Heme Detoxification Protein (PfHDP) is essential for the hemoglobin uptake and metabolism in Plasmodium falciparum.

Hemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP); a protein localized in the parasitophorus vacuole, parasite food vacuole, and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using GFP trap beads showed the association of PfHDP with Hb as well as with the members of PTEX translocon complex. Association of PfHDP with Hb or Pfexp-2, a component of translocon complex was confirmed by protein-protein interaction and immunolocalization tools. Based on these associations, we studied the role of PfHDP in Hb uptake using the PfHDP-HA-GlmS transgenic parasites line. PfHDP knockdown significantly reduced the Hb uptake in these transgenic parasites in comparison to the wild-type parasites. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Transient knockdown of a member of translocon complex, PfHSP101 in HSP101-DDDHA parasites also showed a decreased uptake of Hb inside the parasite. Together, these results advocate an interaction between PfHDP and the translocon complex at the parasitophorus vacuole membrane and also suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

Mixed plantation of wheat and accumulators in arsenic contaminated plots: A novel way to reduce the uptake of arsenic in wheat and load on antioxidative defence of plant.

Wheat (W) and accumulators (A) were planted in plots (arsenic amended soil and without arsenic) designed with ecotoxicological concern for arsenic safe-grains. For the study sixteen plots of 2 × 2 × 0.5 m (l × b × h) size were prepared. Arsenic (As) in the form of sodium arsenate was applied at 50 mg/kg in plots. Out of these sixteen plots eight plots had arsenic amended soil and rest 8 without any arsenic (C). Accumulator's viz. Pteris vittata (PV), Phragmites australis (PA) and Vetiveria zizanioides (VZ) were planted along with wheat in combination (W + PV, W + PA and W + VZ) in twelve plots (6 AWAs plots and 6 AWC plots). In the rest 4 plots (2 WAs plots and 2 WC plots), only wheat was planted. The study was conducted for two cropping seasons, where accumulators were left in the plots between the cropping seasons except that before 2nd cropping accumulators were properly pruned and extra tillers were removed. The germination % of wheat in WAs in 1st and the 2nd cropping season was found to be 55 and 57%, while in AWAs and AWC plots it was between 86 and 92% (W + VZ, 56 and 73%). The physiological activity was found to be reduced in WAs plots compared to AWAs (except for vetiver combination) and AWC plots in both cropping seasons. The antioxidant activity was enhanced in WAs compared with AWAs. The arsenic concentration in grains of wheat was within the permissible limit set by WHO and GOI in AWAs plots while it exceeded the limit in W + VZ (in 1st cropping) and WAs in both cropings.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Exploring Heme and Hemoglobin Binding Regions of Plasmodium Heme Detoxification Protein for New Antimalarial Discovery.

Hemoglobin degradation/hemozoin formation, essential steps in the Plasmodium life cycle, are targets of existing antimalarials. The pathway still offers vast possibilities to be explored for new antimalarial discoveries. Here, we characterize heme detoxification protein, PfHDP, a major protein involved in hemozoin formation, as a novel drug target. Using in silico and biochemical approaches, we identified two heme binding sites and a hemoglobin binding site in PfHDP. Treatment of Plasmodium falciparum 3D7 parasites with peptide corresponding to the hemoglobin binding domain in PfHDP resulted in food vacuole abnormalities similar to that seen with a cysteine protease inhibitor, E-64 (I-1). Screening of compounds that bound the modeled PfHDP structure in the heme/hemoglobin-binding pockets from Maybridge Screening Collection identified a compound, ML-2, that inhibited parasite growth in a dose-dependent manner, thus paving the way for testing its potential as a new drug candidate. These results provide functional insights into the role of PfHDP in Hz formation and further suggest that PfHDP could be an important drug target to combat malaria.

Rice planted along with accumulators in arsenic amended plots reduced arsenic uptake in grains and shoots.

An experiment was designed using phytoremadiation technology to obtain grains of rice safe for consumption. Sixteen plots of size 2 × 2 m were prepared (8 plots were treated with 50 mg kg-1 of sodium arsenate and rest 8 without any treatment). The study was done for two plantations (1st and 2nd plantation). Rice was planted with three accumulators (Phragmites australis, Vetiveria zizanioides and Pteris vitatta) in treated and untreated plot. Arsenic in grains of Actr (R + Pt, R + Ph and R + Vt) for 1st plantation was 0.4, 0.2 and 0.2 mg kg-1 where as in the case of wActr (Ras) it was 3 mg kg-1. In 2nd plantation the concentration of arsenic in grain of Actr (R + Pt, R + Ph and R + Vt) was 0.1, 0.1 and 0.1 mg kg-1 where as in the case of wActr (Ras) it was 2 mg kg-1. Significant differences in growth and yield parameters of rice between Actr and wActr in 1st plantation, while for 2nd plantation the activity was reduced in combinations except R + Pt and no significant difference was observed between Actr, Acntr and wActr. The study concluded that combinations of accumulators with crops could be useful for the survival and safe grains in As-contaminated soils but with some amendments in long-term remediation.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Host ICAMs play a role in cell invasion by Mycobacterium tuberculosis and Plasmodium falciparum.

Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and participate in diverse cellular processes including host-pathogen interactions. ICAM-1 is expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: a molecular dynamics and normal mode analysis study.

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.

Studies on active site mutants of P. falciparum adenylosuccinate synthetase: insights into enzyme catalysis and activation.

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²+ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K(m) values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k(cat) value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.

Adenine metabolism in Plasmodium falciparum.

Plasmodium falciparum lacks the de novo purine biosynthesis pathway and relies entirely on the salvage pathway to meet its purine nucleotide requirements. The entire flux for purine nucleotide biosynthesis in the parasite is believed to be through hypoxanthine guanine phosphoribosyltransferase (HGPRT), with the enzymes, adenosine kinase and adenine phosphoribosyltransferase (APRT) being unannotated in the Plasmodium genome database. This manuscript reports on the studies carried out to explore bypass mechanisms, if any, for AMP synthesis in the intraerythrocyitc stages of the parasite life cycle. Uptake and subsequent incorporation of radiolabel adenine in the nucleotide pool of saponin released erythrocyte free parasites implicated the role of parasite encoded enzymes in adenine metabolism. To explore the route for AMP synthesis in the parasite, we have monitored adenine mediated supplementation of metabolic viability in saponin released hadacidin (N-formyl-N-hydroxyglycine) treated parasites. Our results implicate the role of an APRT like activity that enables parasite survival when the flux through the HGPRT pathway is blocked.

Methanocaldococcus jannaschii adenylosuccinate synthetase: studies on temperature dependence of catalytic activity and structural stability.

Two important attributes of enzymes produced by thermophilic organisms are thermophilicity and structural stability. This manuscript discusses the characterization of these two aspects in adenylosuccinate synthetase from the thermophilic archaeon, Methanocaldococcus jannaschii. Adenylosuccinate synthetase catalyzes the formation of succinyl-AMP from IMP and aspartate with the simultaneous conversion of GTP to GDP. Temperature dependence of M. jannaschii AdSS (MjAdSS) catalysis exhibited a biphasic Arrhenius Plot with a transition at 40 degrees C. Pre-steady-state kinetics as a function of temperature indicated a change in rate determining step of the reaction across the inflection point. Slow release of products from the enzyme active site probably accounts for the thermophilicity of MjAdSS. Thermal unfolding of MjAdSS exhibited a T(m) of 85 degrees C, with the process being only partially reversible. Stability of MjAdSS assessed by equilibrium unfolding revealed the robustness of the secondary and tertiary structure of the enzyme which remained intact even at 8 M concentration of urea. Guanidinium chloride induced denaturation of MjAdSS permitted estimation of thermodynamic parameters. The unfolding profiles could be described as a composite of atleast two distinct transitions, with a stable intermediate in the unfolding pathway.

Kinetic characterization of adenylosuccinate synthetase from the thermophilic archaea Methanocaldococcus jannaschii.

Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.

Unique kinetic mechanism of Plasmodium falciparum adenylosuccinate synthetase.

Adenylosuccinate synthetase (AdSS) catalyses the Mg(2+) dependent formation of adenylosuccinate from IMP and aspartate, the reaction being driven by the hydrolysis of GTP to GDP. All characterized AdSS thus far exhibit a random kinetic mechanism. We present here kinetic evidence that unlike all other AdSS, Plasmodium falciparum AdSS (PfAdSS) has ordered substrate binding. Inhibition studies show that binding of GTP requires IMP binding while aspartate binds to the enzyme-IMP-GTP complex. A structural basis for this difference in mechanism is presented. Kinetically, PfAdSS is closer to the mouse acidic isozyme rather than to the mouse basic isozyme. The mouse acidic isozyme is thought to play a role in the purine nucleotide biosynthetic pathway. Regulation of PfAdSS in vivo can therefore, be expected to be similar to the mouse acidic isozyme, in agreement with the role of PfAdSS as the only pathway for the synthesis of adenine nucleotides in the parasite. However, PfAdSS differs from both the mammalian homologs in that fructose-1,6-bisphosphate, a potent inhibitor of the mammalian enzyme, is an activator of PfAdSS. The differences highlighted here are promising in terms of species-specific drug design, targeting this essential enzyme in the parasite.