Figure 5. The B-box regulates the activity of pyrin.

(A) Schematic representations of the domain structure of pyrin and Trim5α, and the chimeric pyrin-Trim5α mutants used in B, C and F below.

(B) Western blots showing caspase-1, PSTPIP1 and pyrin or chimeric pyrin mutants in 293-caspase-1-ASC cells 28h after transfection with an empty vector (1^st lane), or co-transfection with the indicated pyrin or chimeric pyrin-Trim5α mutants plus an empty vector (vec) or PSTPIP1-A230T mutant plasmid (A230).

(C) Percentages of ASC pyroptosomes in 293-ASC-EGFP-N1 cells similarly transfected as in B (mean ± SD; n = 5).

(D-F) Pyrin or pyrin mutants were immunoprecipitated (IP) from HEK293 cell lysates 24h after transfection with the indicated plasmids for full-length (FL) pyrin or pyrin mutants plus the indicated Flag-tagged PSTPIP1 plasmids. The lysates and IPs were fractionated by SDS-PAGE and immunoblotted (IB) with anti-pyrin or anti-Flag (detects PSTPIP1) antibodies.

(G) The PYD of pyrin interacts with its B-box. GST or GST-PYD fusion protein (pyrin PYD residues 1-100) were incubated with ³⁵S-labeled pyrin-LN-BB-CC (left panels) or pyrin-LN-ΔBB-CC (right panels) in the absence (2^nd and 3^rd lanes) or the presence of dIAP as a non-specific control (4^th lane) or PSTPIP1 A230T (5^th lane). The bound proteins were fractionated by SDS-PAGE and detected by autoradiography (top panels). The corresponding immobilized GST and GST-PYD proteins are shown in the lower panels.