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PubMed2004

Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery.

The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood. Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain. To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay. We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton. The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners. Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected. Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery.

Amino Acid Sequence↗
PubMed1996

Mechanism of interferon action. Biochemical and genetic evidence for the intermolecular association of the RNA-dependent protein kinase PKR from human cells.

The interferon-inducible protein kinase (PKR) is activated by an RNA-dependent autophosphorylation. Structure-function studies of the 551 amino acid PKR kinase from human cells have revealed that catalytic-deficient PKR mutants such as PKR(1-551)K296R display a dominant negative behavior when expressed in transfected cells. The potential for PKR to form protein multimers has therefore been examined. Three types of studies, including both genetic and biochemical analyses, demonstrated that PKR from human cells undergoes an intermolecular association that is not dependent upon RNA. First, the intermolecular association of PKR in vitro was demonstrated in the context of an enzyme-substrate interaction. Purified recombinant histidine-tagged PKR(1-551)K296R mutant protein was phosphorylated by purified wild-type PKR; this intermolecular phosphorylation of PKR was dependent on double-stranded RNA. At a fixed RNA concentration, high concentrations of the HIS-PKR(1-551)K296R mutant both impaired the autophosphorylation of wild-type PKR and blocked the trans-phosphorylation of itself. Second, the yeast two-hybrid system was used to probe the intermolecular association of PKR in vivo. Coexpression of the full-length catalytic-deficient phosphotransfer mutant PKR(1-551)K296R as a fusion protein with the Gal4 activation domain and the Gal4 DNA binding domain resulted in the expression of two Gal4-responsive reporter genes, HIS3 and lacZ. The full-length RNA-binding deficient PKR(1-551)K64E/K296R double mutant also interacted with PKR(1-551)K296R sufficiently to activate Gal4-responsive reporter genes; however, other PKR mutants including PKR(1-280)wt and PKR(281-551)K296R as well as p53, RAS, and BCL2 did not. Third, both PKR(1-551)K296R and PKR(1-551)K64E/K296R enhanced the expression of the reovirus S1 gene and S1/S4 chimeric gene in cotransfected COS cells. By contrast, the expression of the reovirus S4 gene was not enhanced by cotransfection with either PKR(1-551)K296R or PKR(1-551)K64E/K296R. These results indicate that PKR interacts with itself in an intermolecular manner both in vivo and in vitro, and that RNA binding is neither necessary nor sufficient for PKR multimerization.

Animals↗
PubMed2006

Discovery of a nanomolar inhibitor of the human murine double minute 2 (MDM2)-p53 interaction through an integrated, virtual database screening strategy.

An integrated, virtual database screening strategy has led to 7-[anilino(phenyl)methyl]-2-methyl-8-quinolinol (4, NSC 66811) as a novel inhibitor of the murine double minute 2 (MDM2)-p53 interaction. This quinolinol binds to MDM2 with a Ki of 120 nM and activates p53 in cancer cells with a mechanism of action consistent with targeting the MDM2-p53 interaction. It mimics three p53 residues critical in the binding to MDM2 and represents a promising new class of non-peptide inhibitors of the MDM2-p53 interaction.

Aniline Compounds↗
PubMed2006

Multivortex micromixing.

The ability to mix liquids in microchannel networks is fundamentally important in the design of nearly every miniaturized chemical and biochemical analysis system. Here, we show that enhanced micromixing can be achieved in topologically simple and easily fabricated planar 2D microchannels by simply introducing curvature and changes in width in a prescribed manner. This goal is accomplished by harnessing a synergistic combination of (i) Dean vortices that arise in the vertical plane of curved channels as a consequence of an interplay between inertial, centrifugal, and viscous effects, and (ii) expansion vortices that arise in the horizontal plane due to an abrupt increase in a conduit's cross-sectional area. We characterize these effects by using confocal microscopy of aqueous fluorescent dye streams and by observing binding interactions between an intercalating dye and double-stranded DNA. These mixing approaches are versatile and scalable and can be straightforwardly integrated as generic components in a variety of lab-on-a-chip systems.

Animals↗
PubMed2001

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase.

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.

Adenosine Triphosphatases↗
PubMed1999

Relinquishing the need to "fix it": medical intervention with domestic abuse.

