N-terminal Truncation of Prion Protein Affects Both Formation and Conformation of Abnormal Protease-resistant Prion Protein Generatedin Vitro
2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês
10.1074/jbc.m103799200
ISSN1083-351X
AutoresVictoria Lawson, Suzette A. Priola, K Wehrly, Bruce Chesebro,
Tópico(s)Trace Elements in Health
ResumoTransmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34–94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation. Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34–94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation. transmissible spongiform encephalopathy prion protein proteinase K hamster PrP protease-sensitive prion protein protease-resistant prion protein glycophosphatidylinositol polyacrylamide gel electrophoresis Transmissible spongiform encephalopathy (TSE)1 diseases are a family of fatal neurodegenerative disorders, which affect both humans and animals. These diseases are characterized by the accumulation of an abnormal form of prion protein (PrP), which is associated with the pathogenic process and differs from normal PrP in its secondary structure and chemical properties. The susceptibility of PrP to digestion with proteinase K (PK) is generally used to distinguish the normally protease-sensitive PrP (PrP-sen) from the abnormal protease-resistant form (PrP-res) (1Meyer R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (514) Google Scholar). The C-terminal globular domain of PrP-sen, residues 125–231, is composed of three α-helices and two β-strands connected by loops and turns (2Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 3Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar) and has two potentialN-linked glycosylation sites at residues 181 and 197 (4Caughey B. Race R.E. Ernst D. Buchmeier M.J. Chesebro B. J. Virol. 1989; 63: 175-181Crossref PubMed Google Scholar, 5Bolton D.C. Meyer R.K. Prusiner S.B. J. Virol. 1985; 53: 596-606Crossref PubMed Google Scholar, 6Manuelidis L. Valley S. Manuelidis E.E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4263-4267Crossref PubMed Scopus (74) Google Scholar). In contrast, the N-terminal portion of PrP-sen, residues 23–124, has the properties of a flexible random coil polypeptide (2Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 3Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar, 7Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar) although some bends and turns could be associated with residues 90–119 (8Liu H. Farr-Jones S. Ulyanov N.B. Llinas M. Marqusee S. Groth D. Cohen F.E. Prusiner S.B. James T.L. Biochemistry. 1999; 38: 5362-5377Crossref PubMed Scopus (197) Google Scholar). Following conversion of PrP-sen to PrP-res, the β-sheet content of the molecule increases (9Safar J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. J. Biol. Chem. 1993; 268: 20276-20284Abstract Full Text PDF PubMed Google Scholar, 10Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar, 11Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar), and residues 90–231 become protease-resistant, whereas residues 23–89 remain susceptible to protease digestion (12Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (372) Google Scholar, 13Hope J. Hunter N. Ciba Found. Symp. 1988; 135: 146-163PubMed Google Scholar).It is apparent from several in vitro studies that the amyloidogenic region of PrP from residues 113–120 is important for the generation of PrP-res. First, PrP-sen with this region deleted is not converted to a protease-resistant form when expressed in scrapie-infected neuroblastoma cells (14Holscher C. Delius H. Burkle A. J. Virol. 1998; 72: 1153-1159Crossref PubMed Google Scholar). Furthermore, peptides composed of amino acid residues within this region inhibit conversion of PrP-sen to PrP-res in both cell-free conversion reactions (15Chabry J. Caughey B. Chesebro B. J. Biol. Chem. 1998; 273: 13203-13207Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 16Chabry J. Priola S.A. Wehrly K. Nishio J. Hope J. Chesebro B. J. Virol. 1999; 73: 6245-6250Crossref PubMed Google Scholar) and scrapie-infected neuroblastoma cells (16Chabry J. Priola S.A. Wehrly K. Nishio J. Hope J. Chesebro B. J. Virol. 