Skip to main content
SpringerLink
Log in

In Vitro Prion Amplification Methodology for Inhibitor Screening

  • Protocol
  • First Online:
Protein Misfolding Diseases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1873))

Abstract

Prion (PrPC) is an endogenous protein found mainly in the nervous system, and its misfolded isoform (PrPSc) is associated with a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or simply prion diseases. The PrPSc isoform shows an intriguing ability to self-perpetuate, acting as template for PrPC misfolding and consequent aggregation. Aggregation in vitro and in vivo follows a fibrillation processes that is associated with neurodegeneration. Therefore, it is important to investigate and understand the molecular mechanisms involved in this process; such understanding also allows investigation of the action of possible candidate molecules to inhibit this process. Here, we highlight useful in vitro methodologies and analyses that were developed using PrP as a protein model but that, as other amyloid proteins also exhibit the same behavior, may be applied to understand other “prion-like” diseases such as Alzheimer’s and Parkinson’s disease.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Prusiner SB (1998) Prions. Proc Natl Acad Sci U S A 95(23):13363–13383. https://doi.org/10.1073/pnas.95.23.13363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Silva JL, Cordeiro Y (2016) The “Jekyll and Hyde” actions of nucleic acids on the prion-like aggregation of proteins. J Biol Chem 291(30):15482–15490. https://doi.org/10.1074/jbc.R116.733428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Eraña H, Venegas V, Moreno J, Castilla J (2017) Prion-like disorders and transmissible spongiform Encephalopathies: an overview of the mechanistic features that are shared by the various disease-related misfolded proteins. Biochem Biophys Res Commun 483(4):1125–1136. https://doi.org/10.1016/j.bbrc.2016.08.166

    Article  CAS  PubMed  Google Scholar 

  4. Soto C (2012) Transmissible proteins: expanding the prion heresy. Cell 149(5):968–977. https://doi.org/10.1016/j.cell.2012.05.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rangel LP, Costa DCF, Vieira TCRG, Silva JL (2014) The aggregation of mutant p53 produces prion-like properties in cancer. Prion 8(1):75–84. https://doi.org/10.4161/pri.27776

    Article  CAS  PubMed  Google Scholar 

  6. Morris AM, Watzky MA, Finke RG (2009) Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature. Biochim Biophys Acta 1794(3):375–397. https://doi.org/10.1016/j.bbapap.2008.10.016

    Article  CAS  PubMed  Google Scholar 

  7. Vieira TC, Cordeiro Y, Caughey B, Silva JL (2014) Heparin binding confers prion stability and impairs its aggregation. FASEB J 28(6):2667–2676. https://doi.org/10.1096/fj.13-246777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ferreira NC, Ascari LM, Hughson AG, Cavalheiro GR, Góes CF, Fernandes PN, Hollister JR, da Conceição RA, Silva DS, Souza AMT, Barbosa MLC, Lara FA, Martins RAP, Caughey B, Cordeiro Y (2017) A promising anti-prion trimethoxychalcone binds to the globular domain of PrPC and changes its cellular location. Antimicrob Agents Chemother. https://doi.org/10.1128/AAC.01441-17

  9. Ferreira NC, Marques IA, Conceição WA, Macedo B, Machado CS, Mascarello A, Chiaradia-Delatorre LD, Yunes RA, Nunes RJ, Hughson AG, Raymond LD, Pascutti PG, Caughey B, Cordeiro Y (2014) Anti-prion activity of a panel of aromatic chemical compounds: in vitro and in silico approaches. PLoS One 9(1):e84531. https://doi.org/10.1371/journal.pone.0084531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Burchell JT, Panegyres PK (2016) Prion diseases: immunotargets and therapy. Immunotargets Ther 5:57–68. https://doi.org/10.2147/ITT.S64795

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Martins PM (2013) True and apparent inhibition of amyloid fibril formation. Prion 7(2):136–139. https://doi.org/10.4161/pri.23111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, Vyas S, Uversky VN, Fink AL (2001) Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry 40(20):6036–6046. https://doi.org/10.1021/bi002555c

