Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized. Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA. Conclusion: MtRecD possesses strong 5 3- and weak 3 5-helicase activities. Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5 overhangs relative to the 3 overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having 18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3 overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5 overhangs, it could also catalyze significant unwinding of substrates containing 3 overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5 3 and weak 3 5 unwinding activities. The extent of unwinding of Y-shaped DNA structures was approximate to 3-fold lower compared with duplexes with 5 overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented Delta mimG: Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant Msm Rv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-kappa B, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Synthesis and evaluation of the biological activity of N `-2-oxo-1,2 dihydro-3H-indol-3-ylidene] benzohydrazides as potential anticancer agents

New N'-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzohydrazide derivatives were synthesized and evaluated for their cytotoxic properties against murine leukemia, L1210, human leukemia, REH and K562, human T-cell leukemia, CEM and human cervix carcinoma, HeLa cells. Among the tested compounds, the 3,4,5-trimethoxy-N'-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]ben zohydrazide derivative (5t) emerged as the most potent inhibitor against all the tumor cell lines evaluated. To investigate the mechanism of action, 5t was further studied by cell cycle analysis, mitochondrial membrane potential analysis, DNA fragmentation and Annexin V-FITC flow cytometric analysis, which suggested that 5t was able to induce apoptosis at submicromolar range.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

An enolato-bridged dinuclear Cu(II) complex with a coumarin-assisted precursor: a spectral, magnetic and biological study

A new, phenoxo-bridged Cu-II dinuclear complex Cu-2(L)(2)(DMF)(2)] (1) has been obtained by employing the coumarin-assisted tridentate precursor, H2L, benzoic acid(7-hydroxy-4-methyl-2-oxo-2H-chromen-8-ylmethylene)-hydrazide]. Complex 1 has been systematically characterized by FTIR, UV-Vis, fluorescence and PR spectrometry. The single crystal X-ray diffraction analysis of 1 shows that the geometry around each copper ion is square pyramidal, comprising two enolato oxygen atoms belonging to different ligands (which assemble the dimer bridging the two metal centers), one imine-N and one phenolic-O atoms of the Schiff base and one oxygen atom from the DMF molecule. The temperature dependent magnetic interpretation agrees with the existence of weak ferromagnetic interactions between the bridging dinuclear Cu(II) ions. Both the ligand and complex 1 exhibit anti-mycobacterial activity and considerable efficacy towards M. tuberculosis H37Rv ATCC 27294 and M. tuberculosis H37Ra ATCC 25177 strains. The cytotoxicity study on human adenocarcinoma cell lines (MCF7) suggests that the ligand and complex 1 have potential anticancer properties. Molecular docking of H2L with the enoyl acyl carrier protein reductase of M. tuberculosis H37R(v) (PDB ID: 4U0K) is examined and the best docked pose of H2L shows one hydrogen bond with Thr196 (1.99 angstrom).

A novel benzimidazole derivative binds to the DNA minor groove and induces apoptosis in leukemic cells

DNA minor groove binders are an important class of chemotherapeutic agents. These small molecule inhibitors interfere with various cellular processes like DNA replication and transcription. Several benzimidazole derivatives showed affinity towards the DNA minor groove. In this study we show the synthesis and biological studies of a novel benzimidazole derivative (MH1), that inhibits topoisomerase II activity and in vitro transcription. UV-visible and fluorescence spectroscopic methods in conjunction with Hoechst displacement assay demonstrate that MH1 binds to DNA at the minor groove. Cytotoxic studies showed that leukemic cells are more sensitive to MH1 compared to cancer cells of epithelial origin. Further, we find that MH1 treatment leads to cell cycle arrest at G2/M, at early time points in Molt4 cells. Finally multiple cellular assays demonstrate that MH1 treatment leads to reduction in MMP, induction of apoptosis by activating CASPASE 9 and CASPASE 3. Thus our study shows MH1, a novel DNA minor groove binder, induces cytotoxicity efficiently in leukemic cells by activating the intrinsic pathway of apoptosis.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90 degrees light scattering and FtsZ polymer pelleting assays. The gamma 32P-GTP synthesised by NDK from GDP and gamma 32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound P-32-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

Clean localization super-resolution microscopy for 3D biological imaging

We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells. (C) 2016 Author(s).

Electron transfer amongst flavo- and hemo-proteins: diffusible species effect the relay processes, not protein-protein binding

Hitherto, electron transfer (ET) between redox proteins has been deemed to occur via donor-acceptor binding, and diffusible reactive species are considered as deleterious side-products in such systems. Herein, ET from cytochrome P450 reductase (CPR, an animal membrane flavoprotein) and horseradish peroxidase (HRP, a plant hemoprotein) to cytochrome c (Cyt c, a soluble animal hemoprotein) was probed under diverse conditions, using standard assays. ET in the CPR-Cyt c system was critically inhibited by cyanide and sub-equivalent levels of polar one-electron cyclers like copper ions, vitamin C/Trolox and superoxide dismutase. In the presence of lipids, inhibition was also afforded by amphipathic molecules vitamin E, palmitoyl-vitamin C and the membrane hemoprotein, cytochrome b(5). Such nonspecific inhibition (by diverse agents in both aqueous and lipid phases) indicated that electron transfer/relay was effected by small diffusible agents, whose lifetimes are shortened by the diverse radical scavengers. When CPR was retained in a dialysis membrane and Cyt c presented outside in free solution, ET was still observed. Further, HRP (taken at nM levels) catalyzed oxidation of a phenolic substrate was significantly inhibited upon the incorporation of sub-nM levels of Cyt c. The findings imply that CPR-Cyt c or HRP-Cyt c binding is not crucial for ET. Further, fundamental quantitative arguments (based on diffusion/collision) challenge the erstwhile protein-protein binding-assisted ET hypothesis. It is proven beyond reasonable doubt that mobile and diffusible electron carriers (ions and radicals) serve as ``redox-relay agents'' in the biological ET models/setup studied.