Plasmodium falciparum Hep1 Is Required to Prevent the Self Aggregation of PfHsp70-3

Abstract

The majority of mitochondrial proteins are encoded in the nucleus and need to be imported from the cytosol into the mitochondria, and molecular chaperones play a key role in the efficient translocation and proper folding of these proteins in the matrix. One such molecular chaperone is the eukaryotic mitochondrial heat shock protein 70 (Hsp70); however, it is prone to self-aggregation and requires the presence of an essential zinc-finger protein, Hsp70-escort protein 1 (Hep1), to maintain its structure and function. PfHsp70-3, the only Hsp70 predicted to localize in the mitochondria of P. falciparum, may also rely on a Hep1 orthologue to prevent self-aggregation. In this study, we identified a putative Hep1 orthologue in P. falciparum and co-expression of PfHsp70-3 and PfHep1 enhanced the solubility of PfHsp70-3. PfHep1 suppressed the thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were indeed essential for maintaining the function of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Soluble and functional PfHsp70-3, acquired by co-expression with PfHep-1, will facilitate the biochemical characterisation of this particular Hsp70 protein and its evaluation as a drug target for the treatment of malaria.

Fig 1. Primary structure analysis and homology model of the zinc binding domain (zf-DNL) of PfHep1.

(A) Alignment of full-length PfHep1 with selected Hep orthologues from Leishmania braziliensis (LbHep1;XP_001565573.1), Homo sapiens (HsHep1; NM_001080849), Arabidopsis thaliana (AtZR3; AAO64784.1), Chlamydomonas reinhardtii (CrHep2; XP_001700157.1) and Saccharomyces cerevisiae (yHep1; NP_014089.2), where the mitochondrial/chloroplast signalling peptide (M/CSP) for each protein is shown in dark grey, and the zinc binding domain (zf-DNL) is shown in black. Degree of amino acid conservation is symbolized by the following: (*) all fully conserved residues; (:) one of the residues is fully conserved and (.) residues are weakly conserved. The conserved cysteine residues are highlighted with red boxes, and the residues implicated in facilitating interaction with their Hsp70 chaperone partner are highlighted with green boxes. (B) Structure of the zf-DNL of PfHep1 was modelled using the yeast Hep1 (Zim17/Tim15) structure (PBD accession no. 2E2Z) as the template and generated using the online Swiss Model program [37]. (C) Structure of the zf-DNL of yeast Hep1. The zinc ion is shown in red. Models were rendered using PyMol [38]. The tetracysteine motifs implicated in zinc chelation are shown as ball and stick.

Fig 2. PfHep1 enhanced the solubility of PfHsp70-3 and facilitated native purification.

(A) SDS-PAGE (10%) analysis of the solubility of PfHsp70-3 in the presence and absence of PfHep1. Lane M: protein markers in kDa, lane 1: E. coli BL21(DE3) [pQE30-PfHsp70-3] 4 hrs post IPTG induction (total protein), lanes 2–3: supernatant and pellet fractions of cells harvested and lysed 4 hrs post IPTG induction, lane 4: E. coli BL21(DE3) [pQE30-PfHsp70-3; pACYCDuet1-PfHep1] 4 hrs post IPTG induction (total protein), lanes 5–6: supernatant and pellet fractions from lysed cells co-transformed with pQE30-PfHsp70-3 and pACYCDuet-1-PfHep1 4 hrs post IPTG induction. (B) Western analysis using anti-His antibody. (C) SDS-PAGE (10%) analysis of the purification of PfHsp70-3, after co-expression with PfHep1, by nickel affinity chromatography. Lane M: protein markers in kDa, lane 1: E. coli BL21 (DE3) [pQE30-PfHsp70-3; pACYCDuet1-PfHep1] 4hrs post IPTG induction, lane 2: fraction unbound to the cOmplete His-tag purification resin, lanes 3–5: washes containing 50 mM imidazole, lanes 6–8: elutions of PfHsp70-3 and PfHep1 using 750 mM imidazole, lane 9: bead fraction. (D) Western analysis for detection of PfHsp70-3 (top) and PfHep1 (bottom) using anti-His antibodies.

Fig 3. Solubilization and purification of PfHep1.

(A) SDS-PAGE (10%) analysis of the solubility of PfHep1 before and after addition of sarcosyl. Lane M: protein markers in kDa, lanes 1–2: supernatant and pellet fractions of cells not treated with sarcosyl, lanes 3–4: supernatant and pellet fraction of cells treated with 3% sarcosyl. (B) Western analysis using anti-His antibody. (C) Purification of PfHep1 after solubilization with 3% sarcosyl, lane M: protein markers in kDa, lane 1: E. coli M15 ([pREP4]; pQE30-PfHep1) 4 hrs post IPTG induction, lane 2: fraction unbound to the cOmplete His-tag purification resin, lanes 3–5: washes using 50 mM imidazole, lanes 6–8: elutions of PfHep1 using 750 mM imidazole, and lane 9: bead fraction. (D) Western analysis for detection of PfHep1 using anti-His antibodies.

Fig 4. PfHep1 prevented the thermal aggregation of PfHsp70-3.

The thermal aggregation of PfHsp70-3 (2 μM) was initiated by incubation at 50°C for 30 min and monitored by light scattering at 360 nm. Varying concentrations of PfHep1 were added to PfHsp70-3. PfHep1 suppressed the aggregation of PfHsp70-3 in a dose-dependent manner, with equimolar concentrations of PfHep1 and PfHsp70-3 resulting in complete aggregation suppression. PfHep1 after chelation of zinc ions by EDTA (E_PfHep1) aggregated and consequently was unable to suppress the aggregation of PfHsp70-3. The combined data of three independent experiments conducted in triplicate on three independent batches of protein are shown. The bars represent standard deviations. A statistically significant difference between a reaction and PfHsp70-3 alone is indicated by * (p>0.05) above the reaction using a Student’s t-test.

Fig 5. PfHep1 did not prevent the aggregation of MDH or citrate synthase.

(A) MDH aggregation suppression assays were initiated by the addition of 0.72 μM MDH to assay buffer at 45°C for 30 min and monitored by light scattering at 360 nm. Varying concentrations of PfHep1 resulted in less than 10% suppression of MDH aggregation. (B) 0.15μM of citrate synthase was incubated at 45°C for 30 mins in both the absence and presence of varying concentrations of PfHep1 and PfHsp70-3. PfHep1 resulted in less than 3% suppression of CS aggregation. PfHsp70-3 suppressed the aggregation of both MDH and CS in a dose-dependent manner. PfHep1 did not enhance the ability of PfHsp70-3 to suppress MDH or CS aggregation and did not display intrinsic co-chaperone activity. The combined data of three independent experiments conducted in triplicate on three independent batches of protein are shown for both MDH and CS. The bars represent standard deviations. A statistically significant difference between a reaction and MDH alone is indicated by * (p>0.05) above the reaction using a Student’s t-test. The addition of PfHep1 to PfHsp70-3 and substrate did not produce a statistically different decrease in aggregation compared to PfHsp70-3 and substrate.