85 research outputs found
Role of the Hsp40 Family of Proteins in the Survival and Pathogenesis of the Malaria Parasite
No full textThe role of molecular chaperones in Escherichia coli cells subjected to gold nanoparticles induced stress
No full textPhD (Biochemistry)Department of Biochemistry and MicrobiologyColloidal suspensions of gold particles of nanometre (nm) sizes are termed gold nanoparticles (AuNPs). Although stable, AuNPs have been reported to be toxic to E. coli cells by collapsing the bacterial cell membranes and promoting protein misfolding. An understanding of biodistribution in drug delivery and the effects AuNPs have on the function and structure of proteins such as heat shock proteins is important. Heat shock proteins facilitate protein folding and are particularly important during cellular stress. At high concentrations, AuNPs are thought to promote protein aggregation. Heat shock proteins are thought to alleviate cell stress induced by AuNPs. This study explored the role of heat shock proteins in conferring cytoprotection to E. coli against the effects of AuNPs. Citrate-AuNPs were synthesized and their integrity was validated at 520 nm by ultraviolet-visible-near infrared spectroscopy (UV-Vis-NIR). Crystallinity was confirmed by X-Ray diffraction (XRD), while dynamic light scattering (DLS) estimated the size distribution at 13 nm. Furthermore, transmission electron microscopy (TEM) and scanning electron microscope (SEM) revealed the spherical shape and crystal lattice surface morphology of citrate-AuNPs respectively. A complementation assay was conducted using cells deficient of DnaK function (E. coli ΔdnaK52). E. coli ΔdnaK52 was transformed with a recombinant dnaK before examining both DnaK deficient and transformed cells using TEM. E. coli O157:H7 was exposed to citrate-AuNPs (0 – 50 μg/ml) and allowed to grow at 37 oC before protein expression was analysed using electrophoresis followed by LC-MS analysis. This led to the identification of highly expressed proteins such as DnaK, GAPDH, ClpX, DnaJ, and GroEL. Subsequent co-affinity assay revealed possible interaction protein partners of DnaK. These were identified as ClpB, HtpG, GroEL, DnaJ, and SurA proteins. Furthermore, circular dichroism and fluorescence spectroscopy established that recombinant DnaK is stable at citrate-AuNPs concentrations less than 10 μg/ml and the protein was unstable at concentrations beyond 10 μg/ml citrate-AuNPs. In addition, the ATPase activity of recombinant DnaK increased in the presence of citrate-AuNPs at 2.5 μg/ml. The ability of DnaK to suppress aggregation of MDH in vitro was abrogated by the presence of >10 μg/ml citrate-AuNPs. The findings suggest that at low concentrations (10 μg/ml), citrate-AuNPs destabilizes protein conformation and function. Altogether the findings suggest that DnaK in cooperation with its network partners is implicated in E. coli cytoprotection against citrate-AuNPs toxicity.NR
Characterization of heat shock protein 70-z (PfHsp70-z) from plasmodium falciparium
No full textPhD (Biochemistry)Department of BiochemistryMalaria 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
Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and protease
No full textPhD (Microbiology)Department of MicrobiologyHigh genetic diversity is a major contributory factor in the development of drug resistance, in
addition to challenges in diagnosis and treatment monitoring in the therapeutics of human
immunodeficiency virus (HIV) .Within the wide HIV-1 diversity, differences in mutational
frequency, disease progression, drug response and transmission amongst HIV-1 subtypes
have been shown. In spite HIV-1 subtype C (HIV-1C) being the most prevalent variant globally,
none of the available drugs nor screening assays for inhibitory molecules have been developed
targeting the genetics of this important subtype. This study therefore aimed to overexpress and
biophysically characterize HIV-1C reverse transcriptase and protease to serve as reagents in
the development of assays for routine screening of molecules inhibitory to HIV-1C.
Heterologous expression of HIV-1C reverse transcriptase and protease isolates that are
prevalent in South Africa was carried out in Escherichia coli (E. coli (BL21-DE3). The secondary
and tertiary structures of the proteins were determined using, circular dichroism (CD) and
fluorescence spectroscopy respectively. Thereafter, interaction studies to delineate interaction
properties of natural products for possible inhibition of protease were conducted. Furthermore,
in silico studies to determine binding interactions, further confirmed by in vitro binding assays
of a pepsin inhibitor homolog (Bm-33) from Brugia malayi , against protease were also
conducted. Expressed reverse transcriptase and protease from the globally prevalent HIV-1C
were shown to be structurally and functionally intact for application in downstream HIV-1
inhibition assays. Interaction studies on the other hand revealed successful inhibition of the
expressed HIV-1C PR with gallotanin. Furthermore, binding interactions of Bm-33 and HIV-1
PR revealed the first intermolecular interactions of the two molecules displaying possible
inhibition of HIV-1 PRNR
Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70
No full textHeat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action
Role of the J Domain Protein Family in the Survival and Pathogenesis of Plasmodium falciparum
No full textPlasmodium falciparum has dedicated an unusually large proportion of its genome to molecular chaperones (2% of all genes), with the heat shock protein 40 (Hsp40) family (now called J domain proteins, JDPs) exhibiting evolutionary radiation into 49 members. A large number of the P. falciparum JDPs (PfJDPs) are predicted to be exported, with certain members shown experimentally to be present in the erythrocyte cytosol (PFA0660w and PFE0055c) or erythrocyte membrane (ring-infected erythrocyte surface antigen, RESA). PFA0660w and PFE0055c are associated with an exported plasmodial Hsp70 (PfHsp70-x) within novel mobile structures called J-dots, which have been proposed to be dedicated to the trafficking of key membrane proteins such as erythrocyte membrane protein 1 (PfEMP1). Well over half of the PfJDPs appear to be essential, including the J-dot PfJDP, PFE0055c, while others have been found to be required for growth under febrile conditions (e.g. PFA0110w, the ring-infected erythrocyte surface antigen protein [RESA]) or involved in pathogenesis (e.g. PF10_0381 has been shown to be important for protrusions of the infected red blood cell membrane, the so-called knobs). Here we review what is known about those PfJDPs that have been well characterised, and may be directly or indirectly involved in the survival and pathogenesis of the malaria parasite
The Role of Non-Canonical Hsp70s (Hsp110/Grp170) in Cancer
No full textAlthough cancers account for over 16% of all global deaths annually, at present, no reliable therapies exist for most types of the disease. As protein folding facilitators, heat shock proteins (Hsps) play an important role in cancer development. Not surprisingly, Hsps are among leading anticancer drug targets. Generally, Hsp70s are divided into two main subtypes: canonical Hsp70 (Escherichia coli Hsp70/DnaK homologues) and the non-canonical (Hsp110 and Grp170) members. These two main Hsp70 groups are delineated from each other by distinct structural and functional specifications. Non-canonical Hsp70s are considered as holdase chaperones, while canonical Hsp70s are refoldases. This unique characteristic feature is mirrored by the distinct structural features of these two groups of chaperones. Hsp110/Grp170 members are larger as they possess an extended acidic insertion in their substrate binding domains. While the role of canonical Hsp70s in cancer has received a fair share of attention, the roles of non-canonical Hsp70s in cancer development has received less attention in comparison. In the current review, we discuss the structure-function features of non-canonical Hsp70s members and how these features impact their role in cancer development. We further mapped out their interactome and discussed the prospects of targeting these proteins in cancer therapy
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