Moaz Ahmad

Plasmodium falciparum DOZI, an RNA helicase interacts with eIF4E.

DEAD box RNA helicases play crucial roles in RNA metabolism such as splicing, ribosome biogenesis, RNA transport, degradation and translation. DDX6/DOZI (development of zygote inhibited) is one of the well characterized member of the DEAD box family and is highly conserved from humans to malaria parasite. DDX6 is involved in a variety of biological processes, which include the sexual development of the protozoan parasite. In the present manuscript we report that P. falciparum DOZI (DDX6 homologue); PfDZ50 contains the characteristic DNA and RNA binding, nucleic acid-dependent ATPase and RNA unwinding activities. Enzymatic characterization of truncated derivatives of PfDZ50 such as PfDZ50T1 (domain 1) and PfDZ50T2 (domain 2) shows that none of them contains ATPase activity. Furthermore, we confirmed that PfDZ50 interacts with PfeIF4E mainly through domain 1. Using in vitro translation assays we show that PfDZ50 inhibits translation. With the same assays we further report that externally added PfeIF4E restores ~70% of translation. Using immunofluorescence assays we demonstrate that PfDZ50 is localized mainly in the cytoplasm in the asexual intraerythrocytic developmental stages of P. falciparum. The localization pattern further suggests that PfDZ50 appears typically in granular bodies throughout the cytoplasm. Thus these studies will advance our knowledge regarding the function of PfDZ50/DDX6 in general.

Plasmodium falciparum UvrD helicase translocates in 3' to 5' direction, colocalizes with MLH and modulates its activity through physical interaction.

Malaria is a global disease and a major health problem. The control of malaria is a daunting task due to the increasing drug resistance. Therefore, there is an urgent need to identify and characterize novel parasite specific drug targets. In the present study we report the biochemical characterization of parasite specific UvrD helicase from Plasmodium falciparum. The N-terminal fragment (PfUDN) containing UvrD helicase domain, which consists of helicase motifs Q, Ia–Id, II, III and most of motif IV, and the C-terminal fragment (PfUDC1) containing UvrD helicase C terminal domain, consisting of remaining part of motif IV and motifs IVa–IVc and 161 amino acids of intervening sequence between motif IV and V, possess ssDNA-dependent ATPase and DNA helicase activities in vitro. Using immunodepletion assays we show that the ATPase and helicase activities are attributable to PfUDN and PfUDC1 proteins. The helicase activity can utilize the hydrolysis of all the nucleotide and deoxynucleotide triphosphates and the direction of unwinding is 3′ to 5′. The endogenous P. falciparum UvrD contains the characteristic DNA helicase activity. PfUDN interacts with PfMLH (P. falciparum MutL homologue) and modulates the endonuclease activity of PfMLH and PfMLH positively regulates the unwinding activity of PfUDN. We show that PfUvrD is expressed in the nucleus distinctly in the schizont stages of the intraerythrocytic development of the parasite and it colocalizes with PfMLH. These studies will make an important contribution in understanding the nucleic acid transaction in the malaria parasite.

Novel RuvB nuclear ATPase is specific to intraerythrocytic mitosis during schizogony of Plasmodium falciparum.

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.

Plasmodium falciparum RuvB proteins: Emerging importance and expectations beyond cell cycle progression.

The urgent requirement of next generation antimalarials has been of recent interest due to the emergence of drug-resistant parasite. The genome-wide analysis of Plasmodium falciparum helicases revealed three RuvB proteins. Due to the presence of helicase motif I and II in PfRuvBs, there is a high probability that they contain ATPase and possibly helicase activity. The Plasmodium database has homologs of several key proteins that interact with RuvBs and are most likely involved in the cell cycle progression, chromatin remodeling, and other cellular activities. Phylogenetically PfRuvBs are closely related to Saccharomyces cerevisiae RuvB, which is essential for cell cycle progression and survival of yeast. Thus PfRuvBs can serve as potential drug target if they show an essential role in the survival of parasite.