1999; 73: 6245-6250Crossref PubMed Google Scholar).The in vitro relevance of the N-terminal region of PrP-sen is supported in vivo by the altered disease susceptibility of PrP knockout mice expressing a transgene encoding various truncated PrP-sen molecules. In these studies, mice expressing PrP-sen with a deletion of residues 32–93 were susceptible to mouse scrapie, albeit with reduced levels of detectable PrP-res, altered clinical signs, and pathology (17Flechsig E. Shmerling D. Hegyi I. Raeber A.J. Fischer M. Cozzio A. von Mering C. Aguzzi A. Weissmann C. Neuron. 2000; 27: 399-408Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In contrast, mice expressing further truncated PrP-sen (residues 32–106) were not susceptible to infection (18Weissmann C. J. Biol. Chem. 1999; 274: 3-6Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, it appears that residues upstream of amino acid 93 influence PrP-res formation and scrapie pathogenesis in mice, whereas residues immediately downstream (94) appear to be important for susceptibility to scrapie infection.In the present study, a cell-free conversion assay was used to study the biochemical influence of the flexible N-terminal tail of PrP on the formation of protease-resistant PrP. This assay generates de novo PrP-res (19Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (788) Google Scholar) and has been shown to mimic the species and strain-specific interactions that occur between PrP-sen and PrP-res to form protease-resistant PrP (20Kocisko D.A. Priola S.A. Raymond G.J. Chesebro B. Lansbury Jr., P.T. Caughey B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3923-3927Crossref PubMed Scopus (327) Google Scholar, 21Bessen R.A. Kocisko D.A. Raymond G.J. Nandan S. Lansbury P.T. Caughey B. Nature. 1995; 375: 698-700Crossref PubMed Scopus (457) Google Scholar, 22Bossers A. Belt P. Raymond G.J. Caughey B. de Vries R. Smits M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4931-4936Crossref PubMed Scopus (158) Google Scholar, 23Raymond G.J. Hope J. Kocisko D.A. Priola S.A. Raymond L.D. Bossers A. Ironside J. Will R.G. Chen S.G. Petersen R.B. Gambetti P. Rubenstein R. Smits M.A. Lansbury Jr., P.T. Caughey B. Nature. 1997; 388: 285-288Crossref PubMed Scopus (237) Google Scholar, 24Bossers A. de Vries R. Smits M.A. J. Virol. 2000; 74: 1407-1414Crossref PubMed Scopus (119) Google Scholar, 25Raymond G.J. Bossers A. Raymond L.D. O'Rourke K.I. McHolland L.E. Bryant III, P.K. Miller M.W. Williams E.S. Smits M. Caughey B. EMBO J. 2000; 19: 4425-4430Crossref PubMed Scopus (234) Google Scholar). The conversion of a series of PrP-sen molecules with progressive deletions within the N-terminal tail was studied in the presence or absence of post-translational modifications such as glycosylation and the addition of the C-terminal glycophosphatidylinositol (GPI) anchor. Our results show that deletions of residues 34–94 and 95–113 have distinct consequences for conversion of PrP-sen to a protease-resistant form and suggest an underlying role for the flexible N-terminal domain of PrP-sen in scrapie pathogenesis.DISCUSSIONDeletion of residues 34–94 and 34–113 from within the N-terminal tail of PrP-sen influenced the quantity and conformation of PrP-res generated in a cell-free assay. It is unclear how these residues may be influencing PrP conversion. However, the primary amino acid sequence within the 34–94 deletion is composed of five octapeptide repeats that may influence intramolecular and intermolecular interactions between PrP molecules and/or other cellular factors that are required for efficient conversion (39Priola S.A. Chesebro B. J. Biol. Chem. 1998; 273: 11980-11985Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 40Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1900) Google Scholar). Extra copies of the octapeptide repeat are associated with heritable TSE disease in both humans (41Pocchiari M. Mol. Aspects Med. 1994; 15: 195-291Crossref PubMed Scopus (70) Google Scholar) and transgenic mice (42Chiesa R. Piccardo P. Ghetti B. Harris D.A. Neuron. 1998; 21: 1339-1351Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 43Chiesa R. Drisaldi B. Quaglio E. Migheli A. Piccardo P. Ghetti B. