Functional differences of two forms of the inhibitor of caspase-activated DNase, ICAD-L, and ICAD-S.
Caspase-activated DNase (CAD) is responsible for the DNA fragmentation that occurs during apoptosis. CAD is complexed with an inhibitor of CAD (ICAD) in non-apoptotic, growing cells. Here, we report that mouse WR19L and human Jurkat T lymphoma cells express two alternative forms of ICAD, ICAD-L and ICAD-S, at similar levels. CAD was predominantly associated with ICAD-L in these cell lines. When CAD was expressed alone in Sf9 cells, it was found in insoluble fractions. However, when CAD was co-expressed with ICAD-L and ICAD-S, it was recovered as a soluble protein complexed predominantly with ICAD-L. In vitro transcription and translation of CAD cDNA did not produce a functional protein. Addition of ICAD-L but not ICAD-S to the assay mixture resulted in the synthesis of functional CAD. These results indicated that ICAD-L but not ICAD-S works as a specific chaperone for CAD, facilitating its correct folding during synthesis. Recombinant CAD, as a complex with ICAD-L, was then produced in Sf9 cells. The complex was treated with caspase 3, and CAD was purified to homogeneity. The purified CAD had DNase activity with a high specific activity.
Sakahira
H
H
Department of Genetics, Osaka University Medical School, Technology Corporation, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Enari
M
M
Nagata
S
S
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D016367:Baculoviridae / Q000235:genetics; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014407:Tumor Cells, Cultured
D051017:Apoptosis Regulatory Proteins; D018832:Molecular Chaperones; D011506:Proteins; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease