Figure 2. Effect of caspase 3 and 7 on proteolytic maturation of _pIL-33 and ST2-interacting _mIL-33 forms.

A. Immunoblot analysis of recombinant IL-33 (100 ng) incubated for 2 hours with control medium or 10, 30, or 100 units of recombinant human active caspases 3, 7 or 8 (100 units). B. Immunoblot analysis of necrotic supernatants of TE-7 cells overexpressing _pIL-33 (1–270). Cells were pre-incubated with control medium or Poly (I:C) (10 nM) for 8 hours. Control supernatants were incubated with medium or 100 units of recombinant human active caspases 3, 7, or 8 for 2 hours. C. Immunoblot analysis of TE-7 cells overexpressing wildtype (1–270), single-point mutated (D175N; D178N), and double-point mutated (D175N/D178N) _pIL-33. Necrotic supernatants were incubated with control medium or 100 units of recombinant human active caspases 3 or 7 for 2 hours. D. X-ray complex structure of ST2 receptor (S117-S268; PDB ID: 4KC3) juxtaposed with the NMR-based structure of IL-33 (S111-T270; PDB ID: 2KLL) showing IL-33 minimal binding domain interacting with ST2 and the non-interacting IL-33 flexible loop with a.a. D175 and D178 (yellow); D179-T270 is posterior to the IL-33 and is not interacting with ST2 receptor binding interface (grey). E. Schematic representation of (D) inclusive of the M1-H109 and caspase cleavage site (D175, D178) as determined by MS/tandem LC-MS. F. Immunoblot analysis of coimmunoprecipitation of IL-33 and ST2: TE-7 cells overexpressing _pIL-33 (1–270) were incubated with Poly (I:C) (10 nM) for 8 hours and lysed. Lysate input control (left) and co-immunoprecipitation of IL-33 with ST2-Fc (middle) or normal goat IgG (right) are shown. A-C, F. Data are representative of n = 3 independent experiments. Left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (cl, cleaved; fl, full length; m, mature; p, precursor).