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5574-5579Crossref PubMed Scopus (138) Google Scholar) and have been shown to induce PrP aggregation and alter PrP processing in tissue culture cells (39Priola S.A. Chesebro B. J. Biol. Chem. 1998; 273: 11980-11985Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 44Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 45Lehmann S. Harris D.A. J. Biol. Chem. 1996; 271: 1633-1637Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In the current study, a considerable amount of HaWT PrP was pelleted by centrifugation in the absence of PrP-res, whereas deletion of the octapeptide repeat region in deletion mutant HaΔ94 significantly reduced the amount of this PrP-sen self-aggregation (Fig.4 a). The reduced ability of PrP to form self-aggregates was associated with a parallel decline in conversion efficiency. The role of these residues might be to enhance PrP polymerization, which might be beneficial for conversion to a protease-resistant form (40Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1900) Google Scholar). Deletion of additional residues 95–113 (HaΔ113) caused a further decrease in PrP polymerization and a concurrent decrease in cell-free conversion. Therefore, residues within the N-terminal tail required for PrP-PrP interactions and efficient conversion include both the octapeptide repeat region and amino acids between residue 95–113. Interestingly, insertions and point mutations within this region (residues 95–113) are known to lead to genetic TSE diseases in humans (41Pocchiari M. Mol. Aspects Med. 1994; 15: 195-291Crossref PubMed Scopus (70) Google Scholar) and have been shown to confer biochemical properties reminiscent of PrP-res on the mutated PrP-sen molecule (46Lehmann S. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5610-5614Crossref PubMed Scopus (97) Google Scholar).In our experiments, deletion of residues 95–113 influenced not only the efficiency of PrP conversion in the cell-free assay but also the nature of the PrP-res produced. There was no apparent shift in molecular weight of the largest bands derived from PK digestion of PrP-res generated from unglycosylated HaΔ94, HaΔ113, and HaΔ120 (Fig. 2 b). Therefore, in the presence of PrP-res, these constructs could adopt a fully protease-resistant conformation, which probably reflects the absence of the primary digestion site at residue 89. However, PK digestion of HaΔ113, HaΔ120, and HaΔ124 also resulted in the formation of lower molecular mass bands between 7 and 14 kDa. These bands are likely to be the result of protease digestion at multiple sites downstream of the primary digestion site, since antibody mapping indicated that they were a result of progressive PK digestion from the N terminus. 2V. Lawson, unpublished observations. In PrP-res derived from HaWT, residues 95–113 might protect these secondary cleavage sites from protease digestion. Alternatively, deletion of these residues may alter the conformation of the final protease-resistant product such that these secondary sites are more readily exposed to protease digestion. The significance of these altered forms of protease-resistant PrP in neurodegeneration and clinical disease remains to be determined. However, their formation in the cell-free conversion assay was template-dependent, since they were not detected in the absence of a PrP-res seed. Furthermore, similarly sized low molecular weight protease-resistant PrP forms have been detected in inherited TSE diseases of humans and in brain homogenates of scrapie-infected rodents (13Hope J. Hunter N. Ciba Found. Symp. 1988; 135: 146-163PubMed Google Scholar, 47Kocisko D.A. Lansbury Jr., P.T. Caughey B. Biochemistry. 1996; 35: 13434-13442Crossref PubMed Scopus (86) Google Scholar, 48Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 49Parchi P. Chen S.G. Brown P. Zou W. Capellari S. Budka H. Hainfellner J. Reyes P.F. Golden G.T. Hauw J.J. Gajdusek D.C. Gambetti P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8322-8327Crossref PubMed Scopus (183) Google Scholar, 50Capellari S. Parchi P. Russo C.M. Sanford J. Sy M.S. Gambetti P. Petersen R.B. Am. J. Pathol. 