Characterization of caspase-8L: a novel isoform of caspase-8 that behaves as an inhibitor of the caspase cascade

Caspase-8 (Fas-associating protein with death domain-like interleukin-1beta- converting Enzyme [FLICE]/MACH/Mch5) belongs to a family of cysteine proteases presumed to be the apex of the apoptotic signaling pathways. We recently reported the presence of a novel isoform of Caspase-8, named caspase-8L, generated by the alternative splicing of human Caspase-8 gene, from human peripheral blood lymphocytes by reverse transcriptase-polymerase chain reaction. We herein report a functional analysis of caspase-8L in the Fas-mediated apoptotic pathway. Caspase-8L is missing the catalytic site of Caspase-8 but retains 2 N-terminal repeats of the death-effector domain. The caspase-8L messenger RNA was detected in various tissues but not in any cell lines examined. In human peripheral blood lymphocytes, caspase-8L was strongly suggested to be expressed at the protein level. In MCF-7 cells, caspase-8L transfection itself did not affect cell viability but instead inhibited the Apoptosis induced by the cotransfection of Caspase-8 in a dominant negative manner. Moreover, Fas-mediated Apoptosis was inhibited in caspase-8L-transfected Jurkat cells, which were associated with a reduction in the Caspase-8 catalytic activity. In vitro binding assays demonstrated that caspase-8L bound to FADD (Fas-associating protein with death domain) and caspase-8a and blocked the binding of Caspase-8 to FADD. In in vivo binding assays, transfected caspase-8L bound to endogenous FADD. Thus, caspase-8L acts as an inhibitor of Caspase-8 by interfering with the binding of Caspase-8 to FADD and is involved in the regulation of Fas-mediated Apoptosis.