Studies have revealed that upon recognition by cytotoxic T lymphocytes (CTL), apoptosis of the target cells is initiated by at least two distinct mechanisms; the Fas-Fas ligand (Fas-L) pathway, in which surface Fas-L on CTL cross-links Fas on target cells, and the granule exocytosis pathway whose major components consist of perforin, a putative pore-forming protein, and serine protease granzymes, particularly granzyme B (GrB). The DNA fragmentation into nucleosomal fragments is the most recognizable biochemical feature of apoptotic cell death that is regulated mainly by the DNA fragmentation factor (DFF) complex. DFF is composed of an inhibitor molecule (DFF-45/ICAD) tightly associated with a nuclease known as caspase-activated DNase (CAD/DFF-40/CPAN). Cleavage of the two caspase-3 specific sites on DFF45 during apoptosis was shown to result in the release of DFF-40 and the induction of DNA fragmentation. To investigate the ability of GrB to induce cell death in a caspase-independent fashion, we first analyzed the importance of putative caspase-3 cleavage sites within the DFF45 protein during apoptosis. In our study we provide direct evidence that the cleavage of DFF45 after aspartic acid 117 (D117) is both necessary and sufficient to induce DNA fragmentation through several apoptotic stimuli. Site directed mutation of D117E completely blocked oligonucleosomal DNA fragmentation without affecting other morphological and biochemical features of apoptotic cell death. / GrB shares with caspases the cleavage specificity for aspartic acid at the P1 cleavage site. Based on our observations that (i) GrB cleaves DFF45 at the same site that is necessary and sufficient for the induction of DNA fragmentation (D117) and (ii) DFF complex is constitutively localized in the nucleus, we next investigated the ability of GrB to induce DNA fragmentation in the absence of caspase activity. Using caspase-3 specific inhibitors, we show that GrB indeed can directly target DFF45 protein and induces DNA fragmentation. More importantly, we show the ability of GrB to directly cleave DFF45 and liberate CAD endonuclease activity. Taken together, these results revealed a new apoptotic pathway that provides a critical back-up system when the caspase pathway is inhibited in the target cells by viral or tumor proteins. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84841 |
| Date | January 2003 |
| Creators | Sharif-Askari, Ehsan |
| Contributors | Sekaly, Rafick-Pierre (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002141401, proquestno: AAINQ98371, Theses scanned by UMI/ProQuest. |
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