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Ligand binding to distinct states diverts aggregation of an amyloid-forming protein

Nature Chemical Biology volume 7, pages 730–739 (2011)Cite this article

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Abstract

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β2 microglobulin (β2m) fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β2m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation and diverting the course of assembly to the formation of spherical aggregates. The results demonstrate the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers toward alternative assembly pathways.

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Figure 1: Fibrillogenesis of β2m at pH 2.5 in the presence of different small molecules.
Figure 2: Inhibition of fibril formation of β2m by rifamycin SV does not occur by a colloidal mechanism.
Figure 3: Structural properties of spherical aggregates of β2m formed in the presence of rifamycin SV.
Figure 4: ESI-IMS-MS 3D Driftscope plots of β2m monomeric conformers and their ligand binding capabilities at pH 2.5.
Figure 5: 1H-15N HSQC NMR spectra of β2m and its variant F62A,Y63A,Y67A in the presence of rifamycin SV.
Figure 6: β2m assembly at pH 2.5, 37 °C monitored using ThT fluorescence and ESI-IMS-MS.
Figure 7: Rifamycin SV dissociates β2m oligomers when added at different times throughout the lag phase.
Figure 8: Model of the inhibition of β2m aggregation by rifamycin SV at pH 2.5.

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References

  1. Sipe, J.D. et al. Amyloid fibril protein nomenclature: 2010 recommendations from the nomenclature committee of the International Society of Amyloidosis. Amyloid 17, 101–104 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Chiti, F. & Dobson, C.M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Bernstein, S.L. et al. Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease. Nat. Chem. 1, 326–331 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Glabe, C.G. Structural classification of toxic amyloid oligomers. J. Biol. Chem. 283, 29639–29643 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Necula, M., Kayed, R., Milton, S. & Glabe, C.G. Small molecule inhibitors of aggregation indicate that Aβ oligomerization and fibrillization pathways are independent and distinct. J. Biol. Chem. 282, 10311–10324 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Gosal, W.S. et al. Competing pathways determine fibril morphology in the self-assembly of β2-microglobulin into amyloid. J. Mol. Biol. 351, 850–864 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Carrell, R.W. Cell toxicity and conformational disease. Trends Cell Biol. 15, 574–580 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Martins, I.C. et al. Lipids revert inert Aβ amyloid fibrils to neurotoxic protofibrils that affect learning in mice. EMBO J. 27, 224–233 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Lee, H.G. et al. Challenging the amyloid cascade hypothesis: Senile plaques and amyloid-β as protective adaptations to Alzheimer disease. Ann. NY Acad. Sci. 1019, 1–4 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Porat, Y., Abramowitz, A. & Gazit, E. Inhibition of amyloid fibril formation by polyphenols: Structural similarity and aromatic interactions as a common inhibition mechanism. Chem. Biol. Drug Des. 67, 27–37 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Regazzoni, L. et al. A combined high-resolution mass spectrometric and in silico approach for the characterization of small ligands of β2-microglobulin. ChemMedChem 5, 1015–1025 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Cohen, F.E. & Kelly, J.W. Therapeutic approaches to protein-misfolding diseases. Nature 426, 905–909 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Conway, K.A., Rochet, J.C., Bieganski, R.M. & Lansbury, P.T. Kinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adduct. Science 294, 1346–1349 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Feng, B.Y. et al. Small-molecule aggregates inhibit amyloid polymerization. Nat. Chem. Biol. 4, 197–199 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lendel, C. et al. On the mechanism of non-specific inhibitors of protein aggregation: Dissecting the interactions of α-synuclein with Congo red and lacmoid. Biochemistry 48, 8322–8334 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. McGovern, S.L., Caselli, E., Grigorieff, N. & Shoichet, B.K. A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J. Med. Chem. 45, 1712–1722 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Ladiwala, A.R., Dordick, J.S. & Tessier, P.M. Aromatic small molecules remodel toxic soluble oligomers of amyloid β through three independent pathways. J. Biol. Chem. 286, 3209–3218 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Armstrong, A.H., Chen, J., McKoy, A.F. & Hecht, M.H. Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aβ peptide. Biochemistry 50, 4058–4067 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Platt, G.W., Routledge, K.E., Homans, S.W. & Radford, S.E. Fibril growth kinetics reveal a region of β2-microglobulin important for nucleation and elongation of aggregation. J. Mol. Biol. 378, 251–263 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Routledge, K.E., Tartaglia, G.G., Platt, G.W., Vendruscolo, M. & Radford, S.E. Competition between intra-molecular and inter-molecular interactions in an amyloid forming protein. J. Mol. Biol. 389, 776–786 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Meng, F., Marek, P., Potter, K.J., Verchere, C.B. & Raleigh, D.P. Rifampicin does not prevent amyloid fibril formation by human islet amyloid polypeptide but does inhibit fibril thioflavin-T interactions: Implications for mechanistic studies of β-cell death. Biochemistry 47, 6016–6024 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Lieu, V.H., Wu, J.W., Wang, S.S.S. & Wu, C.H. Inhibition of amyloid fibrillization of hen egg-white lysozymes by rifampicin and p-benzoquinone. Biotechnol. Prog. 23, 698–706 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Li, J., Zhu, M., Rajamani, S., Uversky, V.N. & Fink, A.L. Rifampicin inhibits α-synuclein fibrillation and disaggregates fibrils. Chem. Biol. 11, 1513–1521 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Tomiyama, T., Kaneko, H., Kataoka, K., Asano, S. & Endo, N. Rifampicin inhibits the toxicity of pre-aggregated amyloid peptides by binding to peptide fibrils and preventing amyloid-cell interaction. Biochem. J. 322, 859–865 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Carazzone, C. et al. Sulfonated molecules that bind a partially structured species of β2-microglobulin also influence refolding and fibrillogenesis. Electrophoresis 29, 1502–1510 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Smith, A.M., Jahn, T.R., Ashcroft, A.E. & Radford, S.E. Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry. J. Mol. Biol. 364, 9–19 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Smith, D.P., Radford, S.E. & Ashcroft, A.E. Elongated oligomers in β2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc. Natl. Acad. Sci. USA 107, 6794–6798 (2010).

