Eriocalyxin B Inhibits STAT3 Signaling by Covalently Targeting STAT3 and Blocking Phosphorylation and Activation of STAT3
Fig 5
EB covalently targeted STAT3 Cys712.
(A) A549 cells were transfected with plasmids encoding the myc-tagged wild-type, C418S, C426S, C468S, C542S, C550S, C687S, C712S, or C718S mutation of STAT3 for 24 h. The cells were then treated with EB at indicated concentrations for 2 h before stimulation with IL-6 for 15 min. Whole cell lysates were processed for western blot analysis with anti-STAT3 or anti-p-STAT3 Tyr705 antibodies. The molecular weight of the exogenous myc-tagged STAT3 is 95 kDa, while the endogenous STAT3 is 87 kDa. (B) The sequence comparison of all human STAT family members showing that the cysteine at position 712 of STAT3 is unique (highlighted in gray). (C) EB (10 mM, 3.75 μL) was incubated with the STAT3 Cys712-containing peptide FT-8 (FICVTPTT) (500 μM, 75 μL) at 37°C for 2 h, and the products were resolved by mass spectrometry. The molecular weight of peptide FT-8 is 881.3 and the molecular weight of the covalent product between EB and FT-8 is 1225.5. (D) EB (10 mM, 3.75 μL) was incubated with the peptide FT-8 (FICVTPTT) (500 μM, 75 μL) at 37°C for 2 h, and the products were analyzed by MS/MS. b3, b4, b5, b6, b7 represent the fragmented EB-containing peptides. C* represents the Cys bound by EB. (E) EB (20 mM, 3.75 μL) was incubated with peptide QP-10 (QFTKCCPPKP) (500 μM, 75 μL) at 37°C for 2 h, and the products were analyzed by mass spectrometry. (F) Computational modeling of the interaction between EB and STAT3. The crystal structure of STAT3 was obtained from PDB (Protein Data Bank). Oxygen atoms of EB were shown in red. Hydrogen bonds between Gln644, Asn647, and EB were shown in purple. The predicted distance between the α, β-unsaturated carbonyl and the thiol of Cys712 is 4.3 À as indicated.