Identification of heat shock factor binding protein in Plasmodium falciparum

Abstract

Background: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum.

Methods: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum.

Results: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite.

Conclusions: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

Figure 1

Bioinformatics analysis of PfHSBP. (A) Sequence alignment of PfHSBP with related HSBPs from other organisms. (B) Phylogenetic tree for HSBP, shows PfHSBP is an ancestral protein to both plants and animal HSBPs. Optimal tree with sum of branch length = 2.72 is shown and bootstrap value is shown next to the branch. (C)PfHSBP models (Red), predicted using I-TASSER server online, aligned with HsHSBP (green).

Figure 2

In vitro characterization of PfHSBP. (A) 15% SDS-PAGE gel and immunoblot showing purified recombinant PfHSBP. Lanes:- TL - total lysate, SP - pellet remaining after sonication, SS -supernatant after sonication, FT -flow through, 20 mM - wash with 20 mM imidazole, 50 mM - wash with 50 mM imidazole, and 200 mM - elution with 200 mM imidazole. (B) 2-D-electrophoresis of purified recombinant protein. (C) MS-based identification of PfHSBP. (D) Circular dichroism spectrum of PfHSBP.

Figure 3

In vivo PfHSBP expression. (A) PfHSBP exists in P. falciparum as a trimer and hexamer (Lanes 1 and 3 -P. falciparum lysate; lanes 2 and 4 - purified recombinant protein. Lanes 1 and 2 were probed with polyclonal antibody generated against PfHSBP, whereas, lanes 3 and 4 were probed with pre-immune serum). (B) Size exclusion chromatography of PfHSBP. Immunoblot shows the fractions of P. falciparum lysate containing PfHSBP (top panel). The graph represents quantitation of PfHSBP signal obtained (bottom panel). (C) Immunoprecipitation by PfHSBP antiserum shows pull down of hexameric PfHSBP, Lane 1-Protein-G control, Lane 2- Pre immune serum control, Lane 3- PfHSBP IP. (D)PfHSBP expression pattern in different blood stages of P. falciparum. (R - Ring stage, T - Trophozoite stage, S - Schizont stage). β-actin was used as a loading control. Bar diagram shows quantitative amounts of PfHSBP present in different stages of P. falciparum.

Figure 4

PfHSBP undergoes nuclear translocation upon heat shock. (A) immunoblot analysis to examine PfHSBP expression under normal and heat shock conditions reveals that PfHSBP does not get heat induced; (B) immunoblot analysis of cytoplasmic and nuclear fractions to determine the localization of PfHSBP under normal and heat shock conditions; (C) relative amount of PfHSBP in cytoplasmic and nuclear compartments in normal and heat shock conditions. Upon heat shock amount of PfHSBP in nucleus increases; (D) IFA to determine the localization of PfHSBP under normal and heat shock conditions. (Cl - Control condition (37°C), HS - heat shock condition (42°C); C - cytoplasm, N - nucleus) reveals nuclear translocation of PfHSBP upon heat shock.

Figure 5

Co-immunoprecipitation with α-PfHSBP antiserum confirms PfHSBP-PfHsp70 interaction. (A) immunoprecipitation performed on P. falciparum lysate using α-HSBP antibody followed by immunoblot analysis with α-PfHSP70-1 and α-PfHSBP antibody. Signal for PfHSP70-1 in the immunoblot indicates interaction between the two proteins; (B) immunoprecipitation performed on nuclear and cytoplasmic fractions of P. falciparum using α-PfHSBP followed by α-PfHSP70-1 immunoblot. PfHSP70-1 signal is obtained in the cytoplasmic fraction.