Mechanism of Bcl-2 and Bcl-X(L) inhibition of NLRP1 inflammasome: loop domain-dependent suppression of ATP binding and oligomerization.

NLRP1 (NLR family, pyrin domain-containing 1) is a contributor to innate immunity involved in intracellular sensing of pathogens, as well as danger signals related to cell injury. NLRP1 is one of the core components of caspase-1-activating platforms termed "inflammasomes," which are involved in proteolytic processing of interleukin-1beta (IL-1beta) and in cell death. We previously discovered that anti-apoptotic proteins Bcl-2 and Bcl-X(L) bind to and inhibit NLRP1 in cells. Using an in vitro reconstituted system employing purified recombinant proteins, we studied the mechanism by which Bcl-2 and Bcl-X(L) inhibit NLRP1. Bcl-2 and Bcl-X(L) inhibited caspase-1 activation induced by NLRP1 in a concentration-dependent manner, with K(i) approximately 10 nM. Bcl-2 and Bcl-X(L) were also determined to inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and Bcl-X(L) was demonstrated to interfere with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-X(L), which are located between the first and second alpha-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-X(L) inhibit NLRP1.