Nucleic acid-binding specificity of human FUS protein

FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with K_d^app values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners.     

Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands

Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABA(A) receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an unbiased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design.     

Nucleic acid-binding properties of the RRM-containing protein RDM1

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L119GF --> AAA mutation affects the mode of RDM1 binding to single-stranded DNA.     

Use of a fluorescent polarization based high throughput assay to identify new calmodulin ligands

In order to develop a fluorescence polarization (FP) assay for calcium binding proteins, a fluorescent peptides based library of 1328 compounds has been synthesized. The use of this library has been validated by setting up a FP-high-throughput screening (FP-HTS) assay for calmodulin using the synthetic gene product (synCaM). With this assay, a set of 880 FDA approved compounds was screened. Besides the promazine class, we discovered two new classes of compounds that interact with calmodulin in a calcium dependent manner. One class has compounds with anti-histaminic/spasmolytic activities, and the other one are detergents with antibacterial activities.     

Strategies to identify ligands for orphan G-protein-coupled receptors

G-protein-coupled receptors are the most tractable class of protein targets for small molecule drug design. Sequencing of the human genome allied to bio-informatic analysis has identified a large number of putative receptors for which the natural ligands remain undefined. A range of currently employed and developing strategies to identify ligands that interact with these orphan receptors and to validate them as drug targets are described and discussed.     

Nucleic acid-binding metabolic enzymes: Living fossils of stereochemical interactions?

Recently, a series of intriguing observations expanded the list of a number of metabolic enzymes known to be associated with various forms of nucleic acids, including single- and double-stranded DNA, cognate and noncognate RNAs, and specific tRNAs. There is no clear reason why such a phenomenon should take place in contemporary cell physiology, or, further, why such a property has evolved at all. Sixteen known cases are presented in an attempt to delineate any common features of these enzymes. Apart from their ancient nature, as judged by their wide distribution and their participation in fundamental biochemical pathways, it appears that these enzymes do not share any structural or functional characteristics. Given that most of these proteins require nucleotide-based cofactors for their activity, it is proposed that they may represent genuine molecular fossils of the transition from an RNA to a protein world. Their nucleic acid-binding properties are in keeping with previously proposed hypotheses regarding the origins and evolution of nucleotide-based cofactors. The mode of interaction between these proteins and their nucleic acid substrates remains unclear, but it may represent an extended form of stereochemical interactions that have been proposed for the origins of the genetic code.Correspondence to: C.A. Ouzounis     

Preparation and biological activity of molecular probes to identify and analyze jasmonic acid-binding proteins

Several types of jasomonic acid (JA) derivatives, including JA--amino acid conjugates, a JA--biotin conjugate, a JA--dexamethasone heterodimer, and a JA-fluoresceine conjugate, were prepared as candidates for molecular probes to identify JA--binding proteins. These JA derivatives, excepting the JA--fluoresceine conjugate, exhibited significant biological activities in a rice seedling assay, a rice phytoalexin-inducing assay, and/or a soybean phenylalanine ammonia-lyase-inducing assay. These JA derivatives could therefore be useful probes for identifying JA--binding proteins. The activity spectra of the prepared compounds were different from each other, suggesting that different types of JA receptors were involved in the perception of JA derivatives in the respective bioassays.     

Fluorescence-based duplex-quadruplex competition test to screen for telomerase RNA quadruplex ligands

RNA and DNA guanine-rich sequences can adopt unusual structures called Guanine quadruplexes (G4). A quadruplex-prone RNA sequence is present at the 5'-end of the 451-nt-long RNA component of telomerase, hTERC. As this quadruplex may interfere with P1 helix formation, a key structural element for this RNA, we are seeking molecules that would alter this RNA duplex-quadruplex equilibrium. In this work, we present a fluorescence-based test designed to identify G4 ligands specific for the hTERC G-rich motif and that can prevent P1 helix formation. From an initial panel of 169 different molecules, 11 were found to be excellent P1 duplex inhibitors. Interestingly, some of the compounds not only exhibit a strong selectivity for quadruplexes over duplexes, but also demonstrated a preference for G4-RNA over all other quadruplexes. This test may easily be adapted to almost any quadruplex-forming sequence and converted into HTS format.     

Benzoindoloquinolines interact with DNA tetraplexes and inhibit telomerase

Telomeric G-rich single-stranded DNA can adopt a G-tetraplex structure which has been shown to inhibit telomerase activity. We have examined benzoindoloquinolines derivatives for their ability to stabilize an intramolecular G-quadruplex. The increase in T(m) value of the G-quadruplex was associated with telomerase inhibition in vitro.     

