Agnieszka Szyk

Experimental Models of Protein-RNA Interaction: Isolation and Analyses of tRNAPhe and U1 snRNA-Binding Peptides from Bacteriophage Display Libraries

Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNAPhe (tRNAACPhe) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd ≍ 0.1–5.0 μM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein–RNA complexes.

Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation responsive element with trans-activator of transcription protein (49–57): Capillary electrophoresis mobility shift assay

A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested that a single, conserved arginine residue (presumably Arg52) within the arginine-rich region (ARR) of Tat plays the major role for the Tat-TAR recognition. To study structural requirements of the Arg52 position, Tat(49-57)-NH2 analogues substituted with nonencoded amino acids at the Arg52 position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49-57) a significant shift of migration time of TAR from 18.66 min (RSD=1.4%) to 20.12 min (RSD=2.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg52 [L-Arg52-->Cit, -->Orn, -->Arg(NO2), -->Arg(Me2)] strongly disrupted or abolished the TAR-Tat peptide interaction. Enantiomeric substitution, L-Arg52-->D-Arg was the only one which notably promoted TAR-Tat peptide interaction. The results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg52 plays a crucial role for TAR-Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA-peptide complex formation. The methodology presented should be generally applicable to study RNA-peptide (protein) interaction.

Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay

A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2-O-methylcytidine (Cm32), 2-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions.

Effects of Diabetes mellitus on the Contractile Activity of Carbachol and Galanin in Isolated Gastric Fundus Strips of Rats

The role of the cholinergic and peptidergic pathways in the impairment of gastric motility associated with diabetic gastroparesis was assessed at the postsynaptic level using isolated fundus smooth muscle strips. Maximal contractile responses to carbachol and galanin were significantly decreased in fundus strips isolated from rats rendered diabetic by a single intraperitoneal injection of streptozotocin (STZ, 70 mg/kg) 1, 4 and 8 weeks before experiments. We also observed notable decrements in the slopes and Hill’s coefficients without conspicuous changes in the EC50 of the respective galanin concentration-response curves measured in strips obtained from STZ animals after 4 and 8 weeks. L-NAME reversed the above-mentioned alterations in an L-arginine-sensitive manner in STZ rats after 4 weeks but not in STZ rats after 8 weeks. The blood plasma nitrite/nitrate levels in STZ animals after 4 and 8 weeks were increased by 44.6 and 61.9%, respectively. Ca2+-independent nitric oxide synthase activity in gastric fundus strips and stomach corpus mucosa from STZ rats after 4 weeks was markedly enhanced by 37.4 and 31.9%, respectively, suggesting an enhanced nitric oxide production. In vivo insulin treatment prevented diabetes-induced alterations in smooth muscle contractility. We conclude that the smooth muscle dysfunction evoked by experimental diabetes causing diminished contractions of fundus strips to carbachol and galanin is at least partly due to the increased nitric oxide synthesis.

Inhibition of gastric acid secretion by galanin in rats: Relation to endogenous histamine release

Inhibitory effect of galanin on basal and secretagogs-stimulated gastric acid secretion was investigated in urethane-anesthetized rats. A rat stomach was mounted in an ex-vivo chamber, perfused with saline, and either gastric acid or alkaline secretion was determined by titrating the perfusate. Gastric mucosal blood flow (GMBF) was measured by a laser Doppler flowmeter. Intravenous infusion of galanin dose-dependently inhibited the increase of acid secretion induced by pentagastrin and carbachol but not by histamine, without any influence on the GMBF response. Galanin also reduced basal acid secretion while increasing GMBF, but did not evoke any change in basal gastric alkaline secretion. M15, which is a galanin receptor antagonist in the central nervous system but acts as a full agonist in the gastrointestinal smooth muscle, also suppressed pentagastrin-induced acid secretion, similar to galanin. In addition, pentagastrin increased the release of histamine into the gastric lumen, and this response was significantly inhibited by galanin. These results suggest that the inhibitory effect of galanin on acid secretion is mediated by suppression of endogenous histamine release from enterochromaffin-like cells and that the process may be related to the activation of the same subtype of galanin receptors as in the central nervous system and pancreatic beta-cells.

