Identification of heat shock factor binding protein in Plasmodium falciparum
- Syed K Sayeed†1,
- Varun Shah†1,
- Shweta Chaubey1,
- Meetali Singh1,
- Shuba V Alampalli1 and
- Utpal S Tatu1Email author
© Sayeed et al.; licensee BioMed Central Ltd. 2014
Received: 31 January 2014
Accepted: 12 March 2014
Published: 27 March 2014
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.
Pf HSBP 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 Pf HSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum.
Pf HSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. Pf HSBP 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. Pf HSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of Pf HSBP with Pf HSP70-1 in the cytoplasm of the parasite.
This study emphasizes the structural and biochemical conservation of Pf HSBP 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.
KeywordsHSBP HSF Malaria Plasmodium falciparum Heat shock response
The human malaria parasite, Plasmodium falciparum is exposed to wide ranges of temperature fluctuation during its life cycle. During transmission from the arthropod vector to the human host, it encounters around 12˚C switch in the environmental temperature. Moreover, the parasite has to adapt to temperature fluctuations due to the febrile episodes that occur during clinical manifestation of the disease. Considering the repeated heat stress conditions encountered by the parasite during its life cycle, presence of a robust heat shock response machinery is essential for its survival.
In eukaryotes, there are three main factors regulating heat shock response: (i) heat shock factors (HSF), which are transcription factors regulating heat shock protein (hsp) genes; (ii) heat shock elements (HSE), DNA binding motif for HSF binding, present upstream of heat inducible genes; and, (iii) heat shock proteins (HSP), which protect other cellular proteins and also help in the regulation of HSF. The regulation of heat shock response is also supported by certain accessory factors such as heat shock factor binding protein (HSBP), which are known to be involved in attenuation of the heat shock response. While P. falciparum is endowed with the presence of a repertoire of HSPs which play a critical role in the life cycle of the parasite, the mechanism of their induction is only partly understood[2–5]. For example, the heat shock transcription factor is yet to be identified in the parasite. Despite the seeming absence of HSF, we show here that P. falciparum possesses an HSBP, a known negative regulator of HSF.
Under stress conditions such as heat shock, HSF undergoes transition from monomer to active functional phosphorylated trimer and consequently leads to the induction of HSPs. Attenuation of heat shock response is believed to be mediated by binding of HSBP. In order to attenuate the heat shock response, HSF dissociates from active trimeric form to monomers and thus loses its DNA-binding activity. This shift in oligomeric status is brought about by binding of HSBP and HSP70 to HSF. In initial phase of attenuation, HSBP undergoes transition from hexameric to trimeric form and binds to active trimer of HSF and thus negatively regulates its activity[1, 6, 7]. Thereafter, HSBP also associates with HSP70. However, the precise roles of HSBP and Hsp70 in heat shock response remain obscure.
HSBP was first identified in a yeast two-hybrid screen using HSF as the bait. The HSBP domain is highly conserved across all species. The protein has been identified in all species except the budding yeast, Saccharomyces cerevisiae. Moreover, in plants, HSBP has also been implicated in seed and endosperm development and embryogenesis[8–10].
The HSBP family of proteins is highly conserved and contains hydrophobic heptad repeats characteristic of coiled-coil proteins and also self-associates leading to oligomerization. Changes in protein oligomerization status are known to be associated with regulation of various cellular processes. This particular phenomenon plays an important role in HSBP-HSF interactions. HSBP is usually localized in the nucleus[1, 6, 7]. Hsu and colleagues have reported that Arabidopsis thaliana HSBP localizes to the cytoplasm and translocates to the nucleus to participate in attenuation of heat shock response.
Despite the critical role of heat shock response in life cycle of malaria parasite, regulation of heat shock response in Plasmodium is poorly understood. Therefore, studies on Pf HSBP in absence of a canonical HSF can provide new insights into our understanding of mechanism of heat shock response in Plasmodium. In this study, characterization of Pf HSBP was performed to gain insights into its functional roles. Pf HSBP (PF3D7_1120900/PF11_0216) is an evolutionarily conserved protein with a high helical content. It shows 28% identity to human HSBP1 protein with the presence of characteristic HSBP core domain. This protein was found to be present as a homo-oligomer in P. falciparum and translocates to nucleus upon heat shock. The study also shows that Pf HSBP is capable of interacting with Pf HSP70-1. These results suggest that Pf HSBP is an integral part of heat shock response machinery of P. falciparum and its study will address the gap in our understanding of heat shock response in this parasite.
