Adjacent Tyrosine Residues Research Articles

Characterization and analysis of a novel diguanylate cyclase PA0847 from Pseudomonas aeruginosa PAO1.

Background: As a central signaling molecule, cyclic diguanylate (c-di-GMP) is found to regulate various bacterial phenotypes, especially those involved in pathogen infection and drug resistance. Noticeably, many microbes have up to dozens of proteins that are involved in c-di-GMP metabolism. This apparent redundancy and the relevant functional specificity have become the focus of research. While a number of these proteins have been identified and investigated, the functions of PA0847, a PAS and GGDEF domain-containing protein from Pseudomonas aeruginosa PAO1, remain unclear.Materials and methods: In the current study, microbiology, biochemistry and structural biology methods were applied to characterize the gene/protein of PA0847.Results: We showed that PA0847 affects bacterial motility but not biofilm formation. We recorded the phenotypic influences of amino acids and compounds, and found that PA0847 is involved in response to various environmental nutrients and factors, suggesting its possible role in sensing environmental cues. Both in-vitro and in-vivo studies showed that PA0847 is an active diguanylate cyclase (DGC), whose activity depends on the neighboring PAS domain. Interestingly, PA0847 demonstrates no significant product inhibition, though the key residues of two I-sites for c-di-GMP binding are conserved in its GGDEF domain. A local structural change imposed by an adjacent tyrosine residue was identified, which indicates the structural and functional diversities of the GGDEF family proteins.Conclusion: Our data provide evidence for understanding the signaling mechanism of the unique c-di-GMP metabolizing protein PA0847.

Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.

Simulations of FGFR2 Kinase Activation Loop Dynamics and their Effects on Catalytic Activity

Unregulated activity of tyrosine kinases (TKs) is responsible for numerous developmental, oncologic and musculoskeletal diseases, yet the regulatory mechanisms of TKs are still in need of elucidation. Fibroblast growth factor receptor (FGFR) kinases are a TK family whose catalytic activity appears to be regulated by phosphorylation of two adjacent tyrosine residues in the kinase “activation loop.” However, there is no clear mechanism to explain how tyrosine phosphorylation in the activation loop enables substrate catalysis. Using enhanced-sampling molecular dynamics (MD) simulations of FGFR2 kinase, we demonstrate by simulation that phosphorylation of Tyr657 in FGFR2 does not disable an inhibitory mechanism, as seen in other homologous structures such as FGFR1 kinase, but actively promotes substrate catalysis by altering the conformational dynamics of the kinase. Phosphorylation of Tyr657 allows for greater coordination of the kinase's N-lobe and C-lobe, facilitating enhanced coordination of the enzyme and substrate for catalysis. Moreover, inward motion of Tyr657, promoted by phosphorylation, stabilizes the presence of Arg664 in the enzyme's active site. We propose that the presence of Arg664 near ATP and the substrate tyrosine residue, previously observed in a crystal structure of FGFR3 kinase, locks the substrate tyrosine in a precise position, sandwiched by two arginine residues, favoring catalysis. Additionally, Arg664 helps to coordinate the position of ATP, and may also lower the activation energy for the catalytic phosphorylation reaction. Our study of activation loop dynamics in the pTyr657-in state of FGFR2 kinase may provide avenues for targeted therapies to counteract pathologic over-activation of FGFR2 kinase leading to uncontrolled signal transduction and uninhibited cell growth.

Binding of oxo-Cu2 clusters to ferric ion-binding protein A from Neisseria gonorrhoeae: a structural insight