    Article  CAS  PubMed  Google Scholar 

  28. Armstrong, D.W., Schneiderheinze, J., Nair, U., Magid, L.J. & Butler, P.D. Self-association of rifamycin B: Possible effects on molecular recognition. J. Phys. Chem. B 103, 4338–4341 (1999).

    Article  CAS  Google Scholar 

  29. Kayed, R. et al. Annular protofibrils are a structurally and functionally distinct type of amyloid oligomer. J. Biol. Chem. 284, 4230–4237 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. O'Nuallain, B. & Wetzel, R. Conformational Abs recognizing a generic amyloid fibril epitope. Proc. Natl. Acad. Sci. USA 99, 1485–1490 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Xue, W.F. et al. Fibril fragmentation enhances amyloid cytotoxicity. J. Biol. Chem. 284, 34272–34282 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Smith, D.P. et al. Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur. J. Mass Spectrom. (Chichester, Eng.) 15, 113–130 (2009).

    Article  CAS  Google Scholar 

  33. Smith, D.P., Giles, K., Bateman, R.H., Radford, S.E. & Ashcroft, A.E. Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry. J. Am. Soc. Mass Spectrom. 18, 2180–2190 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Platt, G.W., McParland, V.J., Kalverda, A.P., Homans, S.W. & Radford, S.E. Dynamics in the unfolded state of β2-microglobulin studied by NMR. J. Mol. Biol. 346, 279–294 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Tomiyama, T. et al. Rifampicin prevents the aggregation and neurotoxicity of Aβ protein in vitro. Biochem. Biophys. Res. Commun. 204, 76–83 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Borysik, A.J., Radford, S.E. & Ashcroft, A.E. Co-populated conformational ensembles of β2-microglobulin uncovered quantitatively by electrospray ionisation mass spectroscopy. J. Biol. Chem. 279, 27069–27077 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Smith, D.P., Jones, S., Serpell, L.C., Sunde, M. & Radford, S.E. A systematic investigation into the effect of protein destabilization on β2-microglobulin amyloid formation. J. Mol. Biol. 330, 943–954 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Xue, W.F., Homans, S.W. & Radford, S.E. Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly. Proc. Natl. Acad. Sci. USA 105, 8926–8931 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. Ladner, C.L. et al. Stacked sets of parallel, in-register beta-strands of β2-microglobulin in amyloid fibrils revealed by site-directed spin labeling and chemical labeling. J. Biol. Chem. 285, 17137–17147 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Debelouchina, G.T., Platt, G.W., Bayro, M.J., Radford, S.E. & Griffin, R.G. Magic angle spinning NMR analysis of β2-microglobulin amyloid fibrils in two distinct morphologies. J. Am. Chem. Soc. 132, 10414–10423 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gazit, E. A possible role for π-stacking in the self-assembly of amyloid fibrils. FASEB J. 16, 77–83 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Taniguchi, S. et al. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J. Biol. Chem. 280, 7614–7623 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Ehrnhoefer, D.E. et al. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 15, 558–566 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Bieschke, J. et al. EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity. Proc. Natl. Acad. Sci. USA 107, 7710–7715 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Ladiwala, A.R.A. et al. Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Aβ off-pathway conformers. J. Biol. Chem. 285, 24228–24237 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Grabenauer, M., Wu, C., Soto, P., Shea, J.E. & Bowers, M.T. Oligomers of the prion protein fragment 106–126 are likely assembled from beta-hairpins in solution, and methionine oxidation inhibits assembly without altering the peptide's monomeric conformation. J. Am. Chem. Soc. 132, 532–539 (2010).

    Article  CAS  PubMed  Google Scholar 

  47. Dupuis, N.F., Wu, C., Shea, J.E. & Bowers, M.T. Human islet amyloid polypeptide monomers form ordered beta-hairpins: A possible direct amyloidogenic precursor. J. Am. Chem. Soc. 131, 18283–18292 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ashcroft, A.E. Mass spectrometry and the amyloid problem–how far can we go in the gas phase? J. Am. Soc. Mass Spectrom. 21, 1087–1096 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Laurén, J., Gimbel, D.A., Nygaard, H.B., Gilbert, J.W. & Strittmatter, S.M. Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers. Nature 457, 1128–1132 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank A. Kalverda for advice on NMR spectroscopy, D.P. Smith for advice about α-synuclein, K. Ainley for technical assistance, C. Glabe for the gift of OC and A11 antibodies and Aβ1-40 oligomers, J. Rushworth for creating fibrillar oligomers of Aβ1-42, R. Wetzel for the gift of WO1 antibodies and the Radford and Ashcroft groups for helpful discussions and advice. We acknowledge the Wellcome Trust (grant numbers 075675 and 062164) and BBSRC (Grant Number BB/526502/1) for funding. The Synapt HDMS mass spectrometer was purchased with funding from the Biotechnology and Biological Sciences Research Council (Research Equipment Initiative grant BB/E012558/1).

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  1. Lucy A Woods and Geoffrey W Platt: These authors contributed equally to this work.

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  1. Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom

    Lucy A Woods, Geoffrey W Platt, Andrew L Hellewell, Eric W Hewitt, Steve W Homans, Alison E Ashcroft & Sheena E Radford

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Contributions

L.A.W. designed and carried out MS experiments, performed MS data analysis, carried out colloidal inhibition assays and wrote the paper. G.W.P. designed and carried out small-molecule screen, designed NMR experiments and performed NMR data analysis and wrote the paper. A.L.H. carried out immunological assays, cell toxicity assays and wrote the paper. S.E.R., A.E.A., S.W.H. and E.W.H. designed experiments, performed data interpretation and wrote the paper.

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Correspondence to Alison E Ashcroft or Sheena E Radford.

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Woods, L., Platt, G., Hellewell, A. et al. Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nat Chem Biol 7, 730–739 (2011). https://doi.org/10.1038/nchembio.635

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