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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
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A novel DEAD box helicase Has1p from Plasmodium falciparum: N-terminal is essential for activity Krishna Prakash, Renu Tuteja ⁎ Malaria Group, International Centre for Genetic Engineering and Biotechnology, P. O. Box 10504, Aruna Asaf Ali Marg, New Delhi-110067, India
a r t i c l e
i n f o
Article history: Received 24 November 2009 Received in revised form 2 February 2010 Accepted 4 February 2010 Available online 11 February 2010 Keywords: DEAD box Helicase Malaria Plasmodium falciparum Unwinding
a b s t r a c t Helicases catalyze the opening of nucleic acid duplexes and are implicated in many nucleic acid metabolic cellular processes that require single stranded DNA or reorganization of RNA structure. Previously we have reported that Plasmodium falciparum genome contains a number of DEAD box helicases. In the present study we report the cloning, expression and characterization of one of the novel members of DEAD box family from P. falciparum. Our results indicate that it is a homologue of Has1p from yeast and it contains DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities. This enzyme can utilize all the nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs) for its unwinding activity. Using a truncated derivative of this protein we further report that the N-terminal region of the protein is essentially required for its activity. These studies suggest that besides the conserved helicase domain the highly variable N-terminal region also contributes in the activity of the protein. © 2010 Elsevier Ireland Ltd. All rights reserved.
Helicases are responsible for the unwinding of nucleic acid duplexes in an ATP-dependent manner [1,2]. The energy for this unwinding is provided by the intrinsic nucleic acid-dependent NTPase activity of helicases. Depending on the type of substrate the helicases are classified as DNA or RNA helicases. RNA helicases are implicated in many cellular processes that require reorganization of RNA structure, such as transcription, mRNA splicing, translation initiation, RNA editing, export, and degradation. These enzymes are identified by the presence of nine different conserved motifs [3]. Due to the presence of sequence DEAD in one of the motifs, these are commonly known as DEAD box helicases [4]. Members of this family are present in almost all the organisms and are involved in many different biological processes including DNA repair, transcription, pre-rRNA processing, ribosome biogenesis and splicing [5]. Plasmodium falciparum is a parasite, which causes the most lethal form of malaria [6]. A malaria vaccine would be the ultimate weapon to fight this deadly disease but unfortunately despite encouraging advances a vaccine against malaria is not available yet. Moreover the parasite and the mosquito vector have developed drug resistance gradually therefore controlling this disease is a daunting task [7]. The rational development of novel and affordable anti-malarial drugs for the treatment of malaria and the identification of new drug targets is an important goal. The recent completion of malaria genome project and availability of new technologies for genome wide comparison of genomes is helpful in identifying key targets in biochemical pathways
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that are parasite specific and can be interrupted without deleterious consequences for the host. One of the promising targets could be helicases, which are key enzymes and required for almost all the nucleic acid transactions in malaria parasite [8]. Previously we have reported that P. falciparum genome contains a number of helicases and at least 22 DEAD box helicases [9,10]. In the present study we report the cloning, purification and characterization of one of the helicases of DEAD box family from the malaria parasite P. falciparum. Our studies reveal that this enzyme is homologous to Has1p, contains DNA and RNA helicases, ssDNA and RNA-dependent ATPase and RNA binding activities. We also report that the N-terminal region of the protein is essentially required for all its activities. Using bioinformatics analysis it has been reported previously that P. falciparum genome contains a number of putative DEAD box helicase genes [9–11]. We reported that a putative RNA helicase with PlasmoDB number PFF1500c contains all the characteristic features of DEAD box family [9,10]. Therefore this helicase was selected for cloning and characterization. P. falciparum (strain 3D7) was cultured as described earlier [12]. Total RNA was isolated and was used for the preparation of cDNA using a cDNA synthesis kit (Superscript firststrand synthesis system from Invitrogen, Carlsbad, CA, USA). This cDNA was used as a template because the genomic DNA contains one intron. The sequence was obtained from PlasmoDB (http://PlasmoDB. org) [13] and the complete open reading frame (1806 base pair) of helicase gene of P. falciparum was PCR amplified using the forward primer PfH69F (5′-GGGATCCATGATGGATGATGATAAT-3′) and the reverse primer PfH69R (5′-CCTCGAGTTATTTAAATTTTTTTTTTTTG-3′). The restriction sites are written in italics. The PCR product of ∼1.8 kb was gel purified and cloned into the pGEM-T vector from Promega
