Browsing by Author "Masamba, Priscilla"

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    Structural Characterization of the interaction between divalent cations and the novel Schistosoma mansoni Universal Stress Protein G4LZI3
    (University of Zululand, 2017) Masamba, Priscilla; Kappo, A.P
    Approximately 200 000 people in sub-Saharan Africa lose their lives to schistosomiasis every year. This coupled with other severe Neglected Tropical Diseases, HIV/AIDS, malaria, tuberculosis and poverty, does not make the situation any better. Vaccines targeting this disease are still yet to be discovered and current treatment is becoming a problem as drug resistance has been reported. Thus, looking for an alternative treatment regimen is fast becoming a priority. This study took an interest in Universal Stress Proteins and focused on a novel G4LZI3 USP, which has been put forward as a possible candidate vaccine. A sufficient amount (0.518mg/ml) of recombinant G4LZI3 protein was successfully expressed using 0.5mM IPTG and subjected to a Nickel-NTA column for purification. The fractions were pooled together, concentrated down and used to investigate the binding of the protein with 3 metallic divalent cations using Isothermal Titration Calorimetry (ITC). Additionally, bioinformatics was employed to predict the secondary structure of the protein, generate a 3-dimensional model of the protein and use it for docking the divalent cations. Moreover, the interacting partners of the G4LZI3 protein were determined, thus giving a clue towards the function of the protein. Bioinformatics tools assisted in confirming Ca2+, Mg2+ and Zn2+ as putative ligands and additionally identified ATP, AMP and U20 as possible molecules that interact with the G4LZI3 USP protein. However, in vivo studies only showed interaction between Mg2+ and the protein. These results provide prospects for future studies toward drug discovery for schistosomiasis.
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    Structural Determination of the novel Universal Stress Protein G4LZI3 from Shistosoma mansoni using X-ray Crystallography
    (University of Zululand, 2020) Masamba, Priscilla
    Structural determination of the novel Universal Stress Protein G4LZI3 from Schistosoma mansoni using X-ray Crystallography Priscilla Masamba PhD (Biochemistry) thesis, Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand Schistosoma mansoni is a parasitic multicellular helminth of the Schistosomatidae family and is one of the three main causative agents responsible for the multifactorial disease schistosomiasis, a debilitating and chronic vascular infection of paramount socio-economic importance and international public health concern. Recent estimates approximate the disease to account for the loss of 70 million disability-adjusted life years (DALYs) thus acknowledging schistosomiasis a more debilitating infection than malaria and a huge competitor with HIV/AIDS and TB. Mortality rates result in nothing less than 250 000 deaths per annum in sub-Saharan Africa alone where more than 90% of all schistosome infections stem from. Treatment for the disease has now become an issue of gross concern worldwide without the development of a vaccine as well as growing evidence of drug resistant strains against Praziquantel, which has been the backbone of treatment for all schistosome strains for over three decades. Furthermore, the elusiveness surrounding the mechanism of action of Praziquantel (PZQ), its lack of success in killing larval schistosomes 2-4 weeks post-infection as well as its failure to disrupt the transmission cycle of 3 the worm, has thereby caused wide interest in the design and development of alternative treatment against schistosomiasis. It has been proposed that the cascade of events encompassing the transition of the worm throughout its developmental cycle, leading to the various changes in its morphological and chemical composition allowing the worm evade the various defense mechanism of humans, as well as its tolerance to sudden environmental changes and conditions of stress, is to a large extend due the over-expression of heat shock proteins (HSPs) and universal stress proteins (USPs). This current study therefore focuses on the G4LZI3 USP whose over-expression has been displayed throughout all developmental forms of the parasite during conditions of stress and has therefore been identified as a ‘lead’ molecule in the design of alternative treatment against schistosomiasis. However, the mechanism of action of USPs is not well understood. Therefore, the main aim of this study was to determine the structure and infer biological function of this important druggable protein. Before the structure was solved, a wide range of polyphenols, which have been proposed as potential inhibitors were docked onto the homology model of the protein and subjected to molecular dynamics (MD) simulations. Results revealed strong interaction between two polyphenols namely curcumin and catechin, suggesting the inhibitory potential of these polyphenols. The ability of polyphenols to scavenge oxidative stress associates these compounds with the prevention of diseases or infections that are linked to ROS production, which is one of the features of schistosomiasis. Hence, investigating polyphenols as potential nutraceuticals for schistosomiasis may be an interesting venture towards alternative treatment. 4 The structure of the G4LZI3 protein was thereafter solved using X-ray crystallography; the protein consists of 4 parallel β-sheets and 3 α-helices with the presence of a short 310 helix positioned close to the C-terminal of the protein. A generated Ramachandran plot showed that though there is still room for improvement, the solved structure is a relatively good model with 92% of the residues present in the favored and allowed regions. Inferred function points towards the protein binding ATP for regulation or even acting as an ATPase. Finally, another set of molecular dynamic simulations were conducted on small molecule inhibitors docked onto the solved structure of the G4LZI3 protein using a pharmacophore generated from curcumin. Two of the molecules identified exhibited inhibitory activity against the protein by demonstrating future studies would therefore consider synthesizing these compounds for both in vivo and in vitro studies towards alternative anti-schistosomidals.