Papers by Addmore Shonhai
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of t... more Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the costeffective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem ® . In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.
Molecular Genetics and Genomics, Jun 23, 2005
Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that e... more Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp 70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp 70 was able to suppress the thermosensitivity of an Escherichia coli dnaK 756 strain, but not that of the corresponding deletion strain (DeltadnaK 52) or dnaK 103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp 70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp 70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK 756 strain. We also identified two key amino acid residues (V 401 and Q 402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp 70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp 70 and PfHsp 70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp 40 chaperone machinery.
The International Journal of Biochemistry & Cell Biology
Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establish... more Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp7...
American Journal of Biochemistry and Biotechnology, 2014
ABSTRACT Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups o... more ABSTRACT Heat 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.
Protein Expression and Purification, 2011
Molecular chaperones have been used for the improved expression of target proteins within heterol... more Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93 nmol/hr/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.
Heat Shock Proteins of Malaria, 2013
Protein & Peptide Letters, 2008
The spatial orientation of domains of the heat shock protein 70 from Plasmodium falciparum (PfHsp... more The spatial orientation of domains of the heat shock protein 70 from Plasmodium falciparum (PfHsp70) were mapped based on a three-dimensional model of the protein. Purified PfHsp70 displayed chaperone activity in vitro. Amino acid substitutions introduced in the chaperone's substrate binding cavity compromised the protein's chaperone function.
Heat Shock Proteins of Malaria, 2013
Zimbabwe Veterinary Journal, 2004
Toxicology Letters, 2014
Nanoparticles that are made from zinc and titanium oxide have found widespread applications, incl... more Nanoparticles that are made from zinc and titanium oxide have found widespread applications, including their use in sunscreens. However, there is little information regarding their effects on immune cells. In the current study, we synthesized charge stabilized and "ligand free" colloid stable ZnO₂ and TiO₂ nanoparticles. Most previous published studies commonly used ZnO and TiO₂ nanoparticles. In the current study we investigated the comparative toxicity of ZnO₂ and TiO₂ nanoparticles. Therefore, our results based on ZnO₂ which is more oxidative than ZnO provides novel data on the possible toxicity of this species of nanoparticles. First, we investigated the immunomodulatory action of these nanoparticles on human peripheral blood mononuclear cells and their effects on DNA and protein integrity. A minimum concentration of ZnO₂ nanoparticles of 1 μg/mL inhibited the production of two inflammatory cytokines: interleukin-1-β and interleukin 6 by peripheral blood mononuclear cells in the presence of lipopolysaccharides. On the other hand, TiO₂ nanoparticles at a concentration range of 0.1-100 μg/mL did not present apparent toxicity to the peripheral blood mononuclear cells. ZnO₂ nanoparticles at a minimum concentration of 2 μg/mL caused DNA damage in vitro. TiO₂ nanoparticles at a concentration range of 25-100 μg/mL only caused marginal DNA damage. ZnO₂ nanoparticles at a minimum concentration of 5 μg/mL were capable of promoting aggregation of malate dehydrogenase, and facilitated its degradation at higher concentrations. Exposure of malate dehydrogenase to TiO₂ at a concentration range of 2.5-15 μg/mL did not alter the solubility of malate dehydrogenase. Altogether, our findings suggest that charge stabilized ZnO₂ nanoparticles are nascent and interact with DNA and protein and may be harmful to immune cells. In addition, the propensity of ZnO₂ nanoparticles to promote protein aggregation could facilitate the production of protein complexes that may interfere with normal immune functions.
The International Journal of Biochemistry & Cell Biology, 2008
Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establish... more Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp70-1, were both localised to the parasites nucleus and cytoplasm. In some cases, Pfj4 was also detected in the erythrocyte cytoplasm of infected erythrocytes. Immunoprecipitation studies and size exclusion chromatography indicated that Pfj4 and PfHsp70-1 may directly or indirectly interact. Our results suggest a possible involvement of Pfj4 together with PfHsp70-1 in cytoprotection, and therefore, parasite survival inside the erythrocyte.
Protein Science, 2007
It is becoming increasingly apparent that heat shock proteins play an important role in the survi... more It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.
Protein & Peptide Letters, 2011
Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan 37 parasites (Plas... more Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan 37 parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading 38 human cells as part of their pathology. These parasites appear to have evolved a 39 relatively expanded or diverse complement of genes encoding molecular chaperones. 40
Molecular Genetics and Genomics, 2005
Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that e... more Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp 70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp 70 was able to suppress the thermosensitivity of an Escherichia coli dnaK 756 strain, but not that of the corresponding deletion strain (DeltadnaK 52) or dnaK 103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp 70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp 70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK 756 strain. We also identified two key amino acid residues (V 401 and Q 402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp 70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp 70 and PfHsp 70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp 40 chaperone machinery.
Malaria Journal, 2008
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of t... more Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the costeffective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem ® . In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.
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Papers by Addmore Shonhai