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(Madison, WI, USA) and the positive clones were sequenced by automated DNA sequencing. The nucleotide sequence was submitted to the GenBank and the accession number is FJ641053. The nested forward primer PfH69F1 (5′-GGGATCCATGTTTGAAGAATTAAATATATGTG-3′) and the reverse primer PfH69R were used for the amplification of the nested gene (1359 base pair), which lacked first
149 amino acids. The DNA bands were excised using BamHI and XhoI enzymes (New England Biolabs, Beverly, MA, USA) and gel purified for subcloning into the protein expression vector pET-28a (Novagen, Madison, WI, USA). The sequence analysis indicated that this gene encodes a polypeptide of 601 amino acid residues with a predicted molecular
Fig. 1. A. Comparison of amino acid sequence. The sequence of P. falciparum homologue (PfH69) was compared with different proteins from Plasmodium vivax (XP_001616317), Schizosaccharomyces pombe (NP_594488), Cryptosporidium hominis (XP_666241) and Homo sapiens (NP_006764). Multiple alignments were done using ClustalW program. The accession numbers of the aligned sequences are written in brackets. B. Alignment of the N-terminal region of Has1p homologues. C. Domain wise comparison of PfH69 of P. falciparum (i) with its human homologue (ii). The conserved sequences of each domain are written inside the boxes. The numbers refer to the amino acids separating the various domains and the length of N- and C-terminal extensions. This figure is not drawn to scale. Lower part of each panel shows the amino acid position and structure of each domain. The domain analysis was done by using ‘Scan Prosite’ at http://expasy.org/tools/scanprosite/. The text in bracket is the name of the domain and the numbers are position of respective domains in the protein. D. Structure modeling. PfH69 sequence was submitted to Swissmodel server and the structure was obtained. The molecular graphic images were produced using the UCSF Chimera package from the resource for Biocomputing, Visualization, and Informatics (http://www.cgl.ucsf.edu/chimera) at the University of California, San Francisco (supported by NIH P41 RR-01081). a. PfH69; b. template H1V8 and c. superimposed image.
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Fig. 1 (continued).
weight of ∼69 kDa. The homology search revealed that the encoded protein is highly homologous to DBP18 (human) or Has1p (yeast) and thus it has been designated as PfH69 (P. falciparum helicase 69) [14]. A multiple alignment of amino acid sequence homology search using NCBI database revealed that PfH69 aligned contiguously and showed the highest homology with its counterpart from Plasmodium vivax (Fig. 1A). But the multiple alignments of only the N-terminal region indicated that it is least conserved in all these proteins (Fig. 1B). A detailed comparison of all the conserved motifs of PfH69 and human homologue showed that the sequence in all the motifs is almost same (Fig. 1C (i) and (ii)). The detailed sequence analysis using ScanProsite (http://expasy.org/tools/scanprosite/) showed that it contains the characteristic helicase motifs including the Q motif (Fig. 1C (i)). The positions of these motifs are slightly variable. It is interesting to note that both PfH69 and the human homologue do not contain the Walker-A like motif (GtkGkGKS) downstream of motif VI as reported previously for yeast Has1p [14]. The sequence analysis also showed that the N-terminal region of PfH69 is enriched in asparagine residues while the same region of human homologue is enriched in lysine residues. The expression profile of PFF1500c in PlasmoDB shows that
it is expressed in all the life cycle stages of development of P. falciparum [13]. For structural modeling the sequence of PfH69 was submitted to the Swissmodel homology-modeling server (http://swissmodel. expasy.org/) [15]. The structural model obtained was based on the crystal structure of a DEAD box protein from Methanococcus jannaschii [16] (Fig. 1D (i) and (ii)). It has been shown that unlike other helicases, this protein existed as a dimer in the crystal [16] (Fig. 1D (ii)). When the modeled structure of PfH69 was superimposed, it is clear that this structure superimposes with only one subunit of the dimer (Fig. 1D (iii)). Molecular graphic images were produced using the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera) from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR01081) [17]. In vitro ATPase activity has been demonstrated for a large number of helicases however, an in vitro ATP-dependent nucleic acid unwinding (helicase) activity has been demonstrated for only a limited number of them [5,18]. The sequence alignment analysis indicated that the N-terminal region of PfH69 is highly variable and
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Fig. 1 (continued).
