Molecular Analysis of the TRAIL DISC and its signalling pathways

The TNF-receptor family plays a decisive role during the ontogenesis and homeostasis of multicellular organisms. This importance is apparent by the multitude of diseases which are caused by defects in either the ligands, receptors, or the associated signal transduction processes of the TNF family.The receptors of the TNF family exert their effects mainly via two groups of intracellu-lar signal transduction pathways. Stimulation of the receptors of the TNF family in many cases leads to changes in gene expression. One important pathway in this process is the activation of the NF-kB signalling cascade. The second, equally important path-way utilized by members of the TNF family is the triggering of the caspase cascade. This pathway is utilized mainly by a subgroup of the TNF-receptor family, the death receptors. The death receptors are characterized by the presence of an intracellular protein domain, the death domain (DD). This domain is necessary for direct coupling of the death receptors to activation of the caspase cascade. Caspase activation occurs in a protein complex termed DISC (death inducing signalling complex). Amongst the death receptors, the TRAIL system stands out due to its complex receptor system. This consists of two death-receptors, TRAIL-R1 and TRAIL-R2 as well as two receptors con-taining no or a truncated death domain, TRAIL-R3 and R4, respectively. The cytokine TRAIL is of special interest due to its ability to kill tumor cells while normal cells are resistant to TRAIL. These properties make TRAIL a promising drug candidate for tumor therapy. Yet, preceding the clinical development, it is obligatory to better under-stand the molecular basis of the action and signalling of TRAIL. This knowledge will help to evaluate possible risks as well as fields of application.At the beginning of this work, the composition of the TRAIL-DISC was controversially discussed. This was caused by the protein overexpression utilized in previous experiments.In this work, a method was devised to isolate the native TRAIL-DISC for the first time. Using this method it was shown that the composition of the TRAIL-DISC identical to the CD95-DISC, regarding the already known components. It was shown that the initiator caspase, caspase-8 as well as the adaptor protein FADD are components of the TRAIL-DISC. Using cell lines deficient in either one of these two proteins it could further be shown that these two proteins are essential for normal apoptosis induction by TRAIL. It could further be shown that no qualitative difference exists between the TRAIL-R1 and TRAIL-R2 DISC.The involvement of caspase-10 in TRAIL induced signal transduction has been discussed controversially as well. In this work, it could be shown that a majority of commercially available antibodies against this protein are unspecific. Using a specific anti-body, which was identified in this work, it was demonstrated that three caspase-10 isoforms are expressed in cell lines. For the first time it could be demonstrated that caspase-10, like caspase-8, is recruited to the native TRAIL- and CD95-DISC. Further, it was shown that caspase-10 cannot substitute for caspase-8 and thus, possibly serves a function different from apoptosis induction.The results of this work are the basis for further studies of the signal transduction in the TNF-Receptor family, where still some fundamental questions are unresolved. One of these is unquestionably the function and of caspase-10 and the pathways, it is involved in