Palmitoylation of human FasL modulates its cell death-inducing function

Abstract

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas-FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.

Figure 1

Metalloprotease, but not SPPL2a-mediated, FasL processing decreases FasL-induced cell death. WSU cells were pre-treated with inhibitors of either MMP or SPPL2a before co-culture with the Fas-bearing JH6.2 cells (inhibitors remained in media during the co-culture period). Effective inhibition of ADAM10 or SPPL2 was confirmed by western blot. G247 antibody was used to detect the full-length FasL and the antibody Ab-3 was employed for the detection of the N-terminal APL and SPA fragments, ezrin was used as loading control. Cell death was quantified by flow cytometric quantification of the subG1 population of propidium iodide-stained ethanol-fixed cells. (a) JH6.2 cells were co-cultured for 6 hours with WSU cells stably transfected with either hFasL or mock transfected, and were either pre-treated with the SPPL2 inhibitor (Z-LL)₂ (2 μM, 2 h) or left untreated. The graph represents the average of two independent experiments, with error bars indicating the S.D. (b) JH6.2 cells were co-cultured for 4 h with WSU cells stably transfected with hFasL or mock transfected and either pre-treated with the MMP inhibitor TAPI-2 (50 μM, 2 h) or left untreated. The graph represents the average of four independent experiments, with error bars indicating the S.D.

Figure 2

hFasL processing depends on the lipid nanodomain integrity and on the binding to Fas receptor. (a) Stable WSU-hFasL cells were treated with cholesterol oxidase (CO; 4 U/ml) for 4 h or left untreated (NT). Cells were then solubilized in Brij 98 detergent and subjected to sucrose gradient separation before analysis of the heavy fraction (HF) and the light fraction (LF) by western blot analysis. (b) Cell lysates of WSU cells stably transfected with wild-type hFasL or hFasLA247E were analyzed by western blotting. G247 antibody was used to detect the full-length FasL and the antibody Ab-3 was used to measure the N-terminal APL fragment; Ezrin was used as loading control. (c) Stable WSU hFasL cells were treated with the metalloprotease inhibitors TAPI-2 (50 μM), GI254023x (10 μM) or INCB-3619 (10 μM) for 4 h or left untreated (NT). Cells were then solubilized in Brij 98 detergent and subjected to sucrose gradient separation before western blot analysis of the heavy and light fractions (identified respectively by Rab5 and Fyn). G247 antibody was used to detect the full-length FasL and the antibody Ab-3 was used to detect the N-terminal APL fragment. Ezrin was used as loading control

Figure 3

Palmitoylation of hFasL is required for optimal hFasL processing and cytotoxic activity. (a) Diagram of the hFasL transmembrane domain, highlighting the proposed palmitoylation site at aa 82, which we mutated to serine. (b) WSU cells stably transfected with constructs encoding for hFasL, hFasLC82S or hFasLA247E were subjected to an acyl-biotinyl exchange protocol as described in Materials and Methods. Specificity of the experiment was controlled by omitting the hydroxylamine (HA) treatment and Fyn was used as an internal control for reaction efficiency. (c) JH6.2 cells were co-cultured for the indicated time points with WSU cells stably transfected with hFasL, hFasLC82S or mock. Cell death was then quantified by flow cytometric analysis of the subG1 population of propidium iodide-stained ethanol-fixed cells. The graph represents the average of four independent experiments with error bars indicating the S.D. The level of expression of wild-type hFasL or hFasLC82S at the cell surface of WSU transfected cells was comparable (see the inset). (d) Cell lysates of WSU cells stably transfected with constructs encoding for hFasL, hFasLC82S or mock transfected were analyzed by western blotting. (e) Levels of soluble FasL were measured in the supernatant of WSU cells stably transfected with wild-type hFasL or hFasLC82S. The graph represents the average of two independent experiments with error bars indicating the S.D.

Figure 4

Palmitoylation of hFasL and receptor-binding are not required for hFasL-ADAM10 interaction. (a) ADAM10 is localized both inside and outside the rafts' nanodomains. Cells were solubilized in Brij 98 detergent and subjected to sucrose gradient separation before immunoblot analysis of the heavy fraction (HF) and the light fraction (LF) to detect the different forms of hFasL and ADAM10. (b) Co-immunoprecipitation of hFasL with ADAM10. Cell lysates prepared from WSU cells stably transfected with hFasL, hFasLC82S, hFasLA247E or mock were subjected to immunoprecipitation using an anti-ADAM10 antibody and were analyzed by western blot. (c) Stable WSU-hFasL cells were treated with TAPI-2 (50 μM) or Dec-CMK (5 μM, 15 μM) for 4 hours or left untreated (NT) before cell lysates were analyzed by western blot. (d) Levels of sFasL in the supernatant of cells treated with TAPI-2 (50 μM) or Dec-CMK (15 μM) for 4 hours or left untreated (NT) were quantified by ELISA. (e) Stable WSU-hFasL cells were either treated for 4 h with TAPI-2 (50 μM) or Dec-CMK (15 μM), or left untreated (NT) before cell lysates were subjected to immunoprecipitation using an anti-ADAM10 antibody and were analyzed by western blot

Figure 5

Wild-type hFasL is confined to nanodomains whereas the palmitoylation- and receptor-deficient mutants are not. Diffusion behavior of hFasL-GFP, hFasLC82S-GFP and hFasLA247E-GFP in COS-7 cells was measured by FCS at 37°C; t₀ was determined from the position at which the diffusion curves intersect the time axis (diffusion time). Error bars indicate the S.D. of 10 independent experiments

Figure 6

Schematic representation of the ADAM10-mediated processing mechanism of hFasL. At leat two different populations of hFasL were found in our study, one being inside the rafts, the other outside the rafts. Both population can interact with ADAM10, but only the palmitoylated raft localized one might optimally interacts with the receptor and mediates Fas-cell death. Only in this population FasL can be processed; a procedure which downregulates Fas-mediated cell death on the target cell