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Parameters that Affect Macromolecular Self-Assembly of Prion Protein

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Abstract

Amyloidogenesis of prion protein (PrP) is closely associated with the pathobiology of prion diseases. To understand details on formation of PrP amyloids, we investigated various conditions that influence the process in vitro, using full length and truncated recombinant PrP. Disrupted agitation and fluctuated temperature resulted in prolongation of lag phase during PrP amyloid formation. With the same conditions and material for the assay, fluorescence microplate readers of different manufacturers, which are assumed to have incongruent level of mechanical performance, demonstrated variations for the length of lag phase and the level of fluorescence detection. Presence of preformed amyloid seeds accelerated PrP amyloid formation. Similarly, recombinant proteins of different species affected effectual generation of amyloids. This process was also influenced by the concentrations and truncation of recombinant PrP. By investigating several conditions to perform PrP amyloid formation assay, our study addresses the factors that determine how much and how rapidly PrP amyloids are formed.

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Abbreviations

Gdn-HCl:

Guanidine hydrochloride

MoPrP:

Mouse prion protein

PrP:

Prion protein

PrPC :

Cellular prion protein

PrPSc :

Scrapie prion protein

PAFA:

PrP amyloid formation assay

PBS:

Phosphate-buffered saline

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SHaPrP:

Syrian hamster prion protein

TEM:

Transmission electron microscopy

ThT:

Thioflavin T

References

  1. Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95:13363–13383

    Article  CAS  Google Scholar 

  2. Ryou C (2007) Prions and prion diseases: fundamentals and mechanistic details. J Microbiol Biotechnol 17(7):1059–1070

    CAS  Google Scholar 

  3. Novitskaya V, Bocharova OV, Bronstein I, Baskakov IV (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J Biol Chem 281(19):13828–13836

    Article  CAS  Google Scholar 

  4. Corsaro A, Thellung S, Villa V, Nizzari M, Florio T (2012) Role of prion protein aggregation in neurotoxicity. Int J Mol Sci 13(7):8648–8669

    Article  CAS  Google Scholar 

  5. Bucciantini M, Nosi D, Forzan M, Russo E, Calamai M, Pieri L, Formigli L, Quercioli F, Soria S, Pavone F, Savistchenko J, Melki R, Stefani M (2012) Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1. FASEB J 26(2):818–831. doi:10.1096/fj.11-189381

    Article  CAS  Google Scholar 

  6. Caughey B, Baron GS, Chesebro B, Jeffrey M (2009) Getting a grip on prions: oligomers, amyloids, and pathological membrane interactions. Ann Rev Biochem 78(1):177–204

    Article  CAS  Google Scholar 

  7. Ghetti B, Dlouhy SR, Giaccone G, Bugiani O, Frangione B, Farlow MR, Tagliavini F (1995) Gerstmann-Strässler-Scheinker disease and the Indiana kindred. Brain Pathol 5:61–75

    Article  CAS  Google Scholar 

  8. Piccardo P, Manson JC, King D, Ghetti B, Barron RM (2007) Accumulation of prion protein in the brain that is not associated with transmissible disease. Proc Natl Acad Sci USA 104(11):4712–4717

    Article  CAS  Google Scholar 

  9. Sim VL, Caughey B (2009) Ultrastructures and strain comparison of under-glycosylated scrapie prion fibrils. Neurobiol Aging 30(12):2031–2042

    Article  CAS  Google Scholar 

  10. Wille H, Michelitsch MD, Guenebaut V, Supattapone S, Serban A, Cohen FE, Agard DA, Prusiner SB (2002) Structural studies of the scrapie prion protein by electron crystallography. Proc Natl Acad Sci USA 99:3563–3568

    Article  CAS  Google Scholar 

  11. Govaerts C, Wille H, Prusiner SB, Cohen FE (2004) Evidence for assembly of prions with left-handed b-helices into trimers. Proc Natl Acad Sci USA 101:8342–8347

