Identification of ICAD-derived peptides capable of inhibiting caspase-activated DNase.
The DNA fragmentation factor is a heterodimeric complex that consists of caspase-activated DNase (CAD) and its inhibitor (ICAD). As only partial structural information on this nuclease/inhibitor complex is available, understanding of how its subunits interact on the molecular level remains largely elusive, particularly how CAD inhibition is achieved by ICAD. In this study, we used the SPOT (peptide array) method to identify protein-protein interaction sites in the DNA fragmentation factor complex, focusing on those possibly involved in CAD inhibition. We observed a particularly strong interaction of ICAD with the dimerization (C2) domain of CAD. Additional interactions with the Zn(2+) -binding site close to the catalytic centre and the catalytic centre itself in the C3 domain of CAD were detected, suggesting that prevention of CAD homodimerization and local structural perturbation or blocking of the active site together constitute a dual inhibitory mechanism to effectively inhibit CAD. The results obtained by the SPOT method were validated by performing inhibition assays employing selected soluble ICAD-derived peptides. In these assays, two ICAD-derived peptides were identified that are capable of efficiently and specifically inhibiting CAD activity in solution.
Institute of Biochemistry, Justus-Liebig-University Giessen, Germany.
Daniel
D
Kutscher
Alfred
A
Pingoud
Albert
A
Jeltsch
Gregor
G
Meiss
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry; D001665:Binding Sites; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors*; D010455:Peptides; D040081:Protein Array Analysis; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification
D051017:Apoptosis Regulatory Proteins; D010455:Peptides; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease