Therapeutics Program, National Cancer Institute (Rockville, MD). RC was supplied by the Pharmaceutical Research Institute (Kyowa Hakko Kogyo, Shizuoka, Japan). These drugs were dissolved as a 1 mM stock in DMSO. TU was from Sigma (St. Louis, MO), and PNGase F (E.C. 3.5.1.52) was from Roche (Mannheim, Germany). Anti-PrPc mouse monoclonal antibody 3F4 was a generous gift from Prof. Prusiner (University of California, San Francisco) and from Dr. Huber (Robert Koch Institute, Berlin). Rabbit polyclonal antibody N-12 was a generous gift from Prof. Prusiner (University of California, San Francisco). Mouse monoclonal anti-actin antibody AC-40 was from Sigma.

Western blot
Subconfluent cells were lysed on ice in TNESV (50 mM Tris–HCl pH 7.4, 1% Nonidet P-40, 2 mM ethylenediamine tetra-acetic acid [EDTA], 100 mM NaCl, 1 mM orthovanadate) containing 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride, and the lysate was cleared by centrifugation for 10 min at 12,000 x g. Protein determinations were made using the BCA-protein assay (Pierce, Rockford, IL). Protein aliquots were boiled for 5 min in Laemmli buffer (Laemmli, 1970Go) and resolved by SDS–PAGE. Semi-dry transfer onto activated polyvinylidene fluoride-based Immobilon-P membrane (Millipore, Bedford, MA) was followed by blocking for 1 h at room temperature in 5% nonfat dry milk dissolved in phosphate buffered saline (PBS) with 0.05% (v/v) Tween 20 (PBST). Incubations with primary and horseradish-peroxidase-linked secondary antibodies (Amersham, Arlington Heights, IL) were for 1 h at room temperature, followed by five washes in PBST. Enhanced chemiluminescence with Supersignal (Pierce) was applied according to manufacturer's instructions using Kodak X-Omat AR films (Kodak, Rochester, NY).

Digestion with PNGase F
Subconfluent PC3M cells were washed twice with PBS, scraped into phosphate buffer (Na2HPO4, pH 7.1; NaCl, 150 mM; EDTA, 10 mM) and then lysed by repeated aspirations through a fine needle (22G x 1/4, Microlance 3). Afterward, 50 µg of precleared lysate was boiled along with 50 µg fetuin for 5 min followed by digestion with 10 U PNGase F at 37°C for 4 h. After resolution by SDS–PAGE and wet transfer, PrPc was detected using 3F4 as the primary and horseradish-peroxidase-linked sheep anti-mouse monoclonal antibody as the secondary antibody.


    Acknowledgements
 
We are grateful to Prof. Prusiner, Department of Neurology, University of California, San Francisco, and Dr. Huber, Robert-Koch-Institute, Berlin, for supplying antiprion antibodies. We also want to thank Dr. Lendeckel, Clinic of Experimental Medicine, Otto-von-Guericke-University, Magdeburg, Germany, for the generous permission to use the isotope laboratory.

1 To whom correspondence should be addressed; e-mail: hans-joachim.ochel{at}medizin.uni-magdeburg.de Back


    Abbreviations
 
BSE, bovine spongiform encephalopathy; DMSO, dimethyl sulfoxide; EDTA, ethylenediamine tetra-acetic acid; FAK, focal adhesion kinase; GA, geldanamycin; GE, geldampicin; HSP, heat shock protein; PBS, phosphate buffered saline; PNGase F, peptide:N-glycosidase F; PrPc, cellular prion protein; PrPres, protease-resistant isoform of the prion protein; PrPSc, scrapie-associated isoform of the prion protein; RC, radicicol; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TSE, transmissible spongiform encephalopathy; TU, tunicamycin


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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