Sequential processing of human ProIL-1 beta by caspase-1 and subsequent folding determined by a combined in vitro and in silico approach

Purpose. Interleukin-lp is a multifunctional cytokine produced by activated monocytes and macrophages that requires caspase-1 (IL-1 converting enzyme/ ICE) to process the 31kDa inactive precursor protein to the biologically ac tive 17kDa peptide. This activation event involves ICE cleavage at Asp(27) (site 1) and Asp(116) (site 2). To address the sequential processing at ICE cut sites we combined in vitro analysis and molecular modeling to investig ate the sequence of molecular events. Methods. Pulse chase labeling followed by immunoprecipitation of IL-1 beta in activated human monocyte lysates demonstrated sequential cutting by ICE at site I before site 2 in vitro. To corroborate these findings, we constru cted a homology model of proIL-1 beta after the crystal structure of anothe r ICE substrate, human al-antitrypsin (23% sequence identity). Results. Comparative modeling revealed that site I on proIL-1 beta is acces sible to ICE but site 2 is not. Molecular dynamics simulations following IC E cleavage at site 1 and removal of the 3kDa aminoterminal fragment, render ed site 2 accessible to ICE. Conclusions. The close agreement between the in vitro and modeled behavior of IL-1 beta support our contention that IL-1 beta may be structurally rela ted to al-antitrypsin and also predicts that proIL-1 beta requires sequenti al processing for activation. These findings may facilitate the development of novel pharmacological agents that control posttranslational proIL-1 bet a modification, thereby preventing excessive production of this potent infl ammatory cytokine.