MSc (Biochemistry) / Department of Biochemistry / Malaria is a disease that claims about half a million lives annually, mainly children. There are 5 Plasmodium species that cause malaria; namely, P. falciparum, P. ovale, P. malariae, P. knowlesi and P. vivax. P. falciparum is the most virulent of them all. The parasite upregulates some heat shock proteins (Hsps) in response to stress it encounters during its life cycle. These Hsps play a major role in proteostasis. The drug resistance of P. falciparum to traditionally used remedies has led to a need for the development of novel drugs. Hsps have been implicated as antimalarial drug targets. Hsps act as molecular chaperones and some make complexes, which are important in facilitating protein folding. As an example, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) form a functional complex through an adaptor protein, Hsp70-Hsp90 organizing protein (Hop). P. falciparum expresses six Hsp70s that are localized in different subcellular compartments. Amongst them, P. falciparum Hsp70-x (PfHsp70-x), is exported to the erythrocyte where it is implicated in host cell remodeling. PfHsp70-x possesses an ATPase domain, substrate binding domain and a C-terminal subdomain. PfHsp70-x possesses an EEVN motif on its C-terminus which is implicated in interactions with co-chaperones amongst them, Hop. Although some of the chaperone functions of PfHsp70-x have been reported, its interaction with human chaperones has not been investigated. The availability of PfHsp70-x in the infected erythrocyte cytosol presents a possibility that this protein may functionally cooperate with human Hsp90 via human Hop (human Hop). This hypothesis that PfHsp70-x interacts with human chaperones is strengthened by the absence of Hsp90 and Hop of parasite origin in the infected erythrocytes. The main aim of this study was to explore the chaperone activity of PfHsp70-x and its functional co-operation with human Hop. Recombinant PfHsp70-x (full length and EEVN deletion mutant) proteins were expressed in E. coli XL1 Blue cells and purified using nickel affinity chromatography. PfHsp70-x was found to be structurally comprised of mostly alpha helices and demonstrated heat stability based on circular dichroism (CD) spectrometry studies. It was established that the EEVN motif may be important for the ATPase activity of PfHsp70-x. However, it was established that the EEVN motif was not important in regulating the holdase chaperone (protein aggregation suppression) function of PfHsp70-x. Furthermore, PfHsp70-x and its mutant preferentially bound to asparagine-rich peptides. Parasite proteins have high asparagine repeat regions as compared to human proteins. In addition, preference for asparagine-rich proteins
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could signify that PfHsp70-x is biased towards binding proteins of parasitic origin. Surface plasmon resonance (SPR) analysis suggested that PfHsp70-x interacts with human Hop with relatively higher affinity compared to its EEVN minus derivative. In conclusion, the removal of the EEVN motif of PfHsp70-x does not affect the chaperone function of PfHsp70-x. However, the EEVN motif is essential for the interaction of PfHsp70-x with human Hop.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:univen/oai:univendspace.univen.ac.za:11602/934 |
| Date | 09 1900 |
| Creators | Mabate, Blessing |
| Contributors | Shonhai, A., Zininga, T. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | 1 online resource (xiii, 78 leaves : color illustrations) |
| Rights | University of Venda |
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