Plasmodium falciparum cultures
Plasmodium falciparum 3D7 strain was cultured in human O+ erythrocytes at 5% haematocrit in RPMI 1640 medium supplemented with 200 μM hypoxanthine, 0.2% (w/v) sodium bicarbonate, 0.2% (w/v) glucose and 0.5% (w/v) Albumax II. For stage-specific studies, parasites were tightly synchronized by 5% sorbitol treatment as described previously and isolated at ring (2–12 hours post infection/hpi), trophozoite (24–30 hpi) and schizont (36–48 hpi) stages. Control P. falciparum cultures were grown at 37°C. For heat shock, P. falciparum cultures were incubated at 42°C for one hour.
Bacterial strains, plasmids and growth conditions
Escherichia coli strains DH5α and BL21 (DE3) pLysS were cultured at 37°C in Luria broth. Recombinant strains were also cultured under similar conditions with appropriate antibiotics (ampicillin-100 μg/ml and chloramphenicol -34 μg/ml). The plasmid pRSETA was used for expression studies of the hsbp gene.
α-His-tag and α-histone antibodies were commercially purchased. α-His-tag antibody was used at 1:10,000 dilution; α-histone antibody was used at dilution of 1:500 and Horse radish peroxidase conjugated secondary antibody was used at 1:5,000 dilution for western blotting. α-Pf Hsp60 antibody was used in dilution 1:1,000. α-Pf HSBP polyclonal antiserum was raised in rat against the peptide ‘LSDNLLNKVDNMEKYLDELE’ from Pf HSBP sequence. α-Pf HSBP antibody was used at 1:500 dilution and anti-rat secondary antibody was used at 1:3,000 dilution for western blotting. For IFA, α-Pf HSBP antibody was used at 1:50 dilution and FITC conjugated anti-rat secondary antibody was used at 1:300 dilution. Animal handling was done adhering to the guidelines for animal handling at the Indian Institute of Science.
HSBP protein sequences were downloaded from NCBI database and aligned using MUSCLE algorithm in MEGA 6 suite. A phylogenetic analysis using aligned sequences was performed by the neighbor joining algorithm in MEGA 6.0 software. Bootstrap test with 500 replicates was performed and all positions containing gaps were deleted[11, 12]. Secondary structures of Pf HSBP and Hs HSBP were predicted using Coils, which calculates the probability of a structure to adopt a coiled-coil conformation. A three-dimensional model of the Pf HSBP was constructed by homology modelling. The human HSBP model prediction was performed using the I-TASSER (iterative threading assembly refinement) server online. The PyMOL program (The PyMOL Molecular Graphics System, Version 22.214.171.124 Schrödinger, LLC) was used to generate the figure for the model structure.
Cloning and purification of hsbp gene
Amplification of hsbp gene was carried out using primers Pf HSBP_BamHI_F (5′-CCGCGGGATCC ATGAATTTTAACGAAATGTTAAAGAG-3′) and Pf HSBP_SacI_R (5′-CCGCGGAGCTC TTACTGTAAATTATTTTGAGTTGTATAG-3′). The obtained 342 bp product was cloned into pRSETA (BamHI and SacI sites) and transformed into DH5α. To obtain higher expression the recombinant plasmid was transformed in E. coli BL21 (DE3) pLysS strain. The protein was over-expressed by induction with 0.5 mom IPTG for 16 hours at 16°C. The culture was lysed by sonication in 6 M Urea, 50 mM Tris-Cl (pH 7.5), 500 mM NaCl, 10% glycerol and 5 mM imidazole with appropriate protease inhibitors. His-tagged Pf HSBP was purified using nickel-nitrilotriacetic acid affinity chromatography. Protein was renatured by step-wise dialysis in buffer containing no urea.
