Novel role of an ER-resident chaperone pathway in cancer signalling
Mohtar, Mohamad Aimanuddin
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Anterior gradient-2 (AGR2) is an endoplasmic reticulum (ER)-resident protein that belongs to a member of protein disulphide isomerase (PDI) superfamily. AGR2 initially emerged as a dominant effector of basic biological properties in vertebrates such as specifying forebrain integrity and limb generation. Subsequent studies in mammals implicated the role of AGR2 as a pro-metastatic protein essential to cancer progression and drug resistance, asthma and inflammatory bowel disease. AGR2 protein is mainly overexpressed in a number of human cancers and involved in pathways for ER stress, protein folding, transcription regulation, and exosome formation. Hence, AGR2 protein represents a clinically-relevant oncoprotein in tumour emergence and survival. The aim of this thesis is to shed more light on the role of AGR2 in cancer development. AGR2 was previously shown that it could bind sequence-specifically to a linear peptide motif. In this study, hydrogen/deuterium exchange mass spectrometry was used to identify the dominant peptide-binding site on AGR2 by comparing the deuterium uptake between AGR2 and AGR2 with its ligand (linear peptide motif). The binding of the peptide was probed by making mutant series in the identified peptide ‘docking site’ region on AGR2. A consensus peptide-binding motif was then developed to identify potential cellular proteins that harbour this motif as potential AGR2 client proteins. Database mining using this consensus binding peptide demonstrated that transmembrane proteins were dominant class of proteins. Epithelial cell adhesion molecule (EpCAM), an oncogenic transmembrane protein, was chosen as putative AGR2 client proteins and their interaction was verified using both cell-free and cell-based assays. The AGR2 and EpCAM pathway dynamics were reconstituted and investigated in cells that do not endogenously express both proteins. Further, the expression of AGR2 and EpCAM were assessed in clinical tumour samples using immunohistochemistry. Proteomics screen using quantitative tandem mass tag (TMT) mass spectrometry on cells transiently overexpressing AGR2 were used to identify potential AGR2 client proteins and to find relevant dominant pathways affected by AGR2 signalling. Additionally, synthetic tools were devised to further dissect the function, regulation and ‘druggability’ of AGR2 protein. These tools include: i) isolation of high-affinity AGR2-binding synthetic antibodies from a phage-scFv (single-chain variable fragment) library; ii) engineering synthetic mini-protein (synPRO) containing copies of wild-type and mutated AGR2 linear peptide motif and; iii) engineering synthetic membrane protein model that bind to AGR2. In conclusion, the data presented hereby demonstrated a novel role of an ER-resident protein AGR2 which possess an intrinsic sequence-specific peptide binding for a subset of its client proteins and one function of this motif is to ensure proper maturation of client proteins to their final destination. Development of synthetic tools in this study can be further manipulated to disrupt AGR2 signalling and the fate of its binding proteins which in turn highlights a potentially ‘druggable’ stage in the oncogenic secretory pathway.