Igor Mett

Targeted Disruption of the Mouse Caspase 8 Gene Ablates Cell Death Induction by the TNF Receptors, Fas/Apo1, and DR3 and Is Lethal Prenatally

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

A protein related to a proteasomal subunit binds to the intracellular domain of the p55 TNF receptor upstream to its ‘death domain’

A novel protein that binds specifically to the intracellular domain of the p55 tumor necrosis factor (TNF) receptor was cloned by two‐hybrid screening of a HeLa cell cDNA library. Data bank searches revealed high sequence similarity of the protein (55.11) to yeast, nematode and plant proteins, whose functions are yet unknown. Significant similarity was also found between 55.11 and SEN3, the yeast equivalent of the p112 subunit of the 26S proteasome. Deletion analysis showed that the protein binds to the p55 receptor upstream to the region involved in induction of cell death.

THE YEAST TWO-HYBRID SCREENING TECHNIQUE AND ITS USE IN THE STUDY OF PROTEIN-PROTEIN INTERACTIONS IN APOPTOSIS

The yeast two-hybrid technique provides a general approach for cloning cDNAs merely by exploiting the ability of their encoded proteins to bind to a protein of interest. The technique therefore offered a useful access to the analysis of the mechanisms of cell death at the initial stage of their study, when only a few of the proteins involved and very little about their mode of action were known. Conversely, the knowledge of cell death mechanisms gained by this technique provided a useful insight into both the potential and the limitations of this technique.