Protein was acetone precipitated and dissolved in 2D lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2% ampholytes (pH 3 to 10) and 5% DTT). Isoelectric focusing (IEF) gel was polymerized in tube (7 cm x 1.5 mm) followed by pre-focusing of IEF tube gel at 250 V for 30 min. Protein was then loaded onto tube gel and resolved at 500 V for four hours. Tube gels were incubated in equilibration buffer (125 mM Tris-Cl, (pH 8.8), 2% SDS, 5 mM DTT and 10% glycerol) for 20 min. Second dimension was carried out on 12% SDS-PAGE gel.
The samples were resolved on SDS-PAGE and blotted on nitrocellulose membranes. Immunoblot was performed. The blots probed with HRP-conjugated secondary antibody were developed by chemiluminiscence, whereas blots probed with non-HRP conjugated antibody were developed by nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) method.
Circular dichroism (CD) spectrum was recorded on a Jasco J-810 spectropolarimeter. Pf HSBP (in buffer with 50 mM Tris-Cl pH 7.5 and 75 mM NaCl) was scanned from 240 to 200 nm at a scan rate of 50 nm/min. Data were corrected for the baseline with respect to buffer and analysed by program K2D2, which estimates protein secondary structure from CD spectra.
In-gel digestion and mass spectrometry
A narrow slice corresponding to HSBP size was cut from the stained SDS-PAGE gel and further sliced into smaller gel pieces. After several washes with 100 mM ammonium bicarbonate (NH4HCO3) in 50% acetonitrile, the gel pieces were subjected to a reduction step using 10 mM dithiothreitol in 100 mM NH4HCO3 buffer (45 min at 56°C). Alkylation was performed with a solution of 55 mM iodoacetamide in 100 mM NH4HCO3 (30 min at room temperature in the dark) followed by in-gel digestion with 20 μl of trypsin (10 ng/μl) in 50 mM NH4HCO3 (overnight at 37°C). Subsequently, the peptides were extracted in NH4HCO3 buffer with 5% formic acid. Samples were vacuum-dried and reconstituted in buffer with 5% formic acid. The protein digest spectrum was acquired on a Q-STAR Elite (QTOF) mass spectrometer equipped with Applied Biosystems Nano Spray II ion source. For identification of proteins, the processed data were searched against NCBI non-reduntant database using the Protein Pilot 4.0 software (threshold 10%) with precursor and fragment mass tolerances of 0.15 Da, cysteine carbamidomethylation as fixed modification and methionine oxidation as variable modification.
Metabolic labelling and immunoprecipitation
Plasmodium falciparum in culture was labelled metabolically with [35S] cysteine and -methionine at 150 μCi/ml (BRIT) culture for 12 hours. Plasmodium falciparum-infected erythrocytes were lysed with 0.05% saponin (saponin lysis) to obtain the parasites (saponin pellet (SP)), which were lysed with 1% Triton X-100 and the obtained lysate was used for immunoprecipitations (IPs). α-Pf HSBP antisera was used at 1:25 dilution and incubated at 4°C for 12 hours on an end-to-end rotator. Protein G beads were then added and incubated for three hours, at the end of which the beads were washed five times (20 min) with wash buffer (1% Triton X-100 in PBS). After the washes, the immunoprecipitates were eluted by boiling in Laemmli buffer and analysed by SDS-PAGE. The gels were vacuum-dried and exposed in a phosphoimager cassette. The film was scanned after 48 hours using a phosphoimager (GE Healthcare).
Cells were lysed (saponin lysis) to obtain parasites. Cytoplasmic and nuclear fractionation was carried out using Lanzeret et al. method. Briefly, SP was resuspended in lysis buffer (20 mM HEPES (pH 7.8), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.65% NP-40 and protease inhibitors) and incubated on ice for 5 min. After centrifugation at 2,000 g for 10 min, the supernatant obtained is the cytoplasmic extract. The pellet was resuspended in nuclear extraction buffer (20 mM HEPES (pH 7.8), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.65% NP-40 and protease inhibitors) and vortexed for 10 min. After centrifugation, the supernatant obtained is the nuclear extract.
Indirect immunofluorescence assay
Immunofluorescence assay was performed as previously described by Tonkin et al.. Briefly, cells were washed once with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde in PBS for 30 min. Fixed cells were washed once with PBS and then permeabilized with 0.1% Triton X-100 in PBS for 2 min. Cells were then incubated in blocking solution (3% BSA in PBS) for one hour. α-HSBP antibody (1:50 dilution; 3% BSA in PBS) was added and allowed to bind for one hour. Cells were washed with PBS for three times (10 min each) to remove excess primary antibody. FITC-conjugated anti-rat secondary antibody (1:300 dilution; 3% BSA in PBS) was added and allowed to bind for one hour. Cells were washed three times with PBS and mounted in 70% glycerol with 2% DABCO. The coverslips were then inverted onto a glass microscope slide, mounted and sealed.
