Structural Characterization of the Switch domain regions of Schistosoma mansoni druggable Adenylate cyclase-stimulating Gα-protein
Loading...
Date
2017
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Zululand
Abstract
Among the neglected tropical diseases (NTDs), schistosomiasis, a common human parasitic disease; continues to rank high as one of the major causes of morbidity and mortality among the labour forces in known endemic regions. Recently, the socio-economic impact of Schistosoma infections have been reported to surpass that of malaria in sub-Saharan Africa, as schistosomiasis is known to cause severe damage to various organs thereby impairing childhood development and adult productivity. Schistosoma mansoni one of the two main widely distributed causative species parasitizing human, is believed to be responsible for over 90 % of all human schistosomiasis. In the last three decades, praziquantel (PZQ) remains the first-line drug for the treatment of schistosomiasis. Due to drug pressure, some strains of Schistosoma mansoni has exhibited worrisome signs of resistance to treatment with PZQ. In search for a novel and/or new anti-schistosomal agent or molecule as an alternative to praziquantel treatment, the two molecular switch domain regions (switch I and switch II) of S. mansoni has been identified as a promising druggable target against the parasite. The broad focus of this study was to explore the solubility, stability and druggability of the Gα protein from the switch domain regions of S. mansoni. In-silico analysis of the switch domain regions using various bioinformatics tools has revealed the existence of two promising functional motifs which are highly conserved in nature. Homology modeling of the switch domain regions and overall stereochemical parameters as evaluated by the Ramachandran plot analysis in RAMPAGE reveals that the modelled structure is stable and of good quality. Docking studies revealed the binding and interaction patterns of the switch domains protein with guanosine-5'-triphosphate, magnesium, calcium and zinc. Recombinant GST-tagged switch domain was heterologously expressed in Escherichia coli (E. coli) JM109 and subsequently purified using glutathione-agarose beads. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis the recombinant GST-Switch domain protein shows that a high yield soluble fusion protein was obtained, as visible prominent bands (~31.84 kDa) corresponding to the GST-Switch domain protein were observed in the region of 25 – 37 kDa. Preliminary characterization of the GST-Switch domain protein was carried out using fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectrophotometry, fluorescence spectroscopy, differential scanning calorimeter (DSC), thermogravimetric analysis (TGA), nuclear magnetic resonance spectroscopy (NMR) and isothermal titration calorimetry (ITC). The results obtained from this study, has given us a considerable insight into the nature of the protein in terms of its purity, functional groups, structural and thermal stability as well as its folding state and its binding interaction with two of the hypothesized ligands in literature. In conclusion, the overall results from the computational studies, wet-laboratory experiments and characterization studies are impressive and suggest the possible druggability of the switch domain regions. These findings will enhance our understanding in selective identification of small molecule inhibitors or peptides, which could be developed as novel therapeutic candidates in the control and treatment of human Schistosoma infections.
Description
A thesis submitted to the Faculty of Science and Agriculture in fulfilment of the requirements for the Degree of Doctor of Philosophy (Ph.D) in Biochemistry in the Department of Biochemistry and Microbiology at the University Of Zululand, South Africa, 2017
Keywords
antimicrobial peptides --characterization --docking --drug resistance --human schistosomiasis --neglected tropical diseases -- praziquantel ---recombinant protein --Schistosoma mansoni --trematode