Abstract
In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an α-helical state, which undergoes aggregation at 37°C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of α-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.
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Abbreviations
- AFM:
-
Atomic force microscopy
- ANS:
-
8-Anilino-1-naphthalenesulfonate
- ApoA-I:
-
Apolipoprotein A-I
- [1-93]ApoA-I:
-
Recombinant 93-residue N-terminal fragment of ApoA-I
- CD:
-
Circular dichroism
- DOTAP:
-
1,2,-Dioleoyl-3-trimethylammonium-propane
- GST:
-
Glutathione-S-transferase
- HDL:
-
High-density lipoproteins
- POPC:
-
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
- POPS:
-
1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine
- ThT:
-
Thioflavin T
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Acknowledgments
The authors wish to thank Prof. A. Gliozzi for helpful discussions. This work was supported by MIUR, Ministero dell’Università e della Ricerca Scientifica, Italy (PRIN 2005, Project N. 2005053998_004 and PRIN 2006, Project N. 2006058958_002) and by the University of Genoa (Fondi di Ateneo).
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D. M. Monti and F. Guglielmi contributed equally to the paper.
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Monti, D.M., Guglielmi, F., Monti, M. et al. Effects of a lipid environment on the fibrillogenic pathway of the N-terminal polypeptide of human apolipoprotein A-I, responsible for in vivo amyloid fibril formation. Eur Biophys J 39, 1289–1299 (2010). https://doi.org/10.1007/s00249-010-0582-2
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DOI: https://doi.org/10.1007/s00249-010-0582-2