This is the authors' abstract. We don't add key points for this paper.
The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPΔ112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPΔ112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPΔ112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPΔ112-119 indicated altered interaction properites. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation. The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPΔ112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPΔ112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPΔ112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPΔ112-119 indicated altered interaction properites. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation. The prion diseases, the best known of which include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cows, result from the generation and propagation of a conformational variant (PrPSc) of the normal prion protein (PrPC), a predominantly brain-derived glycoprotein of unknown function (1Mastrianni J.A. Roos R.P. Semin. Neurol. 2000; 20: 337-352Crossref PubMed Scopus (33) Google Scholar). Once PrPSc is introduced or spontaneously generated in the brain, it can replicate by complexing with, and templating its conformation onto, additional PrPC (2Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 3Caughey B. Trends Biochem. Sci. 2001; 26: 235-242Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). PrPSc is distinguished from PrPC in that it is insoluble in non-ionic detergents, displays a relative resistance to proteinase K (PK), 1The abbreviations used are: PK, proteinase K; aa, amino acids; PBS, phosphate-buffered saline; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ER, endoplasmic reticulum. and carries a significantly higher content of β-sheet structure (4Pan 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 (2076) Google Scholar). The molecular mechanisms underlying the conversion and replication of PrPSc are unknown. A greater understanding of the specific regions of PrP that critically participate in the de novo generation and replication of PrPSc is a major goal of prion research, both in terms of understanding the well recognized species barrier to transmission and as a prelude to developing therapeutic interventions. While the C-terminal globular domain of PrPC is dominated by α-helical structure, the N-terminal region of the protein is unstructured by NMR (5Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wuthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1128) Google Scholar). However, within the N-terminal half of PrP is the so-called “toxic peptide,” a highly conserved segment comprised of amino acids (aa) 105-125 (mouse sequence numbers). Synthetic peptides corresponding to this segment form fibrils in solution with β-sheet structure and are toxic to cultured mouse hippocampal cells (6Gasset M. Baldwin M.A. Lloyd D.H. Gabriel J.M. Holtzman D.M. Cohen F. Fletterick R. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10940-10944Crossref PubMed Scopus (316) Google Scholar, 7Forloni G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, 8Brown D.R. Herms J. Kretzschmar H.A. Neuroreport. 1994; 5: 2057-2060Crossref PubMed Scopus (184) Google Scholar), suggesting that this segment may feature in PrPSc-PrPC associations. Within the toxic peptide, extending from aa 112-119 is a palindrome sequence (AGAAAAGA), also known as the “hydrophobic core” (Fig. 1). This segment of PrP was found to be the most fibrilogenic motif within the toxic peptide (6Gasset M. Baldwin M.A. Lloyd D.H. Gabriel J.M. Holtzman D.M. Cohen F. Fletterick R. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10940-10944Crossref PubMed Scopus (316) Google Scholar), and later work showed that altering the sequence could modulate its structure and toxicity (9Jobling M.F. Stewart L.R. White A.R. McLean C. Friedhuber A. Maher F. Beyreuther K. Masters C.L. Barrow C.J. Collins S.J. Cappai R. J. Neurochem. 