Proteomic and Molecular Characterisation of TRAIL-Induced Signalling Complexes

TRAIL selectively induces apoptosis in cancerous cells through the formation of the death-inducing signalling complex (DISC). Apoptosis can progress through either a Type I (direct procaspase-3 cleavage) or Type II (Bid cleavage) route. However, factors acting at the DISC, which may determine the selectivity of TRAIL and/or which pathway (Type I/II) is activated, are currently ill-defined. Therefore, the aim of this thesis was to investigate the initial phase of TRAIL signalling through protein purification, mass spectrometry and functional assays. A tagged variant of FADD (DISC component) was stably expressed, allowing interactions with calpain small subunit 1 (calpain S1) and heat shock cognate 71 kDa protein (HSC70) to be identified by mass spectrometry. It was speculated that FADD and calpains may interact via calpain S1, whilst HSC70 may contribute to TRAIL- induced subcellular redistribution of FADD. Mass spectrometry of the TRAIL DISC identified the known components and a potential interaction between transferrin receptor and TRAIL-R1/-R2. It was hypothesised that this interaction may help target TRAIL-R1/-R2 and DISC for endocytosis. Increased TRAIL DISC formation in Type I versus Type II cells was unlikely to be the result of other proteins, since no significant differences in DISC composition were detected. Further studies indicated that low TRAIL DISC formation in the Type II Jurkat cell line was likely related to the absence of TRAIL-R1 expression and inefficient recruitment of FADD to TRAIL-R2. Functional assays suggested that Type I and Type II TRAIL DISC have a similar substrate preference. Therefore, in contrast to previous reports, these data suggest that the lower level of DISC formed in a Type II cell line does not preferentially cleave Bid to activate the intrinsic pathway. Instead lower caspase-8 activation at the Type II DISC results in overall less substrate cleavage, delaying apoptosis and thus enabling the intrinsic pathway to contribute to caspase-3 activation.