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Title: | Structural Interaction of the tumour suppressor p53 with manganese superoxide dismutase |
Authors: | Dalli, Abigail |
Keywords: | Enzymes Mitochondria Tumor suppressor proteins p53 protein |
Issue Date: | 2020 |
Citation: | Dalli, A. (2020). Structural Interaction of the tumour suppressor p53 with manganese superoxide dismutase (Bachelor's dissertation). |
Abstract: | p53, exhibiting a non-canonical function, was found to translocate into the mitochondrial matrix and interact with manganese superoxide dismutase (MnSOD), possibly inhibiting its antioxidant function, leading to oxidative stress and induction of rapid p53-dependent apoptosis (Zhao et al., 2005). Although there is evidence for immunoprecipitation of p53-MnSOD, this interaction has not yet been analysed in vitro by biophysical techniques. The aim of this dissertation was to observe the putative association between the tumour suppressor p53 and the antioxidant enzyme MnSOD. Preliminary results from SOD activity gels in this study show that MnSOD activity is decreased at 25oC and 37oC in the presence of p53. Thus, the biological relevance of this inhibition implies that oxidative stress leads to cytoplasmic stimulation of apoptosis. The proteins were expressed in Escherichia coli cultures and purified using metal chelate affinity chromatography (MCAC), with a poly-histidine tag at the Nterminus of human MnSOD (hMnSOD) and of human p53 (His6p53N), and at the Cterminus of human p53 (His6p53C), which was cloned. hMnSOD was purified to high homogeneity. A protocol was established to express and purify p53 to reduce its propensity for aggregation, where His6p53C fared better than His6p53N. A cytochrome c SOD activity assay was conducted to observe any changes in MnSOD activity when using p53 as an effector, but no changes in kinetic parameters were observed. Future work will involve purification of human p53 into separate domains to possibly improve its stability, and the use of biophysical techniques such as isothermal titration calorimetry (ITC) to observe the putative p53-MnSOD interaction. |
Description: | B.SC.MEDICAL BIOCHEMISTRY |
URI: | https://www.um.edu.mt/library/oar/handle/123456789/67037 |
Appears in Collections: | Dissertations - FacM&S - 2020 Dissertations - FacM&SPB - 2020 |
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