2000; 157: 613-622Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), suggesting that these proteins and/or the PrP-res from which they are generated may play a role in TSE pathogenesis in vivo.The conversion efficiency of unglycosylated PrP-sen was not decreased by deletion of residues within the N-terminal region (Fig.2 c). This was in contrast to the effect of these same deletions in the glycosylated protein (Fig. 1 c). Interestingly, the binding of the N-terminally truncated PrP-sen to PrP-res was also improved in the absence of glycosylation. However, conversion of unglycosylated PrP appeared to result in a larger proportion of the lower molecular weight species in the protease-resistant PrP. Quantification of the percentage conversion excluding these lower molecular weight species gave a similar pattern for conversion of the PrP deletion mutants (Fig. 2 d) as was seen previously for glycosylated PrP (Fig. 1 c). Glycosylation may therefore modulate interactions between PrP-sen and PrP-res and promote conversion to a more protease-resistant conformation. Furthermore, the presence of a longer N-terminal region appears to overcome this glycosylation effect, since HaWT and HaΔ94 were predominantly converted to higher molecular weight protease-resistant forms regardless of glycosylation state. Interactions of the flexible N-terminal tail of PrP-sen with antibodies (51Li R. Liu T. Wong B.S. Pan T. Morillas M. Swietnicki W. O'Rourke K. Gambetti P. Surewicz W.K. Sy M.S. J. Mol. Biol. 2000; 301: 567-573Crossref PubMed Scopus (106) Google Scholar) or other parts of PrP-sen itself (7Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar) have been reported to alter the conformation of the glycosylated C-terminal domain of PrP. Therefore, residues 34–113, which affected the conversion of glycosylated PrP-sen, might act by modulating the conformation of the C-terminal domain, thus enabling efficient interactions between PrP-sen and PrP-res.A further post-translational modification of PrP-sen involved expression of the molecule without the signal sequence for the GPI anchor. This modification resulted in a molecule that was mostly unglycosylated, which may have contributed to the improved conversion efficiency of HaΔ113, HaΔ120, and HaΔ124. However, efficient conversion of GPI-negative PrP was still dependent on residues 34–94, which supported our earlier hypothesis that intermolecular interactions between residues in the PrP octapeptide repeat region may be necessary for efficient conversion of PrP-sen. However, for unglycosylated PrP containing a GPI anchor, residues 34–94 were not required for efficient conversion (Fig. 2 c), suggesting that in the absence of glycosylation, the hydrophobic fatty acids of the GPI anchor of PrP-sen might promote the PrP-PrP interactions required for conversion. Thus, in the absence of a GPI anchor, the N-terminal region may be required to mediate interactions between PrP-sen molecules to promote efficient conversion to a protease-resistant form.Residues 34–93 are not required for scrapie susceptibility, since transgenic mice expressing PrP-sen lacking these residues remain susceptible to scrapie infection, albeit with delayed kinetics, reduced levels of detectable PrP-res, and an altered pattern of neurodegeneration (17Flechsig E. Shmerling D. Hegyi I. Raeber A.J. Fischer M. Cozzio A. von Mering C. Aguzzi A. Weissmann C. Neuron. 2000; 27: 399-408Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The decrease in PrP-res produced from cell-free conversion of HaΔ94 could correspond with the decreased PrP-res formation and delayed onset of disease observed in these transgenic mice. However, the cause of the altered pattern of neurodegeneration is unknown. Transgenic mice expressing further truncated PrP-sen lacking residues 34–106 are reportedly not susceptible to scrapie infection (18Weissmann C. J. Biol. Chem. 1999; 274: 3-6Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This is in contrast with the current study, in which PrP-sen truncated between residues 34 and 113 could be converted to a protease-resistant form. This discrepancy may reflect a simple difference between sequences of PrP-sen that are essential for conversion of mouse PrP versus the hamster PrP conversion presented here. Certainly, different amino acid residues have been shown to influence species-specific conversion of mouse and hamster PrP (52Priola S.A. Chesebro B. J. Virol. 