    Article  CAS  PubMed  Google Scholar 

  13. Kamal Zaidi F, Bhat R (2018) Resveratrol interferes with an early step in the fibrillization pathway of human lysozyme and modulates it towards less-toxic, off-pathway aggregates. Chembiochem 19(2):159–170. https://doi.org/10.1002/cbic.201700207

    Article  CAS  PubMed  Google Scholar 

  14. Hyeon JW, Kim SY, Lee SM, Lee J, An SS, Lee MK, Lee YS (2017) Anti-prion screening for Acridine, dextran, and tannic acid using real time-quaking induced conversion: a comparison with PrPSc-infected cell screening. PLoS One 12(1):e0170266. https://doi.org/10.1371/journal.pone.0170266

    Article  PubMed  PubMed Central  Google Scholar 

  15. Wilham JM, Orru CD, Bessen RA, Atarashi R, Sano K, Race B, Meade-White KD, Taubner LM, Timmes A, Caughey B (2010) Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog 6(12):e1001217. https://doi.org/10.1371/journal.ppat.1001217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Schmitz M, Cramm M, Llorens F, Müller-Cramm D, Collins S, Atarashi R, Satoh K, Orrù CD, Groveman BR, Zafar S, Schulz-Schaeffer WJ, Caughey B, Zerr I (2016) The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases. Nat Protoc 11(11):2233–2242. https://doi.org/10.1038/nprot.2016.120

    Article  CAS  PubMed  Google Scholar 

  17. Baskakov IV, Bocharova OV (2005) In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features. Biochemistry 44(7):2339–2348. https://doi.org/10.1021/bi048322t43

    Article  CAS  PubMed  Google Scholar 

  18. Bocharova OV, Breydo L, Salnikov VV, Gill AC, Baskakov IV (2005) Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob disease. Protein Sci 14(5):1222–1232. https://doi.org/10.1110/ps.041186605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Naiki H (2017) Thioflavin T: not an all-rounder, but a trustworthy friend for over 27 years. Amyloid 24(sup1):9. https://doi.org/10.1080/13506129.2017.1278688

    Article  PubMed  Google Scholar 

  20. Serio TR, Cashikar AG, Kowal AS, Sawicki GJ, Moslehi JJ, Serpell L, Arnsdorf MF, Lindquist SL (2000) Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 289(5483):1317–1321. https://doi.org/10.1126/science.289.5483.1317

    Article  CAS  PubMed  Google Scholar 

  21. Lee J, Culyba EK, Powers ET, Kelly JW (2011) Amyloid-Œ≤ forms fibrils by nucleated conformational conversion of oligomers. Nat Chem Biol 7(9):602–609. https://doi.org/10.1038/nchembio.624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nizynski B, Dzwolak W, Nieznanski K (2017) Amyloidogenesis of tau protein. Protein Sci 26(11):2126–2150. https://doi.org/10.1002/pro.3275

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are thankful to Lucas M. Ascari (Faculty of Pharmacy-UFRJ) for revision of the in vitro-produced fibril assay.

Author information

Authors and Affiliations

  1. Instituto Federal do Rio de Janeiro, IFRJ, Rio de Janeiro, Brazil

    Tuane Cristine R. G. Vieira

  2. Instituto Nacional de Ciência Tecnologia de Biologia Estrutural e Bioimagem, UFRJ, Rio de Janeiro, Brazil

    Tuane Cristine R. G. Vieira & Jerson L. Silva

  3. Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, Rio de Janeiro, Brazil

    Jerson L. Silva

Authors
  1. Tuane Cristine R. G. Vieira
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jerson L. Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tuane Cristine R. G. Vieira .

Editor information

Editors and Affiliations

  1. BioISI – Biosystems and Integrative Sciences Institute, Faculty of Sciences University of Lisbon, Lisbon, Portugal

    Cláudio M. Gomes

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Vieira, T.C.R.G., Silva, J.L. (2019). In Vitro Prion Amplification Methodology for Inhibitor Screening. In: Gomes, C. (eds) Protein Misfolding Diseases. Methods in Molecular Biology, vol 1873. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8820-4_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8820-4_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8819-8

  • Online ISBN: 978-1-4939-8820-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation