Extended Data Fig. 5: STING associated ADSL-generated fumarate competes with cGAMP for binding to STING.
From: ADSL-generated fumarate binds and inhibits STING to promote tumour immune evasion
(a) FLAG/strep-STING was purified from BT-549 cells was incubated with 0.2 μCi of 14C-α -KG, 14C-fumarate, 14C-succinate, for 30 min and relative radiometric metabolite-protein binding was detected by scintillation counting (n = 4 biological experiments). (b) Specific frame of binding modes of STING in complex Fumarate, α-KG and Succinate in 250 ns simulations. Fumarate, α-KG and Succinate are shown in yellow, cyan and pink respectively. (c) Evolution of the backbone RMSD of the STING-ligand complex from the initial frame in 100 ns MD simulations. RMSD of the Fumarate, α-KG and are shown in green, purple and blue respectively. (d) Parental BT-549 and MDA-MB-231 cells were treated with biotin-cGAMP (0.1 μg/mL) in the presence of 5 μM and 20 μM α-KG, fumarate and succinate respectively for 6 h. Total lysates were harvested for immunoprecipitation and immunoblotting analyses as indicated. (e) Purified His-STING CBD was incubated with 0.2 μCi 32P cGAMP in the presence of 10 μM and 50 μM α-KG, fumarate and succinate respectively. Relative cGAMP bound to STING was detected by scintillation counting (n = 4 biological experiments). (f) GST-ADSL WT and A291V was incubated with purified His-STING CBD and beads coupled with cGAMP in the presence of 0.1 mM and 0.5 mM S-AMP respectively. Pulldown assay and immunoblotting analyses were performed. (g) GST-ADSL WT and A291V was incubated with purified His-STING CBD and beads coupled with 0.2 μCi 32P cGAMP in the presence of 0.1 mM and 0.5 mM S-AMP respectively. Relative cGAMP bound to STING was detected by scintillation counting (n = 4 biological experiments). (h) Cellular thermal shift assay using BT-549 and MDA-MB-231 cells expressing ADSL shRNA with reconstituted expression of the indicated ADSL protein (left). STING in the soluble fraction was quantified by densitometric analysis of the blots (n = 3 biological experiments) (right). All data are presented as mean ± SD; Statistical significance was determined by two-tailed Student’s t-test. Experiments were repeated three times (d and f) with similar results.
