Advertisement

Characterization of Amyloid-Like Properties in Bacterial Intracellular Aggregates

  • Anna Villar-Pique
  • Susanna Navarro
  • Salvador VenturaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1258)

Abstract

Protein aggregation into amyloid conformations is associated with more than 50 different human disorders. Recent studies demonstrate that the expression in bacteria of amyloid proteins results in the formation of intracellular aggregates structurally related to those underlying human diseases. The ease with which prokaryotic organisms can be genetically and biochemically manipulated makes them useful systems for studying how and why protein aggregates inside the cell, providing a tractable environment to rationally model in vivo amyloid formation. In this chapter we present an overview of the methods used to characterize the kinetic, structural, and functional properties of amyloid-like bacterial intracellular aggregates and how they can be employed to screen for lead compounds that might modulate amyloid deposition.

Key words

Protein aggregation Inclusion bodies Amyloid Bacteria 

Notes

Acknowledgments

This work was supported by grants BFU2010-14901 from Ministerio de Ciencia e Innovación (Spain), 2009-SGR-760 from AGAUR (Generalitat de Catalunya). S.V. has been granted an ICREA Academia award (ICREA).

References

  1. 1.
    Invernizzi G, Papaleo E, Sabate R et al (2012) Protein aggregation: mechanisms and functional consequences. Int J Biochem Cell Biol 44:1541–1554PubMedCrossRefGoogle Scholar
  2. 2.
    Calamai M, Kumita JR, Mifsud J et al (2006) Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes. Biochemistry 45:12806–12815PubMedCrossRefGoogle Scholar
  3. 3.
    Fernandez-Busquets X, de Groot NS, Fernandez D et al (2008) Recent structural and computational insights into conformational diseases. Curr Med Chem 15:1336–1349PubMedCrossRefGoogle Scholar
  4. 4.
    Nelson R, Eisenberg D (2006) Recent atomic models of amyloid fibril structure. Curr Opin Struct Biol 16:260–265PubMedCrossRefGoogle Scholar
  5. 5.
    Ventura S, Villaverde A (2006) Protein quality in bacterial inclusion bodies. Trends Biotechnol 24:179–185PubMedCrossRefGoogle Scholar
  6. 6.
    de Groot NS, Sabate R, Ventura S (2009) Amyloids in bacterial inclusion bodies. Trends Biochem Sci 34:408–416PubMedCrossRefGoogle Scholar
  7. 7.
    Sabate R, de Groot NS, Ventura S (2010) Protein folding and aggregation in bacteria. Cell Mol Life Sci 67:2695–2715PubMedCrossRefGoogle Scholar
  8. 8.
    Garcia-Fruitos E, Sabate R, de Groot NS et al (2011) Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J 278:2419–2427PubMedCrossRefGoogle Scholar
  9. 9.
    Carrio M, Gonzalez-Montalban N, Vera A et al (2005) Amyloid-like properties of bacterial inclusion bodies. J Mol Biol 347:1025–1037PubMedCrossRefGoogle Scholar
  10. 10.
    Morell M, Bravo R, Espargaro A et al (2008) Inclusion bodies: specificity in their aggregation process and amyloid-like structure. Biochim Biophys Acta 1783:1815–1825PubMedCrossRefGoogle Scholar
  11. 11.
    Dasari M, Espargaro A, Sabate R et al (2011) Bacterial inclusion bodies of Alzheimer’s disease beta-amyloid peptides can be employed to study native-like aggregation intermediate states. Chembiochem 12:407–423PubMedCrossRefGoogle Scholar
  12. 12.
    de Groot NS, Ventura S (2006) Protein activity in bacterial inclusion bodies correlates with predicted aggregation rates. J Biotechnol 125:110–113PubMedCrossRefGoogle Scholar
  13. 13.
    de Groot NS, Aviles FX, Vendrell J et al (2006) Mutagenesis of the central hydrophobic cluster in Abeta42 Alzheimer’s peptide. Side-chain properties correlate with aggregation propensities. FEBS J 273:658–668PubMedCrossRefGoogle Scholar
  14. 14.
    Villar-Pique A, Espargaro A, Sabate R et al (2012) Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors. Microb Cell Fact 11:55PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Villar-Pique A, de Groot NS, Sabate R et al (2012) The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity. J Mol Biol 421:270–281PubMedCrossRefGoogle Scholar
  16. 16.
    Jahn TR, Radford SE (2008) Folding versus aggregation: polypeptide conformations on competing pathways. Arch Biochem Biophys 469:100–117PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Garcia-Fruitos E, Gonzalez-Montalban N, Morell M et al (2005) Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact 4:27PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239PubMedCrossRefGoogle Scholar
  19. 19.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544PubMedCrossRefGoogle Scholar
  20. 20.
    Waldo GS, Standish BM, Berendzen J et al (1999) Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17:691–695PubMedCrossRefGoogle Scholar
  21. 21.
