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Structure/Function analysis of p55 tumor necrosis factor receptor and fas-associated death domain. Effect on necrosis
E Boone1, T Vanden Berghe, G Van Loo
1Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, 9000 Ghent, Belgium.
Abstract:
Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.
Insights
Tumor necrosis factor (TNF) triggers cell death via its receptor (TNF-R55). The receptor's death domain (DD) initiates necrosis in L929sA cells, independent of FADD's death effector domain, revealing an active necrotic pathway.
Area of Science:
- Cellular biology
- Molecular mechanisms of cell death
Background:
- Tumor necrosis factor (TNF) binding to p55 TNF receptor (TNF-R55) typically induces apoptosis.
- Fibrosarcoma cell line L929sA exhibits necrosis upon TNF triggering.
- The intracellular domain of TNF-R55 includes a death domain (DD) crucial for apoptosis.
Purpose of the Study:
- To investigate the role of the TNF-R55 death domain (DD) in TNF-induced necrosis.
- To elucidate the mechanisms underlying TNF-R55-mediated necrosis in L929sA cells.
Main Methods:
- Structure/function analysis of TNF-R55 mutants in L929sA cells.
- Assessment of cell death characteristics: swelling, membrane permeabilization, nuclear condensation, DNA hypoploidy, and reactive oxygen intermediates.
- Evaluation of synergistic effects with caspase inhibitors and dominant-negative FADD mutants.
Main Results:
- TNF-R55-mediated necrosis initiation is confined to the death domain (DD).
- Necrosis involves cell swelling, membrane permeabilization, and mitochondrial reactive oxygen intermediates, without nuclear condensation or DNA hypoploidy.
- Caspase inhibitors synergize with DD-containing receptors in necrosis.
- Fas-associated death domain (FADD) deletion mutants lacking the death effector domain do not inhibit necrosis.
Conclusions:
- The death domain (DD) of TNF-R55 is necessary and sufficient for initiating necrotic cell death.
- The death domain of FADD can independently elicit an active necrotic cell death pathway.