The grounded theory method was used to study obstetricians/gynecologists' interactions with victims of domestic abuse. Double-bind conflict emerged as the basic social psychological problem. Physicians struggled between approaching domestic abuse as a legitimate medical problem or retreating from it as an overwhelming social issue. Resolution occurred through a three-stage process that changed the doctors' objective from "fixing" the abuse to supporting the patient. This goal revision required education on the dynamics of domestic abuse, confrontation of personal issues, and formation of community partnerships. Implications include development of curricula and protocols to create a united front against domestic abuse within health care, social service, legal, and law enforcement communities.

Attitude of Health Personnel↗
PubMed1995

NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.

Amino Acid Sequence↗
PubMed2004

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels.

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.

Binding Sites↗
PubMedDate not supplied

Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA.

Bovine pancreatic deoxyribonuclease I (DNase I), an endonuclease that degrades double-stranded DNA in a nonspecific but sequence-dependent manner, has been used as a biochemical tool in various reactions, in particular as a probe for the structure of chromatin and for the helical periodicity of DNA on the nucleosome and in solution. Limited digestion by DNase I, termed DNase I 'footprinting', is routinely used to detect protected regions in DNA-protein complexes. Recently, we have solved the three-dimensional structure of this glycoprotein (relative molecular mass 30,400) by X-ray structure analysis at 2.5 A resolution and have subsequently refined it crystallographically at 2.0 A. Based on the refined structure and the binding of Ca2+-thymidine 3',5'-diphosphate (Ca-pTp) at the active site, we propose a mechanism of action and present a model for the interaction of DNase I with double-stranded DNA that involves the binding of an exposed loop region in the minor groove of B-DNA and electrostatic interactions of phosphates from both strands with arginine and lysine residues on either side of this loop. We explain DNase I cleavage patterns in terms of this model and discuss the consequences of the extended DNase I-DNA contact region for the interpretation of DNase I footprinting results.

Binding Sites↗
PubMed2006

Characterization of the reactivity determinants of a novel hairpin substrate of yeast RNase III.

RNase III enzymes form a conserved family of proteins that specifically cleave double-stranded (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. Yeast RNase III (Rnt1p) selects its substrate by recognizing the structure generated by a conserved NGNN tetraloop (G2-loop). Mutations of the invariant guanosine stringently inhibit binding and cleavage of all known Rnt1p substrates. Surprisingly, we have found that the 5' end of small nucleolar RNA 48 is processed by Rnt1p in the absence of a G2-loop. Instead, biochemical and structural analyses revealed that cleavage, in this case, is directed by a hairpin capped with an AAGU tetraloop, with a preferred adenosine in the first position (A1-loop). Chemical probing indicated that A1-loops adopt a distinct structure that varies at the 3' end where Rnt1p interacts with G2-loops. Consistently, chemical footprinting and chemical interference assays indicate that Rnt1p binds to G2 and A1-loops using different sets of nucleotides. Also, cleavage and binding assays showed that the N-terminal domain of Rnt1p aids selection of A1-capped hairpins. Together, the results suggest that Rnt1p recognizes at least two distinct classes of tetraloops using flexible protein RNA interactions. This underscores the capacity of double-stranded RNA binding proteins to use several recognition motifs for substrate identification.

Base Sequence↗
PubMed1999

Two regions of simian virus 40 T antigen determine cooperativity of double-hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding.

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

Antigens, Polyomavirus Transforming↗
PubMed2000

Ligand-inducible interaction of the DRIP/TRAP coactivator complex with retinoid receptors in retinoic acid-sensitive and -resistant acute promyelocytic leukemia cells.

Retinoic acid (RA) signaling is mediated by its nuclear receptors RXR and RAR, which bind to their cognate response elements as a heterodimer, RXR/RAR, and act in concert with coregulatory factors to regulate gene transcription on ligand binding. To identify specific cofactors that interact with the RXR/RAR heterodimer in acute promyelocytic leukemia (APL) cells, a double cistronic construct was used that allowed coexpression of the RXR LBD (ligand binding domain) with the RAR LBD as an affinity matrix to pull down interacting proteins from nuclear extracts prepared from a human APL cell line, NB4. A group of proteins was detected whose interaction with RXR/RAR is ligand inducible. The molecular weight pattern of these proteins is similar to that of a complex of proteins previously identified as DRIP or TRAP, which are ligand-dependent transcription activators of VDR and TR, respectively. The RXR/RAR-interacting proteins from NB4 were confirmed to be identical to the DRIP subunits by comparative electrophoresis, Western blot analysis, and in vitro protein interaction assay. In addition to RXR/RAR, the DRIP component can interact directly with the APL-specific PML-RARalpha fusion protein. The same DRIP complex is present in RA-resistant APL cells and in a variety of cancer cell lines, supporting its global role in transcriptional regulation.