contains very little sequence conservation. Therefore in order to check the role of this N-terminal region in the activity of the protein we deleted this region and analyzed the truncated protein also for all the activities. For biochemical characterization of the encoded proteins the full-length amplified product and the truncated product were cloned into the bacterial expression vector pET28a and the protein was produced after an induction with IPTG. The recombinant histagged PfH69 and its truncated version PfH69T, which lacked the amino acid 1 to 149 from N-terminal, were purified using Ni2+-NTA (Qiagen, GmbH, Germany) affinity chromatography. The western blot analysis using anti-his antibodies detected the protein in various elutes of varying imidazole concentration. The final purification step yielded purified PfH69 (∼ 69 kDa) and PfH69T (∼ 52 kDa) enzymes and SDS-PAGE analysis followed by silver staining showed that the purified proteins are homogeneous and do not contain any contaminating protein (lane 1 of Fig. 2A and D respectively). The freshly purified PfH69 and PfH69T were used for all the activity analysis assays described in the following sections because on storage the proteins degraded and lost their activity. All the helicases reported till date contain intrinsic nucleic aciddependent ATPase activity and the energy released is required for the translocation of the protein on the nucleic acid [1,2]. The ATPase activity of PfH69 and PfH69T was determined by measuring the percentage release of radioactive Pi from [γ-32P] ATP by using a method described earlier [19]. The reaction was performed at 37 °C both in the presence and absence of 100 ng of M13 mp19 ssDNA and RNA from P. falciparum using a method described earlier [19]. The reaction was stopped by chilling to 0 °C. One µl of the mixture was spotted onto a polyethyleneimine-cellulose thin-layer strip (Sigma (St. Louis, MO, USA) and ascending chromatogrphy was performed in
0.5 M LiCl, 1 M formic acid at room temperature for about 15 min. The strip was dried at room temperature and exposed to Amersham hyperfilm (Amersham Biosciences, Pittsburgh, PA, USA) to identify the radioactive spots of ATP and Pi. For quantitation these spots were cut from the strip and counted with liquid scintillation fluid. The ATPase activity of both PfH69 and PfH69T was tested under standard assay conditions. It was interesting to note that RNA stimulated the ATPase activity more as compared to ssDNA. Therefore the ATPase activity of various concentrations of PfH69 (100, 200 and 300 ng) and PfH69T (100, 200 and 300 ng) was tested in the presence of RNA. The results showed that the activity of full-length PfH69 increased in concentration dependent manner (Fig. 2B, lanes 2–4). But there was no increase in the activity of PfH69T with increase in the concentration and it remained more or less constant (Fig. 2E, lanes 2–4). These results suggest that the activity of the truncated protein is not concentration dependent and the truncation has caused a loss of the ATPase activity. The ATPase activity of the same concentration of both PfH69 and PfH69T was tested in the presence of RNA at various time points (0, 5, 10, 20, 60 and 120 min). The results showed that the ATPase activity of PfH69 was detectable after 10 min of reaction (Fig. 2C, lane 3) but PfH69T showed detectable activity only after 60 min of reaction (Fig. 2F, lane 5). On comparison of the activity of PfH69 and PfH69T at different time points, the maximum activity was observed at 120 min. It is interesting to note that the activity of PfH69T was only ∼ 15% of the activity of PfH69 (Fig. 2C, lane 6 versus Fig. 2F, lane 6). It has been shown previously that yeast Has1p contains RNAdependent ATPase and DNA/RNA unwinding activity [14] but DNA/ DNA unwinding has not been reported for Has1p. After establishing the intrinsic nucleic acid-dependent ATPase activity of PfH69 and PfH69T, the helicase activity was determined using the standard
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Fig. 1 (continued).
