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-1β (IL-1β) 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 $ ≈ 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 BCI-$X_L $, which are located between the first and second α-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-$X_L $ inhibit NLRP1.