Specific induction of Z-DNA conformation by a nuclear localization signal peptide of lupin glutaminyl tRNA synthetase

Recently we have sequenced cDNA of plant glutaminyl-tRNA synthetase (GlnRS) from Lupinus luteus. At the N terminal part the protein contains a lysine rich polypeptide (KPKKKKEK), which is identical to a nuclear localization signal (NLS). In this paper we showed that two synthetic peptides (20 and 8xa0amino acids long), which were derived from lupin GlnRS containing the NLS sequence interact with DNA, but one of them (8aa long) changing its conformation from the B to the Z form. This observation clearly suggests that the presence of the NLS polypeptide in a leader sequence of GlnRS is required not only for protein transport into nucleus but also for regulation of a gene expression. This is the first report suggesting a role of the NLS signal peptide in structural changes of DNA.

LYSINE14GALANIN(1-15)-NH2 : A PARTIAL AGONIST AT GALANIN RECEPTORS IN RAT ISOLATED GASTRIC FUNDUS

The study was undertaken to characterize the effects of the porcine galanin [pGal(1-29)-NH2] analogue [Lys14]pGal(1-15)-NH2 on rat gastric fundus. [Lys14]pGal(1-15)-NH2 is a less potent contractile agent than pGal(1-29)-NH2 (EC50 74.1 vs. 43.7 nmol/l, respectively) and shows a significantly lower maximal response than pGal(1-29)-NH2. Concentration-contraction curves were constructed for pGal(1-29)-NH2 alone (control) and pGal(1-29)-NH2 in the presence of 10, 100, and 1,000 nmol/l of [Lys14]pGal(1-15)-NH2. [Lys14]pGal(1-15)-NH2 shifted the concentration-contraction curves of pGal(1-29)-NH2 significantly to the right, whereas their linear portions remained parallel to that for the pGal(1-29)-NH2 control. [Lys14]pGal(1-15)-NH2 markedly increased the EC50 of the respective pGal(1-29)-NH2 concentration-contraction curves. It did not substantially change the maximal response of the muscles to pGal(1-29)-NH2 and the form of the respective concentration-contraction curves. Schilds plot gave a straight line with a slope of 0.84. The pA2 value for [Lys14]pGal(1-15)-NH2 was 8.23. [Lys14]pGal(1-15)-NH2 seems to be a partial Gal receptor agonist. Since the lack of specific Gal receptor antagonists in the gastrointestinal tract makes a precise characterization of its role as a motility modulator difficult, the position 14 in the pGal(1-29)-NH2 molecule looks as an attractive target in the search of a pure Gal receptor antagonist in the smooth muscles of the gut.

Pharmacological characterization of the contractile effects of galanin(l‐29)‐NH2, galantide and galanin(l‐14)‐(α‐aminobutyric acid8)scyliorhinin‐I in the rat gastric fundus†

