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).Heart amyloid fibrils were found to be mainly constituted by the 93-residue N-terminal fragment of ApoA-I, named [1-93]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: Next, we analysed the fate of the internalized polypeptide in cardiomyoblasts. After a prolonged exposure to FITC-[1–93]ApoA-I (24 hrs), the complete disappearance of intracellular fluorescent signals associated to the polypeptide was observed (Fig. 6A and C), suggestive of polypeptide massive degradation. To investigate the degradation pathway, we used specific inhibitors of proteasomal and lysosomal activities. When cells were pre-incubated with the proteasome inhibitor MG132, we observed the persistence of [1–93]ApoA-I intracellular fluorescence at 24 hrs (Fig. 6B), indicative of proteasome involvement in [1–93]ApoA-I degradation. To test whether [1–93]ApoA-I is targeted to lysosomes, we incubated cells with FITC-[1–93]ApoA-I in the presence of ammonium chloride, an inhibitor of intralysosomal catabolism. Following incubation, lysosomes were labelled with LysoTracker red. As shown in Fig. 6D, a strong fluorescence signal associated to the polypeptide (green) was found to co-localize with lysosomes (red), suggesting that lysosomes play a role in [1–93]ApoA-I catabolism. Different results were obtained instead with full-length ApoA-I, as even at 24 hrs incubation with FITC-ApoA-I, the persistency of protein fluorescence within the cells was observed (Fig. 6E and G). This is in agreement with recent reports indicating that in different cell types ApoA-I is not significantly degraded . Moreover, as shown in Fig. 6G, ApoA-I was found to co-localize with lysosomes, in line with reports indicating that lysosomes are an intracellular station of ApoA-I [3, 4]. In the presence of either MG132 or ammonium chloride, fluorescence signals associated to ApoA-I did not significantly increase with respect to cells untreated with the inhibitors, as shown in Fig. 6F and H, respectively. Furthermore, by using different inhibitors, such as ALLN for the proteasome, and chloroquine for the lysosomes, we confirmed the data reported above (data not shown). Taken together, our results indicate that the inhibition of lysosomal or proteasomal activity does not significantly alter the amount of intracellular ApoA-I, whereas both pathways seem to be involved in the degradation of the fibrillogenic polypeptide.
Affiliation: Department of Structural and Functional Biology, University of Naples Federico II, School of Biotechnological Sciences, Naples, Italy.