    Belli M, Ramazzotti M, Chiti F (2011) Prediction of amyloid aggregation in vivo. EMBO Rep 12:657–663PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Castillo V, Grana-Montes R, Sabate R et al (2011) Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes. Biotechnol J 6:674–685PubMedCrossRefGoogle Scholar
  23. 23.
    Guidolin D, Agnati LF, Albertin G et al (2012) Bioinformatics aggregation predictors in the study of protein conformational diseases of the human nervous system. Electrophoresis 33:3669–3679PubMedCrossRefGoogle Scholar
  24. 24.
    Conchillo-Sole O, de Groot NS, Aviles FX et al (2007) AGGRESCAN: a server for the prediction and evaluation of “hot spots” of aggregation in polypeptides. BMC Bioinformatics 8:65PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Woulfe J (2008) Nuclear bodies in neurodegenerative disease. Biochim Biophys Acta 1783:2195–2206PubMedCrossRefGoogle Scholar
  26. 26.
    Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10:524–530PubMedCrossRefGoogle Scholar
  27. 27.
    Lindner AB, Madden R, Demarez A et al (2008) Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc Natl Acad Sci U S A 105:3076–3081PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Kim W, Kim Y, Min J et al (2006) A high-throughput screen for compounds that inhibit aggregation of the Alzheimer’s peptide. ACS Chem Biol 1:461–469PubMedCrossRefGoogle Scholar
  29. 29.
    Martinez-Alonso M, Vera A, Villaverde A (2007) Role of the chaperone DnaK in protein solubility and conformational quality in inclusion body-forming Escherichia coli cells. FEMS Microbiol Lett 273:187–195PubMedCrossRefGoogle Scholar
  30. 30.
    Vera A, Gonzalez-Montalban N, Aris A et al (2007) The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng 96:1101–1106PubMedCrossRefGoogle Scholar
  31. 31.
    de Groot NS, Ventura S (2006) Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett 580:6471–6476PubMedCrossRefGoogle Scholar
  32. 32.
    Espargaro A, Sabate R, Ventura S (2012) Thioflavin-S staining coupled to flow cytometry. A screening tool to detect in vivo protein aggregation. Mol Biosyst 8:2839–2844PubMedCrossRefGoogle Scholar
  33. 33.
    Rajan RS, Illing ME, Bence NF et al (2001) Specificity in intracellular protein aggregation and inclusion body formation. Proc Natl Acad Sci U S A 98:13060–13065PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Hart RA, Rinas U, Bailey JE (1990) Protein composition of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli. J Biol Chem 265:12728–12733PubMedGoogle Scholar
  35. 35.
    Wang L, Maji SK, Sawaya MR et al (2008) Bacterial inclusion bodies contain amyloid-like structure. PLoS Biol 6:e195PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Cano-Garrido O, Rodriguez-Carmona E, Diez-Gil C et al (2013) Supramolecular organization of protein-releasing functional amyloids solved in bacterial inclusion bodies. Acta Biomater 9:6134–6142PubMedCrossRefGoogle Scholar
  37. 37.
    Hubbard SJ (1998) The structural aspects of limited proteolysis of native proteins. Biochim Biophys Acta 1382:191–206PubMedCrossRefGoogle Scholar
  38. 38.
    Kong J, Yu S (2007) Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin (Shanghai) 39:549–559CrossRefGoogle Scholar
  39. 39.
    Tycko R (2006) Solid-state NMR as a probe of amyloid structure. Protein Pept Lett 13:229–234PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89:271–277PubMedCrossRefGoogle Scholar
  41. 41.
    Wasmer C, Benkemoun L, Sabate R et al (2009) Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218–289) are amyloids. Angew Chem Int Ed Engl 48:4858–4860PubMedCrossRefGoogle Scholar
  42. 42.
    Garrity SJ, Sivanathan V, Dong J et al (2010) Conversion of a yeast prion protein to an infectious form in bacteria. Proc Natl Acad Sci U S A 107:10596–10601PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Espargaro A, Villar-Pique A, Sabate R et al (2012) Yeast prions form infectious amyloid inclusion bodies in bacteria. Microb Cell Fact 11:89PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Liebman SW, Derkatch IL (1999) The yeast [PSI+] prion: making sense of nonsense. J Biol Chem 274:1181–1184PubMedCrossRefGoogle Scholar
  45. 45.
    Tanaka M, Weissman JS (2006) An efficient protein transformation protocol for introducing prions into yeast. Methods Enzymol 412:185–200PubMedCrossRefGoogle Scholar
  46. 46.
    Tanaka M (2010) A protein transformation protocol for introducing yeast prion particles into yeast. Methods Enzymol 470:681–693PubMedCrossRefGoogle Scholar
  47. 47.
    Chernoff YO, Lindquist SL, Ono B et al (1995) Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science 268:880–884PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anna Villar-Pique
    • 1
  • Susanna Navarro
    • 1
  • Salvador Ventura
    • 1
    Email author
  1. 1.Departament de Bioquímica i Biologia Molecular, Institut de Biotecnologia i de BiomedicinaUniversitat Autònoma de BarcelonaBarcelonaSpain

Personalised recommendations