Dong-Eun Kim

Hexachlorophene inhibits Wnt/beta-catenin pathway by promoting Siah-mediated beta-catenin degradation.

Aberrant activation of Wnt/β-catenin signaling and subsequent up-regulation of β-catenin response transcription (CRT) is a critical event in the development of human colon cancer. Thus, Wnt/β-catenin signaling is an attractive target for the development of anticancer therapeutics. In this study, we identified hexachlorophene as an inhibitor of Wnt/β-catenin signaling from cell-based small-molecule screening. Hexachlorophene antagonized CRT that was stimulated by Wnt3a-conditioned medium by promoting the degradation of β-catenin. This degradation pathway is Siah-1 and adenomatous polyposis colidependent, but glycogen synthase kinase-3β and F-box β-transducin repeat-containing protein-independent. In addition, hexachlorophene represses the expression of cyclin D1, which is a known β-catenin target gene, and inhibits the growth of colon cancer cells. Our findings suggest that hexachlorophene attenuates Wnt/β-catenin signaling through the Siah-1-mediated β-catenin degradation.

T7 DNA Helicase: A Molecular Motor that Processively and Unidirectionally Translocates Along Single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.

Protein-kinase-C-mediated β-catenin phosphorylation negatively regulates the Wnt/β-catenin pathway

Normally, the Wnt/β-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/β-catenin pathway is the amino-terminal phosphorylation of β-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated β-catenin phosphorylation as a novel mechanism regulating the Wnt/β-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/β-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of β-catenin, which promoted β-catenin degradation. Moreover, the depletion of PKCα inhibited the phosphorylation and degradation of β-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the β-catenin level outside of the Wnt/β-catenin pathway.

Mechanism of DNA Adsorption and Desorption on Graphene Oxide

Graphene oxide (GO) adsorbing a fluorophore-labeled single-stranded (ss) DNA serves as a sensor system because subsequent desorption of the adsorbed probe DNA from GO in the presence of complementary target DNA enhances the fluorescence. In this study, we investigated the interaction of single- and double-stranded (ds) DNAs with GO by using a fluorescently labeled DNA probe. Although GO is known to preferentially interact with ssDNA, we found that dsDNA can also be adsorbed on GO, albeit with lower affinity. Furthermore, the status of ssDNA or dsDNA previously adsorbed on the GO surface was investigated by adding complementary or noncomplementary DNA (cDNA or non-cDNA) to the adsorption complex. We observed that hybridization occurred between the cDNA and the probe DNA on the GO surface. On the basis of the kinetics driven by the incoming additional DNA, we propose a mechanism for the desorption of the preadsorbed probe DNA from the GO surface: the desorption of the GO-adsorbed DNA was facilitated following its hybridization with cDNA on the GO surface; when the GO surface was almost saturated with the adsorbed DNA, nonspecific desorption dominated the process through a simple displacement of the GO-adsorbed DNA molecules by the incoming DNA molecules because of the law of mass action. Our results can be applied to design appropriate DNA probes and to choose proper GO concentrations for experimental setups to improve specific signaling in many biosensor systems based on the GO platform.

The Kinetic Pathway of RNA Binding to the Escherichia coli Transcription Termination Factor Rho

The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures. Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP. These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho. The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 × 108 m − 1s− 1, resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s− 1. The PR2 isomerizes to PR3 with a rate constant of 32 s− 1 in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate. The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s− 1; thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step. The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity. The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel. These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner.

Selection of an Antiviral RNA Aptamer Against Hemagglutinin of the Subtype H5 Avian Influenza Virus

Avian influenza is an acute viral respiratory disease caused by RNA viruses of the family Orthomyxoviridae. The influenza A virus subtype H5 can cause severe illness and results in almost 100% mortality rate among livestock. Hemagglutinin (HA) present in the virus envelope plays an essential role in the initiation of viral infection. In this study, we investigated the efficacy of using HA as a target for antiviral therapy through nucleic acid aptamers. After purification of the receptor binding domain (HA1) of HA protein, activity of recombinant HA1 was confirmed by using hemagglutination assay. We selected RNA aptamer candidates after 15 rounds of iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) targeting the biologically active HA protein. The selected RNA aptamer HAS15-5, which specifically binds to HA1, exhibited significant antiviral efficacy according to the results of a hemagglutination inhibition assay using egg allantoic fluids harboring the virus. Thus, the RNA aptamer HAS15-5, which acts by blocking and inhibiting the receptor-binding domain of viral HA, can be developed as a novel antiviral agent against type H5 avian influenza virus.

