Comparative characterisation of<i>Plasmodium falciparum</i>Hsp70-1 relative to<i>E. coli</i>DnaK reveals functional specificity of the parasite chaperone

Lebepe, Charity Mekgwa; Matambanadzo, Pearl Rutendo; Makhoba, Xolani Henry; Achilonu, Ikechukwu; Zininga, Tawanda; Shonhai, Addmore

doi:10.1101/2020.03.09.984104

2020

DOI: 10.1101/2020.03.09.984104

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Comparative characterisation ofPlasmodium falciparumHsp70-1 relative toE. coliDnaK reveals functional specificity of the parasite chaperone

Charity Mekgwa Lebepe

Pearl Rutendo Matambanadzo

Xolani Henry Makhoba

et al.

Abstract: Hsp70 is one of the most prominent molecular chaperones. Although Hsp70s from various organisms are generally conserved, they exhibit specialised cellular functions. It remains to be fully understood how these highly conserved molecules exhibit specialised functional features. Plasmodium falciparum Hsp70-1 (PfHsp70-1) is a cytosol localised molecular chaperone that is implicated in the cyto-protection and pathogenicity of the malaria parasite. In the current study, we investigated the comparative structure-fun… Show more

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Cited by 2 publications

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Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

Makumire

Dongola

Chakafana

et al. 2021

IJMS

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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 elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.

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Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

Makumire

Dongola

Chakafana

et al. 2021

IJMS

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Characterization of an Atypical Trypanosoma brucei Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue

Burger

Macucule-Tinga

Bentley

et al. 2021

IJMS

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Trypanosoma brucei (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against T. brucei.

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Comparative characterisation ofPlasmodium falciparumHsp70-1 relative toE. coliDnaK reveals functional specificity of the parasite chaperone

Cited by 2 publications

References 63 publications

Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

Characterization of an Atypical Trypanosoma brucei Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue

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