New insights into the mechanisms whereby low molecular weight CCR5 ligands inhibit HIV-1 infection

CC chemokine receptor 5 (CCR5) is a G-protein-coupled receptor for the chemokines CCL3, -4, and -5 and a coreceptor for entry of R5-tropic strains of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T-cells. We investigated the mechanisms whereby nonpeptidic, low molecular weight CCR5 ligands block HIV-1 entry and infection. Displacement binding assays and dissociation kinetics demonstrated that two of these molecules, i.e. TAK779 and maraviroc (MVC), inhibit CCL3 and the HIV-1 envelope glycoprotein gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that they bind to regions of CCR5 distinct from the gp120- and CCL3-binding sites. We observed that TAK779 and MVC are full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins. Dissociation of [(125)I]CCL3 from CCR5 was accelerated by TAK779, to a lesser extent by MVC, and by GTP analogs, suggesting that inverse agonism contributes to allosteric inhibition of the chemokine binding to CCR5. TAK779 and MVC also promote dissociation of [(35)S]gp120 from CCR5 with an efficiency that correlates with their ability to act as inverse agonists. Displacement experiments revealed that affinities of MVC and TAK779 for the [(35)S]gp120-binding receptors are in the same range (IC(50) ～6.4 versus 22 nm), although we found that MVC is 100-fold more potent than TAK779 for inhibiting HIV infection. This suggests that allosteric CCR5 inhibitors not only act by blocking gp120 binding but also alter distinct steps of CCR5 usage in the course of HIV infection.     

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

The ends of human chromosomes are protected from the degradation associated with cell division by 15-20 kb long segments of hexameric repeats of 5'-TTAGGG-3' termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea catechins, in MCF-7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF-7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF-7 cells when treated with EGCG. Furthermore, down-regulation of hTERT gene expression in MCF-7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF-7 cells with EGCG resulted in a time-dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F-1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF-7 and HL60 cancer cell lines and may work through different pathways involving both anti-oxidant effects and epigenetic modulation.     

Fatty acid-binding proteins inhibit hydration of epoxyeicosatrienoic acids by soluble epoxide hydrolase

Epoxyeicosatrienoic acids (EETs) are potent regulators of vascular homeostasis and are bound by cytosolic fatty acid-binding proteins (FABPs) with K(d) values of approximately 0.4 microM. To determine whether FABP binding modulates EET metabolism, we examined the effect of FABPs on the soluble epoxide hydrolase (sEH)-mediated conversion of EETs to dihydroxyeicosatrienoic acids (DHETs). Kinetic analysis of sEH conversion of racemic [(3)H]11,12-EET yielded K(m) = 0.45 +/- 0.08 microM and V(max) = 9.2 +/- 1.4 micromol min(-1) mg(-)(1). Rat heart FABP (H-FABP) and rat liver FABP were potent inhibitors of 11,12-EET and 14,15-EET conversion to DHET. The resultant inhibition curves were best described by a substrate depletion model, with K(d) = 0.17 +/- 0.01 microM for H-FABP binding to 11,12-EET, suggesting that FABP acts by reducing EET availability to sEH. The EET depletion by FABP was antagonized by the co-addition of arachidonic acid, oleic acid, linoleic acid, or 20-hydroxyeicosatetraenoic acid, presumably due to competitive displacement of FABP-bound EET. Collectively, these findings imply that FABP might potentiate the actions of EETs by limiting their conversion to DHET. However, the effectiveness of this process may depend on metabolic conditions that regulate the levels of competing FABP ligands.     

Statistical methods to identify mechanisms in studies of eco-evolutionary dynamics

While the reciprocal effects of ecological and evolutionary dynamics are increasingly recognized as an important driver for biodiversity, detection of such eco-evolutionary feedbacks, their underlying mechanisms, and their consequences remains challenging. Eco-evolutionary dynamics occur at different spatial and temporal scales and can leave signatures at different levels of organization (e.g., gene, protein, trait, community) that are often difficult to detect. Recent advances in statistical methods combined with alternative hypothesis testing provides a promising approach to identify potential eco-evolutionary drivers for observed data even in non-model systems that are not amenable to experimental manipulation. We discuss recent advances in eco-evolutionary modeling and statistical methods and discuss challenges for fitting mechanistic models to eco-evolutionary data.     

Dynamics in microbial communities: unraveling mechanisms to identify principles

Diversity begets higher-order properties such as functional stability and robustness in microbial communities, but principles that inform conceptual (and eventually predictive) models of community dynamics are lacking. Recent work has shown that selection as well as dispersal and drift shape communities, but the mechanistic bases for assembly of communities and the forces that maintain their function in the face of environmental perturbation are not well understood. Conceptually, some interactions among community members could generate endogenous dynamics in composition, even in the absence of environmental changes. These endogenous dynamics are further perturbed by exogenous forcing factors to produce a richer network of community interactions and it is this ‘system'' that is the basis for higher-order community properties. Elucidation of principles that follow from this conceptual model requires identifying the mechanisms that (a) optimize diversity within a community and (b) impart community stability. The network of interactions between organisms can be an important element by providing a buffer against disturbance beyond the effect of functional redundancy, as alternative pathways with different combinations of microbes can be recruited to fulfill specific functions.     

Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor

The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.