    Article  CAS  Google Scholar 

  12. DeMarco ML, Silveira J, Caughey B, Daggett V (2006) Structural properties of prion protein protofibrils and fibrils: an experimental assessment of atomic models. Biochemistry 45(51):15573–15582

    Article  CAS  Google Scholar 

  13. Ryou C, Mays CE (2008) Prion propagation in vitro: are we there yet? Int J Mol Sci 5:347–353

    Article  CAS  Google Scholar 

  14. Shin W, Lee B, Hong S, Ryou C, Kwon M (2008) Cloning and expression of a prion protein (PrP) gene from Korean bovine (Bos taurus coreanae) and production of rabbit anti-bovine PrP antibody. Biotechnol Lett 30(10):1705–1711

    Article  CAS  Google Scholar 

  15. Ryou C, Prusiner SB, Legname G (2003) Cooperative binding of dominant-negative prion protein to kringle domains. J Mol Biol 329:323–333

    Article  CAS  Google Scholar 

  16. Kang H-E, Weng CC, Saijo E, Saylor V, Bian J, Kim S, Ramos L, Angers R, Langenfeld K, Khaychuk V, Calvi C, Bartz J, Hunter N, Telling GC (2012) Characterization of conformation-dependent prion protein epitopes. J Biol Chem 287(44):37219–37232

    Article  CAS  Google Scholar 

  17. Baskakov IV, Legname G, Baldwin MA, Prusiner SB, Cohen FE (2002) Pathway complexity of prion protein assembly into amyloid. J Biol Chem 277:21140–21148

    Article  CAS  Google Scholar 

  18. Colby DW, Zhang Q, Wang S, Groth D, Legname G, Riesner D, Prusiner SB (2007) Prion detection by an amyloid seeding assay. Proc Natl Acad Sci 104(52):20914–20919

    Article  CAS  Google Scholar 

  19. Halfmann R, Lindquist S (2008) Screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis. J Vis Exp 17:e838

    Google Scholar 

  20. Herczenik E, Gebbink MFBG (2008) Molecular and cellular aspects of protein misfolding and disease. FASEB J 22(7):2115–2133

    Article  CAS  Google Scholar 

  21. Dobson CM (2003) Protein folding and misfolding. Nature 426:884–890

    Article  CAS  Google Scholar 

  22. Härd T, Lendel C (2012) Inhibition of amyloid formation. J Mol Biol 421(4–5):441–465

    Article  Google Scholar 

  23. Cobb NJ, Apetri AC, Surewicz WK (2008) Prion protein amyloid formation under native-like conditions involves refolding of the C-terminal α-helical domain. J Biol Chem 283(50):34704–34711

    Article  CAS  Google Scholar 

  24. McKinley MP, Meyer RK, Kenaga L, Rahbar F, Cotter R, Serban A, Prusiner SB (1991) Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis. J Virol 65:1340–1351

    CAS  Google Scholar 

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Acknowledgments

This work was supported in part by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012R1A1A2043356).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Biology, College of Arts and Sciences, University of Kentucky, 675 Rose St., Lexington, KY, 40506, USA

    Seon-Gu Kim

  2. Department of Pharmacy, College of Pharmacy, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan, Gyeonggi-do, 426-791, Republic of Korea

    Hye-Mi Lee & Chongsuk Ryou

  3. Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan, Gyeonggi-do, 426-791, Republic of Korea

    Chongsuk Ryou

Authors
  1. Seon-Gu Kim
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  2. Hye-Mi Lee
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Corresponding author

Correspondence to Chongsuk Ryou.

Additional information

Seon-Gu Kim and Hye-Mi Lee have contributed equally for this work.

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Kim, SG., Lee, HM. & Ryou, C. Parameters that Affect Macromolecular Self-Assembly of Prion Protein. Protein J 33, 243–252 (2014). https://doi.org/10.1007/s10930-014-9556-z

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