Plasmodium falciparum MLH is schizont stage specific endonuclease

Malaria is one of the most important infectious diseases in many regions around the world including India. Plasmodium falciparum is the cause of most lethal form of malaria while Plasmodium vivax is the major cause outside Africa. Regardless of considerable efforts over the last many years there is still no commercial vaccine against malaria and the disease is mainly treated using a range of established drugs. With time, the malaria parasite is developing drug resistance to most of the commonly used drugs. This drug resistance might be due to defective mismatch repair in the parasite. Previously we have reported that the P. falciparum genome contains homologues to most of the components of mismatch repair (MMR) complex. In the present study we report the detailed biochemical characterization of one of the main component of MMR complex, MLH, from P. falciparum. Our results show that MLH is an ATPase and it can incise covalently closed circular DNA in the presence of Mn(2+) or Mg(2+) ions. Using the truncated derivatives we show that full length protein MLH is required for all the enzymatic activities. Using immunodepletion assays we further show that the ATPase and endomuclease activities are attributable to PfMLH protein. Using immunofluorescence assay we report that the peak expression of MLH in both 3D7 and Dd2 strains of P. falciparum is mainly in the schizont stages of the intraerythrocytic development, where DNA replication is active. MMR also contributes to the overall fidelity of DNA replication and the peak expression of MLH in the schizont stages suggests that MLH is most likely involved in correcting the mismatches occurring during replication. This study should make a significant contribution in our better understanding of DNA metabolic processes in the parasite.

Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth

BackgroundHuman malaria parasite infection and its control is a global challenge which is responsible for ~0.65 million deaths every year globally. The emergence of drug resistant malaria parasite is another challenge to fight with malaria. Enormous efforts are being made to identify suitable drug targets in order to develop newer classes of drug. Helicases play crucial roles in DNA metabolism and have been proposed as therapeutic targets for cancer therapy as well as viral and parasitic infections. Genome wide analysis revealed that Plasmodium falciparum possesses UvrD helicase, which is absent in the human host.ResultsRecently the biochemical characterization of P. falciparum UvrD helicase revealed that N-terminal UvrD (PfUDN) hydrolyses ATP, translocates in 3’ to 5’ direction and interacts with MLH to modulate each other’s activity. In this follow up study, further characterization of P. falciparum UvrD helicase is presented. Here, we screened the effect of various DNA interacting compounds on the ATPase and helicase activity of PfUDN. This study resulted into the identification of daunorubicin (daunomycin), netropsin, nogalamycin, and ethidium bromide as the potential inhibitor molecules for the biochemical activities of PfUDN with IC50 values ranging from ~3.0 to ~5.0 μM. Interestingly etoposide did not inhibit the ATPase activity but considerable inhibition of unwinding activity was observed at 20 μM. Further study for analyzing the importance of PfUvrD enzyme in parasite growth revealed that PfUvrD is crucial/important for its growth ex-vivo.ConclusionsAs PfUvrD is absent in human hence on the basis of this study we propose PfUvrD as suitable drug target to control malaria. Some of the PfUvrD inhibitors identified in the present study can be utilized to further design novel and specific inhibitor molecules.

Emerging importance of mismatch repair components including UvrD helicase and their cross-talk with the development of drug resistance in malaria parasite

Human malaria is an important parasitic infection responsible for a significant number of deaths worldwide, particularly in tropical and subtropical regions. The recent scenario has worsened mainly because of the emergence of drug-resistant malaria parasites having the potential to spread across the world. Drug-resistant parasites possess a defective mismatch repair (MMR); therefore, it is essential to explore its mechanism in detail to determine the underlying cause. Recently, artemisinin-resistant parasites have been reported to exhibit nonsynonymous single nucleotide polymorphisms in genes involved in MMR pathways such as MutL homolog (MLH) and UvrD. Plasmodium falciparum MLH is an endonuclease required to restore the defective MMR in drug-resistant W2 strain of P. falciparum. Although the role of helicases in eukaryotic MMR has been questioned, the identification and characterization of the UvrD helicase and their cross-talk with MLH in P. falciparum suggests the possible involvement of UvrD in MMR. A comparative genome-wide analysis revealed the presence of the UvrD helicase in Plasmodium species, while it is absent in human host. Therefore, PfUvrD may emerge as a suitable drug target to control malaria. This review study is focused on recent developments in MMR biochemistry, emerging importance of the UvrD helicase, possibility of its involvement in MMR and the emerging cross-talk between MMR components and drug resistance in malaria parasite.