1995; 69: 7754-7758Crossref PubMed Google Scholar, 53Priola S.A. Chabry J. Chan K. J. Virol. 2001; 75: 4673-4680Crossref PubMed Scopus (57) Google Scholar). However, a more attractive explanation is that residues between positions 94 and 106 are required for initial infection with a TSE agent but are not essential for the subsequent formation of PrP-res. We have shown that the protease-resistant products associated with conversion of N-terminally deleted PrP-sen lack a large portion of the N-terminal region. Thus, it may also be that the antibodies used to detect PrP-res in the transgenic mouse model were specific to regions of PrP not present in the lower molecular weight protease-resistant products described here (17Flechsig E. Shmerling D. Hegyi I. Raeber A.J. Fischer M. Cozzio A. von Mering C. Aguzzi A. Weissmann C. Neuron. 2000; 27: 399-408Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar).Expression of a foreign PrP species in mouse scrapie-infected neuroblastoma cells can block the generation of mouse PrP-res (32Priola S.A. Caughey B. Race R.E. Chesebro B. J. Virol. 1994; 68: 4873-4878Crossref PubMed Google Scholar). In the present studies, interference with mouse PrP-res formation in Sc+N2a cells was reduced by deletion of residues 34–94 from hamster PrP-sen and completely eliminated by deletion of residues 34–113. There was a strong correlation between the effect of deletion of these regions of the PrP molecule on interference in Sc+N2a cells and conversion in the cell-free assay (Fig. 1). It is therefore possible that residues 34–113 play a similar role in both processes. Previous cell-free conversion studies suggested that interference with PrP-res generation by the expression of a foreign PrP species was primarily caused by inhibition of the acquisition of protease resistance rather than by preventing binding of the homologous PrP (54Horiuchi M. Priola S.A. Chabry J. Caughey B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5836-5841Crossref PubMed Scopus (135) Google Scholar). Consistent with this conclusion, the ability of the hamster-derived PrP-sen to bind to mouse PrP-res was unaffected by the deletions within the N-terminal tail. This suggests that residues in the flexible N-terminal tail of PrP-sen may prevent the homologous PrP species from acquiring protease resistance by interfering with a step of conversion process subsequent to binding. Interestingly, in the system described here, molecules that have been shown to interfere with PrP-res generation in Sc+N2a cells have contained one or both residues of the 3F4 epitope (32Priola S.A. Caughey B. Race R.E. Chesebro B. J. Virol. 1994; 68: 4873-4878Crossref PubMed Google Scholar, 52Priola S.A. Chesebro B. J. Virol. 1995; 69: 7754-7758Crossref PubMed Google Scholar), and deletion of this epitope in the HaΔ113 mutant ablated the ability of HaPrP to induce interference. The noninterfering PrP-sen molecules may also be less able to interact with other components of the conversion process, such as glycosaminoglycans. Zulianello et al. (55Zulianello L. Kaneko K. Scott M. Erpel S. Han D. Cohen F.E. Prusiner S.B. J. Virol. 2000; 74: 4351-4360Crossref PubMed Scopus (86) Google Scholar) proposed that PrP residues 23–34 may bind to an auxiliary molecule required for conversion. However, as residues 23–34 were present in both interfering and noninterfering deletion mutants described in the present paper, these residues do not appear to account for the interference observed here. Transmissible spongiform encephalopathy (TSE)1 diseases are a family of fatal neurodegenerative disorders, which affect both humans and animals. These diseases are characterized by the accumulation of an abnormal form of prion protein (PrP), which is associated with the pathogenic process and differs from normal PrP in its secondary structure and chemical properties. The susceptibility of PrP to digestion with proteinase K (PK) is generally used to distinguish the normally protease-sensitive PrP (PrP-sen) from the abnormal protease-resistant form (PrP-res) (1Meyer R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (514) Google Scholar). The C-terminal globular domain of PrP-sen, residues 125–231, is composed of three α-helices and two β-strands connected by loops and turns (2Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 3Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar) and has two potentialN-linked glycosylation sites at residues 181 and 197 (4Caughey B. Race R.E. Ernst D. Buchmeier M.J. Chesebro B. J. Virol. 