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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: conformation / geldanamycin / glycosylation / Hsp90 / prion
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1996). 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., 1994
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After resolution of total cell lysates by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), 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.
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1995), 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., 1992
; Mayer et al., 1992
). 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., 1996
). A concentration-dependent role for HSP104 for the maintenance of the prion-like state [PSI+] in yeast was also reported (DebBurman et al., 1997
). 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., 1985; Haraguchi et al., 1989
), and the glycosylation of PrPc differs from the glycosylation pattern of PrPSc (Rudd et al., 1999
). 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, 1997
; Ma and Lindquist, 1999
). 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 SDSPAGE. 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., 1996), mutant P53 (Blagosklonny et al., 1996
), Raf-1 (Schulte et al., 1995
), and FAK (Ochel et al., 1999
), 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, 2001
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 TrisHCl 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, 1970) and resolved by SDSPAGE. 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 SDSPAGE 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 |
---|
1 To whom correspondence should be addressed; e-mail: hans-joachim.ochel{at}medizin.uni-magdeburg.de
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blagosklonny, M.V., Toretsky, J., and Neckers, L. (1995) Geldanamycin selectively destabilizes and conformationally alters mutated p53. Oncogene, 11, 933939.[ISI][Medline]
Blagosklonny, M.V., Toretsky, J., Bohen, S., and Neckers, L. (1996) Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc. Natl Acad. Sci. USA, 93, 83798383.
Bolton, D.C., Meyer, R.K., and Prusiner, S.B. (1985) Scrapie PrP 2730 is a sialoglycoprotein. J. Virol., 53, 596606.[ISI][Medline]
Brandner, S., Isenmann, S., Raeber, A., Fischer, M., Sailer, A., Kobayashi, Y., Marino, S., Weissman, C., and Aguzzi, A. (1996) Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature, 379, 339343.[CrossRef][ISI][Medline]
Bruce, M.E., Will, R.G., Ironside, J.W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J., Birkett, C., and others. (1997) Transmissions to mice indicate that "new variant" CJD is caused by the BSE agent. Nature, 389, 498501.[CrossRef][ISI][Medline]
Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M., Weissman, C. (1993) Mice devoid of PrP are resistant to scrapie. Cell, 73, 13391347.[ISI][Medline]
Chavany, C., Mimnaugh, E., Miller, P., Bitton, R., Nguyen, P., Trepel, J., Whitesell, L., Schnur, R., Moyer, J., and Neckers, L. (1996) p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2. J. Biol. Chem., 271, 49744977.
Chesebro, B. (1998) BSE and prions: uncertainties about the agent. Science, 279, 4243.
Collinge, J., Sidle, K.C.L., Meads, J., Ironside, J., and Hill, A.F. (1996) Molecular analysis of prion strain variation and the aetiology of "new variant" CJD. Nature, 383, 685690.[CrossRef][ISI][Medline]
DebBurman, S.K., Raymond, G.J., Caughey, B., and Lindquist, S. (1997) Chaperone-supervised conversion of prion protein to its protease-resistant form. Proc. Natl Acad. Sci. USA, 94, 1393813943.
Duksin, D. and Mahoney, W.C. (1982) Relationship of the structure and biological activity of the natural homologues of tunicamycin. J. Biol. Chem., 257, 31053109.
Edenhofer, F., Rieger, R., Famulok, M., Wendler, W., Weiss, S., and Winnacker, E.-L. (1996) Prion protein PrPc interacts with molecular chaperones of the HSP60 family. J. Virol., 70, 47244728.[Abstract]
Endo, T., Groth, D., Prusiner, S.B., and Kobata, A. (1989) Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry, 28, 83808388.[ISI][Medline]
Fan, J.-Q. and Lee, Y.C. (1997) Detailed studies on substrate structure requirements of glycoamidases A and F. J. Biol. Chem., 43, 2705827064.[CrossRef]
Farquhar, C.F., Somerville, R.A., and Bruce, M.E. (1998) Straining the prion hypothesis. Nature, 391, 345346.[CrossRef][ISI][Medline]
Grenert, J.P., Sullivan, W.P., Fadden, P., Haystead, T.A.J., Clark, J., Mimnaugh, E., Krutzsch, H., Ochel, H.J., Schulte, T.W., Sausville, E., and others. (1997) The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J. Biol. Chem., 272, 2384323850.
Haraguchi, T., Fisher, S., Olofsson, S., Endo, T., Groth, D., Tarentino, A., Borchelt, D.R., Teplow, D., Hood, L., Burlingame, A., and others. (1989) Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Arch. Biochem. Biophys., 274, 113.[ISI][Medline]
Haywood, A.M. (1997) Transmissible spongiform encephalopathies. N. Engl. J. Med., 337, 18211828.
Kellings, K., Meyer, N., Mirenda, C., Prusiner, S.B., and Riesner, D. (1993) Analysis of nucleic acids in purified scrapie prion preparations. Arch. Virol., 7(suppl), 215225.
