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.
Himeji
Daisuke
D
Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Japan.
Horiuchi
Takahiko
T
Tsukamoto
Hiroshi
H
Hayashi
Kenshi
K
Watanabe
Takeshi
T
Harada
Mine
M
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms; D019556:COS Cells; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000201:enzymology / Q000276:immunology; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology
D061945:Caspase Inhibitors; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases