Browsing by Author "Mathenjwa, Makhosazana Siduduzile"

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    Recombinant expression, purification and structural characterization of Saccharomyces cerevisiae RING finger domain using Nuclear Magnetic Resonance (NMR) spectroscopy
    (University of Zululand, 2018) Mathenjwa, Makhosazana Siduduzile
    RING finger domain is a domain that is incorporated with other domains to form a large protein known as RBBP6. This is a 250kDa protein that stands for Retinoblastoma binding domain protein 6. It is classified according to different homologues namely RBQ1, PACT, SNAMA, Mpe-1 and P2P-R depending on the organisms in which they are found. P2P-R (a RING finger containing protein) was found to be highly up-regulated in the oesophageal cells compared to normal epithelial cells. This was a pre-indication that P2P-R might serve as an antigen against cancer or be used in the diagnosis of cancer. Saccharomyces cerevisiae RING finger domain is a protein that is represented by the richness of cysteine sequence motif that binds to two zinc atoms. It has a cross-brace topology consisting of 70 amino acids and its sequence is defined as Cys1-Xaa2-Cys2-Xaa9–39-Cys3-Xaa1–3-His4-Xaa2-3-Cys/His5-Xaa2-Cys6-Xaa4–48-Cys7-Xaa2-Cys8 (where Xaa represents any amino acid residue). The RING finger domain is essential in proteins involved in a range of diverse cellular processes such as apoptosis, viral infections, oncogenesis and ubiquitination. The study presented in this dissertation reports on the recombinant expression and partial purification S. cerevisiae RING finger protein, as well as its biophysical and biochemical characterization. Structural predictions using the ProtParam tool on ExPASy provided information regarding the high structural stability due to the abundance of leucine in the protein. The protein has a limited ability for light absorption which is indicative of the absence of two important aromatic amino acids; tryptophan and tyrosine. More so, Swiss-Model showed that the 3D homology model structure is composed of two α-helices contributing to the stability of the structure, two β-sheets that prevents proteolysis and one 310 helix that maintains the helix-coiling transitions. The local quality estimate revealed the structure is rigid and stable. The non-redundant set of the Protein Data Bank analysis showed the structural prediction of the RING finger protein is accurate to a larger extent. Validation of the predicted model by Ramachandran plot showed that 88% of the amino acids residues are in the favoured region meaning that the modelled structure is an acceptable representation of the S. cerevisiae RING finger protein.More so, interacting partners for S. cerevisiae RING finger protein were also ascertained and these protein partners were shown to be involved in cleavage and polyadenylation, however, zinc fingers from YTH1 showed similarity with RING finger domain as promoters of DNA binding. Phylogenetics and sequence alignment showed the ancestral proteins are involved in ubiquitination, which is one of the major pathways of RING finger domains. Lastly, the biochemical characterization using a 1D proton NMR spectroscopy showed that S. cerevisiae RING finger protein is well folded and can be further worked upon. These results provide the baseline information for the structural determination of the S. cerevisiae RING finger domain for future studies.
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    ScRING finger protein, Mdm2, MdmX, p53, cancer, ubiquitination, drug discovery, ZINC16046951, University of Zululand, South Africa
    (University of Zululand, 2021) Mathenjwa, Makhosazana Siduduzile; Kappo, Abidemi Paul
    World-wide cancer is rated as the deadliest disease, more than TB, HIV and malaria combined. Parveen et al., 2015 reports that unless drastic measures are taken, cancer will statistically increase by 78% by 2030. Although different cancer therapies have been employed to combat cancer development, immunotherapy remains the current hope as a manipulative therapy to provide immune boost, more specificity and effective drug design against cancer (Vanneman and Dranoff, 2012). Really Interesting New Gene (RING) finger protein is a truncated domain from RBBP6 known to function independently in protein-protein interaction and cellular processing signalling. It is a 10 kDa protein defined by its hallmark residues cysteine and histidine that binds two zinc ions for structural stability and integrity (Dominguez., 2004; Krishna et al., 2003). When human RING finger domain was used as a searching bait to fish out other possible RING finger domains from other organisms, Saccharomyces cerevisiae RING (ScRING) finger domain stood out amongst others as it lacked the first zinc binding site which is abnormal for RING finger domain (Kappo et al., 2012). ScRING finger domain is cysteine rich domain from a Mpe-1 RBBP6 homologue that binds zinc ion for structural stability. Therefore, biochemical, structural and In silico characterisation was done for this study to provide basic insight into the hypothesis that ScRING finger protein is a RING finger-like protein or an abnormal RING finger protein and to determine target inhibitors for RING finger-type E3 namely, Mmd2 and MdmX. Expression, purification, Far-UV CD, Intrinsic tryptophan fluorescence, SE-HPL chromatography and ANS fluorescence were used to biochemically and structurally characterise ScRING finger protein. In silico inhibitory studies, for Mdm2 and MdmX complexes with RO-2443 as a control, and ZINC16046951, as the experimental ligand, were conducted using molecular dynamics tools, namely RMSD, RMSF and Rog. MM/PBSA and MM/GBSA were utilized to calculate binding free energy contributions per complex amino acids. Far-UV CD confirmed ScRING finger domain is a protein dominated by β-sheets which are a major contributing factor in protein stability and integrity. ANS fluorescence indicated the presence of hydrophobic binding pockets required for protein-protein interaction. These results provided direction towards regarding ScRING finger protein as a RING finger domain rather than a RING finger-like domain. In silico inhibitory studies showed ZINC16046951 as the highest energy binding and Table ligand to Mmd2 compared to other complexes. These results provided the baseline information needed for drug discovery and structural determination.