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PubMed2004

Biological activity and delivery of peptide nucleic acids (PNA)-DNA chimeras for transcription factor decoy (TFD) pharmacotherapy.

Peptide nucleic acids (PNAs) are recently described DNA mimics, in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine units. These molecules efficiently hybridize with complementary DNA, forming Watson-Crick double helices. In addition, the interest of PNAs and PNA-based analogs is related to the fact that they are resistant to DNases and proteinases. While applications of PNAs as antisense and antigéne molecules in non-viral gene therapy are well documented, their effects as potential transcription factor decoy (TFD) molecules is not demonstrated. PNA/PNA and PNA/DNA duplex are not suitable for TFD. In fact, PNA/PNA duplex does not recognize transcription factors, while, in the case of PNA/DNA hybrids containing nuclear factor binding sites, the interaction with transcription factors is unstable. By sharp contrast, double stranded molecules based on PNA-DNA chimeras exhibit strong TFD activity, display enzymatic stability in serum and cellular extracts and can be delivered to target cells after complexation with liposomes and microspheres. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments on non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

DNA↗
PubMed1995

Double-stranded RNA binding proteins and their substrates.

Double-stranded RNA (dsRNA) binding proteins (dsRBPs) pose unique questions in regard to how proteins interact with RNA. This article presents a general overview of these questions and also introduces specific dsRBPs that are studied by my laboratory.

Adenosine Deaminase↗
PubMed1995

An interaction between the Escherichia coli RecF and RecR proteins dependent on ATP and double-stranded DNA.

The DNA binding and ATPase activities of RecF protein are modulated by RecR protein. Stoichiometric amounts of RecF protein bind to double-stranded (ds) DNA (about 1 RecF monomer/4-6 base pairs) in the presence of adenosine 5'-O-(3-thio)triphosphate (ATP gamma S), forming a homogeneous protein coating on the DNA. Little or no cooperativity is evident in the binding process. In the presence of ATP, RecF binding to dsDNA is much weaker, and no RecF protein coating forms. Instead, small numbers of RecF protomers are interspersed randomly along the DNA. RecR protein does not bind appreciably to the dsDNA under these same conditions. However, a protein coating, similar to that which was observed with RecF protein alone in the presence of ATP gamma S, was produced when both RecF and RecR proteins were incubated with dsDNA in the presence of ATP. An interaction between RecF and RecR enables both proteins to bind tightly to the dsDNA in an approximately 1:1 molar ratio. We also report a weak ATP hydrolytic activity of RecF which is stimulated by RecR.

Adenosine Triphosphatases↗
PubMed2005

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities.

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein-protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.

Adenosine Triphosphatases↗
PubMed1995

Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA.

An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly.

Base Sequence↗
PubMed2002

RNA-protein interactions.

Recent discoveries have revealed that there is a myriad of RNAs and associated RNA-binding proteins that spatially and temporally appear in the cells of all organisms. The structures of these RNA-protein complexes are providing valuable insights into the binding modes and functional implications of these interactions. Even the common RNA-binding domains (RBDs) and the double stranded RNA binding motifs (dsRBMs) have been shown to exhibit a plethora of binding modes.

Adenosine Deaminase↗
PubMed1996

Synthesis, biological activity, and molecular modeling of selective 5-HT(2C/2B) receptor antagonists.

The synthesis and biological activity are reported for a series of analogues of the previously published indole urea 2 (SB-206553), designed to probe the 5-HT(2C) receptor binding site. Small molecule modeling studies have been used to define a region in space which is allowed at the 5-HT(2C) receptor but disallowed at the 5-HT(2A) receptor. In a complementary approach, docking of 2 into our model of the 5-HT(2C) receptor has allowed us to propose a novel primary binding interaction for this series of diaryl ureas, involving a potential double hydrogen-bonding interaction between the urea carbonyl oxygen of the ligand and two serine residues in the receptor. The difference of two valine residues in the 5-HT(2C) receptor for leucine residues in the 5-HT(2A) receptor is believed to account for the observed 5-HT(2C)/5-HT(2A) selectivity with 2.

Animals↗
PubMed2005

How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures.

Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2. They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units. Despite these similarities, TRF1 and TRF2 have different functions at telomeres. The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 angstroms resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p. Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions. The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.

Amino Acid Sequence↗
PubMed2005

Theoretical studies on hydroquinone-benzene clusters.

High-level ab initio calculations were carried out to evaluate the interaction between the hydroquinone and benzene molecules. The intermolecular interaction energy was calculated using the Møller-Plesset second-order perturbation theory at the complete basis set limit and also at the coupled cluster theory with single, double, and perturbatively triple excitations. The calculated binding energy is larger than the benzene dimer interaction energy. The T-shaped cluster (T-a) and the parallel conformation (P-a) are calculated to be nearly isoenergetic. Owing to the large energy gain in the attraction by electron correlation, the dispersion interaction is important for the attraction.

