Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE.

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Plasmodium falciparum RuvB2 translocates in 5'-3' direction, relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex.

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA+ family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5'-3' direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.

Pisum sativum p68 DEAD-box protein is ATP-dependent RNA helicase and unique bipolar DNA helicase.

DEAD-box helicases play essential role in DNA and RNA metabolism such as replication, repair, recombination, transcription, translation, ribosome biogenesis and splicing which regulate plant growth and development. The presence of helicases in the stress-induced ORFs identified by cDNA microarray indicates that helicases might be playing an important role in stabilizing growth in plants under stress. p68 DEAD-box helicase has been identified and characterized from animal systems but the properties and functions of plant p68 are poorly understood. In this study, the identification, purification and characterization of recombinant p68 from Pisum sativum (Psp68) is presented. Psp68 possesses all the characteristic motifs like DEAD-box ATP-binding and helicase C terminal motifs and is structurally similar to human p68 homologue. Psp68 exhibits ATPase activity in the presence of both DNA and RNA and it binds to DNA as well as RNA. It contains the characteristic RNA helicase activity. Interestingly Psp68 also shows the unique DNA helicase activity, which is bipolar in nature (unwinds DNA in both the 5'-3' and 3'-5' directions). The Km values of Psp68 for ATPase are 0.5126 and 0.9142 mM in the presence of DNA and RNA, respectively. The Km values of Psp68 are 1.6129 and 1.14 nM for DNA helicase and RNA helicase, respectively. The unique properties of Psp68 suggest that it could be a multifunctional protein involved in different aspect of DNA and RNA metabolism. This discovery should make an important contribution to better understanding of nucleic acids metabolism plants.

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

BACKGROUND: Human 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. RESULTS: Recently 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. CONCLUSIONS: As 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.

Molecular Characterization of Community- and Hospital- Acquired Methicillin-Resistant Staphylococcus aureus Isolates during COVID-19 Pandemic.

Methicillin-resistant Staphylococcus aureus (MRSA) is a drug-resistant superbug that causes various types of community- and hospital-acquired infectious diseases. The current study was aimed to see the genetic characteristics and gene expression of MRSA isolates of nosocomial origin. A total of 221 MRSA isolates were identified from 2965 clinical samples. To identify the bacterial isolates, the clinical samples were inoculated on blood agar media plates first and incubated at 37 °C for 18-24 h. For further identification, the Gram staining and various biochemical tests were performed once the colonies appeared on the inoculated agar plates. The phenotypic identification of antibiotic susceptibility patterns was carried out using Kirby-Bauer disk diffusion method by following the Clinical and Laboratory Standards Institute (CLSI) 2019 guidelines. The biofilm-producing potentials of MRSA were checked quantitatively using a spectrophotometric assay. All strains were characterized genotypically by SCCmec and agr typing using the specific gene primers. Furthermore, a total of twelve adhesion genes were amplified in all MRSA isolates. MRSA was a frequently isolated pathogen (44% community acquired (CA)-MRSA and 56% hospital acquired (HA)-MRSA), respectively. Most of the MRSA isolates were weak biofilm producers (78%), followed by moderate (25%) and strong (7%) biofilm producers, respectively. Prominent adhesion genes were clfB (100%), icaAD (91%), fib (91%), sdrC (91%) followed by eno (89%), fnbA (77%), sdrE (67%), icaBC (65%), clfA (65%), fnbB (57%), sdrD (57%), and cna (48%), respectively. The results of the current study will help to understand and manage the spectrum of biofilm-producing MRSA-associated hospital-acquired infections and to provide potential molecular candidates for the identification of biofilm-producing MRSA.

STIM Proteins and Regulation of SOCE in ER-PM Junctions.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways.

Quantitative Craniofacial Analysis and Generation of Human Induced Pluripotent Stem Cells for Muenke Syndrome: A Case Report.

