Insights into the fate of the N-terminal amyloidogenic polypeptide of ApoA-I in cultured target cells.
Bottom Line: Apolipoprotein A-I (ApoA-I) is an extracellular lipid acceptor, whose role in cholesterol efflux and high-density lipoprotein formation is mediated by ATP-binding cassette transporter A1 (ABCA1).In this paper, rat cardiomyoblasts were used as target cells to analyse binding, internalization and intracellular fate of the fibrillogenic polypeptide in comparison to full-length ApoA-I.We provide evidence that the polypeptide: (i) binds to specific sites on cell membrane (K(d) = 5.90 ± 0.70 × 10(-7) M), where it partially co-localizes with ABCA1, as also described for ApoA-I; (ii) is internalized mostly by chlatrin-mediated endocytosis and lipid rafts, whereas ApoA-I is internalized preferentially by chlatrin-coated pits and macropinocytosis and (iii) is rapidly degraded by proteasome and lysosomes, whereas ApoA-I partially co-localizes with recycling endosomes.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.Show MeSH
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Mentions: As it is conceivable that the accumulation of the fibrillogenic polypeptide in the extracellular space leads to fibrils deposition, [1–93]ApoA-I fibrils were obtained by incubating the polypeptide for 2 weeks at pH 6.4 in the presence of the co-solvent TFE. AFM analysis of the incubated sample showed the presence of fibrils with height of 2.4 ± 0.1 nm and length between 0.4 and 1.5 μm (Fig. 7A). Fibrils coexist with prefibrillar aggregates, including annular protofibrils, which form a network in the image background; spheroidal aggregates of variable size (height between 3 and 15 nm) are also present.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.