1999; 73: 1557-1565Crossref PubMed Scopus (152) Google Scholar), spotlighting this minimal segment as an area that may feature intimately in the interaction of PrP molecules and their subsequent conversion to PrPSc. Consistent with this idea, larger peptides (aa 106-141) antagonized the in vitro conversion of PrPC to the protease-resistant state in a cell-free conversion model, and this effect was dependent upon the inclusion of the AGAAAAGA sequence in the peptide (10Chabry J. Caughey B. Chesebro B. J. Biol. Chem. 1998; 273: 13203-13207Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The hydrophobic core appears to undergo a profound conformational change upon conversion to PrPSc, since residues 90-120 are accessible to antibodies in PrPC yet not in PrPSc (11Peretz D. Williamson R.A. Matsunaga Y. Serban H. Pinilla C. Bastidas R.B. Rozenshteyn R. James T.L. Houghten R.A. Cohen F.E. Prusiner S.B. Burton D.R. J. Mol. Biol. 1997; 273: 614-622Crossref PubMed Scopus (319) Google Scholar). In addition, antibodies directed against this region effectively “cured” a neuroblastoma cell line chronically infected with scrapie (12White A.R. Enever P. Tayebi M. Mushens R. Linehan J. Brandner S. Anstee D. Collinge J. Hawke S. Nature. 2003; 422: 80-83Crossref PubMed Scopus (423) Google Scholar). Others have shown that deletion of a segment of PrP that overlaps the hydrophobic core produced a PrP that does not convert to PrPSc (13Holscher C. Delius H. Burkle A. J. Virol. 1998; 72: 1153-1159Crossref PubMed Google Scholar, 14Muramoto T. Scott M. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15457-15462Crossref PubMed Scopus (175) Google Scholar). While the above studies support the palindrome as critical to prion generation, direct mechanistic evidence for this is currently lacking. To provide such evidence and further define the role of the AGAAAAGA palindrome in prion processing, we applied a well documented neuroblastoma cell line chronically infected with prions (ScN2a) to model PrPSc replication and a heterologous yeast expression system (15Ma J. Lindquist S. Science. 2002; 298: 1785-1788Crossref PubMed Scopus (268) Google Scholar) to model de novo PrPSc generation. We additionally applied live cell fluorescence resonance energy transfer (FRET) to the yeast model to assess the role of the palindrome in the assembly of PrP molecules. Our results suggest that the hydrophobic core is not required for PrP aggregation; however, it is critical, not only for the de novo generation of the specific PrPSc conformation but also for the proper association of PrPSc and PrPC molecules necessary for the conformational transfer. These results not only confirm the palindrome segment as an essential component to prion genesis but also the of this approach in the of prion conversion and propagation. of PrP mouse PrP sequence was generated by and the molecular were from this by with and the to be The was and in cells to be expressed in cells were the of the yeast mouse PrP sequence from aa which the for and was the yeast expression and To in the in the a was to the and of PrP were by a expression that is in the of as the and when only is In we used the yeast with a which of yeast were from and to and with the of and for by a and from the of were to in and with for were and in of in with for on PrP was found to effectively for which was used as a of the was and the with an additional of and in of in In the was to to that insoluble PrP was the and to for insoluble of yellow fluorescent protein in cell cells were in and a was as the The was the was and the was in an of with yeast were on from the of PrP or the of of protein was with the indicated of (Fig. or with that are not known to with and were in for indicated that the of PrP to but not resistance in or M. and J. A. and were with an A and were used to was with a and yeast were for the of both cyan fluorescent protein and a of aggregates were with for were in and the of aggregates was the The of the fluorescence was with a and a was generated the The of both and for a was To for in which can the of only were a and direct were with relative of of is to the for which the was the were ScN2a cells on as the To PrP of cells were in and for in with bovine and the a of were and in for cells were with bovine in for and in in were and in in was an with a ScN2a cells from were in of and by for A of of the was with for and with in in and to and as dominant-negative ScN2a cells were with of wtPrP the and or the and were as were in for greater were for and and the were in and or were These were in and for with and in with antibodies a or for or a the were in the for and on which were to be of were were from the for and a was generated from the were used in which from the were in the of were an and was of PrP in a prion-infected mouse neuroblastoma cell line (ScN2a) Scott J.M. D.R. A. Prusiner S.B. J. Virol. PubMed Google Scholar) results in its conversion to PrPSc, it the as PrPSc and the molecules in a that the conformational transfer. this cell line is to study the of prion To the of such as specifically mouse PrP in this mouse cell line (13Holscher C. Delius H. Burkle A. J. Virol. 