Figure 8.
Proteolytic processing of TNFR1 by ADAM17 and γ-secretase enables cell death induction. (A) Amino acid sequence around the ADAM17 cleavage site in human and murine TNFR1. Substitution of the P1’ valine to proline in the cleavage-resistant TNFR1 V202P variant is indicated in red. (B) TNFR1−/− mEFs were retrovirally reconstituted with the indicated FLAG-hTNFR1 variants, and expression was verified by SDS-PAGE and immunoblotting using the indicated antibodies. (C) Cell surface levels of the indicated FLAG-hTNFR1 variants stably expressed in TNFR1−/− mEFs were determined by flow cytometry using anti-TNFR1 antibodies; ctrl (control) indicates unstained cells. (D) TNFR1−/− mEFs reconstituted with the indicated TNFR1 variants or ADAM17ex/ex mEFs were incubated with 100 μM zVAD 15 min before stimulation with 50 ng/ml TNF. Cells with impaired membrane integrity were detected by EthDIII uptake after 4 h. Shown are representative images and the quantification of three independent experiments with three replicates and three to four images per replicate analyzed. Scale bar indicates 50 μm. *, P < 0.05; ***, P < 0.001 by generalized estimating equation model. (E) TNFR1−/− mEFs reconstituted with the indicated TNFR1 variants were incubated with 100 μM zVAD or mock-treated 15 min before stimulation with 50 ng/ml TNF for 2 h. Cells were subsequently lysed, and lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. One representative immunoblot and the quantification of five independent experiments are shown. *, P < 0.05 by generalized estimating equation model. (F) WT or ADAM17ex/ex mEFs were incubated with 100 μM zVAD 15 min before stimulation with 1 μg/ml TNF for the indicated time points and subsequently lysed. FADD was isolated by immunoprecipitation, and coassociated proteins were identified by SDS-PAGE and immunoblotting using the indicated antibodies. One representative immunoblot and the quantification of three independent experiments are shown. Asterisk indicates Ig light chains. **, P < 0.01 by generalized estimation equation model. (G) TNFR1 ectodomain shedding by ADAM17 might be followed by intramembrane proteolysis mediated by the PSEN-containing γ-secretase complex, which would result in the liberation of a C-terminal TNFR1 fragment. (H) mEFs with the indicated genotype were incubated with 100 μM zVAD 15 min before stimulation with 50 ng/ml TNF. Cells with impaired membrane integrity were detected by EthDIII uptake after 4 h. Shown are representative images and the quantification of three independent experiments with three technical replicates per experiment and three to four images per replicate analyzed. **, P < 0.01; unpaired two-tailed Student’s t test. Data represent mean ± SEM. assoc. associated; cl. casp.8, cleaved caspase 8; IP, immunoprecipitation; rel., relative; stim., stimulated; TM, transmembrane; VP, V202P.

Proteolytic processing of TNFR1 by ADAM17 and γ-secretase enables cell death induction. (A) Amino acid sequence around the ADAM17 cleavage site in human and murine TNFR1. Substitution of the P1’ valine to proline in the cleavage-resistant TNFR1 V202P variant is indicated in red. (B) TNFR1−/− mEFs were retrovirally reconstituted with the indicated FLAG-hTNFR1 variants, and expression was verified by SDS-PAGE and immunoblotting using the indicated antibodies. (C) Cell surface levels of the indicated FLAG-hTNFR1 variants stably expressed in TNFR1−/− mEFs were determined by flow cytometry using anti-TNFR1 antibodies; ctrl (control) indicates unstained cells. (D) TNFR1−/− mEFs reconstituted with the indicated TNFR1 variants or ADAM17ex/ex mEFs were incubated with 100 μM zVAD 15 min before stimulation with 50 ng/ml TNF. Cells with impaired membrane integrity were detected by EthDIII uptake after 4 h. Shown are representative images and the quantification of three independent experiments with three replicates and three to four images per replicate analyzed. Scale bar indicates 50 μm. *, P < 0.05; ***, P < 0.001 by generalized estimating equation model. (E) TNFR1−/− mEFs reconstituted with the indicated TNFR1 variants were incubated with 100 μM zVAD or mock-treated 15 min before stimulation with 50 ng/ml TNF for 2 h. Cells were subsequently lysed, and lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. One representative immunoblot and the quantification of five independent experiments are shown. *, P < 0.05 by generalized estimating equation model. (F) WT or ADAM17ex/ex mEFs were incubated with 100 μM zVAD 15 min before stimulation with 1 μg/ml TNF for the indicated time points and subsequently lysed. FADD was isolated by immunoprecipitation, and coassociated proteins were identified by SDS-PAGE and immunoblotting using the indicated antibodies. One representative immunoblot and the quantification of three independent experiments are shown. Asterisk indicates Ig light chains. **, P < 0.01 by generalized estimation equation model. (G) TNFR1 ectodomain shedding by ADAM17 might be followed by intramembrane proteolysis mediated by the PSEN-containing γ-secretase complex, which would result in the liberation of a C-terminal TNFR1 fragment. (H) mEFs with the indicated genotype were incubated with 100 μM zVAD 15 min before stimulation with 50 ng/ml TNF. Cells with impaired membrane integrity were detected by EthDIII uptake after 4 h. Shown are representative images and the quantification of three independent experiments with three technical replicates per experiment and three to four images per replicate analyzed. **, P < 0.01; unpaired two-tailed Student’s t test. Data represent mean ± SEM. assoc. associated; cl. casp.8, cleaved caspase 8; IP, immunoprecipitation; rel., relative; stim., stimulated; TM, transmembrane; VP, V202P.

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