title: Sequential processing of human ProIL-1beta by caspase-1 and subsequent folding determined by a combined in vitro and in silico approach.
abstract: OBJECTIVE
  Interleukin-1beta 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 active 17kDa peptide. This activation event involves ICE cleavage at Asp27 (site 1) and Asp116 (site 2). To address the sequential processing at ICE cut sites we combined in vitro analysis and molecular modeling to investigate the sequence of molecular events.
  
  
  METHODS
  Pulse chase labeling followed by immunoprecipitation of IL-1beta in activated human monocyte lysates demonstrated sequential cutting by ICE at site 1 before site 2 in vitro. To corroborate these findings, we constructed a homology model of proIL-1beta after the crystal structure of another ICE substrate, human alpha1-antitrypsin (23% sequence identity).
  
  
  RESULTS
  Comparative modeling revealed that site 1 on proIL-1beta is accessible to ICE but site 2 is not. Molecular dynamics simulations following ICE cleavage at site 1 and removal of the 3kDa amino-terminal fragment, rendered site 2 accessible to ICE.
  
  
  CONCLUSIONS
  The close agreement between the in vitro and modeled behavior of IL-1beta support our contention that IL-1beta may be structurally related to alpha1-antitrypsin and also predicts that proIL-1beta requires sequential processing for activation. These findings may facilitate the development of novel pharmacological agents that control posttranslational proIL-1beta modification, thereby preventing excessive production of this potent inflammatory cytokine.
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus 43210, USA. swaan.1@osu.edu
authors.forename: P W
authors.initials: PW
authors.lastname: Swaan
authors.forename: D L
authors.initials: DL
authors.lastname: Knoell
authors.forename: F
authors.initials: F
authors.lastname: Helsper
authors.forename: M D
authors.initials: MD
authors.lastname: Wewers
mesh_terms: D000595:Amino Acid Sequence; D020170:Caspase 1 / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D003460:Crystallization; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D009000:Monocytes / Q000378:metabolism; D011233:Precipitin Tests; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D013462:Sulfur Radioisotopes
chemical_list: D007375:Interleukin-1; D011498:Protein Precursors; D013462:Sulfur Radioisotopes; C043830:interleukin 1 precursor; D008715:Methionine; D020170:Caspase 1