1998; 72: 1153-1159Crossref PubMed Google Scholar), and antibodies for PrP that may with the conversion T. Scott M. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15457-15462Crossref PubMed Scopus (175) Google Scholar, R. D. Prusiner S.B. Sci. 1992; PubMed Scopus Google Scholar), we the PrP to include the residues and known not to with the conversion R. D. Prusiner S.B. Sci. 1992; PubMed Scopus Google Scholar). ScN2a cells were with PrP or that lacking the hydrophobic core sequence (PrPΔ112-119) of the cells were for PrPSc by PrP is expressed as molecular and PrPΔ112-119 expressed in cells and as suggesting normal the (Fig. However, wtPrP was converted to PrP, PrPΔ112-119 was In of evidence that PrPC to the cell it is to with, and be converted PrPSc B. D. M. S. Chesebro B. Caughey A. Caughey K. J. Virol. PubMed Google Scholar, K. M. Scott M. S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), we on cells which its to the (Fig. in the of the PrP normal and yet is not converted to PrPSc. The of conversion of PrPΔ112-119 to PrPSc was to result from or interaction with PrPSc or an to the PrPSc To we the of PrPΔ112-119 to the conversion of wtPrP in a dominant-negative K. Scott M. S. D. Cohen F.E. Prusiner S.B. J. Virol. 2000; PubMed Scopus Google Scholar). a feature suggest interaction with PrPSc. ScN2a cells were with of PrPΔ112-119 PrP and wtPrP the latter of which the of were by to results are with of co-expressing and a of wild type conversion by PrPΔ112-119 is (Fig. and a was with PrP the a known dominant-negative inhibitor of wild type prion conversion (Fig. conversion of was significantly in the of the We the above results to that PrPΔ112-119 is a association with PrPSc and in with conversion of In support of we the of wtPrP and expression in ScN2a cells. Both were within the insoluble of ScN2a the of PrPΔ112-119 was that of wtPrP (Fig. and In the of insoluble PrP was significantly when were expressed in which not PrP (Fig. and These support the association of PrPΔ112-119 with PrPSc. We to the de novo generation of PrPSc and the in interaction of wild type and deletion as by the ScN2a cell To we employed a heterologous yeast expression system that the generation of a PrPSc-like protein (15Ma J. Lindquist S. Science. 2002; 298: 1785-1788Crossref PubMed Scopus (268) Google Scholar), as by its and PrP the and was expressed the yeast the of the for which cells were and PrP by this protein is expressed the and does not in a of insoluble wtPrP was to in a (Fig. The was which well with that in the of with prion disease and ScN2a cells. To confirm that of protein the of yeast does not we expressed of expression and of expression and with the of (Fig. This that the yeast model be to study the molecular required for PrPSc generation as a to the ScN2a model of PrPSc We applied the yeast expression system to study the role of the palindrome in the de novo generation of PrPSc. aa was expressed in the yeast since this segment as the toxic of In contrast to wtPrP (Fig. and was highly to (Fig. and resistance the of the region to only the AGAAAAGA palindrome (PrPΔ112-119) not the (Fig. and However, when a significantly PrP, a deletion N-terminal to both the palindrome segment and the core of PrPSc was expressed in the yeast the of resistance was to that of wtPrP (Fig. a effect and a critical role for the palindrome in the generation of the PrPSc-like We the of deletion to the PrPSc conformation from their to the hydrophobic core is for PrP we that PrPΔ112-119 be when expressed in the yeast We from yeast and YFP, and and insoluble by The of PrP and was by analysis of the wtPrP and were highly insoluble, with only from the (Fig. In of expressed in the (Fig. and not that is a specific feature of PrP and not a of the yeast PrPΔ112-119 only greater suggesting that is not dependent on the AGAAAAGA We to confirm that wtPrP PrPΔ112-119 within the yeast and were the aggregates from To we and PrPΔ112-119 on the with to the C-terminal These fluorescent fusion proteins were expressed the yeast for which yeast were by fluorescence In