RESUMO
The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.
Assuntos
COVID-19 , Perileno , Humanos , SARS-CoV-2/fisiologia , Nucleosídeos/farmacologia , RNA , Perileno/farmacologia , Antivirais/farmacologiaRESUMO
We examined the assembly of DNA G-quadruplexes (G4s) into higher-order structures using atomic force microscopy, optical and electrophoretic methods, NMR spectroscopy and molecular modeling. Our results suggest that parallel blunt-ended G4s with single-nucleotide or modified loops may form different types of multimers, ranging from stacks of intramolecular structures and/or interlocked dimers and trimers to wires. Decreasing the annealing rate and increasing salt or oligonucleotide concentrations shifted the equilibrium from intramolecular G4s to higher-order structures. Control antiparallel and hybrid G4s demonstrated no polymorphism or aggregation in our experiments. The modification that mimics abasic sites (1',2'-dideoxyribose residues) in loops enhanced the oligomerization/multimerization of both the 2-tetrad and 3-tetrad G4 motifs. Our results shed light on the rules that govern G4 rearrangements. Gaining control over G4 folding enables the harnessing of the full potential of such structures for guided assembly of supramolecular DNA structures for nanotechnology.
Assuntos
Desoxirribose/análogos & derivados , Quadruplex G , Dobramento de RNA , Pareamento de Bases , Desoxirribose/química , Modelos Moleculares , Motivos de Nucleotídeos , Mutação Puntual , Cloreto de PotássioRESUMO
A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N-acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1B's properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ.
Assuntos
Glicoconjugados/metabolismo , Lectinas/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Linhagem Celular , Galinhas , Glicoconjugados/química , Humanos , Lectinas/química , Dados de Sequência Molecular , Polissacarídeos/química , Homologia de Sequência de AminoácidosRESUMO
Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galß1-3GlcNAc or Galß1-3GalNAc as basic motifs was commonly better than that to canonical Galß1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galß1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.
Assuntos
Amino Açúcares/química , Linfócitos B/química , Galectina 4/química , Galectinas/química , Polissacarídeos/química , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/metabolismo , Amino Açúcares/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Bioensaio , Sequência de Carboidratos , Linhagem Celular , Corantes Fluorescentes , Galectina 4/metabolismo , Galectinas/metabolismo , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade EstáticaRESUMO
Using an example of Galß1-3GlcNAc (Le(C)) related glycans, we here demonstrate a risk of data misinterpretation when polyclonal antibodies are probed for their glycan-binding specificities with help of a printed glycan array (PGA). Affinity isolation of antibodies from human serum using Le(C)-Sepharose or 3'-O-SuLe(C)-Sepharose in conditions of excess of the adsorbents generated identical material regardless of the affinity ligand, with the antibodies equally capable of binding to Le(C) and to 3'-O-SuLe(C) disaccharides, as well as to 3'-O-SiaLe(C) trisaccharide. More detailed profiling has shown that the isolated antibodies bind to the inner part of Galß1-3GlcNAc disaccharide. We therefore conclude that serum does not contain different subsets of antibodies specific either to Le(C) or to 3'-O-SuLe(C), despite their visibly different binding signals to these glycans on PGA.
Assuntos
Anticorpos/sangue , Interpretação Estatística de Dados , Análise em Microsséries/métodos , Polissacarídeos/imunologia , Acetilglucosamina/química , Acetilglucosamina/imunologia , Adulto , Anticorpos/isolamento & purificação , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Sequência de Carboidratos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Polissacarídeos/química , Sulfatos/imunologia , Doadores de TecidosRESUMO
Affinity for G-quadruplex (G4) structures may be a common feature of transcription-facilitating histone chaperons (HCs). This assumption is based on previous unmatched studies of HCs FACT, nucleolin (NCL), BRD3, and ATRX. We verified this assumption and considered its implications for the therapeutic applications of synthetic (exogenous) G4s and the biological significance of genomic G4s. First, we questioned whether exogenous G4s that recognize cell-surface NCL and could trap other HCs in the nucleus are usable as anticancer agents. We performed in vitro binding assays and selected leading multi-targeted G4s. They exhibited minor effects on cell viability. The presumed NCL-regulated intracellular transport of G4s was inefficient or insufficient for tumor-specific G4 delivery. Next, to clarify whether G4s in the human genome could recruit HCs, we compared available HC ChIP-seq data with G4-seq/G4-ChIP-seq data. Several G4s, including the well-known c-Myc quadruplex structure, were found to be colocalized with HC occupancy sites in cancer cell lines. As evidenced by our molecular modeling data, c-Myc G4 might interfere with the HC function of BRD3 but is unlikely to prevent the BRD3-driven assembly of the chromatin remodeling complex. The c-Myc case illustrates the intricate role of genomic G4s in chromatin remodeling, nucleosome remodeling, and transcription.
Assuntos
Quadruplex G , Chaperonas de Histonas/química , Simulação de Acoplamento Molecular , Células A549 , Sítios de Ligação , Células Cultivadas , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Genes myc , Células HT29 , Células Hep G2 , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/metabolismo , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Proteína Nuclear Ligada ao X/química , Proteína Nuclear Ligada ao X/metabolismo , NucleolinaRESUMO
The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.