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: To learn about the mechanism of [1–93]ApoA-I uptake in cardiomyoblasts, we analysed different routes of endocytosis. First, the involvement of chlathrin-coated pits was evaluated using Rab5 as a marker, as this protein regulates vesicular transport from the plasma membrane to the endosomes. We transiently expressed Rab5 fused to the RFP in H9c2 cells. Twenty-four hours after transfection, cells were incubated with the FITC protein under test for 6 hrs at 37°C, to allow internalization. As indicated in Fig. 4A, a significant, albeit partial, co-localization of internalized [1–93]ApoA-I (green) with RFP-Rab5 (red) was observed, indicating that a fraction of the internalized polypeptide is associated to early endosomes. Similar results were obtained when ApoA-I was tested (Fig. 4B), in line with recent findings [4, 5]. Additional experiments were performed with labelled transferrin (FITC-Tf), as a marker of the endocytic pathway. For both proteins we confirmed the results obtained with Rab5, because after 6 hrs incubation partial co-localization with FITC-Tf was observed (data not shown). To further confirm that both [1–93]ApoA-I and ApoA-I are internalized in H9c2 cells by chlatrin-mediated endocytosis, we used specific inhibitors of this internalization pathway, such as MDC and sucrose. Upon pre-incubation of H9c2 cells with either MDC or sucrose, we observed that the amount of internalized polypeptide, and that of the full-length protein, appeared to be reduced (data not shown). Hence, endocytosis of both proteins is slowed down, although not fully blocked, by inhibitors of chlatrin-mediated endocytosis. This indicates either that in our experimental conditions the endocytic pathway still functions, albeit less efficiently, or that endocytosis of [1–93]ApoA-I and ApoA-I does not occur solely via chlathrin-coated pits.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.