Extended Data Fig. 7: HBP can be transformed into ALPK1 activation-competent ADP-heptose 7-P by bacterial or host adenylyltransferase.
From: Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose
a, b, Requirement of ALPK1 kinase activity for cytosolic HBP-induced phosphorylation of TIFA (a) and activation of NF-κB (b). HBP was electroporated into wild-type or ALPK1−/− 293T cells expressing Flag–ALPK1 (wild-type or K/M). c, d, Enzymatically synthesized HBP could induce ALPK1-mediated NF-κB activation (c) and TIFA phosphorylation in vitro (d). S7P was reacted with recombinant GmhA or HldE or both. Following protein denaturing and precipitation, reaction supernatants were added to wild-type or ALPK1−/− 293T cells containing an empty vector or Flag–ALPK1 (c). Enzymatically synthesized HBP product (HBPenzymatic), synthetic ADP-LD-Hep or HBP was incubated with ALPK1-(N+K) in the presence of TIFA–His6 (d). e, Schematic of HldE adenylyltransferase synthesis of ADP-Hep and ADP-heptose 7-P from H1P and HBP, respectively. f, g, NF-κB activation by (f) and LC–MS of (g) enzymatically synthesized HBP product. Indicated reaction products were added to wild-type or ALPK1−/− 293T cells (f) or analysed by LC–MS (g). h, i, NF-κB (h) and in vitro ALPK1 activation (i) by ADP-heptose 7-P. HPLC-purified ADP-heptose 7-P was added to wild-type or ALPK1−/− 293T cells expressing Flag–ALPK1 (wild-type or K/M). i, Equal amounts of the indicated sugars were incubated with ALPK1-(N+K) in the presence of TIFA–His6. j, Ultra-performance liquid chromatography with tandem mass spectrometry of the reaction product of HBP and NMNAT1. Purified ADP-heptose 7-P, synthesized by reacting HBP with HldE, was used as the standard. k, Effect of NMNAT1 overexpression on activation of NF-κB by electroporation of HBP into 293T cells. The immunoblots show NMNAT1 expression. Apo-ALPK1-(N+K) was purified from E. coli BL21 (DE3) ΔhldE and phosphorylation of TIFA was assessed by anti-pT9-TIFA immunoblotting (d, i). b, c, f, h, k, NF-κB activation was assessed by luciferase reporter assay (mean ± s.d. from three technical replicates); two-tailed unpaired Student’s t-test was performed (*P < 0.05; **P < 0.01; ***P < 0.001). Data shown are representative of three (a–d, h, i, k) or two (f, g, j) independent experiments.
