Biomedical subjects

P A Beal

Publications and source records attributed to P A Beal.

15 recordsLinked to original sources

PubMed2001

6-bromopurine nucleosides as reagents for nucleoside analogue synthesis.

Surprisingly facile direct substitution reactions with acetyl-protected 6-bromopurine nucleosides are described. Included in the series of bromonucleosides studied is the guanosine derivative N(2)-2',3',5'-tetraacetyl-6-bromopurine ribonucleoside, the synthesis of which is reported here for the first time. Brominated nucleosides had not previously been considered optimal substrates for S(N)Ar reactions given the general reactivity trend for halogenated aromatic systems (i.e. F > Cl > Br > I). However, even weakly nucleophilic aromatic amines give high yields of the substitution products in polar solvents with these 6-bromopurine nucleosides. For primary aromatic amines, secondary aliphatic amines, and imidazole, reaction takes place only at C6, with no effect on the acetyl-protected ribose. In addition, we report the first synthesis of 3',5'-di-O-acetyl-6-bromopurine-2'-deoxyribonucleoside and its reaction with an arylamine in MeOH in the absence of added metal catalyst. Thus, C6-arylamine derivatives of both adenosine and 2'-deoxyadenosine can be prepared via simple S(N)Ar reactions with the corresponding 6-bromo precursor. We also describe high yielding and C6-selective substitution reactions with 6-bromonucleosides using alcohol and thiol nucleophiles in the presence of added base (DBU). Finally, C6-bromonucleosides are shown to be readily hydrogenated to give purine or 2-aminopurine products in good yield. This work increases the arsenal of reactions and strategies available for the synthesis of nucleoside analogues as potential biochemical tools or new therapeutics.

Amines↗

PubMed2001

Synthesis and analysis of RNA containing 6-trifluoromethylpurine ribonucleoside.

We report the synthesis of a 5'-DMT-2'-TBDMS-protected phosphoramidite of 6-trifluoromethylpurine ribonucleoside ((TFM)P) and its use in the site-specific incorporation of 6-trifluoromethylpurine into RNA. Properties of (TFM)P-substituted RNA suggest it will be valuable in the study of RNA structure and the binding of RNA-modifying enzymes, particularly the RNA-editing adenosine deaminases. Reaction: see text.

Amides↗

PubMed2001

Conformational changes that occur during an RNA-editing adenosine deamination reaction.

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.

2-Aminopurine↗

PubMed2001

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2alpha kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2alpha kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA.Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2alpha.

Base Sequence↗

PubMed2001

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage.

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.

Adenoviridae↗

PubMed2000

Analysis of the RNA-editing reaction of ADAR2 with structural and fluorescent analogues of the GluR-B R/G editing site.

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.

2-Aminopurine↗

PubMed2000

Solid-phase synthesis of acridine-based threading intercalator peptides.

The preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and deprotected under solid-phase peptide synthesis procedures to create acridine peptide conjugates as potential threading intercalators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA consistent with intercalative binding.

Acridines↗

PubMed2000

Regulation of the RNA-dependent protein kinase by triple helix formation.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phos-phorylates the alpha-subunit of the translation initiation factor eIF-2, inhibiting its function. PKR is activated in vitro by binding to double-stranded RNA (dsRNA) molecules of approximately 30 bp or longer. Here we show that triple helix forming oligonucleotides (TFOs) inhibit dsRNA binding to the isolated RNA binding domain of PKR. The inhibition is specific to the targeted RNA and dependent on TFO length. Binding to a 30 bp duplex is inhibited by a 28 nt TFO and a 20 nt TFO with an IC(50) of 35 +/- 2 and 210 +/- 22 nM, respectively. An 18 nt TFO partially inhibits binding. The activation of the kinase domain of PKR by a 30 bp RNA duplex is also inhibited by a 28 nt TFO. Inhibition of binding is most effective when the triple helix is formed prior to addition of the protein. These results indicate that triplex formation can be used to prevent the binding of an RNA binding protein with dsRNA-binding motifs.

Base Sequence↗

PubMed2000

Solid-phase synthesis of acridine-peptide conjugates and their analysis by tandem mass spectrometry.

[reaction--see text] A novel and high-yielding synthesis of 9-anilinoacridine-4-carboxylic acid is reported. This acid has been used in the solid-phase synthesis of a small combinatorial library of acridine-peptide conjugates. Tandem mass spectrometry (ES-MS/MS) can be used for structure determination of these compounds at a sensitivity of approximately 10 pmol. This work makes possible the generation of acridine-peptide libraries for the discovery of structure-specific nucleic acid ligands via affinity chromatography selection with mass spectrometric detection.

Acridines↗

PubMed2000

Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR.

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site-specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable cross-linking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR-RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving.

Base Sequence↗

PubMed2000

FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions.

The FKBP12-rapamycin associated protein (FRAP, also RAFT, mTOR) belongs to a family of phosphatidylinositol kinase-related kinases. These kinases mediate cellular responses to stresses such as DNA damage and nutrient deprivation in a variety of eukaryotes from yeast to humans. FRAP regulates G(1) cell cycle progression and translation initiation in part by controlling the phosphorylation states of a number of translational and cell cycle regulators. Although FRAP is known to be phosphorylated in vivo and to phosphorylate several proteins (including itself) in vitro, FRAP's phosphorylation sites and substrate specificity are unknown. We report here the identification of a FRAP autophosphorylation site. This site, Ser-2481, is located in a hydrophobic region near the conserved carboxyl-terminal FRAP tail. We demonstrate that the COOH-terminal tail is required for FRAP kinase activity and for signaling to the translational regulator p70(s6k) (ribosomal subunit S6 kinase). Phosphorylation of wild-type but not kinase-inactive FRAP occurs at Ser-2481 in vivo, suggesting that Ser-2481 phosphorylation is a marker of FRAP autokinase activity in cells. FRAP autophosphorylation is blocked completely by wortmannin treatment but not by rapamycin treatment, amino acid deprivation, or serum withdrawal, treatments that lead to acute dephosphorylation of eIF4E-binding protein (4E-BP1) and p70(s6k). Ser-2481 phosphorylation increases slightly upon c-Akt/PKB activation and dramatically upon calyculin A treatment of T-cells. These results suggest that FRAP-responsive dephosphorylation of 4E-BP1 and p70(s6k) occurs through a mechanism other than inhibition of intrinsic FRAP kinase activity.

Amino Acid Sequence↗

PubMed1999

Synthetic substrate analogs for the RNA-editing adenosine deaminase ADAR-2.

We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2[prime]-carbon of the edited adenosine. Methylation of the 2[prime]-OH caused a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine were deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction.

Adenosine↗

PubMed1995

Control of p70 s6 kinase by kinase activity of FRAP in vivo.

When complexed with the intracellular protein FKBP12, rapamycin is a potent immunosuppressant and an inhibitor of a mitogen-stimulated signalling pathway that leads to activation of p70 S6 kinase (p70S6k) and cyclin-dependent kinases (CDKs). A recently cloned FKBP12-rapamycin-associated protein (FRAP/RAFT) is the likely mediator of these effects. Using FRAP variants that do not bind FKBP12-rapamycin, we demonstrate here that FRAP is a rapamycin-sensitive regulator of p70S6k in vivo and that the kinase activity of FRAP is required for this regulation. In addition, we show that FRAP autophosphorylates in vitro. Consistent with an essential role for FRAP kinase activity in vivo, autophosphorylation of FRAP is inhibited by FKBP12-rapamycin. Deletion studies indicate that the kinase activity of FRAP alone is not sufficient for control of p70S6k and that an amino-terminal domain in FRAP is also required.

Amino Acid Sequence↗

PubMed1992

The influence of single base triplet changes on the stability of a pur.pur.pyr triple helix determined by affinity cleaving.

The influence of sixteen base triplet changes at a single position within a pur.pur.pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G.GC, A.AT and T.AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25 degrees C). Weaker interactions were detected for the C.AT, A.GC and T.CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented.

Base Sequence↗

PubMed1991

Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation.

Relative orientations of the DNA strands within a purine.purine.pyrimidine triple helix have been determined by affinity cleaving. A purine-rich oligonucleotide bound in the major groove of double-helical DNA antiparallel to the Watson-Crick purine strand. Binding depended upon the concentration of multivalent cations such as spermine or Mg2+, and appeared to be relatively independent of pH. Two models with specific hydrogen-bonding patterns for base triplets (G.GC, A.AT, and T.AT) are proposed to explain the sequence specificity of binding. The two models differ in the conformation about the glycosyl bond (syn or anti) and the location of the phosphate-deoxyribose backbone in the major groove of DNA. This motif broadens the structural frameworks available as a basis for the design of sequence-specific DNA binding molecules.

DNA↗