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Characterisation and Detection of P53 Aggregates



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Wu, Yunzhao 


P53 is a tumour-suppressing protein whose primary function is to protect the integrity of the genome, specifically by regulating DNA repair, cell cycle arrest, cell proliferation, and apoptosis. Wild-type (WT) p53 has multiple aggregation-prone regions and can form amyloid aggregates. Some mutants of the p53 protein, such as the R248Q mutant, exhibit higher aggregation propensity than the WT. P53 aggregation is associated with loss-of-function, gain-of-function, and dominant-negative effects of the protein and thus plays a crucial role in cancer. Understanding the properties of p53 aggregates and their pathological implications provides new insights into cancer biology and may lead to novel diagnostic and therapeutic strategies.

Previous studies on p53 aggregates mainly focused on the properties of bacteria-derived p53 core-domain fragments, which have different post-translational modifications from human p53 and may behave distinctly from full-length p53 proteins. The p53 concentrations used in these studies were also higher than the physiological concentrations. Meanwhile, most kinetic studies on p53 aggregation employed microplate reader-based assays, which are limited in sensitivity and cannot provide morphological information about the aggregates. Hence, the biophysical properties of full-length p53 aggregates at physiological concentrations are not well characterised. Also, the detection of p53 aggregates in biopsies has not been demonstrated.

In this thesis, a series of advances in single-molecule assays are presented to characterise full-length p53 aggregates and detect p53 aggregates in human plasma. Firstly, a single-molecule fluorescence microscope with flat-field illumination was constructed and characterised. Secondly, a Python-based computational suite for automated fluorescence image analysis was implemented. Based on these two techniques, the aggregation kinetics, membrane disruptive ability, and morphological features of insect cell-derived full-length WT and R248Q p53 aggregates were characterised. Specifically, p53 aggregation at physiologically relevant concentrations was found to be dominated by a nucleation-elongation process. Meanwhile, both WT and R248Q p53 aggregates exhibit membrane-disruptive abilities. Furthermore, a single-molecule array assay was developed to detect p53 aggregates in plasma samples, demonstrating p53 aggregates as a promising diagnostic biomarker for glioblastoma. The presence of p53 aggregates in the plasma samples of glioblastoma patients was validated using the single-molecule pull-down assay. The work presented in this thesis extends the understanding of full-length p53 aggregates and highlights the implications of p53 aggregates in cancer diagnosis.





Klenerman, David


biomarker, cancer, imaging, p53 aggregate


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge