Papers by Stanley Makumire
Proposed model illustrating the effects of citrate-coated gold nanoparticles in vitro and in <i>E</i>. <i>coli</i> cells
<p>The model describes the proposed effects of single species versus agglomerated species o... more <p>The model describes the proposed effects of single species versus agglomerated species of citrate-gold nanoparticles on the integrity of proteins in vitro and in <i>E</i>. <i>coli</i> cells that are deficient of DnaK and in which DnaK function is restored.</p
TEM and HRTEM images of synthesised citrate-coated gold nanoparticle 0.3 mM gold salt was reduced with 136 mM tri-sodium citrate
<p>(<b>A</b>) Absorption spectra of citrate capped AuNPs; the insert shows the ... more <p>(<b>A</b>) Absorption spectra of citrate capped AuNPs; the insert shows the red wine suspensions obtained. (<b>B</b>) TEM images of the citrate AuNPs (top panel), and HRTEM images of AuNPs (lower panel). The presence of visibly defined lattice fringes confirmed the crystal morphology of the nanoparticles produced. (<b>C</b>) Bar graph representing the frequency of the citrate capped AuNPs by size.</p
Spectrophotometric analysis for the heat-induced aggregation of MDH in the presence of AuNPs and Hsp70
<p>1 μM MDH was suspended in assay buffer in the absence or mixed with high concentrations ... more <p>1 μM MDH was suspended in assay buffer in the absence or mixed with high concentrations of AuNPs (0–100 μgmL<sup>-1</sup>) (<b>a</b>); the assay was repeated in the presence of 1.3 μM Hsp70 (<b>b</b>). The suspensions were subjected to heat stress at 48°C for 20 minutes. Absorbance values were measured at 340 nm in triplicates using a 96-well micro titre plate. Data are presented as mean and standard deviations.</p

Comparative analysis of the effects of citrate AuNPs on <i>E</i>. <i>coli</i> DnaK<sup>-</sup> and <i>E</i>. <i>coli</i> DnaK<sup>+</sup> cells
<p>TEM images showing <i>E</i>. <i>coli</i> DnaK<sup>-</su... more <p>TEM images showing <i>E</i>. <i>coli</i> DnaK<sup>-</sup> and <i>E</i>. <i>coli</i> DnaK<sup>+</sup> cells exposed to citrate-AuNPs. A comparative population overview of <i>E</i>. <i>coli</i> DnaK<sup>-</sup> cells cultured in the presence of AuNPs is shown (panel A1) versus <i>E</i>. <i>coli</i> DnaK<sup>+</sup> cells cultured under similar conditions (panel A2). Panels B1-B2 and C1-C2 illustrate comparative morphological features of <i>E</i>. <i>coli</i> DnaK<sup>-</sup> cells versus <i>E</i>. <i>coli</i> DnaK<sup>+</sup> cells, respectively. AuNP aggregates are seen as black spots inside the cells. Note the evident delineation zones in panels C2 and D2, associated with <i>E</i>. <i>coli</i> DnaK<sup>+</sup> cells that are missing in <i>E</i>. <i>coli</i> DnaK<sup>-</sup> cells (panels C1and D1).</p

RESEARCH ARTICLE DnaK Protein Alleviates Toxicity Induced by Citrate-Coated Gold Nanoparticles in Escherichia coli
A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are cap... more A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are capable of stabilising proteins against heat stress in vitro. However, it remains to be un-derstood if AuNPs confer stability to proteins against cellular stress in vivo. Heat shock pro-teins (Hsps) are conserved molecules whose main role is to facilitate folding of other proteins (chaperone function). Hsp70 (called DnaK in prokaryotes) is one of the most promi-nent molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E. coli BB1553 cells that possess a deleted dnaK gene. We further investigated the effects of the AuNPs on the solubility of the E. coli BB1553 proteome. E. coli BB1553 cells exposed to AuNPs exhibited cellular defects such as filamentation and plasma membranes pulled off the cell wall. The toxic effects of the AuNPs were alleviated by transforming the E. coli B...