1989; 63: 175-181Crossref PubMed Google Scholar, 5Bolton D.C. Meyer R.K. Prusiner S.B. J. Virol. 1985; 53: 596-606Crossref PubMed Google Scholar, 6Manuelidis L. Valley S. Manuelidis E.E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4263-4267Crossref PubMed Scopus (74) Google Scholar). In contrast, the N-terminal portion of PrP-sen, residues 23–124, has the properties of a flexible random coil polypeptide (2Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (660) Google Scholar, 3Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar, 7Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar) although some bends and turns could be associated with residues 90–119 (8Liu H. Farr-Jones S. Ulyanov N.B. Llinas M. Marqusee S. Groth D. Cohen F.E. Prusiner S.B. James T.L. Biochemistry. 1999; 38: 5362-5377Crossref PubMed Scopus (197) Google Scholar). Following conversion of PrP-sen to PrP-res, the β-sheet content of the molecule increases (9Safar J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. J. Biol. Chem. 1993; 268: 20276-20284Abstract Full Text PDF PubMed Google Scholar, 10Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar, 11Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar), and residues 90–231 become protease-resistant, whereas residues 23–89 remain susceptible to protease digestion (12Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (372) Google Scholar, 13Hope J. Hunter N. Ciba Found. Symp. 1988; 135: 146-163PubMed Google Scholar). It is apparent from several in vitro studies that the amyloidogenic region of PrP from residues 113–120 is important for the generation of PrP-res. First, PrP-sen with this region deleted is not converted to a protease-resistant form when expressed in scrapie-infected neuroblastoma cells (14Holscher C. Delius H. Burkle A. J. Virol. 1998; 72: 1153-1159Crossref PubMed Google Scholar). Furthermore, peptides composed of amino acid residues within this region inhibit conversion of PrP-sen to PrP-res in both cell-free conversion reactions (15Chabry J. Caughey B. Chesebro B. J. Biol. Chem. 1998; 273: 13203-13207Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 16Chabry J. Priola S.A. Wehrly K. Nishio J. Hope J. Chesebro B. J. Virol. 1999; 73: 6245-6250Crossref PubMed Google Scholar) and scrapie-infected neuroblastoma cells (16Chabry J. Priola S.A. Wehrly K. Nishio J. Hope J. Chesebro B. J. Virol. 1999; 73: 6245-6250Crossref PubMed Google Scholar). The in vitro relevance of the N-terminal region of PrP-sen is supported in vivo by the altered disease susceptibility of PrP knockout mice expressing a transgene encoding various truncated PrP-sen molecules. In these studies, mice expressing PrP-sen with a deletion of residues 32–93 were susceptible to mouse scrapie, albeit with reduced levels of detectable PrP-res, altered clinical signs, and pathology (17Flechsig E. Shmerling D. Hegyi I. Raeber A.J. Fischer M. Cozzio A. von Mering C. Aguzzi A. Weissmann C. Neuron. 2000; 27: 399-408Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In contrast, mice expressing further truncated PrP-sen (residues 32–106) were not susceptible to infection (18Weissmann C. J. Biol. Chem. 1999; 274: 3-6Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Thus, it appears that residues upstream of amino acid 93 influence PrP-res formation and scrapie pathogenesis in mice, whereas residues immediately downstream (94) appear to be important for susceptibility to scrapie infection. In the present study, a cell-free conversion assay was used to study the biochemical influence of the flexible N-terminal tail of PrP on the formation of protease-resistant PrP. This assay generates de novo PrP-res (19Kocisko D.A. Come J.H. Priola
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