title: Production of biologically active IL-36 family cytokines through insertion of N-terminal caspase cleavage motifs.
abstract: Recent evidence has strongly implicated IL-36 cytokines as key initiators of inflammation in the skin barrier. IL-36 cytokines belong to the extended IL-1 family and, similar to most members of this family, are expressed as inactive precursors that require proteolytic processing for activation. Because the proteases responsible for activation of members of the IL-36 subfamily have not been reported, we have developed a method for the production of biologically active IL-36 through introduction of a caspase cleavage motif, DEVD, within the N-termini of these cytokines. Here, we show that DEVD-modified IL-36α, IL-36β and IL-36γ cytokines were highly soluble and were readily processed and activated by caspase-3. Caspase-3-processed IL-36 family cytokines exhibited robust biological activity on a range of responsive cell types, including primary keratinocytes. We also generated specific polyclonal antibodies against all three IL-36 family members through immunization with purified recombinant IL-36 cytokines. The modified forms of IL-36 described herein will be useful for production of large quantities of biologically active IL-36 for structure and function studies on these important proinflammatory cytokines.
authors.affiliation: Molecular Cell Biology Laboratory Department of Genetics The Smurfit Institute Trinity College Dublin 2 Ireland.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Clancy
authors.affiliation: Molecular Cell Biology Laboratory Department of Genetics The Smurfit Institute Trinity College Dublin 2 Ireland.
authors.forename: Conor M
authors.initials: CM
authors.lastname: Henry
authors.affiliation: Cellular Biotechnology Laboratory Saint-Petersburg State Institute of Technology Russian Federation.
authors.forename: Pavel B
authors.initials: PB
authors.lastname: Davidovich
authors.affiliation: Molecular Cell Biology Laboratory Department of Genetics The Smurfit Institute Trinity College Dublin 2 Ireland.
authors.forename: Graeme P
authors.initials: GP
authors.lastname: Sullivan
authors.affiliation: Cellular Biotechnology Laboratory Saint-Petersburg State Institute of Technology Russian Federation.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Belotcerkovskaya
authors.affiliation: Molecular Cell Biology Laboratory Department of Genetics The Smurfit Institute Trinity College Dublin 2 Ireland; Cellular Biotechnology Laboratory Saint-Petersburg State Institute of Technology Russian Federation.
authors.forename: Seamus J
authors.initials: SJ
authors.lastname: Martin
keywords: IL‐1 family; IL‐36; caspase; cell death; inflammation; protease