Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists.

BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

Midbrain serotonin transporter binding potential measured with [11C]DASB is affected by serotonin transporter genotype.

BACKGROUND: Homozygote carriers of two long (L) alleles of the serotonin transporter (5-HTT) regulatory region displayed in vitro a twofold increase in 5-HTT expression compared with carriers of one or two short (S) alleles. However, in vivo imaging studies yielded contradictory results. Recently, an A > G exchange leading to differential transcriptional activation of 5-HTT mRNA in lymphobalstoid cell lines was discovered in the 5-HTT regulatory region. In vitro and in vivo evidence suggests that [(11)C]DASB, a new 5-HTT ligand offers some advantages over the ligands used in previous studies in measuring 5-HTT density independent of synaptic levels of serotonin. METHOD: We assessed 5-HTT binding potential (BP (2)) in the midbrain of 19 healthy subjects with positron emission tomography and [(11)C]DASB. Accounting for the hypothesized functional similarity of L (G) and S in driving 5-HTT transcription, we assessed whether L (A) L (A) homozygotes display increased midbrain BP (2) compared with carriers of at least one S allele. RESULTS: BP (2) in the midbrain was significantly increased in L (A) L (A) homozygotes compared with carriers of at least one S allele. Interestingly, the genotype effect on the midbrain was significantly different from that on the thalamus and the amygdala where no group differences were detected. CONCLUSIONS: This in vivo study provides further evidence that subjects homozygous for the L (A) allele display increased expression of 5-HTT in the midbrain, the origin of central serotonergic projections.

Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis.

Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab' fragment. The epitope occupies approximately 10% of the molecular surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid substitution, Glu(45)-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.

Yin Yang 1 is a lipopolysaccharide-inducible activator of the murine 3' Igh enhancer, hs3.

The 3' Igh enhancers, DNase I hypersensitive site (hs) 3B and/or hs4, are required for germline transcription, and hence, class switch recombination for multiple isotypes. A number of hs3-binding transcription factors have been identified by EMSA, including octamer and NF-kappa B family members, and Pax5. We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to hs3 and to the mu E1 site of the intronic enhancer, E mu, is induced in primary splenic B cells after approximately 48 h in response to LPS and other activators of class switch recombination. Transient transfection experiments in B cell lines indicate that YY1 is an activator of hs3. Interestingly, levels of YY1 expression are unchanged in resting and LPS-stimulated B cells. Mixing experiments followed by EMSA showed that a protein present in resting B cells prevented binding of YY1 to DNA. We found that recombinant retinoblastoma protein (Rb) inhibited binding of YY1 to hs3 in a dose-dependent manner, and we have identified complexes of endogenous YY1 with the Rb in resting B cells, but not in LPS-stimulated B cells. A difference in Rb phosphorylation state was also confirmed between resting (G(0)) B cells and LPS-stimulated B cells. These observations suggest that the interaction of YY1 with hypophosphorylated Rb in resting B cells prevents interaction of YY1 with DNA. After stimulation with class-switching activators, such as LPS, Rb becomes hyperphosphorylated and YY1 is released and can then bind to the hs3 enhancer and E mu.

The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme.

Trichomonas vaginalis is a parasitic protozoan and the causative agent of trichomoniasis. Its primary purine salvage system, consisting of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase, presents potential targets for designing selective inhibitors as antitrichomonial drugs because of lack of de novo synthesis of purine nucleotides in this organism. cDNA encoding T. vaginalis PNP was isolated by complementation of an Escherichia coli strain deficient in PNP and expressed, and the recombinant enzyme was purified to apparent homogeneity. It bears only 28% sequence identity with that of human PNP but 57% identity with the E. coli enzyme. Gel filtration showed the enzyme in a hexameric form, similar to the bacterial PNPs. Steady-state kinetic analysis of T. vaginalis PNP-catalyzed reactions gave K(m)'s of 31.5, 59.7, and 6.1 microM for inosine, guanosine, and adenosine in the nucleosidase reaction and 45.6, 35.9, and 12.3 microM for hypoxanthine, guanine, and adenine in the direction of nucleoside synthesis. This substrate specificity appears to be similar to that of bacterial PNPs. The catalytic efficiency of this enzyme with adenine as substrate is 58-fold higher than that with either hypoxanthine or guanine, representing a distinct disparity with the mammalian PNPs, which have negligible activity with either adenine or adenosine. The kinetic mechanism of T. vaginalis PNP-catalyzed reactions, determined by product inhibition and equilibrium isotope exchange, was by random binding of substrates (purine base and ribose 1-phosphate) with ordered release of the purine nucleoside first, followed by inorganic phosphate. Formycin A, an analogue of adenosine known as an inhibitor of E. coli PNP without any effect on mammalian PNPs, was shown to inhibit T. vaginalis PNP with a K(is) of 2.3 microM by competing with adenosine. T. vaginalis PNP thus belongs to the family of bacterial PNPs and constitutes a target for antitrichomonial chemotherapy.

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I.

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.

Identification of fetal liver tyrosine kinase 3 (flt3) ligand domain required for receptor binding and function using naturally occurring ligand isoforms.

We used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.

T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies.