Antineoplastic Agents↗
PubMed1982

Changing patterns of single-stranded-DNA-binding proteins in differentiating brain cortex and cerebellar neurons.

Single-stranded-DNA-binding proteins were analyzed in nuclei of differentiating rat cortex and cerebellar neurons. The developmental period investigated ranged from gestational day 19 (i.e. 3 days before term) to postnatal day 30. During this time both types of neurons undergo transition from proliferating, undifferentiated precursor cells to non-proliferating, terminally differentiated neurons. For comparison, nuclei from mature cortex glia and liver were also examined. Nuclei were isolated according to cell type, the proteins were 14C-labeled in vitro by reductive methylation and were fractionated by affinity chromatography on tandemly arranged columns of double-stranded and single-stranded DNA-cellulose. The columns were uncoupled and the proteins adsorbed to the single-stranded DNA were eluted with salt. They were then analyzed by high resolution two-dimensional gel electrophoresis followed by fluorography. This strategy ensured the selective detection of proteins that recognize single-stranded DNA specifically, and eliminated interference by proteins binding to DNA by simple ionic interaction as well as by proteins with affinity for double-stranded DNA. Many single-stranded-DNA-binding proteins showed conspicuous developmental fluctuations. In cortex neurons these took place around the time of birth and the first postnatal week, whereas in cerebellar neurons they occurred later and in a more protracted fashion. Thus, in both cortex and cerebellar neurons the protein changes followed a time course closely paralleling the arrest of cell division and the beginning of terminal differentiation. It is suggested that this approach may lead to the detection of putative regulatory proteins of the cell nucleus.

Animals↗
PubMed2001

Structure of the DNA-binding domain of human telomeric protein, TRF1 and its interaction with telomeric DNA.

TRF1, a key player in regulation of the telomere length, is a double-stranded telomeric DNA binding factor in vertebrate. The DNA-binding domain of TRF1 shows a sequence similarity to each of the tandem repeat in the DNA-binding domain of the c-Myb protein. Here, the solution structure of the DNA-binding domain of human TRF1 has been determined by NMR. It consists of three helices, and its topological arrangement is very close to that of each c-Myb repeat and also to that of each subdomain of the DNA-binding domain in yeast telomeric protein, Rap1p. The interaction with DNA has been investigated by chemical shift perturbations. The result suggests that TRF1 recognizes the sequence centered on AGGGTTA mainly with its N-terminal arm, N-terminal portion of second helix and both ends of third helix.

Base Sequence↗
PubMed1983

Genetic transformation of Streptococcus pneumoniae by DNA cloned into the single-stranded bacteriophage f1.

A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.

Cloning, Molecular↗
PubMed1998

Rice dwarf phytoreovirus segment S11 encodes a nucleic acid binding protein.

The function of rice dwarf virus segment 11 and the corresponding segments of other phytoreoviruses is not yet determined. The amino acid sequence of Pns11, encoded by segment 11, contains a putative zinc finger and five flanking basic regions at the C-terminus. The full-length Pns11 protein and three truncated derivatives, which lack the N-terminus, the zinc-finger or the C-terminal five basic regions were expressed in Escherichia coli and their nucleic acid binding properties were studied. Pns11 interacts with single- and double-stranded forms of DNA and RNA in a sequence-nonspecific manner. The truncated derivative which contains both the zinc-finger and the C-terminal basic regions has the same binding properties as the full-length Pns11. However, removal of either of these domains prevents binding activity. The binding activity of Pns11 was drastically reduced when the blots were treated with a high concentration of EDTA. Moreover, Pns11 extracted from infected rice also binds to single-stranded RNA. These data suggest that RDV Pns11 binding activity is structure-dependent and it may play an important role in virus replication and/or genome assortment.

Amino Acid Sequence↗
PubMed2006

Lead optimization of [(S)-gamma-(arylamino)prolyl]thiazolidine focused on gamma-substituent: Indoline compounds as potent DPP-IV inhibitors.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-gamma-(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To discover a structure for the gamma-substituent of the proline moiety more suitable for interacting with the S(2) pocket of DPP-IV, optimization focused on the gamma-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selectivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in the S(2) pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease in activity caused by removing the electrophilic nitrile.

Animals↗
PubMed2003

Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries, intrinsic affinities, and cooperativities.

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.

Animals↗