The ferric ion-binding protein A (FbpA), a member of transferrin superfamily, is a periplasmic iron transporter employed by many Gram-negative pathogens. Our experiments indicated copper(ii) could bind with Neisseria gonorrhoeae FbpA (NgFbpA), and the binding constant reached up to (8.7 ± 0.2) × 10(8) M(-1)via UV-vis titration. The crystal structure of recombinant Cu-NgFbpA at 2.1 Å revealed that the oxo-Cu2 clusters (dinuclear centres) assembled in the iron binding cleft and were bound to the two adjacent tyrosine residues (Y195 and Y196) of the protein, two Cu ions coordinated with two tyrosines, Y195 and Y196, respectively, which was different from the binding model of Fe ion with FbpA, in which Y195 and Y196 coordinated together with one Fe ion. While this was similar to the binding of Zr and Hf ion clusters, Y195 and Y196 coordinated with two metal ions and the μ-oxo-bridges linking the metal ions. Structural superimposition demonstrated that oxo-Cu2-NgFbpA still keeping an open conformation, similar to the apo-form of NgFbpA. The structure presented additional information towards an understanding of the function of FbpA, and provided a detailed binding model for FbpA protein with the possible metal ions in a biological system.

A Facile Way to Tune Mechanical Properties of Artificial Elastomeric Proteins-Based Hydrogels

Protein-based hydrogels have attracted considerable interests due to their potential applications in biomedical engineering and material sciences. Using a tandem modular protein (GB1)(8) as building blocks, we have engineered chemically cross-linked hydrogels via a photochemical cross-linking strategy, which is based on the cross-linking of two adjacent tyrosine residues into dityrosine adducts. However, because of the relatively low reactivity of tyrosine residues in GB1, (GB1)(8)-based hydrogels exhibit poor mechanical properties. Here, we report a Bolton-Hunter reagent-based, facile method to improve and tune the mechanical properties of such protein-based hydrogels. Using Bolton-Hunter reagent, we can derivatize lysine residues with phenolic functional groups to modulate the phenolic (tyrosine-like) content of (GB1)(8). We show that hydrogels made from derivatized (GB1)(8) with increased phenolic content show significantly improved mechanical properties, including improved Young's modulus, breaking modulus as well as reduced swelling. These results demonstrate the great potential of this derivatization method in constructing protein-based biomaterials with desired macroscopic mechanical properties.

Tyrosine Sulfation Influences the Chemokine Binding Selectivity of Peptides Derived from Chemokine Receptor CCR3

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

The role of vicinal tyrosine residues in the function of Haemophilus influenzae ferric-binding protein A

The periplasmic FbpA (ferric-binding protein A) from Haemophilus influenzae plays a critical role in acquiring iron from host transferrin, shuttling iron from the outer-membrane receptor complex to the inner-membrane transport complex responsible for transporting iron into the cytoplasm. In the present study, we report on the properties of a series of site-directed mutants of two adjacent tyrosine residues involved in iron co-ordination, and demonstrate that, in contrast with mutation of equivalent residues in the N-lobe of human transferrin, the mutant FbpAs retain significant iron-binding affinity regardless of the nature of the replacement amino acid. The Y195A and Y196A FbpAs are not only capable of binding iron, but are proficient in mediating periplasm-to-cytoplasm iron transport in a reconstituted FbpABC pathway in a specialized Escherichia coli reporter strain. This indicates that their inability to mediate iron acquisition from transferrin is due to their inability to compete for iron with receptor-bound transferrin. Wild-type iron-loaded FbpA could be crystalized in a closed or open state depending upon the crystallization conditions. The synergistic phosphate anion was not present in the iron-loaded open form, suggesting that initial anchoring of iron was mediated by the adjacent tyrosine residues and that alternate pathways for iron and anion binding and release may be considered. Collectively, these results demonstrate that the presence of a twin-tyrosine motif common to many periplasmic iron-binding proteins is critical for initially capturing the ferric ion released by the outer-membrane receptor complex.

Peroxide-Dependent Formation of a Covalent Link between Trp51 and the Heme in Cytochrome c Peroxidase

