Protein Misfolding Cyclic Amplification

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Protein Misfolding Cyclic Amplification (PMCA) is an amplification technique (conceptually like PCR but not involving nucleotides) to multiply misfolded prions originally developed by Soto and colleagues.[1] It is a test for spongiform encephalopathies like BSE.

Technique[edit]

The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions.[1][2] By repeating the cycle, the mass of normal protein is rapidly changed into misfolded prion (termed PrPSc).

Development[edit]

PMCA was originally developed to mimic prion replication in vitro with a similar efficiency to the in vivo process, but with accelerated kinetics.[1] PMCA is conceptually analogous to DNA amplification by PCR. In both systems a template grows at expenses of a substrate in a cyclic reaction, combining growing and multiplication of the template units.

Replication[edit]

PMCA has been applied to replicate the misfolded protein from diverse species.[3][4][5] The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrPSc and strikingly it is infectious to wild type animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions.[6]

Automation[edit]

The technology has been automated, leading to a dramatic increase on efficiency of amplification. Indeed, one round of 144 PMCA cycles results in a 6000-fold increase of sensitivity of detection,[7] whereas 2 and 7 rounds of successive PMCA result in 10 million and 3 billion folds amplification.[7]

Sensitivity[edit]

It has been shown that PMCA is capable of detecting as little as a single molecule of oligomeric infectious PrPSc.[7] PMCA possesses the ability to generate millions infectious units, starting with the equivalent to one PrPSc oligomer; well below the infectivity threshold.[7] This data demonstrates that PMCA has a similar power of amplification as PCR techniques used to amplify DNA. It opens a great promise for development of a highly sensitive detection of PrPSc, and for understanding the molecular basis of prion replication. Indeed, PMCA has been used by various groups to PrPSc in blood of animals experimentally infected with prions during both the symptomatic[8] and pre-symptomatic phases[9] as well as in urine.[10]

Uses[edit]

The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the nature of the infectious agent, the phenomenon of prion strains and species barrier, the effect of cellular components, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication.[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] Recent studies by the groups of Supattapone and Ma were able to produce prion replication in vitro by PMCA using purified PrPC and recombinant PrPC with the sole addition of synthetic polyanions and lipids.[35][36] These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute one of the strongest evidence in favor of the prion hypothesis.

References[edit]

