Characterisation of the novel endoplasmic reticulum chaperone ERDJ5

Communication between cells depends to a large extent on interactions between secreted proteins and membrane receptors. Therefore their production and release must be strictly regulated. Proteins that are destined for the extracellular space assemble and fold in the endoplasmic reticulum (ER). The ER provides the ideal environment for the synthesis and folding of proteins with optimal ionic and redox conditions and the presence of many chaperones and isomerases eg Immunoglobulin heavy chain binding protein (BiP), protein disulphide isomerase (PM), and Calnexin. A tight quality control network also exists within the ER ensuring that only correctly folded proteins are transported out via the Golgi network. Unfolded or misfolded proteins are retrotranslocated to the cytosol for proteasomal degradation. The accumulation of misfolded proteins in the ER leads to the transcriptional up-regulation of many genes encoding ER chaperones and isomerases, in a process known as the Unfolded Protein Response (UPR). The protein folding process and proteins translocation from the ER to cytosol are under intense scrutiny as the pathogenesis of many diseases such as Alzheimer's Disease, Cystic Fibrosis and many prion-associated disorders involve misfolding of proteins and processing abnormalities in the ER. In this thesis, we describe the characterisation of a novel ER protein, ERdJ5, with potential chaperone and isomerase capabilities. ERdj5 was discovered during a screen of EST databases for novel thioredoxin proteins. Initial characterisation of human ERdj5 sequence revealed domains resembling DnaJ, PD1 and Thioredoxin domains. Northern blot and in situ hybridisation analysis showed that ERdj5 is a ubiquitously expressed protein, particularly abundant in secretory tissues. In vitro binding studies with BiP were performed which established their interaction via the N-terminal DnaJ domain in an ATP dependant manner. In addition, ERdj5 transcription was induced during ER stress. Combined, these results suggest potential roles for ERdJ5 in the processes of protein folding and stress within the ER. An immunohistochemical study of thioredoxin 1, thioredoxin reductase 1 and ERdj5 in hepatocellular carcinoma (HCC) yielded interesting findings, showing that all proteins were specifically expressed in the tumour tissue. Excitingly, ERdj5 showed a three-fold increase in expression in tumour tissue compared to internal control, thus suggesting ERdj5 and other members of the thioredoxin family as potential immunohistochemical markers in HCC. Cell viability experiments in an HCC derived cell line with ERdj5 knocked down, showed that upon treatment with an ER stress inducer, Tunicamycin, control cells were more susceptible to cell death. Conversely, following treatment with Doxorubicin, a chemotherapy drug, ERdj5 knock down cells were more vulnerable to cell death, indicating a potential defensive role for ERdJ5 in chemotherapeutic drug resistance. During the initial characterisation studies of ERdj5, it was noted that ERdj5 was expressed in neuronal cells of the hippocampus which are sites of neuronal degeneration in Alzheimer's brains. This finding prompted a study to investigate ERdj5 in Alzheimer's disease. Immunohistochemical staining for ERdj5 confirmed that ERdj5 was expressed in degenerating pyramidal cells of frontal cortex in a lesser extent in control brain and in almost all degenerating cells in Alzheimers brain. Processing of amyloid precursor protein, cleavage of which by β and γ-secretases yields β-amyloid, occurs in the early secretory pathway, including the ER and is aided by molecular chaperones such as BiP. Interaction studies established an interaction between ERdj5 and immature APP, and with the APP cleavage product C99, suggesting a chaperoning role for ERdj5 in the processing of APP in the early secretory pathway. ERdj5 has many orthologues from human to mouse to zebrafish. Characterisation of the mouse ERdJ5 sequence revealed a protein of 793 amino acids, organised genomically into 23 exons. ERdj5 is expressed in the developing nervous system, ectoderm, mesoderm and endoderm of the developing mouse embryo. Further immunohistochemical studies in an asthma mouse model produced an interesting differential expression of ERdJ5 in the lungs of the control and asthma models. In addition, ERdj5 showed consistent expression in the smooth muscle of both asthma and control cases, indicating a possible role as a smooth muscle marker. In summary, the results presented in this thesis, characterising ERdj5 greatly adds to our knowledge of this novel mammalian protein. A wide spectrum of roles for ERdj5 has been shown in this study, suggesting ERdj5 as a potential ER chaperone, involved in many diseases. Determining the precise function of ERdJ5 within the secretory pathway demands further investigation as the significance of the ER organelle in the pathogenesis of disease is expanding rapidly