Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Quaternary association in beta-prism I fold plant lectins: Insights from X-ray crystallography, modelling and molecular dynamics

Dimeric banana lectin and calsepa, tetrameric artocarpin and octameric heltuba are mannose-specific beta-prism I fold lectins of nearly the same tertiary structure. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions that undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Energy minimization and simulations were also carried out on models using all possible oligomers by employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also led to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding.

Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (m beta 1, m beta 1'beta 2, m beta 1-beta 5, m beta 1-beta 6 and m beta 4-beta 5) by transplanting beta 1, beta 1'beta 2, beta 1-beta 5, beta 1-beta 6 and beta 4-beta 5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, m beta 1'beta 2(ESWR) SSB was generated by mutating the MtuSSB specific `PRIY' sequence in the beta 2 strand of m beta 1'beta 2 SSB to EcoSSB specific `ESWR' sequence. Biochemical characterization revealed that except for m beta 1 SSB, all chimeras and a control construct lacking the C-terminal domain (Delta C SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, m beta 1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that m beta 1-beta 6, MtuSSB, m beta 1'beta 2 and m beta 1-beta 5 SSBs complemented E. coli Delta ssb in a dose dependent manner. Complementation by the m beta 1-beta 5 SSB was poor. In contrast, m beta 1'beta 2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Interaction of G-Quadruplexes with Nonintercalating Duplex-DNA Minor Groove Binding Ligands

The enzyme telomerase synthesizes the G-rich DNA strands of the telomere and its activity is often associated with cancer. The telomerase may be therefore responsible for the ability of a cancer cell-to escape apoptosis. The G-rich DNA sequences often adopt tetra-stranded structure, known as the G-quadruplex DNA (G4-DNA). The stabilization of the telomeric DNA into the G4-DNA structures by small molecules has been the focus of many researchers for the design and development of new anticancer agents. The compounds which stabilize the G-quadruplex in the telomere inhibit the telomerase activity. Besides telomeres, the G4-DNA forming sequences are present in the genomic regions of biological significance including the transcriptional regulatory and promoter regions of several oncogenes. Inducing a G-quadruplex structure within the G-rich promoter sequences is a potential way of achieving selective gene regulation. Several G-quadruplex stabilizing ligands are known. Minor groove binding ligands (MGBLs) interact with the double-helical DNA through the minor grooves sequence-specifically and interfere with several DNA associated processes. These MGBLs when suitably modified switch their preference sometimes from the duplex DNA to G4-DNA and stabilize the G4-DNA as well. Herein, we focus on the recent advances in understanding the G-quadruplex structures, particularly made by the human telomeric ends, and review the results of various investigations of the interaction of designed organic ligands with the G-quadruplex DNA while highlighting the importance of MGBL-G-quadruplex interactions.

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps 1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA protein interactions under the same set of conditions.

A Monoclonal Antibody against Human Notch1 Ligand-Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem-like Cells

Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-like4 and consequently, signaling in a dose-dependent manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15. These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44(Hi)/CD24(Low) subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem-like cell subpopulation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative stem-like cells appeared to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated expression of genes associated with stemness and epithelial-mesenchymal transition. Thus, targeting individual Notch receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem-like cell subpopulation. Mol Cancer Ther; 11(1); 77-86. (C) 2011 AACR.

CNT loading into cationic cholesterol suspensions show improved DNA binding and serum stability and ability to internalize into cancer cells

Methods which disperse single-walled carbon nanotubes (SWNTs) in water as `debundled', while maintaining their unique physical properties are highly useful. We present here a family of cationic cholesterol compounds (Chol(+)) {Cholest-5en-3 beta-oxyethyl pyridinium bromide (Chol-PB+), Cholest-5en-3 beta-oxyethyl N-methyl pyrrolidinium bromide (Chol-MPB+), Cholest-5en-3 beta-oxyethyl N-methyl morpholinium bromide (Chol-MMB+) and Cholest-5en-3 beta-oxyethyl diazabicyclo octanium bromide (Chol-DOB+)}. Each of these could be easily dispersed in water. The resulting cationic cholesterol (Chol(+)) suspensions solubilized single-walled carbon nanotubes (SWCNTs) by the non-specific physical adsorption of Chol(+) to form stable, transparent, dark aqueous suspensions at room temperature. Electron microscopy reveals the existence of highly segregated CNTs in these samples. Zeta potential measurements showed an increase in potential of cationic cholesterol aggregates on addition of CNTs. The CNT-Chol(+) suspensions were capable of forming stable complexes with genes (DNA) efficiently. The release of double-helical DNA from such CNT-Chol(+) complexes could be induced upon the addition of anionic micellar solution of SDS. Furthermore, the CNT-based DNA complexes containing cationic cholesterol aggregates showed higher stability in fetal bovine serum media at physiological conditions. Confocal studies confirm that CNT-Chol(+) formulations adhere to HeLa cell surfaces and get internalized more efficiently than the cationic cholesterol suspensions alone (devoid of any CNTs). These cationic cholesterol-CNT suspensions therefore appear to be a promising system for further use in biological applications.

