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Structural and biochemical analysis of the death domain complex formed at the Fas receptor.

Ferguson, B.J.; (2005) Structural and biochemical analysis of the death domain complex formed at the Fas receptor. Doctoral thesis , University of London. Green open access

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Abstract

Fas (CD95) is a member of the death receptor superfamily of proteins that are involved in the initiation of apoptosis as induced by the binding of extracellular ligands. At present, little is known about the precise mechanism by which the signal initiated by the interaction between Fas ligand (FasL) and Fas is transduced across the cell membrane to start the apoptotic signalling cascade. The first step in this pathway is the recruitment of the Fas-associated death domain protein (FADD) to the cytoplasmic death domain of Fas via a homotypic protein/protein interaction. This binding event occurs after receptor ligation apparently without any post translational modification such as phosphorylation. In order to better understand this event we have investigated the interaction between the death domains of the human Fas and FADD proteins both in vitro and in a cellular context. The reported interaction between the Fas and FADD death domains (Fas-DD and FADD-DD) was recapitulated using the yeast 2-hybrid assay. Recombinant proteins were then produced for NMR spectroscopy experiments. FADD-DD is highly expressed, and easy to isolate soluble at physiological pH. This domain is readily expressed as a histidine tagged domain. Fas death domain expresses at low levels and produces soluble aggregates when concentrated at a pH above 4. However, it was found that using a Gbl fusion protein to express Fas-DD overcomes these problems and allowed the production of Fas-DD for NMR experiments. NMR titration experiments showed that when these two proteins interact a large, soluble complex is formed. This may be significant in relation to the increasing evidence for the importance of Fas-receptor clustering in its signalling. Mutational analysis of the Fas death domain was also carried out. Here, various previously described as well as several novel point mutations were made on the surface of the Fas death domain to investigate their effect on FADD-DD binding. These mutations were assayed using yeast 2-hybrid methods, NMR analysis and in a cell based assay. In the cell based assay wild type and mutant Fas receptors were overexpressed in a human cell line with no endogenous surface Fas expression. These cells were then assayed for their ability to undergo FasL-induced apoptosis. It was found that residues from many surface regions of Fas-DD are crucial for the FADD-DD interaction. This observation has potentially important implications for the nature of the organisation of the death domains in the death inducing signalling complex (DISC) formed at the Fas receptor.

Type: Thesis (Doctoral)
Title: Structural and biochemical analysis of the death domain complex formed at the Fas receptor.
Identifier: PQ ETD:591710
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: http://discovery.ucl.ac.uk/id/eprint/1444407
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