Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.1.1.6 (CAD)
 4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the mechanism whereby nuclear DNA fragmentation activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant human DNA fragmentation factor 45 kDa subunit (DFF45/ICAD), an inhibitor of the caspase-activated DNase DFF40/CAD, substituted for healthy cytosol in inhibiting DNA fragmentation. An antiserum against human DFF45 detected 44 and 34 kDa proteins (major and minor, respectively) in the cytosols but not in the nuclear or membrane fractions of various cultured human cells. Cytosols depleted of DFF45/ICAD by immunoadsorption had little or no inhibitor of nuclear DNA fragmentation activity and no caspase-activated DNA fragmentation activity. We conclude that immunoreactive DFF45/ICAD is the principal inhibitor of apoptotic DNase activity in the cytosol of healthy cells.
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PMID:Inhibition of apoptosis-associated DNA fragmentation activity in nonapoptotic cells: the role of DNA fragmentation factor-45 (DFF45/ICAD). 987 36

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.
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PMID:Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. 1052 51

CAD/CPAN/DFF40 is an apoptotic nuclease that is associated with the regulatory subunit ICAD/DFF in healthy cells. ICAD has two forms, ICAD-L/DFF45 and ICAD-S/DFF35, which are transcribed from a single gene by alternative splicing. They differ at the C-terminus: 70 amino acids of ICAD-L are replaced by 4 different amino acids in ICAD-S. We previously showed that both transfected and endogenous ICAD-L are nuclear; however, the localization of ICAD and CAD remains controversial and an important issue to clarify. Here we present the evidence that ICAD-L is nuclear due to the presence of an autonomous nuclear localization signal located in the C-terminal 20 amino acids. This NLS is missing from ICAD-S, which is distributed throughout the cell. We also showed that a GFP:CAD fusion protein is located in the nucleus of transfected cells.
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PMID:Differential localization of ICAD-L and ICAD-S in cells due to removal of a C-terminal NLS from ICAD-L by alternative splicing. 1069 46

ICAD / DFF is a downstream molecule of caspases, participating in nuclear DNA fragmentation during apoptosis. ICAD / DFF binds CAD / DFF40 and inhibits its DNase activity. ICAD / DFF has two alternative isoforms, long isoform (ICAD-L / DFF45) and short isoform (ICAD-S / DFF35). We have studied the presence and functional status of ICAD / DFF in human glioma cell lines. All cell lines tested expressed both ICAD-L and ICAD-S. When the cultured glioma cells were exposed to anti-Fas antibody, these isoforms were degraded prior to the fragmentation of the nuclear DNA, indicating that the ICAD / DFF expressed in cultured glioma cells was potentially functional. In primary brain tumors and normal brain tissues, there was a difference in the expression level between ICAD-L and ICAD-S. In glioblastomas, ICAD-S was more abundant than ICAD-L. In contrast, ICAD-L was more abundant than ICAD-S in medulloblastomas. The present findings suggest that primary brain tumors and normal brain constitutively express ICAD / DFF, and that there is a difference between the expression levels of ICAD-L and ICAD-S.
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PMID:Expression of ICAD-l and ICAD-S in human brain tumor and its cleavage upon activation of apoptosis by anti-Fas antibody. 1147 33

DFF45/ICAD has dual functions in the final stage of apoptosis, by acting as both a folding chaperone and a DNase inhibitor of DFF40/CAD. Here, we present the solution structure of the C-terminal domain of DFF45, which is essential for its chaperone-like activity. The structure of this domain (DFF-C) consists of four alpha helices, which are folded in a novel helix-packing arrangement. The 3D structure reveals a large cluster of negatively charged residues on the molecular surface of DFF-C. This observation suggests that charge complementation plays an important role in the interaction of DFF-C with the positively charged catalytic domain of DFF40, and thus for the chaperone activity of DFF45. The structure of DFF-C also provides a rationale for the loss of the chaperone activity in DFF35, a short isoform of DFF45. Indeed, in DFF35, the amino acid sequence is truncated in the middle of the second alpha helix constituting the structure of DFF-C, and thus both the hydrophobic core and the cluster of negative charges are disrupted.
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PMID:Solution structure of the DFF-C domain of DFF45/ICAD. A structural basis for the regulation of apoptotic DNA fragmentation. 1214 88