cells only YFP, a was the (Fig. in the fusion distinct cytosolic aggregates were evident, in to a of PrP not yet or that are the of of (Fig. The cytosolic were as as of expression and were by from the produced aggregates that were from (Fig. the AGAAAAGA palindrome appears to be essential for generation of the PrPSc it does not to be essential for the of PrP molecules. In of the of its of conversion to PrPSc expression in ScN2a and its dominant-negative we the association of PrPΔ112-119 with PrPSc from that of To the of PrP in the of live we PrP fusion proteins with or and their fluorescent with in PrP fusion molecules and (Fig. In of both proteins were within the yeast with as (Fig. In addition, when and were also to (Fig. While results suggest that the proteins were to form heterologous it was also that molecules were in an to their heterologous aggregates that were the of fluorescence To the specific nature of PrP we applied the of to the yeast expression is a for protein that the transfer of energy from an to an 2001; PubMed Scopus Google Scholar, J. E. A. Mol. Biol. 2001; PubMed Scopus Google Scholar). a in the of the is energy that be used for is to the the of fluorescence in yeast can be as to the relative of The of and fluorescent used in the of the PrP fusion are well for in in live cells I. M. R. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. M. Z. R. J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). To PrP molecules be in this we with in yeast and the of the fluorescence upon This was with that of with YFP, since does not in the yeast and is not to specifically with the is significantly when the is with that and PrP fusion proteins undergo specific within the yeast and that this interaction is (Fig. A and We applied this to and specifically the as a for we found that, the by fluorescent when and were and for the for this not significantly from the (Fig. However, a but in the to molecular by was a we the of with the and found that the for the was significantly that for the we the for the of deletion with the of to the of PrPΔ112-119 are the wtPrP interaction a significantly greater the deletion suggesting that PrPΔ112-119 a interaction with wtPrP and These with of the yeast expression and ScN2a dominant-negative support an essential role for the palindrome segment of PrP, not only in the attainment of the PrPSc conformation but also for the association of PrP molecules necessary for the subsequent conversion to PrPSc. in PrPSc of prion disease is a the prion be generated, by conversion of PrPC or by the of PrPSc, such as with the of bovine spongiform Once within the PrPSc by complexing with which it the PrPSc In the of the structure of the of PrPSc, a model of PrPSc and the of the PrPSc-PrPC required to the conformational transfer is not Defining the or residues within PrP that are essential for the de novo generation of PrPSc the of PrPSc to PrPC is not only to the of prion but also to of prion such as the species barrier and in to in the of therapeutic we on an AGAAAAGA palindrome within the N-terminal region of PrP, in prion genesis. of evidence suggest that, unstructured in the N-terminal region of PrP may be intimately in the generation of PrPSc G. Angeretti N. Chiesa R. Monzani E. Salmona M. Bugiani O. Tagliavini F. Nature. 1993; 362: 543-546Crossref PubMed Scopus (896) Google Scholar, D. Williamson R.A. Matsunaga Y. Serban H. Pinilla C. Bastidas R.B. Rozenshteyn R. James T.L. Houghten R.A. Cohen F.E. Prusiner S.B. Burton D.R. J. Mol. Biol. 1997; 273: 614-622Crossref PubMed Scopus (319) Google Scholar, D. Williamson R.A. K. J. E. G. Mehlhorn G. R.A. Burton D.R. Prusiner S.B. Nature. 2001; PubMed Scopus Google Scholar), and of PrPSc support of β-sheet structure this area H. S. Serban A. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). We the for PrPΔ112-119 to be converted to PrP expression in ScN2a a model of PrPSc propagation. We found that of expression and as PrPΔ112-119 was not converted to PrP in this This well with an report (13Holscher C. Delius H. Burkle A. J. Virol. 