  1. ^ a b c Saborio,G.P., Permanne,B. and Soto,C. (2001) Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature, 411, 810-813.
  2. ^ Soto,C., Saborio,G.P. and Anderes,L. (2002) Cyclic amplification of protein misfolding: application to prion- related disorders and beyond. Trends Neurosci., 25, 390-394.
  3. ^ Soto,C., Anderes,L., Suardi,S., Cardone,F., Castilla,J., Frossard,M.J., Peano,S., Saá,P., Limido,L., Carbonatto,M., Ironside,J., Torres,J.M., Pocchiari,M. and Tagliavini,F. (2005) Pre-symptomatic detection of prions by cyclic amplification of protein misfolding. FEBS Lett., 579, 638-642.
  4. ^ Jones,M., Peden,A.H., Prowse,C.V., Groner,A., Manson,J.C., Turner,M.L., Ironside,J.W., MacGregor,I.R. and Head,M.W. (2007) In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc. J.Pathol., 213, 21-26.
  5. ^ Kurt,T.D., Perrott,M.R., Wilusz,C.J., Wilusz,J., Supattapone,S., Telling,G.C., Zabel,M.D. and Hoover,E.A. (2007) Efficient in vitro amplification of chronic wasting disease PrPRES. J.Virol., 81, 9605-9608.
  6. ^ Castilla,J., Saá,P., Hetz,C. and Soto,C. (2005) In vitro generation of infectious scrapie prions. Cell, 121, 195-206.
  7. ^ a b c d Saa,P., Castilla,J. and Soto,C. (2006) Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J.Biol.Chem., 281, 35245-35252.
  8. ^ Castilla,J., Saa,P. and Soto,C. (2005) Detection of prions in blood. Nat.Med., 11, 982-985.
  9. ^ Saa,P., Castilla,J. and Soto,C. (2006) Presymptomatic detection of prions in blood. Science, 313, 92-94.
  10. ^ Gonzalez-Romero,D., Barria,M.A., Leon,P., Morales,R. and Soto,C. (2008) Detection of infectious prions in urine. FEBS Lett., 582, 3161-3166.
  11. ^ Castilla,J., Gonzalez-Romero,D., Saa,P., Morales,R., De,C.J. and Soto,C. (2008) Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions. Cell, 134, 757-768.
  12. ^ Barria,M.A., Mukherjee,A., Gonzalez-Romero,D., Morales,R. and Soto,C. (2009) De novo generation of infectious prions in vitro produces a new disease phenotype. PLoS.Pathog., 5, e1000421.
  13. ^ Deleault,N.R., Lucassen,R.W. and Supattapone,S. (2003) RNA molecules stimulate prion protein conversion. Nature, 425, 717-720.
  14. ^ Bieschke,J., Weber,P., Sarafoff,N., Beekes,M., Giese,A. and Kretzschmar,H. (2004) Autocatalytic self-propagation of misfolded prion protein. Proc.Natl.Acad.Sci.U.S.A. 101, 12207-12211.
  15. ^ Piening,N., Weber,P., Giese,A. and Kretzschmar,H. (2005) Breakage of PrP aggregates is essential for efficient autocatalytic propagation of misfolded prion protein. Biochem.Biophys.Res.Commun., 326, 339-343.
  16. ^ Kim,N.H., Choi,J.K., Jeong,B.H., Kim,J.I., Kwon,M.S., Carp,R.I. and Kim,Y.S. (2005) Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrPC to PrPres. FASEB J. 19, 783-785.
  17. ^ 17. Haley,N.J., Seelig,D.M., Zabel,M.D., Telling,G.C. and Hoover,E.A. (2009) Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay. PLoS.ONE., 4, e4848.
  18. ^ Shi,S., Dong,C.F., Wang,G.R., Wang,X., An,R., Chen,J.M., Shan,B., Zhang,B.Y., Xu,K., Shi,Q., Tian,C., Gao,C., Han,J. and Dong,X.P. (2009) PrP(Sc) of scrapie 263K propagates efficiently in spleen and muscle tissues with protein misfolding cyclic amplification. Virus Res., 141, 26-33.
  19. ^ Atarashi,R., Moore,R.A., Sim,V.L., Hughson,A.G., Dorward,D.W., Onwubiko,H.A., Priola,S.A. and Caughey,B. (2007) Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat.Methods, 4, 645-650.
  20. ^ Castilla,J., Morales,R., Saa,P., Barria,M., Gambetti,P. and Soto,C. (2008) Cell-free propagation of prion strains. EMBO J., 27, 2557-2566.
  21. ^ Green,K.M., Castilla,J., Seward,T.S., Napier,D.L., Jewell,J.E., Soto,C. and Telling,G.C. (2008) Accelerated high fidelity prion amplification within and across prion species barriers. PLoS.Pathog., 4, e1000139.
  22. ^ Morales,R., Buytaert-Hoefen,K.A., Gonzalez-Romero,D., Castilla,J., Hansen,E.T., Hlavinka,D., Goodrich,R.P. and Soto,C. (2008) Reduction of prion infectivity in packed red blood cells. Biochem.Biophys.Res.Commun., 377, 373-378.
  23. ^ Orem, N.R., Geoghegan, J.C., Deleault, N.R., Kascsak, R. and Supattapone, S. (2006) Copper (II) ions potently inhibit purified PrPres amplification. J Neurochem 96, 1409-1415.
  24. ^ Barret, A., Tagliavini, F., Forloni, G., Bate, C., Salmona, M., Colombo, L., et al. (2003) Evaluation of quinacrine treatment for prion diseases. J Virol 77, 8462-8469.
  25. ^ Haley, N.J., Mathiason, C.K., Zabel, M.D., Telling, G.C. and Hoover, E.A. (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.
  26. ^ Jones, M., Peden, A.H., Yull, H., Wight, D., Bishop, M.T., Prowse, C.V., et al. (2009) Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrP) associated with variant Creutzfeldt-Jakob disease. Transfusion 49, 376-384.
  27. ^ Kim, J.I., Surewicz, K., Gambetti, P. and Surewicz, W.K. (2009) The role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro. FEBS Lett 583, 3671-3675.
  28. ^ Kurt, T.D., Telling, G.C., Zabel, M.D. and Hoover, E.A. (2009) Trans-species amplification of PrP(CWD) and correlation with rigid loop 170N. Virology 387, 235-243
  29. ^ Murayama, Y., Yoshioka, M., Horii, H., Takata, M., Yokoyama, T., Sudo, T., et al. (2006) Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation. Biochem Biophys Res Commun 348, 758-762.
  30. ^ Shikiya, R.A., Ayers, J.I., Schutt, C.R., Kincaid, A.E. and Bartz, J.C. (2010) Coinfecting prion strains compete for a limiting cellular resource. J Virol 84, 5706-5714.
  31. ^ Tattum, M.H., Jones, S., Pal, S., Collinge, J. and Jackson, G.S. (2010) Discrimination between prion-infected and normal blood samples by protein misfolding cyclic amplification. Transfusion 50, 996-1002.
  32. ^ Thorne, L. and Terry, L.A. (2008) In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie. J Gen Virol 89, 3177-3184.
  33. ^ Meyerett, C., Michel, B., Pulford, B., Spraker, T.R., Nichols, T.A., Johnson, T., et al. (2008) In vitro strain adaptation of CWD prions by serial protein misfolding cyclic amplification. Virology 382, 267-276.
  34. ^ Chen, B., Morales, R., Barria, M.A. and Soto, C. (2010) Estimating prion concentration in fluids and tissues by quantitative PMCA. Nat Methods 7, 519-520.
  35. ^ Deleault, N.R., Harris, B.T., Rees, J.R. and Supattapone, S. (2007) Formation of native prions from minimal components in vitro. Proc Natl Acad Sci U S A 104, 9741-9746.
  36. ^ Wang, F., Wang, X., Yuan, C.-G. and Ma, J. (2010) Generating a Prion with Bacterially Expressed Recombinant Prion Protein. Science 327, 1132-1135.