Journal Article↗
PubMed1999

Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease). Oligomerization and direct interaction with histone H1.

DNA fragmentation factor (DFF) is a heterodimeric protein composed of 45-kDa (DFF45) and 40-kDa (DFF40) subunits, a protein that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals. DFF45 is a specific molecular chaperone and an inhibitor for the nuclease activity of DFF40. Previous studies have shown that upon cleavage of DFF45 by caspase-3, the nuclease activity of DFF40 is relieved of inhibition. Here we further investigate the mechanism of DFF40 activation. We demonstrate that DFF45 can also be cleaved and inactivated by caspase-7 but not by caspase-6 and caspase-8. The cleaved DFF45 fragments dissociate from DFF40, allowing DFF40 to oligomerize to form a large functional complex that cleaves DNA by introducing double strand breaks. Histone H1 directly interacts with DFF, confers DNA binding ability to DFF, and stimulates the nuclease activity of DFF40 by increasing its Kcat and decreasing its Km.

Apoptosis↗
PubMed2004

The Drosha-DGCR8 complex in primary microRNA processing.

RNase III proteins play key roles in microRNA (miRNA) biogenesis. The nuclear RNase III Drosha cleaves primary miRNAs (pri-miRNAs) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs. While Dicer (class III) and other simple RNase III proteins (class I) have been studied intensively, the class II enzyme Drosha remains to be characterized. Here we dissected the action mechanism of human Drosha by generating mutants and by characterizing its new interacting partner, DGCR8. The basic action mechanism of Drosha was found to be similar to that of human Dicer; the RNase III domains A and B form an intramolecular dimer and cleave the 3' and 5' strands of the stem, respectively. Human Drosha fractionates at approximately 650 kDa, indicating that Drosha functions as a large complex. In this complex, Drosha interacts with DGCR8, which contains two double-stranded RNA (dsRNA)-binding domains. By RNAi and biochemical reconstitution, we show that DGCR8 may be an essential component of the pri-miRNA processing complex, along with Drosha. Based on these results, we propose a model for the action mechanism of class II RNase III proteins.

Blotting, Northern↗
PubMed1975

[Communicative aspects of studies in schizophrenia].

Family studies of schizophrenics have shown the importance of factors such as a malignant mother-child symbiosis, changes in the family role structure, pseudo-mutuality and techniques on how 'to drive someone crazy'. More recent studies concern themselves with disturbance of communication and interaction within schizophrenic families. In particular, the problems of double-bind and other paradoxical modes of communication are discussed by the author. In contrast to double-bind as a sociological aetiological model which defines schizophrenia as a metacommunicative disturbance and evoked reaction of the self-system, the hypothesis is proposed of an intrapsychic double-bind which the patient is producing wihtin himself.

Family↗
PubMed1995

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of the STAT6 SH2 domain.

The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.

Alanine↗
PubMed1999

Interaction of cationic alpha-helical peptide with phosphorothioate DNA hybrid.

Synthetic amphiphilic alpha-helix peptides were found to bind to stabilize double or triple stranded DNA. The stabilization effect was significant for cationic alpha-helix peptides which indicated the importance of electrostatic interaction of positive charge of peptide and negative charge of DNA. It should be also pointed out that hybrid double or triple helical complexes containing phosphorothioate oligonucleotide were stabilized to a larger extent respect to phosphodiester oligonucleotides. Since it was shown that cationic amphiphilic alpha-helix peptide accelerate membrane permeability of DNA, the present study can provide a solution for the problems of antisense or triplex oligonucleotide in their practical application.

Amino Acid Sequence↗
PubMed2002

Exportin-5 mediates nuclear export of minihelix-containing RNAs.

The adenovirus VA1 RNA (VA1), a 160-nucleotide (nt)-long RNA transcribed by RNA polymerase III, is efficiently exported from the nucleus to the cytoplasm of infected cells, where it antagonizes the interferon-induced antiviral defense system. We recently reported that nuclear export of VA1 is mediated by a cis-acting RNA export motif, called minihelix, that comprises a double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt protruding 3' end. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin) that remained to be determined. Here we show using microinjection in Xenopus laevis oocytes that VA1 is transported to the cytoplasm by exportin-5, a nuclear transport factor for double-stranded RNA binding proteins. Gel retardation assays revealed that exportin-5 directly interacts with VA1 RNA in a RanGTP-dependent manner. More generally, in vivo and in vitro competition experiments using various VA1-derived, but also artificial and cellular, RNAs lead to the conclusion that exportin-5 preferentially recognizes and transports minihelix motif-containing RNAs.

Active Transport, Cell Nucleus↗
PubMed2005

Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures.

Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was ompA.

Adhesins, Bacterial↗
PubMed1988

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication.

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.

Base Sequence↗