Identification and Characterisation of Novel Mechanisms that Regulate TRAIL-Receptor Signalling Complexes
2017-06-12T10:27:42Z (GMT) by
Activation of death receptors by their cognate ligands can induce apoptosis through the formation of death receptor signalling complexes. TRAIL has been demonstrated to selectively induce apoptosis in transformed cells, but not normal cells, by binding to its death receptors, TRAIL-R1 and TRAIL-R2 and inducing DISC formation. However, the use of TRAIL as a cancer therapeutic has been hampered by widespread intrinsic and acquired resistant to TRAIL in tumours. Gaining a better understanding of the mechanisms which regulate TRAIL signalling in both tumour and normal cells, will enable new strategies to overcome tumour resistance to TRAIL-induced apoptosis. Therefore, the aim of this thesis was to identify and characterise novel mechanisms which regulate TRAIL signalling using protein complex isolation, mass spectrometry and functional assays. By isolating the TRAIL DISC and unstimulated TRAIL-R1/R2 using wild-type TRAIL and TRAIL-R1 or -R2 specific TRAIL mutants, novel protein interactors were identified. Oxysterol binding protein related protein 8 (ORP8) was shown to specifically interact with unstimulated TRAIL-R1/R2 and regulate the cell surface expression of TRAIL-R2. An SCF complex, composed of S-phase kinase associated protein 1 (Skp1), cullin-1 (CUL1) and F-box only protein 11 (FBXO11), was shown to interact with unstimulated TRAIL-R1/R2. The role of this complex was hypothesised to regulate ubiquitination and proteasomal degradation of TRAIL-R1/R2. The serine/threonine phosphatase, PP2A was demonstrated to interact with both the TRAIL DISC and unstimulated TRAILR1/R2. PP2A is likely to regulate TRAIL signalling by de-phosphorylating TRAIL-R1/R2 or associated proteins. In addition, the mechanism by which activated PKC inhibits TRAILinduced apoptosis was further investigated. Activation of PKC induced the phosphorylation of the intracellular domain (ICD) of TRAIL-R2. This phosphorylation, most likely on the TRAIL-R2-T298 residue, prevents aggregation of TRAIL-R2 into a high molecular weight (HMW)-TRAIL DISC. Inhibition of HMW-DISC formation prevents FADD recruitment, caspase-8 recruitment and activation, and blocks the induction of both the apoptotic and non-apoptotic arms of TRAIL signalling.