Overexpression, purification and characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) protein.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur i0n the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.This project was through a grant (L1/402/ 14-1) provided to AS by the Deutsche Forchungsgemeinshaft (DFG) under the theme, “German–African Cooperation Projects in Infectology”. The authors are grateful to the Department of Science and Technology/National Research Foundation (NRF) of South Africa for providing an equipment grant (grant UID, 75464) to AS and (grant UID, 78558) to EP. HWD was awarded a research grant (64788) by the NRF (South Africa). AS is a recipient of a Georg Foster researchNCS201

Overexpression, purification and characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) protein.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur i0n the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.This project was through a grant (L1/402/ 14-1) provided to AS by the Deutsche Forchungsgemeinshaft (DFG) under the theme, “German–African Cooperation Projects in Infectology”. The authors are grateful to the Department of Science and Technology/National Research Foundation (NRF) of South Africa for providing an equipment grant (grant UID, 75464) to AS and (grant UID, 78558) to EP. HWD was awarded a research grant (64788) by the NRF (South Africa). AS is a recipient of a Georg Foster researchNCS201

Supporting data on characterisation of linker switch mutants of Plasmodium falciparum heat shock protein 110 and canonical Hsp70

Here, we present data on characterisation of the linker of Plasmodium falciparum Hsp110 (PfHsp70-z) relative to the linker of canonical Hsp70s in support of a co-published article [1]. The linker of PfHsp70-z was switched with that of canonical Hsp70s, represented by PfHsp70–1 (cytosolic counterpart of PfHsp70-z) and E. coli Hsp70/DnaK. The datasets represent comparative analyses of PfHsp70-z, PfHsp70–1, and E. coli DnaK, relative to their linker switch mutants; PfHsp70-zLS, PfHsp70–1LS, DnaKLS, respectively. Intrinsic and extrinsic fluorescence spectroscopic analyses were employed to elucidate effects of the mutations on the structural features of the proteins. The structural conformations of the proteins were analysed in the absence as well as presence of nucleotides. In addition, stability of the proteins to stress (pH changes and urea) was also determined. Surface plasmon resonance (SPR) was employed to determine affinity of the proteins for ATP. The relative affinities of PfHsp70-z and PfHsp70–1 for the parasite cytosol localised, J domain co-chaperone, PfHsp40, was determined by SPR analysis. The effect of the linker of PfHsp70-z on the interaction of DnaKLS with DnaJ (a co-chaperone of DnaK), was similarly determined. These data could be used for future investigations involving protein-protein/ligand interactions as described in [1]. The raw data obtained using the various techniques here described are hosted in the Mendeley Data repository at [2]

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

Abstract Plasmodium falciparum causes the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is thought to facilitate folding of select group of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organising protein (Hop). P. falciparum Hop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70‐1 and PfHsp90. Here, we characterised the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40°C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70‐1, that is reportedly stable at temperatures as high as 80°C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90

Phenolic contents, antioxidant activity and spectroscopic characteristics of <em>Pterocarpus angolensis </em>DC. stem bark fractions

400-406Oxidative stress has been implicated in the damage of biological molecules resulting in aging and diseases such as Alzheimer, cancer, diabetes, cardiovascular disorders. The study aimed at determining the phenolic contents and antioxidant activities of Pterocarpus angolensis crude extract and fractions. The crude extract and fractions of P. angolensis were evaluated for their phenolic contents using Follin-Ciocalteu reagent. The antioxidant activities were evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and reducing power assays. Ultraviolet-Visible and Fourier transform infrared spectroscopy were used to assess the spectroscopic characteristics. We obtained 7 fractions from the crude extracts after column chromatography on silica gel 60. The results showed that fraction two (PaF2a) displayed the highest DPPH radical scavenging activity (18.7 &micro;g/ml) but had the lowest phenolic content while fraction three (PaF2b) exhibited the highest reducing power activity (44.28 &micro;g/ml) with high phenolic content. Spectroscopic details showed that PaF2a had maximum absorbance at 287.1 nm while PaF2b displayed maximum absorbance at 288.2 nm. The infra-red spectroscopy presented four main characteristic fingerprinting at 1606, 1518, 1444 and 1064 cm-1 as fingerprints for P. angolensis. There was a difference in the wave number at C==C and C-O vibrations between PaF2a and PaF2b. In conclusion, this study has shown that PaF2a and PaF2b are the antioxidant rich fractions of P. angolensis stem bark and exhibited different spectroscopic characteristics

