Functional Interaction of DFF35 and DFF45 with Caspase-activated DNA Fragmentation Nuclease DFF40

Abstract

DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.Apoptosis is fundamentally important in a variety of physiological and pathological processes. Apoptotic cells undergo an orchestrated cascade of events characterized by distinct morphological changes including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1, 2). Activation of the caspase cascade is a key molecular event in the process of apoptosis (3, 4). Apoptotic signals, including growth factor and interleukin deprivation, activation of Fas, ionizing radiation, and a series of chemicals acting as upstream signals, can convert the precursors of caspases into the active enzymes (2). Several important downstream substrates of caspase, such as gelsolin (5), p21-activated kinase-2 (PAK-2) (6), and DNA fragmentation factor 45 (DFF45) 1 (7), whose cleavages clearly induce specific well characterized steps in apoptosis, have been recently identified. The cleavage of chromatin into the nucleosomal fragments, which distinguishes apoptosis from oncosis and necrosis, is a key element in the cell death process and is believed to be mediated by Mg 2ϩ /Ca 2ϩ required and Zn 2ϩ -sensitive nuclease (8 -12).We have previously identified a triplet of nuclease proteins named NP 42-50 that causes DNA degradation in vitro when cells undergo apoptosis (13). The similarity in molecular weight and biochemical characteristics between NP 42-50 and the recently identified DFF40 led us to further investigate these molecules. DFF is a heterodimeric protein composed of DFF45 and DFF40 subunits. DFF45 has been found to be the substrate of caspase-3, and DFF40 has also been cloned and found to be a DNA fragmentation nuclease (7,14,15). Cleavage of the DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. Similar findings have also been described recently in the...

Analyses of apoptotic regulators CASP9 and DFFA at 1P36.2, reveal rare allele variants in human neuroblastoma tumours

“…In the short isoform, DFF35, the first 261 amino acids are identical. Both DFF45 and DFF35 carry two caspase-3-recognition sites (amino-acid positions 117 and 224; Sakahira et al, 1998;Gu et al, 1999). As only the fulllength DFF45 functions as a chaperone for DFF40 (Sakahira et al, 2000), amino acids 261 to 331 seem relevant to this function (Gu et al, 1999).…”

“…Both DFF45 and DFF35 carry two caspase-3-recognition sites (amino-acid positions 117 and 224; Sakahira et al, 1998;Gu et al, 1999). As only the fulllength DFF45 functions as a chaperone for DFF40 (Sakahira et al, 2000), amino acids 261 to 331 seem relevant to this function (Gu et al, 1999). Zhou et al (2001) have recently reported that the Nterminal domain (NTD) of DFF45 (residues 12 -100) is homologous to the NTD of DFF40.…”

“…DFF40 (synonymous names CPAN 'Caspase-Activated Nuclease'/CAD 'Caspase-Activated Dnase') is a DNase that triggers both DNA fragmentation and chromatin condensation . DFF45 (also called ICAD 'Inhibition of CAD') is an inhibitor of DFF40, that also functions as a chaperone for native DFF40 (Gu et al, 1999;Sakahira et al, 2000).…”

DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells

“…CDDP-induced nuclear fragmentation in NB-C201 cells expressing wildtype DFF45, whereas DFF45 (1-290), DFF45 (1-231) or Functional implication of DFF45/ICAD in neuroblastoma M Takahashi et al DFF45 (1-96) failed to induce nuclear fragmentation in response to CDDP (Figure 4c), suggesting that COOHterminal region of DFF45 is essential for the induction of nuclear fragmentation in NB-C201 cells. As described by Gu et al (1999), central domain of DFF45 (amino-acid residues 101-180) is required for the interaction with DFF40 and DFF45 can assist in the synthesis of highly active DFF40. In addition, the catalytic domain of DFF40 is located in its COOH-terminal region (aminoacid residues 290-345) (Inohara et al, 1999).…”

Caspase-dependent cleavage of nucleic acids

“…37 D1, D2, and D3 were much less efficient inhibitors of CAD activity which correlates with their low binding capacity. Gu and co-workers 39 mapped the CAD binding domain of ICAD to amino acid residues 101 ± 180, and the inhibitory domain necessary to inhibit the activity of CAD to amino acid residues 23 ± 100. Based on the three published CAD binding domains of ICAD, i.e.…”