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Insights into the fate of the N-terminal amyloidogenic polypeptide of ApoA-I in cultured target cells.

Arciello A, De Marco N, Del Giudice R, Guglielmi F, Pucci P, Relini A, Monti DM, Piccoli R - J. Cell. Mol. Med. (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.

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ABCA1 expression and co-localization with [1–93]ApoA-I and ApoA-I. (A) Western blot analysis with anti-ABCA1 antibodies of cell lysates prepared from HepG2 cells (25 μg total proteins, lane 1) and from H9c2 cells (50 μg, lane 2). Immunostaining for ABCA1 (green) of HepG2 cells (B) and H9c2 cells (C). Nuclei were stained with Hoechst (blue). (D) and (E), co-localization of [1–93]ApoA-I and ApoA-I with ABCA1. H9c2 cells were incubated for 2 hrs either with 3 μM rhodamine-[1–93]ApoA-I (D), or with 1 μM rhodamine-ApoA-I (E), and immunostained for ABCA1 (green). Nuclei were stained with Hoechst (blue). Cells were observed by confocal microscopy.
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fig03: ABCA1 expression and co-localization with [1–93]ApoA-I and ApoA-I. (A) Western blot analysis with anti-ABCA1 antibodies of cell lysates prepared from HepG2 cells (25 μg total proteins, lane 1) and from H9c2 cells (50 μg, lane 2). Immunostaining for ABCA1 (green) of HepG2 cells (B) and H9c2 cells (C). Nuclei were stained with Hoechst (blue). (D) and (E), co-localization of [1–93]ApoA-I and ApoA-I with ABCA1. H9c2 cells were incubated for 2 hrs either with 3 μM rhodamine-[1–93]ApoA-I (D), or with 1 μM rhodamine-ApoA-I (E), and immunostained for ABCA1 (green). Nuclei were stained with Hoechst (blue). Cells were observed by confocal microscopy.

Mentions: It is known that the plasma membrane is the main platform where lipidation of ApoA-I occurs [4], mediated by the ATP-binding cassette transporter A1 (ABCA1). This allows cellular free cholesterol and phospholipids to be transferred to ApoA-I leading to the biogenesis of nascent HDL. It has been demonstrated that ApoA-I is internalized in an ABCA1-dependent manner, because no internalization was observed in cells ABCA1–/–[20]. To test whether ABCA1 is expressed in cardiomyoblasts, cell lysates were analysed by Western blotting with anti-ABCA1 antibodies. HepG2 lysates were used as a positive control, as high levels of this transporter have been found in the liver. As shown in Fig. 3A, an immuno-positive species, with an apparent molecular mass corresponding to that expected for ABCA1 (about 210 kDa), was present in cell extracts prepared from H9c2 (lane 2) and HepG2 (lane 1) cells. These results were confirmed by immuno-fluorescence analyses of H9c2 and HepG2 cells with anti-ABCA1 antibodies, which revealed immuno-positive signals in both cell lines (Fig. 3B and C). We observed that in H9c2 cells ABCA1 is localized both on the plasma membrane and in intracellular compartments, whereas in HepG2 cells it is mainly located on the plasma membrane. Although data reported in the literature indicate that the transporter is mostly localized on cell plasma membrane [21, 22], consistently with its role in cellular lipid efflux, ABCA1 has been also observed in intracellular compartments [5, 23, 24].


Insights into the fate of the N-terminal amyloidogenic polypeptide of ApoA-I in cultured target cells.

Arciello A, De Marco N, Del Giudice R, Guglielmi F, Pucci P, Relini A, Monti DM, Piccoli R - J. Cell. Mol. Med. (2011)

ABCA1 expression and co-localization with [1–93]ApoA-I and ApoA-I. (A) Western blot analysis with anti-ABCA1 antibodies of cell lysates prepared from HepG2 cells (25 μg total proteins, lane 1) and from H9c2 cells (50 μg, lane 2). Immunostaining for ABCA1 (green) of HepG2 cells (B) and H9c2 cells (C). Nuclei were stained with Hoechst (blue). (D) and (E), co-localization of [1–93]ApoA-I and ApoA-I with ABCA1. H9c2 cells were incubated for 2 hrs either with 3 μM rhodamine-[1–93]ApoA-I (D), or with 1 μM rhodamine-ApoA-I (E), and immunostained for ABCA1 (green). Nuclei were stained with Hoechst (blue). Cells were observed by confocal microscopy.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373434&req=5

fig03: ABCA1 expression and co-localization with [1–93]ApoA-I and ApoA-I. (A) Western blot analysis with anti-ABCA1 antibodies of cell lysates prepared from HepG2 cells (25 μg total proteins, lane 1) and from H9c2 cells (50 μg, lane 2). Immunostaining for ABCA1 (green) of HepG2 cells (B) and H9c2 cells (C). Nuclei were stained with Hoechst (blue). (D) and (E), co-localization of [1–93]ApoA-I and ApoA-I with ABCA1. H9c2 cells were incubated for 2 hrs either with 3 μM rhodamine-[1–93]ApoA-I (D), or with 1 μM rhodamine-ApoA-I (E), and immunostained for ABCA1 (green). Nuclei were stained with Hoechst (blue). Cells were observed by confocal microscopy.
Mentions: It is known that the plasma membrane is the main platform where lipidation of ApoA-I occurs [4], mediated by the ATP-binding cassette transporter A1 (ABCA1). This allows cellular free cholesterol and phospholipids to be transferred to ApoA-I leading to the biogenesis of nascent HDL. It has been demonstrated that ApoA-I is internalized in an ABCA1-dependent manner, because no internalization was observed in cells ABCA1–/–[20]. To test whether ABCA1 is expressed in cardiomyoblasts, cell lysates were analysed by Western blotting with anti-ABCA1 antibodies. HepG2 lysates were used as a positive control, as high levels of this transporter have been found in the liver. As shown in Fig. 3A, an immuno-positive species, with an apparent molecular mass corresponding to that expected for ABCA1 (about 210 kDa), was present in cell extracts prepared from H9c2 (lane 2) and HepG2 (lane 1) cells. These results were confirmed by immuno-fluorescence analyses of H9c2 and HepG2 cells with anti-ABCA1 antibodies, which revealed immuno-positive signals in both cell lines (Fig. 3B and C). We observed that in H9c2 cells ABCA1 is localized both on the plasma membrane and in intracellular compartments, whereas in HepG2 cells it is mainly located on the plasma membrane. Although data reported in the literature indicate that the transporter is mostly localized on cell plasma membrane [21, 22], consistently with its role in cellular lipid efflux, ABCA1 has been also observed in intracellular compartments [5, 23, 24].

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.

Show MeSH
Related in: MedlinePlus