Survival strategies of the malarial parasite Plasmodium falciparum

Plasmodium falciparum, the protozoan parasite causing falciparum malaria, is undoubtedly highly versatile when it comes to survival and defence strategies. Strategies adopted by the asexual blood stages of Plasmodium range from unique pathways of nutrient uptake to immune evasion strategies and multiple drug resistance. Studying the survival strategies of Plasmodium could help us envisage strategies of tackling one of the worst scourges of mankind

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding of Ricinus communis agglutinin and Abrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for both Ricinus communis agglutinin and Abrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions

Carbohydrate specificity and salt-bridge mediated conformational change in acidic winged bean agglutinin

Structures of two crystal forms of the dimeric acidic winged bean agglutinin (WBAII) complexed with methyl-α-D-galactose have been determined at 3.0 Å and 3.3 Å resolution. The subunit structure and dimerisation of the lectin are similar to those of the basic lectin from winged bean (WBAI) and the lectin from Erythrina corallodendron (EcorL). The conformation of a loop and its orientation with respect to the rest of the molecule in WBAII are, however, different from those in all the other legume lectins of known structure. This difference appears to have been caused by the formation of two strategically placed salt bridges in the former. Modelling based on the crystal structures provides a rationale for the specificity of the lectin, which is very different from that of WBAI, for the H-antigenic determinant responsible for O blood group reactivity. It also leads to a qualitative explanation for the thermodynamic data on sugar-binding to the lectin, with special emphasis on the role of a tyrosyl residue in the variable loop in the sugar-binding region in generating the carbohydrate specificity of WBAII

Plasmepsin inhibitors: design, synthesis, inhibitory studies and crystal structure analysis

Plasmepsin group of enzymes are key enzymes in the life cycle of malarial parasites. As inhibition of plasmepsins leads to the parasite's death, these enzymes can be utilized as potential drug targets. Although many drugs are available, it has been observed that Plasmodium falciparum, the species that causes most of the malarial infections and subsequent death, has developed resistance against most of the drugs. Based on the cleavage sites of hemglobin, the substrate for plasmepsins, we have designed two compounds (p-nitrobenzoyl-leucine-β -alanine and p-nitrobenzoyl-leucine-isonipecotic acid), synthesized them, solved their crystal structures and studied their inhibitory effect using experimental and theoretical (docking) methods. In this paper, we discuss the synthesis, crystal structures and inhibitory nature of these two compounds which have a potential to inhibit plasmepsins

Multivalency in lectins-a crystallographic,modelling and light-scattering study involving peanut lectin and a bivalent ligand

Multivalency is believed to be important in the activity of lectins, although definitive structural studies on it have been few and far between. We have now studied the complexation of tetravalent peanut lectin with a synthetic compound containing two terminal lactose moieties, using a combination of crystallography, dynamic light scattering and modelling. Light scattering indicates the formation of an apparent dimeric species and also larger aggregates of the tetrameric lectin in the presence of the bivalent ligand. Crystals of presumably crosslinked lectin molecules could be obtained. They diffract poorly, but X-ray data from them are good enough to define the positions of the lectin molecules. Extensive modelling on possible crosslinking modes of protein molecules by the ligand indicated that systematic crosslinking could lead to crystalline arrays. The studies also provided a rationale for crosslinking in the observed crystal structure. The results obtained provide further insights into the general problem of multivalency in lectins. They indicate that crosslinking involving multivalent lectins and multivalent carbohydrates could lead to an ensemble of a finite number of distinct periodic arrays rather than a unique array

Structural plasticity of peanut lectin: an X-ray analysis involving variation in pH, ligand binding and crystal structure

Until recently, it has only been possible to grow crystals of peanut lectin when complexed with sugar ligands. It is now shown that it is possible to grow peanut lectin crystals at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of these peptides as well as lactose. Low-pH crystal forms of the lectin-lactose complex similar to those obtained at neutral pH have also been grown. Thus, crystals of peanut lectin grown under different environmental conditions, at two pH values with and without sugar bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison between different structures shows that the lectin molecule is sturdy and that the effect of changes in pH, ligand binding and environment on it is small. The region involving the curved front β-sheet and the loops around the second hydrophobic core is comparatively rigid. The back β-sheet involved in quaternary association, which exhibits considerable variability, is substantially flexible, as is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing unusual structural features. Small consistent movements occur in the combining site upon sugar binding, although the site is essentially preformed

Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25°C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in α-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line

This article is made available through the Brunel Open Access Publishing Fund. Copyright: © 2013 Mahajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and timedependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SPD in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.Department of Biotechnology, Indi

Insights into the Regulatory Characteristics of the Mycobacterial Dephosphocoenzyme A Kinase: Implications for the Universal CoA Biosynthesis Pathway

Being vastly different from the human counterpart, we suggest that the last enzyme of the Mycobacterium tuberculosis Coenzyme A biosynthetic pathway, dephosphocoenzyme A kinase (CoaE) could be a good anti-tubercular target. Here we describe detailed investigations into the regulatory features of the enzyme, affected via two mechanisms. Enzymatic activity is regulated by CTP which strongly binds the enzyme at a site overlapping that of the leading substrate, dephosphocoenzyme A (DCoA), thereby obscuring the binding site and limiting catalysis. The organism has evolved a second layer of regulation by employing a dynamic equilibrium between the trimeric and monomeric forms of CoaE as a means of regulating the effective concentration of active enzyme. We show that the monomer is the active form of the enzyme and the interplay between the regulator, CTP and the substrate, DCoA, affects enzymatic activity. Detailed kinetic data have been corroborated by size exclusion chromatography, dynamic light scattering, glutaraldehyde crosslinking, limited proteolysis and fluorescence investigations on the enzyme all of which corroborate the effects of the ligands on the enzyme oligomeric status and activity. Cysteine mutagenesis and the effects of reducing agents on mycobacterial CoaE oligomerization further validate that the latter is not cysteine-mediated or reduction-sensitive. These studies thus shed light on the novel regulatory features employed to regulate metabolite flow through the last step of a critical biosynthetic pathway by keeping the latter catalytically dormant till the need arises, the transition to the active form affected by a delicate crosstalk between an essential cellular metabolite (CTP) and the precursor to the pathway end-product (DCoA)