Abstract
Grp78/BiP is a 78kDa protein located within the lumen of the endoplasmic reticulum (ER), where it helps store Ca2+ and acts particularly as a chaperone assisting in the folding and assembly of membrane-bound or secretory proteins. Recent evidence has implicated the protein as a sensor of ER malfunction, regulating the initiation of a signalling pathway, known as the ER stress response or Unfolded Protein Response (UPR). The work undertaken for this thesis has addressed the role of Grp78/BiP in the mammalian UPR signalling pathway and the consequent effects upon cell survival, programmed cell death, and cell cycle regulation during ER stress. Novel Grp78/BiP fluorescent and bioluminescent chimeric proteins were generated in order to study dynamic changes of EGFP-tagged Grp78/BiP proteins located in the cell. This approach simplified and facilitated the generation of stable cell lines over-expressing Grp78/BiP and provided a powerful tool for single cell analysis to help determine its role in survival, apoptosis and cell cycle arrest under ER stress conditions. Use of these fluorescent molecular indicators showed that the apparent nuclear localisation of the Grp78/BiP is due to its presence in nuclear tubes, which are the results of invaginations of the normal ER strands into the nucleus from the nuclear envelope. This localisation was unaltered under ER stress, even at maximum doses of stressor, and so it appears that Grp78/BiP does not re-locate to the nucleus in order to fulfil its signalling functions. Under ER stress, levels of endogenous GRP78/BiP mRNA, as well as those for GRP94 and calreticulin, were induced, supporting previous evidence for their role as stress-inducible chaperones. Similar results were found for the transcription factor GADD153/CHOP. The induced expression of these mRNAs was attenuated in cells transiently over-expressing wild-type Grp78/BiP (wt-Grp78/BiP), indicating that the induction is triggered by a drop in the levels of free Grp78/BiP within the ER lumen. Such attenuation, however, did not occur in cells transiently expressing the Grp78/BiP chimeric proteins and the ramifications of these observations are discussed. To investigate the role of Grp78/BiP as an ER signalling molecule during ER stress, cells stably expressing Grp78/BiP fusion proteins at high levels, either EGFP-BiP or EGFP-BiP-AEQ, were selected using EGFP as an indicator of their expression. Under ER stress, these cells demonstrated increased cell survival and prevented apoptosis by directly attenuating the activation of UPR signalling pathways by modulating the activation of the ER sensors and increasing the ER folding capacity of the cells. Use of different chimeric proteins showed that, in order to do this, Grp78/BiP requires a free C-terminus. The results also demonstrated that Grp78/BiP over-expression attenuated cyclin D1 loss induced by tunicamycin. This slowed the S-phase of the cell cycle, possibly suggesting a novel role for this protein in cell division. These findings underline the role of Grp78/BiP as a key modulator of the UPR in mammalian cells and provide the basis for further investigation into its functions.