Binding of BIS like and other ligands with the GSK-3 beta kinase: a combined docking and MM-PBSA study

Binding of several bisindolylmaleimide (BIS) like (BIS-3, BIS-8 and UCN1) and other ligands (H89, SB203580 and Y27632) with the glycogen synthase kinase-3 (GSK-3 beta) has been studied using combined docking, molecular dynamics and Poisson-Boltzmann surface area analysis approaches. The study generated novel binding modes of these ligands that can rationalize why some ligands inhibit GSK-3 beta while others do not. The relative binding free energies associated with these binding modes are in agreement with the corresponding measured specificities. This study further provides useful insight regarding possible existence of multiple conformations of some ligands like H89 and BIS-8. It is also found that binding modes of BIS-3, BIS-8 and UCN1 with GSK-3 beta and PDK1 kinases are similar. These new insights are expected to be useful for future rational design of novel, more potent GSK-3 beta-specific inhibitors as promising therapeutics.

Synthesis, X-ray crystal structure, DNA binding, antioxidant and cytotoxicity studies of Ni(II) and Pd(II) thiosemicarbazone complexes

New complexes, [Ni(HL)(PPh3)]Cl (1), [Pd(L)(PPh3)](2), and [Pd(L)(AsPh3)](3), were synthesized from the reactions of 4-chloro-5-methyl-salicylaldehyde thiosemicarbazone [H2L] with [NiCl2(PPh3)(2)], [PdCl2(PPh3)(2)] and [PdCl2(AsPh3)(2)]. They were characterized by IR, electronic, H-1-NMR spectral data. Further, the structures of the complexes have been determined by single crystal X-ray diffraction. While the thiosemicarbazone coordinated as binegative tridentate (ONS) in complexes 2 and 3, it is coordinated as mono negative tridentate (ONS) in 1. The interactions of the new complexes with calf thymus DNA was examined by absorption and emission spectra, and viscosity measurements. Moreover, the antioxidant properties of the new complexes have also been tested against DPPH radical in which complex 1 exhibited better activity than that of the other two complexes 2 and 3. The in vitro cytotoxicity of complexes 1-3 against A549 and HepG2 cell lines was assayed, and the new complexes exhibited higher cytotoxic activity with lower IC50 values indicating their efficiency in killing the cancer cells even at very low concentrations.

Chemistry and biology of DNA-binding small molecules

Regulation of the transcription machinery is one of the many ways to achieve control of gene expression. This has been done either at the transcription initiation stage or at the elongation stage. Different methodologies are known to inhibit transcription initiation via targeting of double-stranded (ds) DNA by: (i) synthetic oligonucleotides, (ii) ds-DNA-specific, sequenceselective minor-groove binders (distamycin A), intercalators (daunomycin) combilexins and (iii) small molecule (peptide or intercalator)-oligonucleotide conjugates. In some cases, instead of ds-DNA, higher order G-quadruplex structures are formed at the start site of transcription. In this regard G-quadruplex DNA-specific small molecules play a significant role towards inhibition of the transcription machinery. Different types of designer DNA-binding agents act as powerful sequence-specific gene modulators, by exerting their effect from transcription regulation to gene modification. But most of these chemotherapeutic agents have serious side effects. Accordingly, there is always a challenge to design such DNA-binding molecules that should not only achieve maximum specific DNA-binding affinity, and cellular and nuclear transport activity, but also would not interfere with the functions of normal cells.

Differential Reaction Kinetics, Cleavage Complex Formation, and Nonamer Binding Domain Dependence Dictate the Structure-Specific and Sequence-Specific Nuclease Activity of RAGs

During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the ``nonamer binding region,'' which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease. (C) 2011 Elsevier Ltd. All rights reserved.

DNA-binding and nuclease activity of1,10-phenanthroline-based thiocyanato, acetato, azido and benzoato bridged dinuclear copper(II) complexes

The mononuclear Cu(II) complex [Cu(phen)(H2O)(NO3)(2)] (1), obtained by the reaction of 1,10-phenanthroline with Cu(NO3)(2)center dot 3H(2)O in methanol solution, reacts with anionic ligands SCN-, AcO-, N-3(-) and PhCO2- in MeOH solution to form the stable binuclear complexes [Cu-2(H2O)(2)(phen)(2)(mu-X)(2)](2) (NO3)(2), where X = SCN- (2), AcO- (3), N-3(-) (4) or PhCO2- (5). The molecular structure of complex 3 was determined by single-crystal X-ray diffraction studies. These complexes were characterized by electronic, IR, ESR, magnetic moments and conductivity measurements. The electrochemical behaviour of the complexes was investigated by cyclic voltammetry. The interactions of these complexes with calf thymus DNA have been investigated using absorption spectrophotometry. Their DNA cleavage activity was studied on double-stranded pBR322 plasmid DNA using gel electrophoresis experiments in the absence and presence of H2O2 as oxidant.