Gel permeation chromatography
The size exclusion chromatography was carried in Superdex 200 (10/300 GL column; GE Healthcare) using NGC Chromatography system (Bio-Rad). Column Washes and elution of sample was carried out in PBS buffer.
Pf HSBP is distinct amongst other conserved HSBPs
The Pf HSBP protein has ambiguity in heptad repeats at two positions ((i) residue 67 - ‘abc’ is missing; and, (ii) residue 86 -‘bcd’ is missing). These stutters are in the N-terminus region of the conserved portion. Whereas, the C-terminus of the protein (residues 89–113 of Pf HSBP corresponding to 38–62 of Hs HSBP) comprises of continuous heptad repeats (Figure 1A). Helix probability of Pf HSBP and Hs HSBP sequences was calculated using prediction program, Coils. Pf HSBP has longer helix compared to human homologue. The low helix propensity region at two positions (residues 67 and 86) within Pf HSBP domain coincides with the positions of stutters in heptad repeats.
Phylogenetic tree of HSBP (Figure 1B) shows that within animal and plant kingdoms, HSBP homologues are very similar and are clustered together. Conversely, Pf HSBP is very distant and is ancestor to both the clusters. The Pf HSBP (red) structure, predicted using I-TASSER server online, when aligned with Hs HSBP (green) showed that the structural pattern of HSBP domain was similar (Figure 1C).
Oligomerization propensity for Pf HSBP and Hs HSBP was calculated using prediction program, Multicoil. It is well documented that Hs HSBP forms a trimer[1, 6, 7]. However, the probability of trimerization is higher in Pf HSBP compared to Hs HSBP. The probability of dimer: trimer is 1:3 for Pf HSBP, whereas, the same for Hs HSBP is only 1.5:1. With respect to total probability, 75% of Pf HSBP can form trimer, whereas only 40% of Hs HSBP can form trimer.
In vitro characterization of Pf HSBP
To validate the identity of the protein, MS/MS analysis of purified Pf HSBP was carried out (Figure 2C). Homology-driven searches for protein identification from MS/MS data in Plasmodium database by using Protein Pilot, identified the protein as Pf HSBP. Peptide scores above 50 and a protein score of 1.3 corresponding to a confidence level greater than 95% were used with error tolerance of 100 ppm.
In order to analyse the secondary structure of Pf HSBP, circular dichroism (CD) studies were carried out with purified recombinant Pf HSBP. Knowing the important functional role played by the helical segment, it was important to perform this experiment in order to draw parallels with the known secondary structures of other HSBPs. In addition, the extra N-terminal stretch possessed uniquely by P. falciparum, could contribute to changes in the secondary structure of the protein. The CD spectrum (Figure 2D) showed double minima at 208 and 222 nm characteristic of helical proteins. Spectrum analysis using K2D2 software showed a high helical content (85%).
Pf HSBP is expressed maximally in trophozoites and exists as an oligomer
The Pf HSBP gene expression pattern obtained from PlasmoDB shows that the maximum expression occurs in the trophozoite stage indicated by the peak during 20–32 hours post invasion (hpi). To examine whether the transcription profile correlates with its proteomic expression, immunoblot analysis was carried out for Pf HSBP for the three intra-erythrocytic stages of P. falciparum (Figure 3D). The immunoblot depicting the relative expression of Pf HSBP (normalized by actin) also clearly showed that the protein was maximally expressed in the trophozoite stages followed by rings and schizont. As mentioned previously, Pf HSBP exists as a multimer in vivo. It is expressed as trimeric as well as hexameric form in all the three stages of the parasite’s life cycle.