Proteolytic Cleavage of Extracellular α-Synuclein by Plasmin: IMPLICATIONS FOR PARKINSON DISEASE*

Background: A prion-like spread of α-synuclein might play a role in the pathogenesis of Parkinson disease. Results: Extracellular α-synuclein was cleaved by plasmin. Cultured microglia and astrocytes did not take up plasmin digested extracellular α-synuclein, and were not activated. Conclusion: Plasmin-mediated α-synuclein clearance problems might play a role in the pathogenesis of Parkinson disease. Significance: Therapies aimed at α-synuclein clearance may lead to new therapies for Parkinson disease. Parkinson disease (PD) is the second most common neurodegenerative disease characterized by a progressive dopaminergic neuronal loss in association with Lewy body inclusions. Gathering evidence indicates that α-synuclein (α-syn), a major component of the Lewy body, plays an important role in the pathogenesis of PD. Although α-syn is considered to be a cytoplasmic protein, it has been detected in extracellular biological fluids, including human cerebrospinal fluid and blood plasma of healthy and diseased individuals. In addition, a prion-like spread of α-syn aggregates has been recently proposed to contribute to the propagation of Lewy bodies throughout the nervous system during progression of PD, suggesting that the metabolism of extracellular α-syn might play a key role in the pathogenesis of PD. In the present study, we found that plasmin cleaved and degraded extracellular α-syn specifically in a dose- and time- dependent manner. Aggregated forms of α-syn as well as monomeric α-syn were also cleaved by plasmin. Plasmin cleaved mainly the N-terminal region of α-syn and also inhibited the translocation of extracellular α-syn into the neighboring cells in addition to the activation of microglia and astrocytes by extracellular α-syn. Further, extracellular α-syn regulated the plasmin system through up-regulation of plasminogen activator inhibitor-1 (PAI-1) expression. These findings help to understand the molecular mechanism of PD and develop new therapeutic targets for PD.

Galangin Suppresses the Proliferation of β-Catenin Response Transcription-Positive Cancer Cells by Promoting Adenomatous Polyposis Coli/Axin/Glycogen Synthase Kinase-3β-Independent β-Catenin Degradation

Galangin is a naturally occurring bioflavonoid with anticancer activity against certain human cancers, yet little is known about its mechanism of action. Here, we used a chemical biology approach to reveal that galangin suppresses β-catenin response transcription (CRT), which is aberrantly up-regulated in colorectal and liver cancers, by promoting the degradation of intracellular β-catenin. Inhibition of glycogen synthase kinase-3β (GSK-3β) activity or mutation of the GSK-3β-targeted sequence from β-catenin was unable to abrogate the galangin-mediated degradation of β-catenin. In addition, galangin down-regulated the intracellular β-catenin levels in cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or Axin, which are components of the β-catenin destruction complex. Galangin repressed the expression of β-catenin/T-cell factor-dependent genes, such as cyclin D1 and c-myc, and thus inhibited the proliferation of CRT-positive cancer cells. Structure-activity data indicated that the major structural requirements for galangin-mediated β-catenin degradation are hydroxyl groups at positions 3, 5, and 7. Our findings suggest that galangin exerts its anticancer activity by promoting APC/Axin/GSK-3β-independent proteasomal degradation of β-catenin.

An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells.

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.

Suppression of Hepatitis C Virus Genome Replication in Cells with RNA-Cleaving DNA Enzymes and Short-Hairpin RNA

A class of antisense oligodeoxyribozymes, known as the 10-23 DNA enzymes (DNAzyme), has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. Herein we have utilized a strategy to identify accessible cleavage sites for DNAzyme in the target RNA, the hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of randomized DNAzyme library. The screening procedure identified 18 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA cleavage in vitro. Using positively charged dendrimer nanoparticles, the target RNA-cleaving DNAzymes that are 31-mer oliogonucleotides are delivered into the human hepatoma cells harboring the HCV subgenomic replicon RNA. DNAzymes introduced into the cells efficiently inhibited HCV RNA replication by reducing the expression of HCV NS3. In addition, we designed short-hairpin RNA (shRNA) that targets the same cleavage site for the selected DNAzyme and confirmed that the shRNA also inhibited HCV NS3 gene expression in the HCV replicon cells. These selected DNAzyme and shRNA may be a viable therapeutic intervention to inhibit HCV replication in hepatic cells. We suggest that the method used in this study can be applicable for identification of available sites in any target RNA for antisense oligonucleotides and siRNAs.