Comparative in vitro cytotoxic, anti-inflammatory and anti-microbiological activities of two indigenous Venda medicinal plants

Department of MicrobiologyBackground: The Vhembe region of the Limpopo province has a rich tradition of medicinal plants use. Traditionally, boiled roots of Ziziphus mucronata are used in the treatment of boils, general swelling and other skin infections. A combination of leaf paste and root infusion treats measles, dysentery, chest complains, and gland swelling. Pterocarpus angolensis is famous for the treatment of menorrhagia, infertility in women, wounds and pain management. The purpose of the present study was to compare the cytotoxicity, anti-inflammatory potential and anti-microbial activities of Ziziphus mucronata and Pterocarpus angolensis from the Vhembe region. Method: U937, MeWo, Vero and RAW 264.7 cells were treated to various concentrations (50, 100, or 125 or 250 μg/ml depending on assays) of Ziziphus mucronata and Pterocarpus angolensis. Cytotoxicity assay was done using MTT; Antiinflammatory activity was assessed using NO production; Anti-bacterial activity was done using the Micro-Broth dilution method and Anti-mycobacteria activity was determined using the Alamar Blue Method while RT activity was measured by ELISA. Results: Cytotoxicity results showed that Pterocarpus was more toxic than Ziziphus as observed in the Vero and MeWo cells; however both displayed toxicity towards a Human cancer cell line. Both extracts did not inhibit nitrate production but induced significant increase in macrophage activation. The plant extracts have shown anti-tuberculosis activity at concentrations >500 μg/ml and there was moderation inhibition of HIV replication. Conclusions: The results obtained indicated that the extracts have pro-inflammatory properties, and the observed toxicity on malignant cell lines must be investigated further for promising anti-cancer drug therapy.University of Venda, Research and Innovation Directorate(S/15/MBY10

Extracts Obtained from Pterocarpus angolensis DC and Ziziphus mucronata Exhibit Antiplasmodial Activity and Inhibit Heat Shock Protein 70 (Hsp70) Function

Malaria parasites are increasingly becoming resistant to currently used antimalarial therapies, therefore there is an urgent need to expand the arsenal of alternative antimalarial drugs. In addition, it is also important to identify novel antimalarial drug targets. In the current study, extracts of two plants, Pterocarpus angolensis and Ziziphus mucronata were obtained and their antimalarial functions were investigated. Furthermore, we explored the capability of the extracts to inhibit Plasmodium falciparum heat shock protein 70 (Hsp70) function. Heat shock protein 70 (Hsp70) are molecular chaperones whose function is to facilitate protein folding. Plasmodium falciparum the main agent of malaria, expresses two cytosol-localized Hsp70s: PfHsp70-1 and PfHsp70-z. The PfHsp70-z has been reported to be essential for parasite survival, while inhibition of PfHsp70-1 function leads to parasite death. Hence both PfHsp70-1 and PfHsp70-z are potential antimalarial drug targets. Extracts of P. angolensis and Z. mucronata inhibited the basal ATPase and chaperone functions of the two parasite Hsp70s. Furthermore, fractions of P. angolensis and Z. mucronata inhibited P. falciparum 3D7 parasite growth in vitro. The extracts obtained in the current study exhibited antiplasmodial activity as they killed P. falciparum parasites maintained in vitro. In addition, the findings further suggest that some of the compounds in P. angolensis and Z. mucronata may target parasite Hsp70 function