Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3.
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ABSTRACT:
SUBMITTER: Molnar OR
PROVIDER: S-EPMC8636512 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.
| S-EPMC3680382 | biostudies-literature
Project description:A promising anticancer therapeutic strategy is the stabilization of telomeric G-quadruplexes using G-quadruplex-binding small molecules. Although many G-quadruplex-specific ligands have been developed, their low potency and selectivity to G-quadruplexes over duplex remains unsolved. Recently, a crystal structure of a telomeric 3' quadruplex-duplex hybrid was reported and the quadruplex-duplex interface was suggested to a good target to address the issues. However, there are no high-resolution complex structures reported for G-quadruplex ligands except for a docked BSU6037. In this study, molecular dynamic (MD) binding simulations with a free ligand were used to study binding poses and dynamics of four representative ligands: telomestatin, TMPyP4, BSU6037, and BRACO19. The MD data showed that BSU6037 was able to fully intercalate into the interface whereas TMPyP4 and BRACO19 could only maintain partial intercalation into the interface and telomestatin only binds at the quadruplex and duplex ends. Both linear ligands, BSU6037 and BRACO19, were able to interact with the interface, yet they were not selective over duplex DNA. The DNA geometry, binding modes, and binding pathways were systematically characterized, and the binding energy was calculated and compared for each system. The interaction of the ligands to the interface was by the means of an induced-fit binding mechanism rather than a lock-key mechanism, consisting of the DNA unfolding at the interface to allow entrance of the drug and then the refolding and repacking of the DNA and the ligand to further stabilize the G-quadruplex. On the basis of the findings in this study, modifications were suggested to optimize the interface binding for TMPyp4 and telomestatin.
| S-EPMC6289566 | biostudies-literature
Project description:Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation.
| S-EPMC4041461 | biostudies-literature
Project description:AIM:To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS:HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS:C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION:The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.
| S-EPMC4481485 | biostudies-literature
Project description:Hepatitis delta virus (HDV) is a highly unusual RNA satellite virus that depends on the presence of hepatitis B virus (HBV) to be infectious. Its compact and variable single-stranded RNA genome consists of eight major genotypes distributed unevenly across different continents. The significance of noncanonical secondary structures such as G-quadruplexes (G4s) is increasingly recognized at the DNA and RNA levels, particularly for transcription, replication, and translation. G4s are formed from guanine-rich sequences and have been identified in the vast majority of viral, eukaryotic, and prokaryotic genomes. In this study, we analyzed the G4 propensity of HDV genomes by using G4Hunter. Unlike HBV, which has a G4 density similar to that of the human genome, HDV displays a significantly higher number of potential quadruplex-forming sequences (PQS), with a density more than four times greater than that of the human genome. This finding suggests a critical role for G4s in HDV, especially given that the PQS regions are conserved across HDV genotypes. Furthermore, the prevalence of G4-forming sequences may represent a promising target for therapeutic interventions to control HDV replication.
| S-EPMC10809645 | biostudies-literature
Project description:Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control.
| S-EPMC7851496 | biostudies-literature
Project description:MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3' UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5' UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5' cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5' exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.
| S-EPMC3271899 | biostudies-other
Project description:ObjectiveOsteosarcoma (OS) is characterized by high levels of the tumour-associated inflammatory microenvironment. Moreover, in approximately 60% of OS, telomere length is maintained by alternative lengthening of telomeres (ALT) pathway. Whether the ALT pathway can be exploited for OS therapeutic treatment and how the OS inflammatory microenvironment influences the anti-cancer drug effect remains unknown. Here, we examined the biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of OS cells.Materials and methodsImmunofluorescence in situ hybridization (IF-FISH) and C-circle experiments were used to detect the G-quadruplex and ALT activity. The redox potential of single guanine, G-quadruplex and G-quadruplex/TMPyP4 was evaluated by the lowest unoccupied molecular orbital energy (LUMO), zeta potential and cyclic voltammetry. Cell viability, flow cytometry and apoptosis, Western blot, comet assay, adhesion, transwell and scratch experiments were performed to compare the anti-tumour proliferation and migration effects of TMPyP4 and cisplatin in the inflammatory microenvironment.ResultsThis study indicated that compared with cisplatin, TMPyP4 could induce the formation of human telomeres and FAK G-quadruplex in vitro and in vivo, and TMPyP4-treated OS cells showed fewer extrachromosomal C-circles and fewer ALT-associated promyelocytic leukaemia bodies. Consequently, the ALT activity and FAK-related cell migration were suppressed by TMPyP4. Mechanistically, the formation of G-quadruplex resulted in both lower redox potential than G within the genome and FAK transcription inhibition, and TMPyP4 could enhance this phenomenon, especially in the inflammatory microenvironment.ConclusionsOur results reveal that TMPyP4 is more suitable for OS treatment than cisplatin.
| S-EPMC8450119 | biostudies-literature
Project description:The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)1/DNA complex (K1 = 3.7 × 10(6)) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)1/DNA complex, (K1 = 7.0 × 10(4)). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)3/DNA and (TMPyP4)4/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.
| S-EPMC3748076 | biostudies-literature
Project description:Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.
| S-EPMC5888870 | biostudies-other