strand displacement assay. The helicase assay measures the unwinding of a 32P-labeled nucleic acid fragment from a partially duplex nucleic acid substrate. The reaction mixture (10 μl) contains 20 mM TrisHCl (pH 8.0), 8 mM dithiothreitol, 1.0 mM MgCl2, 1.0 mM ATP, 30 mM KCl, 4% (w⁄v) sucrose, 80 μg/ml BSA, and 32P-labeled helicase substrate (∼1000 c.p.m.) and the helicase fraction to be assayed was incubated at 37 °C for 60 min. The substrate for all the studies contained non-complementary tails of 15 nucleotides on both the 5′ and 3′ ends. The reaction was terminated by the addition of 0.3% SDS, 10 mM EDTA, 5% glycerol and 0.03% bromophenol blue. After further incubation at 37 °C for 5 min, the substrate and products were separated by electrophoresis on a 12% nondenaturing polyacrylamide gel. The gel was dried and exposed to Amersham hyperfilm (Amersham Biosciences, Pittsburgh, PA, USA) with an intensifying screen for autoradiography. DNA unwinding was quantitated as described previously [19]. Varying concentration of PfH69 (ranging from 50 to 250 ng) was used for activity assay and it was observed that PfH69 showed concentration dependent DNA helicase activity (Fig. 2G, lanes 1 to 5) but PfH69T had no measurable DNA helicase activity (data not shown). The results are in agreement with the results of ATPase assay which showed that PfH69T contains only negligible levels of activity and the intrinsic ATPase activity is essential for the unwinding activity. The effect of different nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs)
on helicase activity of PfH69 was also studied. The helicase activity was observed in the presence of ATP and dATP (Fig. 2H, lanes 2 and 3). It is interesting to note that the helicase activity was also observed in the presence of all the other NTPs and dNTPs like GTP, CTP, UTP, dGTP, dCTP and TTP (Fig. 2H, lanes 4–9) but the kinetics of activity was lower (compare lane 2 with lanes 4–9 of Fig. 2H). The utilization of all the NTPs/dNTPs by PfH69 is a peculiar property and is contrary to our previous findings, where we reported that PfH45 could not utilize any other NTP/dNTP besides ATP/dATP [20]. A number of other helicases such as HDH I, calf thymus helicase I, PDH65, PcDDH45 and eukaryotic eIF4A are also able to utilize only ATP/dATP as cofactor [1,2,21,22]. We also checked the RNA helicase activity of PfH69 by using the assay described earlier [19]. The RNA helicase substrate was prepared by using the RNA oligonucleotides 39-mer 5′-GGGAGAAAUCACUCGGUUGAGGCUAUCCGUAAAGCACGC-3′ and 13 mer 5′-AUAGCCUCAACCG-3 synthesized from Primm srl (Milan, Italy). The RNA helicase activity was tested using different concentrations of PfH69. The results suggest that PfH69 contains concentration dependent RNA unwinding activity (Fig. 2I, lanes 1–4). Similar to DNA helicase activity, we determined the RNA helicase activity of PfH69T but on repeated trials we were unable to detect any RNA unwinding activity in PfH69T (data not shown). The RNA binding activity assay of PfH69 and PfH69T was done by using the same method as described previously with slight modifications [20,23].
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Fig. 2. A. A silver-stained gel of purified PfH69. Lane M is the molecular mass marker and lane 1 contains 0.2 µg of purified PfH69. B. Concentration dependence of RNA-dependent ATPase activity. 50, 100, 200 and 300 ng (lanes 1–4 respectively) of purified PfH69 were used for the assay. Lane C is control without enzyme. C. Time dependence of RNA-dependent ATPase activity. 200 ng of purified PfH69 was used for each time-point 0, 5, 10, 20, 60 and 120 min (lanes 1–6 respectively). Lane C is control without enzyme. The positions of ATP and released inorganic phosphate (Pi) are marked on the left-hand side of the autoradiogram in B and C. D. A silver-stained gel of purified PfH69T. Lane M is the molecular mass marker and lane 1 contains 0.2 µg of purified PfH69T. E. Concentration dependence of RNA-dependent ATPase activity. 50, 100, 200 and 300 ng (lanes 1–4 respectively) of purified PfH69T were used for the assay. Lane C is control without enzyme. F. Time dependence of RNA-dependent ATPase activity. 200 ng of purified PfH69T was used for each time-point 0, 5, 10, 20, 60 and 120 min (lanes 1–6 respectively). Lane C is control without enzyme. The positions of ATP and released inorganic phosphate (Pi) are marked on the left-hand side of the autoradiogram in E and F. G. Concentration dependence of DNA helicase activity. 50, 100, 150, 200 and 300 ng (lanes 1–5 respectively) of purified PfH69 were used for the assay. Lane C is control without enzyme and lane B is heat denatured substrate. H. Preference of nucleotide triphosphate (NTP) for the DNA helicase activity of PfH69. Lane C is control without enzyme and lane 1 is reaction without any NTP. Lanes 2–9 are reactions in the presence of one of the NTPs or dNTPs. I. Concentration dependence of RNA unwinding activity of PfH69. 100, 150, 200 and 300 ng (lanes 1–4 respectively) of purified PfH69 were used for the assay. Lane C is control without enzyme and lane B is heat denatured substrate. The structure of the substrate is shown on the left-hand side of the autoradiogram in G, H and I. The upper band is partially duplex substrate and lower band is the unwound DNA or RNA. J. RNA binding activity of PfH69. Spot number B is 1 µg BSA, spot number 1, PfH69 and 2, PfH69T. K. Western blot probed with anti-his antibody. Spot number 1 is PfH69 and 2, PfH69T.
For these equal amounts (1 μg) of BSA, PfH69 and PfH69T were dot-blotted on precharged PVDF membrane. For precharging the membrane was immersed in 100% methanol for 1 min and then washed with distilled water. After sample application on this precharged membrane, it was blocked and incubated in labeled 13 mer RNA oligonucleotide used for the preparation of RNA helicase substrate using the method described earlier [20]. After binding, the membrane was washed thrice with binding buffer and exposed for autoradiography. To check for equal loading of proteins, equal amounts (1 μg) of PfH69 and PfH69T were dot-blotted on another precharged PVDF membrane. This membrane was blocked and probed with alkaline phosphatase conjugated anti-his antibody, washed and developed using standard protocol. The results indicated that equal amount
of both proteins was bound to the membrane (Fig. 2K, spot numbers 1 and 2). The results of the RNA binding assay showed that PfH69 and PfH69T both bind RNA but PfH69T has only ∼55% of the RNA binding activity as compared to PfH69 (Fig. 2J, spot numbers 1 and 2). There are several previous reports which indicate that the conserved amino acids in motifs I (AxxGxGKT), II (DExH/D), III (S/ TAT) and VI (Q/HRxGRxGR) of yeast eIF4A, Ded1p, Prp2p, Prp16p, and Prp22p as well as viral helicases NS3 and NPH-II are important for in vivo function and in vitro ATPase activity and unwinding [18,24–31]. It has been reported previously that the C-terminal region of Has1p is strongly conserved and that this region most probably confers the substrate specificity, which plays a role in interacting with other
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accessory proteins and is required for in vivo function of Has1p [14]. But to the best of our knowledge there are no reports suggesting the role of N-terminal region of the helicases in activity. Our results in the present study clearly indicate that besides the conserved helicase motifs and C-terminal region the highly variable N-terminal region of the protein is essential for its enzyme activity. Till to date only a few members of the DEAD box protein family have been biochemically characterized and most of the members have been identified bioinformatically and reported as putative helicases. Some of the helicases reported to contain DNA unwinding activity are E. coli RecQ gene product, a 74 kDa protein, yeast Rad3, a product of excision repair gene ERCC3, a 72 kDa protein encoded by the human REQL gene, the 172 kDa protein encoded by DNA2 gene in yeast, PDH45 from pea and eIF-4A from P. cynomolgi [1,2,22]. Few examples of RNA helicases characterized from this family are Arabidopsis thaliana DRH1, Drosophila VASA, PDH45 from pea, mouse eIF-4A, Xenopus-an3 and Xp54, human p68, E. coli CsdA, RhlE and SrmB and hepatitis C virus NS3 helicase [32–35]. Recently we have biochemically characterized a few members of this family from P. falciparum such as PfDH60, PfH45 and PfU52 [19,36,37]. We have reported that PfDH60 and PfH45 are dual helicases and PfU52 contains only RNA unwinding activity [19,36,37]. In the present study we report the biochemical characterization of another member of DEAD box family and report that PfH69 contains nucleic acid-dependent ATPase, RNA and DNA unwinding and RNA binding activities. In silico analysis showed that PfH69 is the homologue of Has1p protein of the yeast. The DNA helicase activity of Has1p homologue has not been reported previously. In view of the fact that we deleted 149 amino acids from the N-terminal and the truncated fragment of this protein had all the conserved motifs (Q, I Ia, Ib, II, III, IV, V and VI) intact, we expected similar activity in the truncated protein but all the enzyme activities were lost in this truncated protein. We speculate that the removal of 149 amino acids from the N-terminal might influence the overall structure of this molecule and most likely this truncation causes overall defolding or total denaturation of the structure resulting in severe conformational change. Therefore the truncated protein loses all its activities. In a previous study we have reported that the truncated fragments of PfH45 do not contain all the enzyme activities and all the helicase motifs on a single polypeptide are required for the ATPase and unwinding activities of a protein [20]. Our results in the present study further signify that the highly variable N-terminal region is also essentially required for the enzyme activities of PfH69 in addition to the conserved helicase domains. The studies presented here should make an important contribution in understanding the role of DEAD box proteins in nucleic acid metabolism in the parasite. Acknowledgements The work in R.T.'s laboratory is supported by the Department of Science and Technology, the Department of Biotechnology and the Defence Research and Development Organization grants. Infrastructural support from the Department of Biotechnology, Government of India is gratefully acknowledged. References [1] Tuteja N, Tuteja R. Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery. Eur J Biochem 2004;271:1835–48. [2] Tuteja N, Tuteja R. Unraveling DNA helicases. Motif, structure, mechanism and function. Eur J Biochem 2004;271:1849–63. [3] Tanner NK, Linder P. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol Cell 2001;8:251–62. [4] Linder P, Lasko PF, Ashburner M, Leroy P, Nielsen PJ, Nishi K, et al. Birth of the D-EA-D box. Nature 1989;337:121. [5] Rocak S, Linder P. DEAD-box proteins: the driving forces behind RNA metabolism. Nature Rev Mol Cell Biol 2004;5:232–41.
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