Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity.

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.

Immunomodulatory activity of zinc peroxide (ZnO2) and titanium dioxide (TiO2) nanoparticles and their effects on DNA and protein integrity.

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-ß 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.