Cloning and expression of four novel isoforms of human interleukin-1β converting enzyme with different apoptotic activities

Abstract

To understand the mechanism of interleukin-1β converting enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE mRNA in two human cell lines by reverse transcription-polymerase chain reaction technique. This resulted in the identification and cloning of four alternatively spliced ICE mRNA isoforms. Although all the alternative splicing events were within the coding sequence of ICE, the four ICE isoforms maintained open reading frames and were designated as ICEβ, γ, δ, and ε. In ICEγ, most of the propeptide (amino acids 20-112) is deleted, which suggests that it may function as a catalyst for ICE autoprocessing in vivo. In ICEδ, amino acids 288-335, which contain the cleavage sites between the p20 and p10 subunits of ICE, are deleted thus resulting in its inactivation. Intriguingly, in ICEε amino acids 20-335, which encompass most of the propeptide and the p20 subunit, are deleted resulting in the formation of a molecule that is homologous to the p10 subunit. Examination of the ability of these four ICE isoforms to cause apoptosis revealed that only the parental ICEs and isoforms β and γ, but not isoforms δ and ε, can induce apoptosis when overexpressed in Sf9 insect cells. In addition, coexpression of the p20 and p10 but not the p20 and ICEε in Sf9 cells results in apoptosis. Interestingly, expression of ICEε and to a lesser degree ICEδ resulted in extension of the survival of baculovirus-infected cells in a manner similar to expression of BCL2. The ability of ICEε to extend the survival of Sf9 cells suggests that baculovirus-induced apoptosis in these cells is mediated by an ICE-like protease. We show that ICEε can bind to the p20 subunit of ICE and potentially may compete with the p10 subunit to form an inactive ICE complex. Therefore, by acting as a dominant inhibitor of ICE activity, ICEε may regulate ICE activation in vivo.