ScRING finger protein, Mdm2, MdmX, p53, cancer, ubiquitination, drug discovery, ZINC16046951, University of Zululand, South Africa

Mathenjwa, Makhosazana Siduduzile

ScRING finger protein, Mdm2, MdmX, p53, cancer, ubiquitination, drug discovery, ZINC16046951, University of Zululand, South Africa

Date

2021

Authors

Mathenjwa, Makhosazana Siduduzile

Publisher

University of Zululand

Abstract

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.

Description

A Dissertation submitted to the Faculty of Science, Agriculture and Engineering in fulfilment of the requirements for the Degree of Doctor of Philosophy of Biochemistry in the Department of Biochemistry and Microbiology at the University of Zululand, South Africa [2021].