Effect of Phosphatidylserine and Cholesterol on Membrane-mediated Fibril Formation by the N-terminal Amyloidogenic Fragment of Apolipoprotein A-I

Abstract

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.

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

“…S9). Because ␣-helix formation of the amyloidogenic segments upon lipid binding strongly inhibits ␤-transition and fibril formation by the N-terminal fragment of apoA-I (18,19), it is likely that the second amyloidogenic segment containing residues 50 -58 is not available for fibril formation of apoA-I 1-83/G26R on the membrane surface (19). The inhibitory effect of the deletion of residues 32-40 on the kinetics of nucleation and fibril growth (Fig.…”

“…Studies of synthetic apoA-I fragment peptides demonstrated that the peptides containing either the first (residues 14 -22) or the second (residues 50 -58) amyloidogenic segment have the ability to form amyloid-like fibrils with ␤-transition, whereas the peptide only containing residues 69 -72 does not form fibrils at neutral pH (13,14). Interestingly, because the two major amyloidogenic segments (residues 14 -22 and 50 -58) overlap with the hydrophobic ␣-helix-forming regions upon lipid binding (15,16), lipid membrane environments significantly affect the fibril-forming properties of the N-terminal fragments or peptides of apoA-I (17)(18)(19).…”

“…We previously reported that the Iowa (G26R) point mutation, the first and most common amyloidogenic mutation found in apoA-I (20,21), greatly facilitates fibril formation by the N-terminal 1-83 fragment of apoA-I in solution (12) as well as on lipid membranes (18,19). Secondary structure examinations of the G26R variant of full-length apoA-I by EPR spectroscopy (21) and hydrogen-deuterium exchange MS (22) demonstrated that the G26R mutation induces widespread ␣-helix destabilization in the N-terminal helix bundle domain and promotes a transition of residues 27-56 to ␤-strand-rich structure.…”

Phosphatidylethanolamine accelerates aggregation of the amyloidogenic N‐terminal fragment of apoA‐I

“…The lipid composition of cell membranes is thought to modulate the interactions of amyloidogenic proteins with membranes. For instance, it is well known that negatively charged phosphatidylserine (PS) strongly influences the kinetics of aggregation and fibril formation of many amyloidogenic proteins including apoA-I [23,[28][29][30][31]. In contrast, with the exception of phosphatidylcholine (PC), little is known about the effects of zwitterionic phospholipids (PLs) such as phosphatidylethanolamine (PE) and sphingomyelin (SM), on the membrane-mediated aggregation and fibrillation properties of amyloidogenic proteins [32][33][34][35].…”

“…Heats of binding of apoA-I 1-83/G26R to SUV were measured with a VP-ITC instruments (Malvern) at 25 °C as described previously [24,31]. The SUV suspension was placed in the sample cell (1.33 ml) and titrated with 10 l aliquots of the apoA-I Author Manuscript equilibrium and a finite number of discrete, equivalent, and non-interacting binding sites on the lipid surface, which is mathematically equivalent to the one-site or Langmuir binding model [41][42][43].…”