Browsing by Author "Faro, Andrew"
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Item Investigation of the interactions of retinoblastoma binding protein-6 with transcription factors p53 and Y-Box Binding Protein-1(2011) Faro, Andrew; Pugh, David J.R.Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.Item Recombinant expression and full backbone assignment of the human DWNN using heteronuclear NMR(University of the Western Cape, 2005) Faro, Andrew; Pugh, David J.R.; Dept. of Biotechnology; Faculty of ScienceThe cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (“ubiquitinated”) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called “ubiquitin-like modifiers”, which consist of a single domain that structurally resembles ubiquitin, and “ubiquitin-domain” proteins, which are multi-domain proteins, which include domains that resemble ubiquitin. This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained. Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb.