Microenvironmentally controlled secondary structure motifs of apolipoprotein A-I derived peptides

Abstract

The structure of apolipoprotein A-I (apoA-I), the major protein of HDL, has been extensively studied in past years. Nevertheless, its corresponding three-dimensional structure has been difficult to obtain due to the frequent conformational changes observed depending on the microenvironment. Although the function of each helical segment of this protein remains unclear, it has been observed that the apoA-I amino (N) and carboxy-end (C) domains are directly involved in receptor-recognition, processes that determine the diameter for HDL particles. In addition, it has been observed that the high structural plasticity of these segments might be related to several amyloidogenic processes. In this work, we studied a series of peptides derived from the N- and C-terminal domains representing the most hydrophobic segments of apoA-I. Measurements carried out using circular dichroism in all tested peptides evidenced that the lipid environment promotes the formation of α-helical structures, whereas an aqueous environment facilitates a strong tendency to adopt β-sheet/disordered conformations. Electron microscopy observations showed the formation of amyloid-like structures similar to those found in other well-defined amyloidogenic proteins. Interestingly, when the apoA-I peptides were incubated under conditions that promote stable globular structures, two of the peptides studied were cytotoxic to microglia and mouse macrophage cells. Our findings provide an insight into the physicochemical properties of key segments contained in apoA-I which may be implicated in disorder-to-order transitions that in turn maintain the delicate equilibrium between both, native and abnormal conformations, and therefore control its propensity to become involved in pathological processes.

Fig. 1

Disorder and aggregation predictions for apoA-I. a The disorder profiles were created with PONDR-FIT and the hydrophobic clusters predicted using the HCA server. Propensities to disorder for DRV^(9–24) and VLES^(221–239) overlapped with the apoA-I disorder profile. b Aggregation profiles of apoA-I were obtained using Zyggregator and prediction of propensity to generate amyloid fibrils and globular structures using Zagg propensity and Ztox propensity respectively

Fig. 2

Far-UV CD spectra of apoA-I derived peptides. a–c Spectra of peptide DRV^(9–24), KLL^(45–63), and VLES^(221–239) incubated for various times in water. d–f CD spectra of peptides DRV^(9–24), KLL^(45–63), and VLES^(221–239) incubated for 24 h in 40 % TFE and 20 mM Lyso-C₁₂PC

Fig. 3

TEM and AFM micrographs of peptides DRV^(9–24), KLL^(45–63), and VLES^(221–239). Evolution of peptide aggregation in water was evaluated by electron and atomic force microscopies on peptides VLES^(221–239) (A–F), DRV^(9–24) (G–L), and KLL^(45–63) (M–R). Incubation times correspond to: 0 h (A, G, M), 24 h (B, H, N), 48 h (C, I, O), 72 h (D, J, P), 92 h (E, K, Q), and 120 days (F, L, R). Arrows (A) show protofibrillar structures. Insets correspond to AFM images of samples used in CD experiments and the scale correspond to 1 µm

Fig. 4

Cytotoxic effects of apoA-I derived peptides. MTT reductions experiments performed with macrophage (A) and microglial cells (B). Aa-d Optical microscopy images of macrophages treated for 24 h with peptides previously incubated at 4 °C in water for 120 days (46 µg/mL). Ba-d) Optical microscopy images of microglial cells treated for 24 h with peptides previously incubated at 4 °C in water for 120 days (93 µg/mL). a Control without peptides, b DRV^(9–24), c KLL^(45–63), and d VLES^(221–239)

Fig. 5

ApoA-I aggregation properties. Lipid-poor apoA-I interacts with the ABCA1 receptor and produce discoidal structures following a process still under active investigation. Discoidal particles are transformed into spherical HDLs by the action of the LCAT enzyme [39]. Only the spherical forms of HDL can interact with the SRB1 receptor. Modified apoA-I can not interact properly with the ABCA1 receptor forming smaller abnormal discoidal HDLs [40]. A direct consequence of this condition is the exposure of highly unstructured apoA-I segments prone to enzymatic hydrolysis [41]. Peptides released by proteolysis might form amyloidogenic structures that can be organized first as micelle-like peptides that can evolve to form globular or protofibrillar structures depending on their residue composition [42, 47]. Modified from [33]