ABCMdb

PMID: 24220029

Gulshan K, Brubaker G, Wang S, Hazen SL, Smith JD
Sphingomyelin depletion impairs anionic phospholipid inward translocation and induces cholesterol efflux.
J Biol Chem. 2013 Dec 27;288(52):37166-79. doi: 10.1074/jbc.M113.512244. Epub 2013 Nov 12., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCA1 p.Trp590Ser The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Login to comment 8 ABCA1 p.Trp590Ser Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Login to comment 22 ABCA1 p.Trp590Ser The W590S Tangier mutant allele of ABCA1 retains its ability to bind lipid-free lipoprotein apoAI, but has impaired PS floppase activity and efflux of phospholipids and cholesterol to apoAI (15, 24-26). Login to comment 38 ABCA1 p.Trp590Ser We found that SM depletion by myriocin or sphingomyelinase (SMase) treatment could compensate for the defective PS floppase activity of the mutant W590S-ABCA1 isoform, restoring its cholesterol efflux activity to apoAI. Login to comment 105 ABCA1 p.Trp590Ser RESULTS Myriocin Enhances Cholesterol Efflux and Restores Efflux Activity of the W590S Abca1 Mutation-We assessed the effects of myriocin, a potent inhibitor of the first step in sphingolipid synthesis on cholesterol efflux to apoAI, a process that is mediated by ABCA1, in stably transfected HEK293 cells expressing equivalent levels of wild type (WT) murine and two mutant ABCA1 isoforms (15). Login to comment 107 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg As previously observed (15, 24-26), cholesterol efflux to apoAI from cells expressing either the W590S (PS translocation deficient) or C1477R (apoAI binding deficient) ABCA1 mutant isoforms was partially, but not completely impaired compared with the WT isoform (p b0d; 0.001 compared with control HEK and WT ABCA1 by ANOVA post test). Login to comment 108 ABCA1 p.Trp590Ser Upon treatment with 10 òe;M myriocin, cholesterol efflux was significantly increased in all four cell types, however, the net increase was greatest for the W590S mutant isoform (2.34% increase, 1.72-fold), with the wild type ABCA1 isoform having a 1.44% net increase, and the other two cell lines having b0d;1% net increases. Login to comment 109 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg Thus, myriocin treatment could overcome much of the impaired cholesterol efflux activity of the W590S Abca1 mutation, although not greatly improving efflux from the C1477R mutation. Login to comment 123 ABCA1 p.Trp590Ser We then compared the effects of depleting sphingomyelin on the cholesterol efflux activity of the WT and W590S isoforms of ABCA1 in stably transfected HEK cells. Login to comment 124 ABCA1 p.Trp590Ser Increasing levels of SMase led to increased cholesterol efflux to apoAI for both isoforms, such that at the highest dose of SMase, the W590S isoform had as much efflux activity as the WT isoform in the absence of SMase (Fig. 3G). Login to comment 125 ABCA1 p.Trp590Ser Thus, we could obtain complete restoration of the efflux activity of the ABCA1 W590S isoform FIGURE 1. Login to comment 127 ABCA1 p.Trp590Ser A, myriocin compensates for the W590S-Abca1 mutation. Login to comment 128 ABCA1 p.Trp590Ser ABCA1 p.Cys1477Arg Non-transfected HEK293 cells along with HEK cells transfected with WT-Abca1-GFP, W590S-Abca1-GFP, or C1477R-Abca1-GFP isoforms were incubated with radiolabeled cholesterol and subsequently chased with apoAI (5 òe;g/ml) for 6 h. Login to comment 142 ABCA1 p.Trp590Ser Taken together, our data strongly indicate that Sphingomyelin Depletion Impairs Phospholipid Flip 37170 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 52ߦDECEMBER 27, 2013 decreased SM, rather than increased ceramide, is responsible for the induction of cholesterol efflux, which can compensate for the impaired PS translocation in the W590S ABCA1 mutation. Login to comment 147 ABCA1 p.Trp590Ser Although WT ABCA1 expression increased cholesterol efflux to NaTC to almost 6-fold, expression of the W590S ABCA1 isoform increased efflux to NaTC by only 3.1-fold, as previously observed (15, 25). Login to comment 148 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Myriocin increased cholesterol efflux from cells expressing either ABCA1 isoforms (1.6-fold for the WT and 2.1-fold for the W590S isoform), such that the myriocin-treated W590S-expressing cells exhibited comparable efflux to NaTC as seen in the myriocin-untreated WT-ABCA1 expressing cells (see gray bars in Fig. 4B). Login to comment 149 ABCA1 p.Trp590Ser Thus, the SM depletion effect on cholesterol efflux is observed both in the absence and presence of ABCA1 expression, and can compensate for the W590S Abca1 mutation even with a non-selective acceptor. Login to comment 151 ABCA1 p.Trp590Ser Because the ABCA1 W590S mutation has defective translocation of PS to the outer leaflet of the plasma membrane (11, 24), we assessed whether SM depletion altered the translocation of phospholipids across the plasma membrane. Login to comment 154 ABCA1 p.Trp590Ser As reported earlier, the W590S-Abca1 expressing HEK cells showed decreased cell surface PS as compared with the WT isoform (15, 24, 25), only 1.3-fold higher than control HEK cells. Login to comment 173 ABCA1 p.Trp590Ser HEK cells stably transfected with either WT-Abca1-GFP or W590S-Abca1-GFP isoforms were loaded with radiolabeled cholesterol and chased with apoAI (5 òe;g/ml) for 6 h. Login to comment 180 ABCA1 p.Trp590Ser HEK293 cells or stably transfected lines with either WT or W590S isoform of Abca1 were incubated with radiolabeled cholesterol and subsequently treated with or without 10 òe;M myriocin for 12 h. Login to comment 184 ABCA1 p.Trp590Ser Here, different letters above the bars show p b0d; 0.01 by ANOVA with a Bonferroni post test. Sphingomyelin Depletion Impairs Phospholipid Flip DECEMBER 27, 2013ߦVOLUME 288ߦNUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 37171 at SEMMELWEIS UNIV OF MEDICINE on December , ment of the W590S-ABAC1 cells with myriocin, there was an almost complete restoration of cell surface PS (1.55-fold versus control HEK cells) (Fig. 5, A and B). Login to comment 188 ABCA1 p.Trp590Ser B, primary flow cytometry data showing shift in annexin V signal in HEK293, HEK-Abca1, and HEK-W590S-Abca1 cells with or without treatment with myriocin. Login to comment 290 ABCA1 p.Trp590Ser This was also indicated by our finding that myriocin increased cholesterol efflux to NaTC in HEK cells, but even more so in cells transfected with WT and particularly the W590S mutant isoform of ABCA1 (Fig. 4B). Login to comment 292 ABCA1 p.Trp590Ser In fact, ABCA1 itself also raises cell surface PS, although this activity is greatly diminished in the W590S ABCA1 mutation that leads to Tangier disease (15, 24-26). Login to comment 293 ABCA1 p.Trp590Ser The SM depletion-mediated increase in cell surface PS was sufficient to compensate for the defective PS translocation in the W590S-Abca1 expressing HEK cells, restoring cholesterol efflux to apoAI (Fig. 3G) and NaTC (Fig. 4B), providing direct proof that the ABCA1 PS floppase activity plays a role in ABCA1-mediated cholesterol efflux. Login to comment 297 ABCA1 p.Trp590Ser This increase in cell surface PS could partially restore the activity of the PS-floppase-deficient Abca1 W590S mutation (Figs. Login to comment