1998; 72: 1153-1159Crossref PubMed Google Scholar) in which PrP, a deletion that overlaps the palindrome (aa was not converted to PrPSc in a cell line chronically infected with prions PrPΔ112-119 conversion of wtPrP to PrPSc in a dominant-negative suggesting that it but is not converted PrPSc, a result by the of PrPΔ112-119 in the insoluble of ScN2a cell The of used for the study of prion such as transmission and cell chronically infected with not the of de novo generation of PrPSc. The directed expression of wtPrP the yeast was shown to support the de novo of a PrPSc-like as by the of and resistance J. Lindquist S. Biol. 1999; PubMed Scopus Google Scholar). We the of this model to define the of PrP required for the de novo generation of PrPSc. we that the of and resistance are specific to PrP, since expression in yeast not We found that deletion of the 105-125 or the AGAAAAGA palindrome the generation of PrP, suggesting that this is not an feature of PrP expression in this but it is critically dependent on the of specific This of resistance the palindrome as such segment necessary for the PrPSc The N-terminal not the of PrP nor the of aggregates when expressed as fusion This not only that of PrP is not directed by the but it also that and of PrP are not to also protein in this the of association to the of are C-terminal to the a cell-free PrPSc replication of PrP, found the of C-terminal peptides (aa and to the produced of of PrPSc generation M. Caughey B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and antibodies directed to C-terminal regions PrPSc in ScN2a cells D. Williamson R.A. K. J. E. G. Mehlhorn G. R.A. Burton D.R. Prusiner S.B. Nature. 2001; PubMed Scopus Google Scholar). The of the specific of is in this yeast system and is currently PrP in distinct of the yeast system that of cell in the study of PrP is that a of such as protein and are only the of PrP molecules as the for this an system for the of live cell a of that of the and of the of the in the and of we found this to be specific and was and suggesting a specific association of the of with or showed that were not significantly from of a While this suggest that are the of aggregates of when expressed in yeast suggest an A the of is that only when the and molecules are within of which for the in which is a in the of in the of the of aggregates and of PrPΔ112-119 expressed in yeast support such a the results are combined with the results of ScN2a cell a model in which PrPC and PrPSc the of the PrPSc-PrPC can be In this model, a of association is the palindrome segment of the hydrophobic the deletion of which the of the PrPSc conformation and on its to in association within the C-terminal of PrPΔ112-119 is not to spontaneously PrPSc in the yeast the C-terminal association are to be to PrPSc. However, the C-terminal with the may function to PrPC with PrPSc to facilitate the conformational transfer necessary for prion is a proper PrPSc-PrPC interaction in an altered association that does not support conversion and conversion of PrPC to PrPSc in a dominant-negative by of prion The C-terminal in this interaction may be to that have was as a that prion as a to of prion such as the of prion to and dominant-negative of a PrP K. Scott M. James T.L. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). However, suggest that in its may not to be to such the is to PrPC with greater for effectively it from the conversion K. Scott M. James T.L. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), we that of the palindrome or of the dominant-negative of prion propagation in ScN2a cells. Furthermore, have that a the and an N-terminal deletion to does not dominant-negative K. Scott M. S. D. Cohen F.E. Prusiner S.B. J. Virol. 2000; PubMed Scopus Google Scholar). This when combined with is with the model we that both are required for the proper association and of PrPSc-PrPC to effect the conformational the of to a association that as a dominant-negative inhibitor of propagation. both are interaction is which to a of dominant-negative In we employed a heterologous yeast expression system to study the of the de novo generation of PrPSc, as a to the ScN2a model of PrPSc propagation. Our results support the of the AGAAAAGA not only for the attainment of the PrPSc conformation but also for the of a PrPSc-PrPC that results in the propagation of PrPSc. This of live cell in yeast this as a approach to the nature of prion generation and of and regions of the protein required for conversion also in the of or peptides that could as specific of prion generation. We Lindquist and for their in developing the yeast expression
No takes yet. Share an insight, caveat, or question.
Norstrom et al. (2005) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: