Arun Pradhan

Unraveling the 'DEAD-box' helicases of Plasmodium falciparum.

Abstract The causative agent for the most fatal form of malaria, Plasmodium falciparum, has developed insecticide and drug resistance with time. Therefore combating this disease is becoming increasingly difficult and this calls for finding alternate ways to control malaria. One of the feasible ways could be to find out inhibitors/drugs specific for the indispensable enzymes of malaria parasite such as helicases. These helicases, which contain intrinsic nucleic acid-dependent ATPase activity, are capable of enzymatically unwinding energetically stable duplex nucleic acids into single-stranded templates and are required for all the nucleic acid transactions. Most of the helicases contain a set of nine extremely conserved amino acid sequences, which are called ‘helicase motifs’. Due to the presence of the DEAD (Asp–Glu–Ala–Asp) in one of the conserved motifs, this family is also known as the ‘DEAD-box’ family. In this review, using bioinformatic approach, we describe the ‘DEAD-box’ helicases of malaria parasite P. falciparum. An in depth analysis shows that the parasite contains 22 full-length genes, some of which are homologues of well-characterized helicases of this family from other organisms. Recently we have cloned and characterized the first member of this family, which is a homologue of p68 and is expressed during the schizont stage of the development of the parasite [Pradhan, A., Chauhan, V.S., Tuteja, R., 2005a. A novel ‘DEAD-box’ DNA helicase from Plasmodium falciparum is homologous to p68. Mol. Biochem. Parasitol. 140, 55–60.; Pradhan A., Chauhan V.S., Tuteja R., 2005b. Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. Mol. Biochem. Parasitol. 144, 133–141.]. It will be really interesting to clone and characterize other members of the ‘DEAD-box’ family and understand their role in the replication and transmission of the parasite. These detailed studies may help to identify a parasite-specific enzyme, which could be a potential drug target to treat malaria. The various steps at which this probable drug can act are also discussed.

Isolation and characterization of Plasmodium falciparum UAP56 homolog: Evidence for the coupling of RNA binding and splicing activity by site-directed mutations☆

UAP56 (U2AF65 associated protein) is a member of the DEAD-box helicase family. Helicases are essential enzymes generally involved in the metabolism of nucleic acids. The gene encoding a member of DEAD-box family was cloned and characterized from the human malaria parasite Plasmodium falciparum. PfU52 is homologous to UAP56 and contains the RNA-dependent ATPase, RNA helicase and RNA binding activities. Using the parasite extract we report that PfU52 is involved in splicing reaction. Site-directed mutagenesis studies indicate that the conserved residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this activity is required for the enzymatic activities of PfU52. PfU52 is expressed in all the intraerythrocytic developmental stages of the parasite. In the present study we have reported the detailed characterization of PfU52 from P. falciparum and these results advance the knowledge regarding the function of UAP56 in general.

Plasmodium falciparum DNA helicase 60. dsRNA- and antibody-mediated inhibition of malaria parasite growth and downregulation of its enzyme activities by DNA-interacting compounds.

Helicases are ubiquitous enzymes that play important roles in all types of DNA transaction in the cells. Recently we have reported the characterization of the first DEAD‐box helicase [Plasmodium falciparum DNA helicase 60 (PfDH60)] from Plasmodium falciparum and have shown that it is a unique, dual bipolar helicase expressed in a stage‐specific manner. In this study, we show the further characterization of PfDH60. For analyzing the significance of this enzyme in parasite growth, we studied the effect of dsRNA and specific antibodies on growth of the parasite. The studies indicate that the parasite cultures treated with PfDH60 dsRNA exhibited ∼ 50% growth inhibition when compared with either untreated cultures or cultures treated with unrelated dsRNA. It was interesting to note that purified immunoglobulins against PfDH60 induced ∼ 62% inhibition of in vitro growth of P. falciparum and that this inhibitory effect was associated with morphologic damage to the parasite. DNA‐interacting compounds inhibit DNA helicase and ssDNA‐dependent ATPase activities of PfDH60. Of various compounds tested, only actinomycin, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the enzyme activities of PfDH60, with apparent IC50 values for helicase inhibition of 0.8, 0.3, 2.0, 1.2 and 1.5 µm, respectively. It may be proposed that these compounds form a complex with DNA and specifically inhibit helicases due to obstruction in the translocation of the enzyme. These compounds also inhibited parasite growth in culture. This is the first study to show inhibition of growth of the parasite by the dsRNA of a helicase, and most probably this is due to interference with cognate mRNA expression.

Isolation and functional characterization of eIF4F components and poly(A)-binding protein from Plasmodium falciparum

The multisubunit translation initiation complex eIF4F contains eIF4E, eIF4A and eIF4G. eIF4A is an ATP-dependent RNA helicase. eIF4G provides the platform for binding initiation factors and it contains the binding sites for eIF4A and eIF4E and interacts with poly(A)-binding protein (PABP). Although the genome of Plasmodium falciparum is fully sequenced but the gene annotation is still incomplete. In this manuscript we present the isolation and characterization of components of the eIF4F complex i.e. eIF4E, eIF4G and PABP from P. falciparum. Our studies indicate that PfeIF4E is involved in translation and PfPABP binds poly(A) specifically. We demonstrate the interaction of PfeIF4G with PfeIF4E, PfeIF4A (PfH45) and PfPABP. These studies demonstrate that these factors are structurally and functionally conserved. These studies will contribute to understand the important process and components of translation complex in the malaria parasite.

PfeIF4E and PfeIF4A colocalize and their double-stranded RNA inhibits Plasmodium falciparum proliferation

Using bioinformatics and biochemical methods in the recent past we have reported the isolation and characterization of the main components of translation initiation complex eIF4F from malaria parasite Plasmodium falciparum. We reported that eukaryotic initiation factor 4A (eIF4A), eukaryotic initiation factor 4E (eIF4E), eukaryotic initiation factor 4G (eIF4G) and poly (A) binding protein (PABP) are structurally and functionally conserved in this parasite. In the present study we report further characterization of PfeIF4A and PfeIF4E. We report that PfeIF4A and PfeIF4E are co-localized and predominantly localized in the cytoplasm. The parasite cultures treated with co-addition of PfeIF4A and PfeIF4E double stranded RNA showed ~ 67% growth inhibition suggesting that inhibition of two components of the same pathway is more effective for inhibiting the proliferation of the malaria parasite Plasmodium falciparum. These observations suggest that PfeIF4A and PfeIF4E are critical for parasite growth and survival.

Isolation and characterization of type I signal peptidase of different malaria parasites.

Type I signal peptidases are important membrane-bound serine proteases responsible for the cleavage of the signal peptide of the proteins. These enzymes are unique serine proteases that carry out catalysis using a serine/lysine catalytic dyad. In the present study, we report the isolation of type I signal peptidase from the malaria parasites Plasmodium falciparum, Plasmodium knowlesi, and Plasmodium yoelii and some characterization of type I signal peptidase of Plasmodium falciparum. We show that these enzymes are homologous to signal peptidases from various sources and also contain the conserved boxes present in other type I signal peptidases. The type I signal peptidase from P falciparum is an intron-less and a single-copy gene. The results also show that the enzyme from Plasmodium falciparum is subject to self-cleavage and it has been demonstrated to possess type I signal peptidase activity in E coli preprotein processing in vivo by complementation assay. This study will be helpful in understanding one of the important metabolic pathways “the secretory pathway” in the parasite and should make an important contribution in understanding the complex process of protein targeting in the parasite.