Structural and functional interrogation of Anterior Gradient-2
Gray, Terry Allan
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Anterior Gradient-2 protein (AGR2) has recently been linked to the onset of several pathologies including asthma and inflammatory bowel disease. Most interestingly, it has been discovered to influence the transformation of cells and metastatic growth essential to cancer development, and has subsequently been linked to the development of resistance to anti-cancer therapeutics. AGR2 protein is overexpressed in a diverse range of human cancer types, and has been detected secreted into the extracellular milieu. Thus, AGR2 protein represents a compelling pro-oncogenic signalling intermediate in tumour emergence and endurance. This thesis presents an interdisciplinary approach including structural biology, cell biology and synthetic biology, and clinical studies to shed more light on the role of AGR2 in cancer development. Synthetic cell based reagents were developed to define the dominant pathways that are reprogrammed in a cell as a result of AGR2 synthesis. A cell panel was engineered incorporating the AGR2 (and mutants thereof) allele into the AGR2-null A375 cell line. These tools were then coupled to quantitative proteomics (SILAC) to unravel the mechanism whereby introduction of AGR2 alters cell phenotype, allowing identification of dominant pathways affected by AGR2 signalling. Using pathway analysis tools, the dominant pathway suppressed by wt-AGR2 expression highlighted the p53-signalling axis. DNA damage induced p53 stabilisation and p21 induction by cisplatin treatment confirmed the influence of AGR2 gene expression. Further data analysis identified the outlying protein expression changes identified by SILAC was the anti-viral cell cycle regulator TSG101 (tumour susceptibility gene 101), and confirmed by immunoblotting. Transfection and silencing studies of TSG101 confirmed that TSG101 attenuates p53 function. These data provide a mechanism to explain the most dominant pathways reprogrammed by AGR2 expression, incorporating ER stress response, proliferation markers and p53 pathway attenuation. Further advances were made in analysis of the function, regulation, and drugability of AGR2 protein. Assays were devised to define the subunit structure of AGR2 as a dimer unit; subsequent functional studies defined intrinsically disordered motifs that regulate stability of the dimer. A two-site sandwich microtiter assay (2SMTA) was designed to screen for self-peptides and mutations that regulate oligomer stability. These assays were used to identify the first biochemical property of AGR2 being that the dimer unit is required for maximal binding to the AAA+ protein, and well characterised AGR2 interactor, Reptin. In addition, based on this dimeric structure, a novel solution based dimerisation assay was developed to identify natural products that are able to disrupt the dimer suggesting that AGR2 itself can be targeted in principle with small molecules for therapeutic purposes.