In this case report, we focus on Muenke syndrome (MS), a disease caused by the p.Pro250Arg variant in fibroblast growth factor receptor 3 (FGFR3) and characterized by uni- or bilateral coronal suture synostosis, macrocephaly without craniosynostosis, dysmorphic craniofacial features, and dental malocclusion. The clinical findings of MS are further complicated by variable expression of phenotypic traits and incomplete penetrance. As such, unraveling the mechanisms behind MS will require a comprehensive and systematic way of phenotyping patients to precisely identify the impact of the mutation variant on craniofacial development. To establish this framework, we quantitatively delineated the craniofacial phenotype of an individual with MS and compared this to his unaffected parents using three-dimensional cephalometric analysis of cone beam computed tomography scans and geometric morphometric analysis, in addition to an extensive clinical evaluation. Secondly, given the utility of human induced pluripotent stem cells (hiPSCs) as a patient-specific investigative tool, we also generated the first hiPSCs derived from a family trio, the proband and his unaffected parents as controls, with detailed characterization of all cell lines. This report provides a starting point for evaluating the mechanistic underpinning of the craniofacial development in MS with the goal of linking specific clinical manifestations to molecular insights gained from hiPSC-based disease modeling.

Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking.

Malaria parasites increase their host erythrocyte's permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell's parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation.IMPORTANCE Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane-bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma.

Genome Wide In silico Analysis of the Mismatch Repair Components of Plasmodium falciparum and Their Comparison with Human Host.

Malaria a major parasitic infection globally particularly in tropical and sub-tropical regions of the world is responsible for about 198 million cases and estimated deaths due to this disease are about 0.6 million. The emergence of drug resistance in the malaria parasite is alarming and it is necessary to understand its underlying cause and molecular mechanisms. It has been established that drug resistant malaria parasites have defective mismatch repair (MMR) therefore it is essential to study this pathway and its components in detail. Recently a number of non-synonymous Single Nucleotide Polymorphisms have been reported in genes involved in MMR pathways. PfMLH is an endonuclease essential to restore the MMR in drug resistant strains of Plasmodium falciparum. Considering all these facts about the role of MMR in emergence of drug resistant parasite, in this manuscript we report a genome wide analysis of the components of the MMR pathway such as MLH, Pms1, MSH2-1, MSH2-2, MSH6, and UvrD using in silico bioinformatics based approaches. The phylogenetic analysis revealed evolutionary closeness with the MMR components of various organisms. It is noteworthy that P. falciparum contains two homologs of MSH2, which are located on different chromosomes. The structural modeling of these components showed their similarity with the human/yeast MMR components. The docking studies reveal that PfUvrD and PfMLH interact with each other. The in silico identification of interacting partners of the major MMR components identified numerous P. falciparum specific proteins. In line with our previous studies the present study will also contribute significantly to understand the MMR pathway of 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 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.

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 RuvB1 is an active DNA helicase and translocates in the 5'-3' direction.

RuvB family of protein contains two similar kinds of proteins i.e. RuvB1 and RuvB2 from yeast to human. These proteins belong to the AAA+ class of proteins and are critical components of several multiprotein complexes involved in diverse cellular activities. There are two RuvB proteins annotated in the Plasmodium database but the identification of the third protein recently by our lab has raised the question why Plasmodium falciparum contains three RuvB proteins instead of two. Hence the biochemical characterizations of these proteins have become essential to understand the role of these proteins in the malaria parasite. Recently we have reported the characterization of the recombinant PfRuvB3, which contains ATPase activity but lacks DNA helicase activity. In the present study we report the phylogenetic analysis and detailed biochemical characterization of one of the other RuvB homologue RuvB1 from P. falciparum. PfRuvB1 shows considerable homology with human as well as yeast RuvB1 and contains Walker motif A and Walker motif B. The activity analysis of this protein revealed that PfRuvB1 is an ATPase and this activity increased significantly in the presence of ss-DNA. PfRuvB1 also contains DNA helicase activity and translocates preferentially in 5' to 3' direction. In vivo investigation of PfRuvB1 revealed that it is constitutively expressed during all the stages of intraerythrocytic cycle of P. falciparum and localizes mainly to the nucleus. These studies will make important contribution in understanding the role of RuvB protein in P. falciparum.

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.

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 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.

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 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.