PhD (Biochemistry) / Department of Biochemistry / Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually,
mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae,
P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The
parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response
to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent
groups of chaperones whose role is central to protein homeostasis and determines the fate
of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70
genes encode for proteins that are localised in different sub-cellular compartments. Of these
two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum,
PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red
blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in
prevention of protein aggregation and facilitates protein folding. Little is known about
PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the
folding of proteins possessing asparagine rich repeats. However, based on structural evidence
PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide
exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional
roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study,
PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification
using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was
investigated and its association with PfHsp70-1 was explored using a co-immuno
precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress
and the protein is heat stable based on investigations conducted using Circular Dichroism.
Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1
were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was
observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and
PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence
analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the
conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to
suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro.
Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone
function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that
Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum
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PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z
may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of
functions of this protein, could explain this PfHsp70-z is an essential protein in malaria
parasite survival. This is the first study to show that PfHsp70-z possess independent
chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a
nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable
molecule and that it is capable of forming high order oligomers.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:univen/oai:univendspace.univen.ac.za:11602/619 |
| Date | January 2015 |
| Creators | Zininga, Tawanda |
| Contributors | Shonhai, Addmore, Burger, A. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 1 online resource (xviii, 172 leaves : color illustrations) |
| Rights | University of Venda |
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