Pf HSBP translocates to the nucleus upon heat shock
The localization of Pf HSBP in the parasite was further confirmed by indirect immunofluorescence analysis (IFA). The signal for Pf HSBP in control cells can be seen majorly in cytoplasm and a small fraction inside the nucleus (Figure 4D (Cl)). On subjecting the cells to heat shock, Pf HSBP was found to be localized in both the compartments (Figure 4D (HS)). However, the relative amount in nucleus increases significantly. These results clearly suggest that despite absence of a nuclear localization signal, Pf HSBP undergoes nuclear translocation upon heat shock and thereby pointing out its involvement in the heat shock response.
Pf HSBP interacts with HSP70-1
Having seen that Pf HSBP and Pf HSP70 are present in a common complex, it was of interest to map the localization of this interaction. To this end, nuclear fractionation was carried out to obtain nuclear and cytoplasmic fractions. Immunoprecipitation was thereafter performed on both these fractions with α-Pf HSBP antibody. It can be observed that in P. falciparum, Pf HSBP and Pf HSP70 interaction takes place in the cytoplasmic compartment (Figure 5B).
The canonical pathway of heat shock response in eukaryotes involves a heat shock transcription factor serving as a sensor of heat shock in the cytoplasm. On exposure to heat shock, HSF undergoes trimerization-based activation and translocates to the nucleus. Such activated HSF binds specifically to HSE in the promoter region of hsp genes and induces their transcription.
In addition to the above core components of the heat shock response machinery, a heat shock factor binding protein (HSBP) has been implicated in the regulation of the heat shock response. HSBP was shown to bind and negatively regulate HSF in mammalian cells[1, 6]. Under conditions of stress, HSBP exists in an inactive state but during recovery from stress it associates with HSF and Hsp70 presumably involved in attenuation of the heat shock response.
Despite the relevance of heat shock response in the pathogenesis of malaria, the HSF necessary for up regulating the transcription of hsp genes has not been identified as yet. However other transcription factors belonging to the AP2 family have been implicated in the up regulation of hsp genes[20, 21]. Despite the absence of a classical HSF, malaria parasite does seem to possess a homologue of HSBP.
HSBP are conserved proteins (<10 kDa) with a high helical content. Interestingly, Pf HSBP is longest (13.17 kDa) amongst all HSBP studied thus far by virtue of an extra N-terminal region. The Pf HSBP domain completely aligned with its human counterpart and thus suggesting similar protein function across species. Bioinformatic analysis suggests that the extra N-terminal segment does not correspond to any known domain. Pf HSBP appears to be phylogenetically distant from its animal or plant counterparts. This could be attributed to the extra N-terminal region possessed by Pf HSBP. The recombinant protein predominantly exists in monomeric form. Like HSBP from other organisms Pf HSBP also exhibits anomalous mobility on SDS-PAGE.
Studies carried out on HSBP from mammalian and plant systems have revealed its functional oligomerization. This protein is known to oligomerize in vivo into trimers and hexamers. The hexameric form of the protein gets converted into the active trimeric form which in turn interacts with the HSF trimer during the attenuation of heat shock response. In vivo, Pf HSBP exist as trimer as well as hexamer and these multimers were detergent resistant. Pf HSBP was maximally expressed in trophozoite stage of the parasite. Pf HSBP predominantly localized in the cytoplasm under normal conditions, however, upon heat stress, it translocated to the nucleus. Nuclear translocation has also been observed in Arabidopsis thaliana. Nuclear localization of Pf HSBP upon heat shock is suggestive of its potential role in regulation of heat shock response.
Studies on HSBP have implicated its role as a negative regulator of HSF. The association of HSBP with HSF and HSP70 coincides with attenuation of heat shock response mechanism and conversion of HSF to inactive form. HSBP initially interacts with HSF and then with HSP70 during recovery from heat shock. However, the precise mechanism remains unclear. This study shows that Pf HSBP is also capable of interacting with Pf HSP70-1 in the cytoplasm.
This study, for the first time, reports the presence of a HSF binding partner, HSBP, in P. falciparum. In the absence of a known HSF, this study on Pf HSBP serves as a prelude in understanding heat shock response machinery in the malaria parasite.
Heat shock factor binding protein
Heat shock factor(s)
Heat shock protein
Heat shock elements
Indirect immunofluorescence assay.
We acknowledge funding from DBT- IISc Partnership Programme. SKS and VS acknowledge funding from Department of Biotechnology, Indian Institute of Science. SC and MS acknowledge fellowship from Council of Scientific and Industrial Research.
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