Upon responding to cellular stress, p53 protein becomes stabilised and acts as a transcription factor mainly resulting from phosphorylation and acetylation of the protein. Nitration of p53 protein is poorly characterised by comparison with phosphorylation and acetylation. The main aim of this work was to study the effects of nitration on p53 functional activities and on p53-MDM2 protein-protein interactions. Preliminary work was to characterise the nitration of p53 protein over-expressed in E. coli BL21(DE3) which was then purified by a series of column chromatography. GST-MDM2 protein along with control GST protein were also overexpressed in BL21 which were subsequently purified by a single step batch purification before subjected to nitration. Peroxynitrite, a nitrating agent used in this study, was generated in vitro. Preliminary nitration work was carried out using BSA as a model protein as it is easily nitrated owing to its high number of tyrosine residues (19 residues). The present results showed that p53 and GST-MDM2 proteins were hardly nitrated as no strong nitro-tyrosine signals were obtained. This might be due to these proteins, being overexpressed in E. coli, were not properly folded resulting in hidden/cryptic tyrosine residues of which making nitration difficult to achieve. Peroxynitrite was shown to have a degrading property, reducing protein levels of peroxynitrite-treated p53, GST-MDM2 and GST proteins. Immunoprecipitation studies of cancer cell lysates with different p53 status treated with peroxynitrite showed very weak signals of nitro-p53 protein in mutant p53 cells whereby no nitro-p53 protein signal in wild-type p53 MCF7 cells. In addition, NO donor GSNO-treated MCF7 cells showed weak nitro-p53 protein signals. / Ministry of Science, Technology and Innovation (MOSTI) of Malaysia
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/14788 |
| Date | January 2014 |
| Creators | Husaini, Roslina |
| Contributors | Picksley, Stephen M., Naseem, Khalid M., Brinkworth, Martin H. |
| Publisher | University of Bradford, Department of Medical Biosciences, Faculty of Life Sciences |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, MPhil |
| Rights | <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. |
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