Highly Efficient Protein Misfolding Cyclic Amplification

English version České info

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP^C into PrP^Sc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP^C may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP^C into PrP^Sc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP^Sc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 10¹²-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP^C susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP^Sc in vitro.

Vyšlo v časopise: Highly Efficient Protein Misfolding Cyclic Amplification. PLoS Pathog 7(2): e32767. doi:10.1371/journal.ppat.1001277
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001277

Souhrn

Zdroje

1. SaborioGP

PermanneB

SotoC

2001 Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411 810 813

2. SaaP

CastillaJ

SotoC

2006 Ultra-efficient Replication of Infectious Prions by Automated Protein Misfolding Cyclic Amplification. J Biol Chem 281 35245 35252

3. Gonzalez-RomeroD

BarriaMA

LeonP

MoralesR

SotoC

2008 Detection of infectious prions in urine. FEBS Lett 582 3161 3166

4. MurayamaY

YoshiokaM

OkadaH

TakataM

TokashiY

2007 Urinary excretion and blood level of prions in scrapie-infected hamsters. J Gen Virol 88 2890 2898

5. ShikiyaRA

AyersJI

SchuttCR

KincaidAE

BartzJC

2010 Coinfecting prion strains compete for a limiting cellular resource. J Virol 84 5706 5714

6. SaaP

CastillaJ

SotoC

2006 Presymptomatic Detection of Prions in Blood. Science 313 92 94

7. TattumMH

JonesS

PalS

CollingeJ

JacksonGS

2010 Discrimination between prion-infected and normal blood samples by protein misfolding cyclic amplification. Transfusion 50 996 1002

8. HaleyNJ

MathiasonCK

ZabelMD

TellingGC

HooverEA

2009 Detection of sub-clinical CWD infection in conventional test-negative deer long after oral exposure to urine and feces from CWD+ deer. Plos ONE 4 e7990

9. BarriaMA

MukherjeeA

Gonzalez-RomeroD

MoralesR

SotoC

2009 De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype. PLOS Pathog 5 e1000421

10. DeleaultNR

HarrisBT

ReesJR

SupattaponeS

2007 Formation of native prions from minimal components in vitro. Proc Acad Natl Sci U S A 104 9741 9746

11. WangF

WangX

YuanC-G

MaJ

2010 Generating a Prion Bacterially Expressed Recombinant Prion Protein. Science 327 1132 1135

12. DeleaultNR

GeogheganJC

NishinaK

KascsakR

WilliamsonRA

2005 Protease-resistant Prion Protein Amplification Reconstituted with Partially Purified substrates and Synthetic Polyanions. J Biol Chem 280 26873 26879

13. DeleaultNR

KascsakR

GeogheganJC

SupattaponeS

2010 Species-dependent differences in cofactor utilization for formation of the protease-resistant prion protein in vitro. Biochemistry 49 3928 3934

14. DeleaultNR

LucassenRW

SupattaponeS

2003 RNA molecules stimulate prion protein conversion. Nature 425 717 720

15. MaysCE

RyouC

2010 Plasminogen stimulates propagation of protease-resistant prion protein in vitro. Faseb J in press

16. NishinaK

DeleaultNR

MahalS

BaskakovI

LuhrsT

2006 The Stoichiometry of Host PrPC Glycoforms Modulates the Efficiency of PrPSc formation in vitro. Biochemistry 45 14129 14139

17. CastillaJ

Gonzalez-RomeroD

SaaP

MoralesR

De CastroJ

2008 Crossing the Species Barrier by PrPSc Replication In Vitro Generates Unique Infectious Prions. Cell 134 757 768

18. GreenKM

CastillaJ

SewardTS

NapierDL

JewellJE

2008 Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers. PLOS Pathog 4 e1000139

19. MeyerettC

MichelB

PulfordB

SparkerTR

NicholsTA

2008 In vitro strain adaptation of CWD prions by serial protein misfolding cyclic amplification. Virology 382 267 276

20. MaysCE

TitlowW

SewardT

TellingGC

RyouC

2009 Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source. Biochem Biophys Res Commun 388 306 310

21. KurtTD

PerrottMR

WiluszCJ

WiluszJ

SupattaponeS

2007 Efficient in vitro amplification of chronic wasting disease PrP-res. J Virol 81 9605 9608

22. BocharovaOV

BreydoL

ParfenovAS

SalnikovVV

BaskakovIV

2005 In vitro conversion of full length mammalian prion protein produces amyloid form with physical property of PrPSc. J Mol Biol 346 645 659

23. GregoriL

LambertBC

GurgelPV

GheorghiuL

EdwardsonP

2006 Reduction of transmissible spongiform encephalopathy infectivity from human red blood cells with prion protein affinity ligands. Transfusion 46 1152 1161

24. GregoriL

McCombieN

PalmerD

BirchP

Sowemimo-CokerSO

2004 Effectiveness of leucoreduction for removal of infectivity of transmissible spongiform encephalopathies from blood. Lancet 364 529 531

25. MakaravaN

KovacsGG

BocharovaOV

SavtchenkoR

AlexeevaI

2010 Recombinant prion protein induces a new transmissible prion disease in wild type animals. Acta Neuropathol 119 177 187

26. SunY

MakaravaN

LeeCI

LaksanalamaiP

RobbFT

2008 Conformational stability of PrP amyloid firbils controls their smallest possible fragment size. J Mol Biol 376 1155 1167

27. BreydoL

BocharovaOV

MakaravaN

SalnikovVV

AndersonM

2005 Methionine Oxidation Interferes with Conversion of the Prion Protein into the Fibrillar Proteinase K-Resistant Conformation. Biochemistry 44 15534 15543

28. OstapchenkoVG

MakaravaN

SavtchenkoR

BaskakovIV

2008 The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP. J Mol Biol 383 1210 1224

29. CastillaJ

SaaP

HetzC

SotoC

2005 In vitro generation of infectious scrapie prions. Cell 121 195 206

30. KramerML

BartzJC

2009 Rapid, high-throughput detection of PrPSc by 96-well immunoassay. Prion 3 44 48

31. MakaravaN

BaskakovIV

2008 The same primary structure of the prion protein yields two distinct self-propagating states. J Biol Chem 283 15988 15996