Overexpression, Purification and Characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) Protein

Abstract

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in 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.

Structural and biochemical characterization of Plasmodium falciparum Hsp70‐x reveals functional versatility of its C‐terminal EEVN motif

“…The ATPase activity of PfHsp70‐x was determined by quantifying the amount of released inorganic phosphate based on a previously described approach . Briefly, 0.4 μM PfHsp70‐x was incubated for 5 minutes in buffer HKM (10 mM HEPES‐KOH pH 7.5, 100 mM KCl, 2 mM MgCl 2 , 0.5 mM DTT).…”

“…The chaperone function of PfHsp70‐x and PfHsp70‐x T was further monitored using a second aggregation prone model protein, malate dehydrogenase (MDH) from porcine heart (Sigma–Aldrich, USA) as previously described . The MDH aggregation assay was conducted under the same experimental conditions as described for luciferase above.…”

“…Association of PfHsp70‐x/PfHsp70‐x T with synthetic peptides that represent typical Hsp70 substrates was investigated using surface plasmon resonance (SPR) analysis as previously described . Briefly, PfHsp70‐x and PfHsp70‐x T were immobilized onto respective ProteOn GLC sensor chips as ligands.…”

“…The direct association between PfHsp70‐x and hHop was further explored using SPR analysis. The assay was conducted at 25°C using filter sterilized and degassed PBS‐Tween running buffer [(8 mM Na 2 HPO 4 , 1.46 mM KH 2 PO 4 , 137 mM NaCl, 3 mM KCl, 0.005% [v/v] Tween 20 and 20 mM EDTA; pH 7.4)] as previously described . The recombinant PfHsp70‐x /PfHsp70‐x T (1 μg mL −1 ) and BSA (as control) were immobilized onto respective ProteOn GLC sensor chips as ligands.…”

PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance inPlasmodium falciparum

“…Synchronized ring stage parasites were exposed to two different physiological stress conditions; temperature (40°C) and drug (30 nM artemisinin) exposure for 6 h. In order to confirm the stress response we looked at the expression level of marker genes, which are known to be upregulated during stress conditions in Plasmodium . For temperature stress we looked at the expression level of the heat shock protein, HSP70 (S3A Fig) [13, 41]. Since the artemisinin is known to induce oxidative stress through production of reactive oxygen species, we confirmed the oxidative stress by validating the expression levels of glutathione S-transferase and superoxide dismutase (S3A Fig) [42].…”