Molecules (Basel, Switzerland), Jan 5, 2017
Heat shock proteins (Hsps), amongst them, Hsp70 and Hsp90 families, serve mainly as facilitators ... more Heat shock proteins (Hsps), amongst them, Hsp70 and Hsp90 families, serve mainly as facilitators of protein folding (molecular chaperones) of the cell. The Hsp70 family of proteins represents one of the most important molecular chaperones in the cell., the main agent of malaria, expresses six Hsp70 isoforms. Two (PfHsp70-1 and PfHsp70-z) of these localize to the parasite cytosol. PHsp70-1 is known to occur in a functional complex with another chaperone, PfHsp90 via a co-chaperone,Hsp70-Hsp90 organising protein (PfHop). (-)-Epigallocatechin-3-gallate (EGCG) is a green tea constituent that is thought to possess antiplasmodial activity. However, the mechanism by which EGCG exhibits antiplasmodial activity is not fully understood. A previous study proposed that EGCG binds to the N-terminal ATPase domain of Hsp70. In the current study, we overexpressed and purified recombinant forms of twocytosol localized Hsp70s (PfHsp70-1 and PfHsp70-z), and PfHop, a co-chaperone of PfHsp70-1. Using th...

PloS one, 2015
A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are cap... more A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are capable of stabilising proteins against heat stress in vitro. However, it remains to be understood if AuNPs confer stability to proteins against cellular stress in vivo. Heat shock proteins (Hsps) are conserved molecules whose main role is to facilitate folding of other proteins (chaperone function). Hsp70 (called DnaK in prokaryotes) is one of the most prominent molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E. coli BB1553 cells that possess a deleted dnaK gene. We further investigated the effects of the AuNPs on the solubility of the E. coli BB1553 proteome. E. coli BB1553 cells exposed to AuNPs exhibited cellular defects such as filamentation and plasma membranes pulled off the cell wall. The toxic effects of the AuNPs were alleviated by transforming the E. coli BB15...

PLOS ONE, 2015
Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria... more Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe PLOS ONE |

Proteins: Structure, Function, and Bioinformatics, 2018
Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein ... more Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein 70 (Hsp70) family. Hsp70s serve as protein folding facilitators in the cell. Amongst the six Hsp70 species that P. falciparum expresses, Hsp70-x (PfHsp70-x), is partially exported to the host red blood cell where it is implicated in host cell remodeling. Nearly 500 proteins of parasitic origin are exported to the parasite-infected red blood cell (RBC) along with PfHsp70-x. The role of PfHsp70-x in the infected human RBC remains largely unclear. One of the defining features of PfHsp70-x is the presence of EEVN residues at its C-terminus. In this regard, PfHsp70-x resembles canonical eukaryotic cytosol-localized Hsp70s which possess EEVD residues at their C-termini in place of the EEVN residues associated with PfHsp70-x. The EEVD residues of eukaryotic Hsp70s facilitate their interaction with co-chaperones. Characterization of the role of the EEVN residues of PfHsp70-x could provide insights into the function of this protein. In the current study, we expressed and purified recombinant PfHsp70-x (full length) and its EEVN minus form (PfHsp70-x T ). We then conducted structure-function assays towards establishing the role of the EEVN motif of PfHsp70-x. Our findings suggest that the EEVN residues of PfHsp70-x are important for its ATPase activity and chaperone function. Furthermore, the EEVN residues are crucial for the direct interaction between PfHsp70-x and human Hsp70-Hsp90 organizing protein (hHop) in vitro. Hop facilitates functional cooperation between Hsp70 and Hsp90. However, it remains to be established if PfHsp70-x and hHsp90 cooperate in vivo. asparagine repeat rich peptide, chaperone, EEVN, heat shock proteins, human hop, PfHsp70-x Malaria persists as a major cause of morbidity and deaths around the world, with the latest data showing that it accounts for ~450 000 deaths annually. 1 Plasmodium falciparum is the most virulent of all the species that cause malaria. It is during the development of the parasite at the blood stage that clinical malaria manifests. In addition, the development of clinical malaria is associated with periodic fever conditions. As part of its response to physiological changes, the malaria parasite is thought to employ its arsenal of heat shock proteins (Hsps). Hsps are molecular chaperones that assist in folding of other proteins. Hsp70 constitute one of the main molecular chaperones of the cell. Structurally, Hsp70 is composed of a conserved N-terminal (ATPase) domain and a less conserved C-terminal substrate binding domain (SBD). Most cytosolic Hsp70s, possess an EEVD motif positioned at the end of the SBD. The EEVD motif is thought to play a role in the interaction of Hsp70 with its cochaperones such as members of the Hsp40 family and another distinct co-chaperone, Hsp70-Hsp90
Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers
PLOS ONE
Bioprospecting for Novel Heat Shock Protein Modulators: The New Frontier for Antimalarial Drug Discovery?
Advances in Experimental Medicine and Biology

International Journal of Molecular Sciences
Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised cano... more Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elev...

