Extended Data Fig. 6: ADSL-generated fumarate binds to STING and competitively inhibits cGAMP-STING interaction in a STING succination and BB-Cl-amidine independent manner.
From: ADSL-generated fumarate binds and inhibits STING to promote tumour immune evasion
Immunoprecipitation and immunoblotting assays were performed with the indicated antibodies, representative results from three independent experiments are shown. (a) ITC assays were performed with recombinant His-STING CBD WT or T263A and fumarate. (b) Kinetic binding curves for interaction between STING WT (upper) or T263A (lower) and fumarate were detected by BLI assay. (c, d) Molecular docking of STING (dimer) with cGAMP shows the potential interaction of STING T263 with cGAMP and fumarate. (e) Purified His-STING CBD WT and T263A was incubated with 0.2 μCi 32P cGAMP. Relative cGAMP bound to STING was detected by scintillation counting (n = 4 biological experiments). (f) Cellular thermal shift assay was performed using BT-549 or MDA-MB-231 cells transfected with Flag-STING WT, Flag-STING T263A or Flag-STING Y167F plasmids (upper). Flag-STING in the soluble fraction was quantified by densitometric analysis of the blots (n = 3 biological experiments) (lower). (g) The thermal stability of STING WT, T263A or Y167F were detected by thermal shift assay. RFU, relative fluorescence units. The data were shown as the mean values of RFU (n = 3 biological experiments). (h) Parental MDA-MB-231 cells and the indicated clones with knock-in expression of T350A, E481/L482A, and A291V mutants were treated with or without hypoxia. (i) MDA-MB-231 cells transfected with the indicated plasmids were treated with increased amount of methyl-fumarate. (j) Purified His-STING CBD and His-PTEN were treated with increased amount of fumarate. A Ni-NTA pulldown assay was conducted. (k) MDA-MB-231 cells expressing ADSL shRNA and STING shRNA with reconstituted expression of the indicated ADSL or STING protein were treated with or without 0.25 μg/ml HT-DNA. (l) MDA-MB-231 cells expressing ADSL shRNA and STING shRNA with reconstituted expression of the indicated ADSL or STING protein were treated with or without BB-Cl in the presence of Biotin-cGAMP. A strepadvinadin pulldown assay was conducted. (m) Purified His-STING CBD WT and C148S were treated with or without fumarate and BB-Cl in the presence of Biotin-cGAMP. A strepadvinadin pulldown assay was conducted. All data are presented as mean ± SD; Statistical significance was determined by two-tailed Student’s t-test.
