A structural analysis of G-quadruplex/ligand interactions

This focused review article discusses in detail, all available high-resolution small molecule ligand/G-quadruplex structural data derived from crystallographic and NMR based techniques, in an attempt to understand key factors in ligand binding and to highlight the biological importance of these complexes. In contrast to duplex DNA, G-quadruplexes are four-stranded nucleic acid structures folded from guanine rich repeat sequences stabilized by the stacking of guanine G-quartets and extensive Watson-Crick/Hoogsteen hydrogen bonding. Thermally stable, these topologies can play a role in telomere regulation and gene expression. The core structures of G-quadruplexes form stable scaffolds while the loops have been shown, by the addition of small molecule ligands, to be sufficiently adaptable to generate new and extended binding platforms for ligands to associate, either by extending G-quartet surfaces or by forming additional planar dinucleotide pairings. Many of these structurally characterised loop rearrangements were totally unexpected opening up new opportunities for the design of selective ligands. However these rearrangements do significantly complicate attempts to rationally design ligands against well defined but unbound topologies, as seen for the series of napthalene diimides complexes. Drawing together previous findings and with the introduction of two new crystallographic quadruplex/ligand structures we aim to expand the understanding of possible structural adaptations available to quadruplexes in the presence of ligands, thereby aiding in the design of new selective entities. (C) 2011 Elsevier Masson SAS. All rights reserved.

Sequential C-H and C-Ru bond formation and cleavage during the thermally induced rearrangement of aryl ruthenium(II) complexes with [C6H3(CH2NMe2)(2)-2,6](-) as a bidentate eta(2)-C,N coordinated ligand. The crystal structures of the isomeric pairs [RuCl{eta(6)-C10H14}{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,n}] (n = 4 or 6) and [Ru(eta(5)-C5H5){(eta(2)-C,N-C6H3(CH2NMe2)(2)-2,n} (PPH3)] (n = 4 or 6)

New air-stable ruthenium(II) complexes that contain the aryldiamine ligand [C6H3(CH2-NMe2)(2)-2,6](-) (NCN) are described. These complexes are [RuCl{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,6}(eta(6)-C10H14)] (2; C10H14 = p-cymene = C6H4Me-Pr-i-4), [Ru{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,6}(eta(5)-C5H5)(PPh3)] (5), and their isomeric forms [RuCl{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,4}(eta(6)-C10H14)] (3) and [Ru{eta(2)-C,N-C6H3(CH2NMe2)(2)-2,4}(eta(5)-C5H5)(PPh3)] (6), respectively. Complex 2 has been prepared from the reaction of [Li(NCN)](2) with [RuCl2(eta(6)-C10H14)](2), whereas complex 5 has been prepared by the treatment of [RuCl{eta(3)-N,C,N-C6H3(CH2NMe2)(2)-2,6}(PPh3)] (4) with [Na(C5H5)](n). Both 2 and 5 are formally 18-electron ruthenium(II) complexes in which the monoanionic potentially tridentate coordinating ligand NCN is eta(2)-C,N-bonded, In solution (halocarbon solvent at room temperature or in aromatic solvents at elevated temperature), the intramolecular rearrangements of 2 and 5 afford complexes 3 and 6, respectively. This is a result of a shift of the metal-C-aryl bond from position-1 to position-3 on the aromatic ring of the NCN ligand. The mechanism of the isomerization is proposed to involve a sequence of intramolecular oxidative addition and reductive elimination reactions of both aromatic and aliphatic C-H bonds. This is based on results from deuterium labeling, spectroscopic studies, and some kinetic experiments. The mechanism is proposed to contain fully reversible steps in the case of 5, but a nonreversible step involving oxidative addition of a methyl NCH2-H bond in the case of 2. The solid-state structures of complexes 2, 3, 5, and 6 have been determined by single-crystal X-ray diffraction. A new dinuclear 1,4-phenylene-bridged bisruthenium(II) complex, [1,4-{RuCl(eta(6)-C10H14)}(2){C-6(CH2NMe2)(4)-2,3,5,6-C,N,C',N'}] (9) has also been prepared from the dianionic ligand [C-6(CH2NMe2)(4)-2,3,5,6](2-) (C2N4). The C2N4 ligand is in an eta(2)-C,N-eta(2)-C',N'-bis(bidentate) bonding mode. Compound 9 does not isomerize in solution (halocarbon solvent), presumably because of the absence of an accessible C-aryl-H bond. Complex 9 could not be isolated in an analytically pure form, probably because of its high sensitivity to air and very low solubility, which precludes recrystallization.

Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C-3-C-8) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

Applied aspects of Rhodococcus genetics

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

Fluid Shear Induces Conformation Change in Human Blood Protein von Willebrand Factor in Solution

Many of the physiological functions of von Willebrand Factor (VWF), including its binding interaction with blood platelets, are regulated by the magnitude of applied fluid/hydrodynamic stress. We applied two complementary strategies to study the effect of fluid forces on the solution structure of VWF. First, small-angle neutron scattering was used to measure protein conformation changes in response to laminar shear rates (G) up to 3000/s. Here, purified VWF was sheared in a quartz Couette cell and protein conformation was measured in real time over length scales from 2-140 nm. Second, changes in VWF structure up to 9600/s were quantified by measuring the binding of a fluorescent probe 1,1'-bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate (bis-ANS) to hydrophobic pockets exposed in the sheared protein. Small angle neutron scattering studies, coupled with quantitative modeling, showed that VWF undergoes structural changes at G < 3000/s. These changes were most prominent at length scales <10 nm (scattering vector (q) range >0.6/nm). A mathematical model attributes these changes to the rearrangement of domain level features within the globular section of the protein. Studies with bis-ANS demonstrated marked increase in bis-ANS binding at G > 2300/s. Together, the data suggest that local rearrangements at the domain level may precede changes at larger-length scales that accompany exposure of protein hydrophobic pockets. Changes in VWF conformation reported here likely regulate protein function in response to fluid shear.

The role of SRC-CAS interactions in cellular transformation:ectopic expression of the carboxy terminus of CAS inhibits SRC-CAS interaction but has no effect on cellular transformation

Several lines of evidence indicate that the adapter molecule p130CAS (crk-associated substrate (CAS)) is required for src-mediated cellular transformation. CAS has been shown to be heavily tyrosine-phosphorylated in src-transformed cells, and genetic variants of src that are deficient in CAS binding are also unable to mediate cellular transformation. In this report, we investigated whether CAS phosphorylation and/or its association with src are required elements of the transformation process. Expression of the carboxy-terminal src binding domain of CAS in Rat 1 fibroblasts expressing a temperature-sensitive allele of v-src inhibited the formation of src-CAS complexes and also inhibited tyrosine phosphorylation of CAS. However, expression of this protein had no effect on morphological transformation, src-mediated actin rearrangements, or anchorage-independent growth of these cells when grown at the src-permissive temperature. Thus, the ability of activated src to mediate cellular transformation is either largely independent of endogenous CAS phosphorylation and/or its association with CAS or, alternatively, the carboxy-terminus of CAS may substitute for endogenous CAS in the process of src-mediated transformation.

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III + IV (S) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.

Characterisation of the active/de-active transition of mitochondrial complex I

Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39 kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site. The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability. Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Chromosome synapsis and recombination in simple and complex chromosomal heterozygotes of tuco-tuco (Ctenomys talarum: Rodentia: Ctenomyidae):Rodentia: Ctenomyidae)

The chromosomal speciation hypothesis suggests that irregularities in synapsis, recombination, and segregation in heterozygotes for chromosome rearrangements may restrict gene flow between karyotypically distinct populations and promote speciation. Ctenomys talarum is a South American subterranean rodent inhabiting the coastal regions of Argentina, whose populations polymorphic for Robertsonian and tandem translocations seem to have a very restricted gene flow. To test if chromosomal differences are involved in isolation among its populations, we examined chromosome pairing, recombination, and meiotic silencing of unsynapsed chromatin in male meiosis of simple and complex translocation heterozygotes using immunolocalization of the MLH1 marking mature recombination nodules and phosphorylated histone γH2A.X marking unrepaired double-strand breaks. We observed small asynaptic areas labeled by γH2A.X in pericentromeric regions of the chromosomes involved in the trivalents and quadrivalents. We also observed a decrease of recombination frequency and a distalization of the crossover distribution in the heterozygotes and metacentric homozygotes compared to acrocentric homozygotes. We suggest that the asynapsis of the pericentromeric regions are unlikely to induce germ cell death and decrease fertility of the heterozygotes; however, suppressed recombination in pericentromeric areas of the multivalents may reduce gene flow between chromosomally different populations of the Talas tuco-tuco.

Downregulation of the Wnt inhibitor CXXC5 predicts a better prognosis in acute myeloid leukemia

The gene CXXC5 on 5q31 is frequently deleted in acute myeloid leukemia (AML) with del(5q), suggesting that inactivation of CXXC5 might play a role in leukemogenesis. Here, we investigated the functional and prognostic implications of CXXC5 expression in AML. CXXC5 mRNA was downregulated in AML with MLL rearrangements, t(8;21) and GATA2 mutations. As a mechanism of CXXC5 inactivation, we found evidence for epigenetic silencing by promoter methylation. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P = .007) and a better overall survival (OS, 46% vs 28%; P < .001) and event-free survival (EFS, 36% vs 21%; P < .001) at 5 years, independent of cytogenetic risk groups and known molecular risk factors. In gene-expression profiling, lower CXXC5 expression was associated with upregulation of cell-cycling genes and codownregulation of genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1). Functional analyses demonstrated CXXC5 to inhibit leukemic cell proliferation and Wnt signaling and to affect the p53-dependent DNA damage response. In conclusion, our data suggest a tumor suppressor function of CXXC5 in AML. Inactivation of CXXC5 is associated with different leukemic pathways and defines an AML subgroup with better outcome.

Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches

In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ-PCR) and 3-colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time-points during treatment (93.38%), and a combined use of both approaches allowed a multi time-point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0.01% were identified by RQ-PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ-PCR and FC approach, the evaluation of 367 follow-up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0.04), >0.01% at the end of induction (P = 0.02), >0.01% at the end of consolidation (P = 0.01), >0.01% prior to the first delayed intensification (P = 0.01), and >0.1% prior to the second delayed intensification and continued maintenance (P = 0.001) were all associated with relapse and, based on early time-points (end of induction and consolidation) a significant log-rank trend (P = 0.0091) was noted between survival curves for patients stratified into high, intermediate and low-risk MRD groups.