Heat shock proteins (Hsps) play an important role in the development and pathogenicity of
malaria... more Heat shock proteins (Hsps) play an important role in the development and pathogenicity of
malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of
other proteins. Hsps are thought to play a crucial role when malaria parasites invade their
host cells and during their subsequent development in hepatocytes and red blood cells. It
is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria
parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is
capable of independent folding of some proteins, its functional cooperation with heat shock
protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone
receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs
through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously
characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the
protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the
blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a
stress-inducible protein. We further explored the direct interaction between PfHop and
PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction
of the two proteins was further validated by co-immunoprecipitation studies. We
observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP
or ADP. However, ADP appears to promote the association of the two proteins better than
ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains
and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of
PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted
with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally
eluted as a protein species of twice its predicted size, suggesting that it may occur as a
dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association
in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a
stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition,
the protein is capable of self-association. The findings suggest that PfHop serves as a module
that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional
complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this
study are important towards the development of possible antimalarial inhibitors targeting
the cooperation of these two chaperones.

DnaK Protein Alleviates Toxicity Induced by Citrate-Coated Gold Nanoparticles in Escherichia coli
PloS one, 2015
A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are cap... more A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are capable of stabilising proteins against heat stress in vitro. However, it remains to be understood if AuNPs confer stability to proteins against cellular stress in vivo. Heat shock proteins (Hsps) are conserved molecules whose main role is to facilitate folding of other proteins (chaperone function). Hsp70 (called DnaK in prokaryotes) is one of the most prominent molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E. coli BB1553 cells that possess a deleted dnaK gene. We further investigated the effects of the AuNPs on the solubility of the E. coli BB1553 proteome. E. coli BB1553 cells exposed to AuNPs exhibited cellular defects such as filamentation and plasma membranes pulled off the cell wall. The toxic effects of the AuNPs were alleviated by transforming the E. coli BB15...

A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs)
are cap... more A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs)
are capable of stabilising proteins against heat stress in vitro. However, it remains to be understood
if AuNPs confer stability to proteins against cellular stress in vivo. Heat shock proteins
(Hsps) are conserved molecules whose main role is to facilitate folding of other
proteins (chaperone function). Hsp70 (called DnaK in prokaryotes) is one of the most prominent
molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in
vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E. coli
BB1553 cells that possess a deleted dnaK gene. We further investigated the effects of the
AuNPs on the solubility of the E. coli BB1553 proteome. E. coli BB1553 cells exposed to
AuNPs exhibited cellular defects such as filamentation and plasma membranes pulled off
the cell wall. The toxic effects of the AuNPs were alleviated by transforming the E. coli
BB1553 cells with a construct expressing DnaK. We also noted that cells in which DnaK
was restored exhibited distinct zones to which the nanoparticles were restricted. Our study
suggests a role for DnaK in alleviating nanoparticle induced stress in E. coli.
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Papers by Stanley Makumire
malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of
other proteins. Hsps are thought to play a crucial role when malaria parasites invade their
host cells and during their subsequent development in hepatocytes and red blood cells. It
is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria
parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is
capable of independent folding of some proteins, its functional cooperation with heat shock
protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone
receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs
through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously
characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the
protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the
blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a
stress-inducible protein. We further explored the direct interaction between PfHop and
PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction
of the two proteins was further validated by co-immunoprecipitation studies. We
observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP
or ADP. However, ADP appears to promote the association of the two proteins better than
ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains
and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of
PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted
with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally
eluted as a protein species of twice its predicted size, suggesting that it may occur as a
dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association
in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a
stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition,
the protein is capable of self-association. The findings suggest that PfHop serves as a module
that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional
complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this
study are important towards the development of possible antimalarial inhibitors targeting
the cooperation of these two chaperones.
are capable of stabilising proteins against heat stress in vitro. However, it remains to be understood
if AuNPs confer stability to proteins against cellular stress in vivo. Heat shock proteins
(Hsps) are conserved molecules whose main role is to facilitate folding of other
proteins (chaperone function). Hsp70 (called DnaK in prokaryotes) is one of the most prominent
molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in
vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E. coli
BB1553 cells that possess a deleted dnaK gene. We further investigated the effects of the
AuNPs on the solubility of the E. coli BB1553 proteome. E. coli BB1553 cells exposed to
AuNPs exhibited cellular defects such as filamentation and plasma membranes pulled off
the cell wall. The toxic effects of the AuNPs were alleviated by transforming the E. coli
BB1553 cells with a construct expressing DnaK. We also noted that cells in which DnaK
was restored exhibited distinct zones to which the nanoparticles were restricted. Our study
suggests a role for DnaK in alleviating nanoparticle induced stress in E. coli.