Identification of a caspase-2 isoform that behaves as an endogenous inhibitor of the caspase cascade.
Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli. Two isoforms of human procaspase-2 have been described initially. Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways. In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2s-Pro. The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last alpha-helix of their caspase recruitment domains was deleted. Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines. Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system. In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation. In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-alpha) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade. These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant. Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.
Droin
N
N
Research Centre of the University of Montreal Hospital Centre, Notre Dame Hospital, Montreal Cancer Institute, Quebec, Canada.
Beauchemin
M
M
Solary
E
E
Bertrand
R
R
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D053143:Caspase 2; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D016923:Cell Death; D002460:Cell Line; D002474:Cell-Free System; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008223:Lymphoma / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D019384:Nucleic Acid Synthesis Inhibitors; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D005047:Etoposide; D053143:Caspase 2; D020169:Caspases