Plasmodium falciparum Werner homologue is a nuclear protein and its biochemical activities reside in the N-terminal region

RecQ helicases, also addressed as a gatekeeper of genome, are an inevitable family of genome scrutiny proteins conserved from prokaryotes to eukaryotes and play a vital role in DNA metabolism. The deficiencies of three RecQ proteins out of five are involved in genetic abnormalities like Bloom syndrome (BS), Werner syndrome (WS), and Rothmund–Thomson syndrome (RTS). It is noteworthy that Plasmodium falciparum contains only two members of the RecQ family as opposed to five members present in the host Homo sapiens. In the present study, we report the biochemical characterization of the homologue of Werner (Wrn) helicase from P. falciparum 3D7 strain. Although there are significant sequence conservations between Wrn helicases of both H. sapiens and P. falciparum as well as among all the other Plasmodium species, they contain some peculiar differences also. In silico studies reveal that PfWrn is evolutionarily close to the bacterial RecQ protein. The N-terminal fragment (PfWrnN) contains all the helicase motifs along with all the functional domains and the predicted structure resembles with the human RecQ1 protein, whereas the C-terminal fragment (PfWrnC) contains no significant domain. Biochemical characterization further revealed that purified recombinant PfWrnN shows ATPase and DNA helicase activity in 3′ to 5′ direction, but PfWrnC lacks the ATPase and helicase activities. Immunofluorescence study shows that PfWrn is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain and localizes distinctly in the nucleus. This study can be used for further characterization of RecQ helicases that will aid in understanding the physiological significance of these helicases in the malaria parasite.

Identification of R2TP complex of Leishmania donovani and Plasmodium falciparum using genome wide in-silico analysis.

Recently discovered R2TP complex is an important multiprotein complex involved in multiple cellular process like snoRNP biogenesis, PIKK signaling, RNA polymerase II assembly and apoptosis. Within R2TP complex, Pih1 tightly interacts with Rvb1/Rvb2 and with Tah1 to form R2TP macromolecular complex. R2TP complex further interacts with Hsp90 to form R2TP-Hsp90 complex, which has been found critical in many cellular process. The genome wide screening of Leishmania donovani and Plasmodium falciparum led to the identification of RuvB like1, RuvB like 2, Pih1, and Tah1. Therefore, we speculate that this complex is also important for these parasites as in the yeast. The detailed analysis of crucial components of R2TP complex, Ld-RuvB like 1, and Ld-RuvB like 2, revealed the presence of characteristic motifs like DNA binding motif and ATPase motifs. Hsp90 is also reported from Leishmania donovani and Plasmodium falciparum suggesting that the R2TP complex further interacts with Hsp90 to form R2TP-Hsp90 complex. Recently it has been discovered that RuvB like proteins are overexpressed in many cancers and their ATPase activity is crucial for cancer cell proliferation and the human RuvBs have been proposed as suitable drug target for cancer. Similarly one of the Plasmodium falciparum RuvB like protein (PfRuvB3) has been found to be specific to the stage where nuclear division led multiplication of parasite take place. Considering all these it seems that the R2TP complex may be playing some critical role both in the cancer cell proliferation in human and rapid multiplication of the parasites Leishmania donovani and Plasmodium falciparum.

Identification of inhibitors of Plasmodium falciparum RuvB1 helicase using biochemical assays.

Human malaria is a major parasitic infection, and the situation has worsened mainly due to the emergence of resistant malaria parasites to several anti-malarial drugs. Thus, an urgent need to find suitable drug targets has led to the development of newer classes of anti-malarial drugs. Helicases have been targeted to develop therapeutics for viral, bacterial, and other microorganism infections. Recently, Plasmodium falciparum RuvB ATPases/helicases have been characterized and proposed as a suitable antimalarial drug target. In the present study, the screening of various compounds was done and the results suggest that PfRuvB1 ATPase activity is inhibited considerably by the novobiocin and partially by cisplatin and ciprofloxacin. Helicase assay of PfRuvB1 in the presence of various compounds suggest novobiocin, actinomycin, and ethidium bromide as potent inhibitors. Novobiocin inhibits the helicase activity of PfRuvB1 possibly by blocking the ATPase activity of PfRuvB1. This study is unique in respect to the identification of novobiocin as inhibitor of PfRuvB1, partially by competing with ATP binding at its active site and provides evidence for PfRuvB1 as target of novobiocin after DNA gyrase-B and HSP90. These studies will certainly help the pharmacologist to design and develop some novel inhibitor specific to PfRuvB1, which may serve as suitable chemotherapeutics to target malaria.