Allergen-specific immunotherapy represents one of the few curative approaches toward type I allergy. Up to 25% of allergic patients are sensitized against the major birch pollen allergen, Bet v 1. By genetic engineering we produced two recombinant (r) Bet v 1 fragments comprising aa 1-74 and aa 75-160 of Bet v 1, which, due to a loss of their native-like fold, failed to bind IgE Abs and had reduced allergenic activity. Here we show that both fragments covering the full Bet v 1 sequence induced human lymphoproliferative responses similar to rBet v 1 wild type. The C-terminal rBet v 1 fragment induced higher lymphoproliferative responses than the N-terminal fragment and represented a Th1-stimulating segment with high IFN-gamma production, whereas the N-terminal fragment induced higher IL-4, IL-5, and IL-13 secretion. Immunization of mice and rabbits with rBet v 1 fragments induced IgG Abs, which cross-reacted with complete Bet v 1 and Bet v 1-related plant allergens and strongly inhibited the IgE binding of allergic patients to these allergens. Thus, our results demonstrate that hypoallergenic T cell epitope-containing rBet v 1 fragments, despite lacking IgE epitopes, can induce Abs in vivo that prevent the IgE binding of allergic patients to the wild-type allergen. The overall demonstration of the immunogenic features of the hypoallergenic rBet v 1 fragments will now enable clinical studies for safer and more efficient specific immunotherapy.

Quantitation of GABA transporter 3 (GAT3) mRNA in rat brain by competitive RT-PCR.

Gamma-amino butyric acid is the major inhibitory neurotransmitter in the brain. GABA transporters (GATs) remove GABA from the synaptic cleft. Till now, five distinct GABA transporters have been cloned and termed consecutively GAT1 to GAT4 and vGAT. To study the mechanisms by which tolerance and dependence associated with drugs enhancing GABAergic transmission is brought upon we analysed the mRNA expression levels of GATs in various brain regions under different conditions. In this paper, we describe our protocol for measurement of GAT3 mRNA expression, and its validation through control experiments for the various steps. We performed competitive reverse transcription and polymerase chain reaction (RT-PCR) with a competitor cRNA as internal standard. Different amounts of competitor cRNA were added to total RNA prepared from different tissue samples, reverse-transcribed and PCR amplified. The PCR amplification gave two products: the GAT wild type fragment and the competitor fragment. PCR products were separated by gel electrophoresis and band intensities were determined from which the relative and absolute abundance of GAT3 mRNA was calculated by regression analysis. Validation experiments in our laboratory showed a 6% intra-assay and a 15% inter-assay variability of this method.

Identification of a novel Stat3 recruitment and activation motif within the granulocyte colony-stimulating factor receptor.

Stat3 is essential for early embryonic development and for myeloid differentiation induced by the cytokines granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). Two isoforms of Stat3 have been identified, (p92) and beta (p83), which have distinct transcriptional and biological functions. Activation of both Stat3 and Stat3beta requires the distal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to determine which, if any, of these tyrosine residues participated in Stat3/beta recruitment and activation. We showed that Stat3 and Stat3beta were affinity purified using phosphopeptides containing Y704 and Y744 but not by nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining the ability of these peptides to destabilize and inhibit DNA binding of activated Stat3. Both Y704 and Y744 contributed to optimal activation of Stat3/beta in M1 murine myeloid leukemia cells containing wild-type and Y-to-F mutant G-CSFR constructs. Carboxy-terminal to Y704 at the +3 position is Gln; YXXQ represents a consensus Stat3 recruitment and activation motif. Y744 is followed at the +3 position by Cys (C); YXXC, represents a novel motif implicated in the recruitment and activation of Stat3. Modeling of the SH2 domain of Stat3 based on homologous SH2 domains of known structure revealed polar residues whose side chains contact the +3 position. This substitution may confer specificity for the Y704- and Y744-based ligands by allowing H-bond formation between the binding surface and the Gln or Cys found at the respective +3 position.

Mg2+ mediated sequence-specific binding of transcriptional activator protein C of bacteriophage Mu to DNA.

The contributions from the secondary structure of the transcriptional activator protein C of bacteriophage Mu to its specific DNA binding and the influence of various factors, viz., electrolytes, and minor groove and major groove binders on this protein-DNA interaction have been addressed. Circular dichroism (CD) spectral results suggest that, in the absence of Mg2+, C protein exhibits a beta-pleated sheetlike structure and Mg2+ changes the conformation to a more alpha-helical structure which could provide specific geometrical constraints complementary to those of DNA-helix. Thus, Mg2+ acts as a cofactor for the binding of the C protein to its specific site in DNA by inducing conformational changes in the protein. Competitive binding studies with minor and major groove binding drugs, viz., distamycin A and methyl green, respectively, and the DMS footprinting data indicate that the C protein recognizes the major groove of DNA during complex formation. Further, upon major groove binding, C protein brings about changes in DNA conformation; such conformational changes could have implications in the transcription process.

Interaction of the transcription factor YY1 with human poly(ADP-ribosyl) transferase.

Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear enzyme that catalyzes the synthesis of ADP-ribose polymers from NAD+ as well as the transfer of these polymers onto acceptor proteins. The function of ADPRT is thought to be related to a number of nuclear processes including DNA repair and transcription. The transcription factor Yin Yang 1 (YY1) is a potent regulator of RNA polymerase II (Pol II)-dependent transcription. In this study Alu-retroposon-associated binding sites for YY1 located in the distal region of the promoter of the human ADPRT gene have been identified suggesting a possible involvement of this protein in the regulation of ADPRT-gene expression. In the presence of the recombinant automodification domain of the ADPRT the formation of specific YY1 complexes, detected in gel-shift experiments, was strongly inhibited, indicating that this domain of the enzyme may interact directly with YY1. In accordance with this result YY1 was specifically precipitated from nuclear extracts by ADPRT immobilized on sepharose. These results suggest a direct ADPRT-YY1 interaction which may be of importance in the regulation of Pol II-dependent transcription. They also indicate that in some human promoters this regulation may be mediated by retroposons of the Alu family.