Structure/Function analysis of p55 tumor necrosis factor receptor and fas-associated death domain. Effect on necrosis in L929sA cells.
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.
Boone
E
E
Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, 9000 Ghent, Belgium.
Vanden Berghe
T
T
Van Loo
G
G
De Wilde
G
G
De Wael
N
N
Vercammen
D
D
Fiers
W
W
Haegeman
G
G
Vandenabeele
P
P
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D009336:Necrosis; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D015843:Serpins / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014764:Viral Proteins
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones; D015703:Antigens, CD; D002352:Carrier Proteins; C505533:FADD protein, human; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C074641:interleukin-1beta-converting enzyme inhibitor