Kenward, N., Hope, J., Landon, M., and Mayer, R.J. (1994) Expression of polyubiquitin and heat-shock protein 70 genes increases in the later stages of disease progression in scrapie-infected mouse brains. J. Neurochem., 62, 18701877.[ISI][Medline]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Lasmezas, C.I., Deslys, J.-P., Robain, O., Jaegly, A., Beringue, V., Peyrin, J.-M., Fournier, J.-G., Hauw, J.-J., Rossier, J., and Dormont, D. (1997) Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science, 275, 402405.
Laszlo, L., Lowe, J., Self, T., Kenwards, N., Landon, M., McBride, T., Farquhar, C., McConnell, I., Brown, J., Hope, J., and Mayer, R.J. (1992) Lysosomes as key organelles in the pathogenesis of prion encephalopathies. J. Pathol., 166, 333341.[ISI][Medline]
Lehmann, S. and Harris, D.A. (1997) Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells. J. Biol. Chem., 272, 2147921487.
Ma, J. and Lindquist, S. (1999) De novo generation of a PrPc-like conformation in living cells. Nature Cell Biol., 1, 358361.[CrossRef][ISI][Medline]
Mayer, R.J., Landon, M., Laszlo, L., Lennox, G., and Lowe, J. (1992) Protein processing in lysosomes: the new therapeutic target in neurodegenerative disease. Lancet, 340, 156159.[CrossRef][ISI][Medline]
Mestel, R. (1996) Putting prions to the test. Science, 273, 184189.
Miller, P., DiOrio, C., Moyer, M., Schnur, R.C., Bruskin, A., Cullen, W., Moyer, J.D. (1994) Depletion of the erbB-2 gene product p185 by benzoquinoid ansamycins. Cancer Res., 54, 27242730.[Abstract]
Ochel, H.J., Schulte, T.W., Nguyen, P., Trepel, J., Neckers, L. (1999) The benzoquinone ansamycin geldanamycin stimulates proteolytic degradation of focal adhesion kinase. Mol. Genet. Metab., 66, 2430.[CrossRef][ISI][Medline]
Oesch, B., Westaway, D., Wälchli, M., Mckinley, M.P., Kent, S.B.H., Aebersold, R., Barry, R.A., Tempst, P., Teplow, D.B., Hood, L.E., and others. (1985) A cellular gene encodes scrapie PrP 2730 protein. Cell, 40, 735746.[ISI][Medline]
Patino, M., Liu, J.-J., Glover, J.R., and Lindquist, S. (1996) Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science, 273, 622626.[Abstract]
Plummer, T.H., Elder, J.H., Alexander, S., Phelan, A.W., Tarentino, A.L. (1984) Demonstration of peptide:N-glycosidase F activity in endo-b-N-acetylglucosaminidase F preparations. J. Biol. Chem., 259, 1070010704.
Prusiner, S.B. (1982) Further purification and characterization of scrapie prions. Biochemistry, 21, 69426950.[ISI][Medline]
Prusiner, S.B. (1991) Molecular biology of prion diseases. Science, 252, 15151522.[ISI][Medline]
Prusiner, S.B., Scott, M.R., DeArmond, S.J., Cohen, F.E. (1998) Prion protein biology. Cell, 93, 337348.[ISI][Medline]
Raymond, G.J., Hope, J., Kocisco, D.A., Priola, S.A., Raymond, L.D., Bossers, A., Ironside, J., Will, R.G., Chen, S.G., Petersen, R.B., and others. (1997) Molecular assessment of the potential transmissibilities of BSE and scrapie to humans. Nature, 388, 285288.[CrossRef][ISI][Medline]
Richter, K. and Buchner, J. (2001) Hsp90: chaperoning signal transduction. J. Cell Physiol., 188, 281290.[CrossRef][ISI][Medline]
Rohwer, R.G. (1984) Scrapie infectious agent is virus-like in size and susceptibility to inactivation. Nature, 308, 658662.[ISI][Medline]
Rudd, P.M., Endo, T., Colominas, C., Groth, D., Wheeler, S.F., Harvey, D.J., Wormald, M.R., Serban, H., Prusiner, S.B., Kobata, A., and Dwek, R.A. (1999) Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl Acad. Sci. USA, 96, 1304413049.
Sailer, A., Büeler, H., Fischer, M., Aguzzi, A., and Weissmann, C. (1994) No propagation of prions in mice devoid of PrP. Cell, 77, 967968.[ISI][Medline]
Schulte, T.W., Blagosklonny, M.V., Ingui, C., and Neckers, L. (1995) Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association. J. Biol. Chem., 270, 2458524588.
Tatzelt, J., Zuo, J., Voellmy, R., Scott, M., Hartl, U., Prusiner, S.B., and Welch, W.J. (1995) Scrapie prions selectively modify the stress response in neuroblastoma cells. Proc. Natl Acad. Sci. USA, 92, 29442948.[Abstract]
Will, R.G., Ironside, J.W., Zeidler, M., Cousens, S.N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A., and Smith, P.G. (1996) A new variant of Creutzfeld-Jakob disease in the UK. Lancet, 347, 921925.[ISI][Medline]