Extended Data Fig. 12: A mutagenesis study of ICCB-19/Apt-1 binding with TRADD to inhibit RDA and activate autophagy.

a, Tradd^−/− MEFs were reconstituted with Flag-tagged WT or mutant TRADD as indicated. Expression levels of TRADD were determined by immunoblotting. b, Tradd^−/− MEFs transfected with Flag-tagged WT or indicated TRADD mutants were stimulated by TNF/5z7 for 9 h in the presence or absence of Apt-1 (10 μM). Cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. Two-tailed t-test. c, d, TRADD-N(G121A)/Apt-1 (c), and TRADD-N(G121A)/ICCB-19 (d) samples for STD-NMR analyses were prepared as that of WT TRADD in (10f) with 1 mM Apt-1 (c), 1 mM ICCB-19 (d), and 13 μM TRADD-N(G121A) in 0.5 mL of PBS in D₂O (10%). e, BIAcore SPR analysis of Apt-1 binding to TRADD-N(G121A). The kinetic profile of Apt-1 binding to TRADD-N(G121A) is shown. A series of concentrations of Apt-1 (ranging from 0.15625 to 5 μM) was used to measure the binding kinetics, with TRADD-N(G121A) immobilized on the CM5 chip. f, Tradd^−/− MEFs were reconstituted with Flag-tagged WT or indicated TRADD mutants. The expression levels of TRADD were determined by immunoblotting. g, h, Tradd^−/− MEFs reconstituted with Flag-mutant TRADD (Y16A/F18A or Y16A/I72A/R119A) were stimulated by TNF/5z7 for indicated time in the presence or absence of Apt-1 (10 μM). The cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. i, A model for mechanism by which Apt-1 targets TRADD to inhibit RDA and activate autophagy. In TNF-stimulated cells: Apt-1 binds to TRADD-N to reduce its binding with TRADD-C which stabilizes the binding of TRADD mediated by its DD in TRADD-C with the DD in TNFR1. The binding of Apt-1 with TRADD in complex I modulates the K63/M1-linked ubiquitination of RIPK1 by reducing the binding of TRADD with TRAF2/cIAP1/2 which increases the recruitment of A20 and HOIP to inhibit the activation of RIPK1 kinase. Increased retention of TRADD in complex I also decreases cytosolic availability of TRADD for the formation of complex IIa in which TRADD is known to be a key component. Treatment with Apt-1 also reduces the activation of NF-κB in TNF-stimulated cells. In autophagy pathway: TRADD normally binds to TRAF2 and cIAP1/2 homeostatically. Apt-1 can release TRAF2 and cIAP1/2 from their binding with TRADD. Released TRAF2/cIAP1/2 in turn mediates K63-linked ubiquitination of Beclin 1 to promote the formation of Vps34 complex, production of PtdIns3P, and activation of autophagy. For gel source data, see Supplementary Fig. 1.

Source data