Summary— Porcine galanin (l‐29)‐NH2, galantide (M15) and galanin (l‐14)‐(a‐aminobutyric acid8)‐scyliorhinin‐I used in concentrations of 300, 1,000 and 3,000 nM respectively caused contractions of rat fundus strips. The contractile responses to galanin(1–29)‐NH2 were not modified by atropine (10 μM), guanethidine (10 μM), naloxone (1 μ), a mixture of propranolol (10 μM) and phentolamine (10 μM), indomethacin (10 μM), a mixture of mepyramine (10 μM) and Cimetidine (10 μM), saralasin (10 μM), and spantide (100 μM). The effects of M15 and galanin(l‐14)‐(a‐aminobutyric acid8)‐scyliorhinin‐l were significantly decreased by atropine for 36 and 18% and by spantide for 37 and 26% respectively. Indomethacin inhibited the muscle response to M15 without influence on the galanin (l‐14)‐(a‐aminobutyric acid8)‐scyliorhinin‐I‐induced action. These results support findings that galanin (l‐29)‐NH2 contracts rat gastric fundus strips by stimulating specific receptors localized on the surface of smooth muscle cells. M15 and galanin(l‐14)‐(a‐aminobutyric acid8)‐scyliorhinin‐I seem to contract smooth muscles not only by acting at galanin receptors, but by interacting with muscarinic or tachykinin receptors or modulating the release of acetylcholine and substance P. Diltiazem (EC50 825 nM), dantrolene (EC50 30.2 μM) and the phospholipase C inhibitors U‐73122 (EC50 549 μM) and U‐73343 (EC50 751 fjM) lowered the contraction to galanin(l‐29)‐NH2 in a concentration‐dependent manner. These observations imply that though the extracellular Ca2+ influx plays a major role in the action of galanin(l‐29)‐NH2, the release of Ca2+ ions from the intracellular stores contributes to the response of smooth muscles to galanin(l‐29) NH2. Norepinephrine (30, 60, 100 and 300 nM) concentration‐dependently reduced the Emax to galanin (l‐29)‐NH2 and reduced the slopes of the concentration‐contraction curves, without a notable change in EC50. Pertussis toxin pre‐treatment (10 and 30 mg/kg intravenous[iv]), 120 h before the experiment, notably increased the maximal response of the rat gastric fundus to galanin(l‐29)‐NH2, without a significant change in the properties of the concentration‐contraction curves (EC50, slopes). The observations may suggest that pertussis toxin‐sensitive GTP‐binding proteins are involved in the modulation of the excitatory effects of galanin(l‐29)‐NH2 in the rat gastric fundus.

Circular Dichroism Studies of the Interaction of Tat Analogues Substituted in the Arg52 Position with TAR RNA HIV-1

The Tat wild-type fragment of sequence Arg49-Lys-Lys-Arg 52-Arg-Gln-Arg-Arg-Arg57-NH2 (labelled as Tat1) and three analogues of this fragment with the substitution Arg52»D-Arg52 (labelled as Tat2) or L-citrulline (Cit) (labelled as Tat3) or L-ornithine (Orn) (labelled as Tat4) were synthesized to study Tat-TAR RNA HIV-1 (27-nucleotide fragment of sequence 5′-AGAUCUGAGCCUGGAGCUCUCU-3′) interactions by circular dichroism. α-helical structure was the most readily adopted by the Tat3 analogue with Arg52»Cit substitution. All the peptides investigated caused conformational changes in the TAR structure. The most dramatic changes were observed for the Tat2-TAR complex.

Mechanisms of Galanin-induced Contraction of Isolated Rat Gastric Fundus Strips {

Galanin (Gal) constricts rat gastric fundus by acting on receptors located in the cell membrane. We compared the role of intracellular Ca2+ release with extracellular Ca2+ influx in Gal-stimulated contraction of isolated gastric smooth muscle strips. We also tested if phospholipase C (PLC) or protein kinase C (PKC) participate in the signal transduction cascade. n n n nConcentration-contraction curves were constructed non-cumulatively in the presence of atropine, hexamethonium, guanethidine and tetrodotoxin. The half-maximum effective concentration (EC50) of Gal was 21.62nM and Hills coefficient was 1.02. The effects of Gal were decreased by diltiazem, Ca2+-deficiency in the buffer, Ca2+ removal from the extracellular medium or quercetin. Depletion of intracellular Ca2+-stores, ryanodine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester diminished the contractile effect of Gal concentration-dependently. Trifluoroperazine and phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitors, neomycin and U-73122, attenuated the gastric fundus response to Gal, whereas phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) blockers, D609 and propranolol, were ineffective. The inhibitors of PKC or myosin light chain kinase, calphostin C, chelerythrine, ML-7 and ML-9, lowered the myogenic activity of Gal. n n n nOur data confirmed that the stimulation of Gal receptors in gastric fundus is coupled to Ca2+ influx through voltage-dependent channels and intracellular Ca2+ release from ryanodine- and inositol 1,4,5-triphosphate-sensitive stores. Enzymes such as PI-PLC and PKC, but not PC-PLC or PLD, play a role in the signal transduction cascade. Calmodulin and myosin light chain kinase lie downstream of the increases in intracellular Ca2+ concentration evoked by Gal.