Mechanisms of aggregation and fibril formation of the amyloidogenic N-terminal fragment of apolipoprotein A-I

Abstract

The N-terminal (1-83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14-22 and 50-58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1-83 fragment (apoA-I 1-83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14-22 completely inhibits fibril formation of apoA-I 1-83/G26R, whereas ⌬32-40 and ⌬50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to ␤-sheet structures in all deletion variants except for the ⌬14-22 variant, indicating that residues 14-22 are critical for the ␤-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1-83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the ⌬50-58 variant was entropically unfavorable, indicating that residues 50-58 entropically promote the nucleation step in fibril formation of apoA-I 1-83/G26R. Moreover, a residue-level structural investigation of apoA-I 1-83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N-and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I. Apolipoprotein (apoA-I) 3 is the major structural and functional constituent of plasma high-density lipoprotein (HDL) This work was partly supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant JP17H03979 (to H. S.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S9.

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“…6A) and AFM images (Fig. 6B), the Δ32–40 and Δ50–58 variants form straight fibrils with transition to β‐sheet structure similarly to intact apoA‐I 1–83/G26R, whereas the Δ14–22 variant does not form apparent fibrils but rather form small spherical aggregates without secondary structural transition [38]. Dot blotting analysis (Fig.…”

“…For instance, the first reported amyloidogenic variant, Iowa (G26R) [33,34], greatly enhances formation of amyloid fibrils by the 1–83 fragment of apoA‐I with a conformational transition from random coil to β‐strand‐rich structure [22,35]. Interestingly, the two highly amyloidogenic segments (residues 14–22 and 50–58) in the N‐terminal 1–83 fragment [36–38] overlap with the amphipathic α‐helix‐forming regions of apoA‐I upon binding to lipid [12,16,35].…”

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“…Using circular dichroism, they also showed that the fragment 14-22 allows β-transition and fibrilization [216]. Studies with electron paramagnetic resonance spectroscopy [219], X-ray crystallographic studies [220] and hydrogen-deuterium exchange mass spectroscopy [221] showed that the G26R mutation induces helix destabilization of the protein in the N-terminal domain leading to a transition of residues 14-58 to a β-sheet conformation (Figure 7) [216]. In mature amyloid fibrils, ApoA1 N-terminal fragments are assembled in a parallel, in-register β-sheet structure and the protofilaments of ApoA1 present a β-strand-loop-β-strand structure [205,217].…”

“…Hereditary ApoA-I amyloidosis is characterized by deposition of the N-terminal 80-100-residue fragments as amyloid fibrils in peripheral organs. Mutation seems to perturb the native protein structure, making it more susceptible to proteolysis and thereby to the release of the N-terminal amyloidogenic fragment [216,217].…”

“…Using ThT method and atomic force microscopy, they showed that the fragment 14-22 is essential for the fibril formation, while fragments 32-40 and 50-58 have a role in the nucleation process. Using circular dichroism, they also showed that the fragment 14-22 allows βtransition and fibrilization [216]. Studies with electron paramagnetic resonance spectroscopy [219], X-ray crystallographic studies [220] and hydrogen-deuterium exchange mass spectroscopy [221] showed that the G26R mutation induces helix destabilization of the protein in the N-terminal domain leading to a transition of residues 14-58 to a β-sheet conformation (Figure 7) [216].…”