A causative link between the structure of aberrant protein oligomers and their toxicity


The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphologically and tinctorially similar, as detected with atomic force microscopy and thioflavin T assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the packing of the hydrophobic interactions between adjacent protein molecules in the oligomers. The lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca2+ ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration.

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Figure 1: Morphology and toxicity of HypF-N aggregates.
Figure 2: Structural properties of the oligomers formed under condition B.
Figure 3: Structural properties of the oligomers formed under condition A.
Figure 4: Interaction of the aggregates formed under conditions A and B with cells.
Figure 5: Dysregulation of cytosolic Ca2+ by aggregates formed under conditions A and B.

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This work was supported by the European Union (Project EURAMY), by the Italian Ministero dell'Istruzione, Università e Ricerca (FIRB RBNE03PX83, PRIN 2006058958 and PRIN 2007XY59Z), Fondazione Cariplo, Ente Cassa di Risparmio di Firenze (project “Lipid rafts” 2008) and the European Molecular Biology Organization Young Investigator Programme. We are grateful to S. Torrassa and D. Nichino for assistance with AFM measurements and to I. Shalova for assistance in the purification and labeling of some of the variants used in this work.

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S.C. and F.C. designed the variants and the experiments to investigate the structure of HypF-N oligomers. S.C. (in part also B.M.) produced, purified and labeled all the variants and performed all the fluorescence experiments (PM, ThT and ANS). A.P. and E.E. performed the MTT assays. M.Z. performed the confocal microscopy experiments. C.P. and A.R. designed, performed and analyzed the AFM data. M.S. and C.C. supervised the experiments on cell cultures. F.C. supervised all the experiments. S.C., C.M.D. and F.C. wrote the manuscript with contributions from A.R., C.C. and M.S.

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Correspondence to Fabrizio Chiti.

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Campioni, S., Mannini, B., Zampagni, M. et al. A causative link between the structure of aberrant protein oligomers and their toxicity. Nat Chem Biol 6, 140–147 (2010). https://doi.org/10.1038/nchembio.283

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