Ascorbate peroxidase (APX), cytochrome c peroxidase (CcP), and the catalase-peroxidases (KatG) share very similar active site structures and are distinguished from other peroxidases by the presence of a distal tryptophan residue. In KatG, this distal tryptophan forms a covalent link to an adjacent tyrosine residue, which in turn links to a methionine residue. We have previously shown [ Pipirou, Z. et al. ( 2007 ) Biochemistry 46 , 2174 - 2180 ] that reaction of APX with peroxide leads, over long time scales, to formation of a covalent link with the distal tryptophan (Trp41) in a mechanism that proceeds through initial formation of a compound I species bearing a porphyrin pi-cation radical followed by radical formation on Trp41, as implicated in the KatG enzymes. Formation of such a covalent link in CcP has never been reported, and we proposed that this could be because compound I in CcP uses Trp191 instead of a porphyrin pi-cation radical. To test this, we have examined the reactivity of the W191F variant of CcP with H(2)O(2), in which formation of a porphyrin pi-cation radical occurs. We show, using electronic spectroscopy, HPLC, and mass spectroscopy, that in W191F partial formation of a covalent link from Trp51 to the heme is observed, as in APX. Radical formation on Trp51, as seen for KatG and APX, is implicated; this is supported by QM/MM calculations. Collectively, the data show that all three members of the class I heme peroxidases can support radical formation on the distal tryptophan and that the reactivity of this radical can be controlled either by the protein structure or by the nature of the compound I intermediate.

Regulation of Chemokine Recognition by Site-Specific Tyrosine Sulfation of Receptor Peptides

Sulfation of tyrosine is a common posttranslational modification of secreted proteins that influences numerous physiological and pathological processes. Studies of tyrosine sulfation have been hindered by the difficulty of introducing sulfate groups at specific positions of peptides and proteins. Here we report a general strategy for synthesis of peptides containing sulfotyrosine at one or more specific position(s). The approach provides a substantial improvement in both yield and convenience over existing methods. Using synthetic sulfopeptides derived from the chemokine receptor CCR3, we demonstrate that sulfation enhances affinity for the chemokine eotaxin by approximately 7-fold or more than 28-fold, depending on which of two adjacent tyrosine residues is sulfated. The synthetic methodology will substantially enhance efforts to understand the functional and structural consequences of protein tyrosine sulfation.

Enhanced Complexity and Catalytic Efficiency in the Hydrolysis of Phosphate Diesters by Rationally Designed Helix‐Loop‐Helix Motifs

HJ1, a 42-residue peptide that folds into a helix-loop-helix motif and dimerizes to form a four-helix bundle, successfully catalyzes the cleavage of "early stage" DNA model substrates in an aqueous solution at pH 7.0, with a rate enhancement in the hydrolysis of heptyl 4-nitrophenyl phosphate of over three orders of magnitude over that of the imidazole-catalyzed reaction, k(2)(HJ1)/k(2)(Im) = 3135. The second-order rate constant, k(2)(HJ1) was determined to be 1.58x10(-4) M(-1) s(-1). The catalyst successfully assembles residues that in a single elementary reaction step are capable of general-acid and general-base catalysis as well as transition state stabilization and proximity effects. The reactivity achieved with the HJ1 polypeptide, rationally designed to catalyze the hydrolysis of phosphodiesters, is based on two histidine residues flanked by four arginines and two adjacent tyrosine residues, all located on the surface of a helix-loop-helix motif. The introduction of Tyr residues close to the catalytic site improves efficiency, in the cleavage of activated aryl alkyl phosphates as well as less activated dialkyl phosphates. HJ1 is also effective in the cleavage of an RNA-mimic substrate, uridine-3'-2,2,2-trichloroethyl phosphate (leaving group pK(a) = 12.3) with a second-order rate constant of 8.23x10(-4) M(-1) s(-1) in aqueous solution at pH 7.0, some 500 times faster than the reaction catalyzed by imidazole, k(2)(HJ1)/k(2)(Im) = 496.

Detection of specific nitrotyrosine-modified proteins as a marker of oxidative stress in cardiovascular disease

the oxidative modification of proteins is a natural consequence of aerobic life and is also recognized to play a crucial role in the pathological response of cells to increased oxidative stress. Several oxidative modifications of a protein can occur as a result of oxidative stress. These can range

Primary Acute Myeloid Leukemic Blasts Display Constitutive Serine 585 Phosphorylation within the 14-3-3 Binding Motif of the GM-CSF/IL-3 Receptor Required for Survival Signalling.

Acute myeloid leukemia (AML) is characterised by defects in the regulation of hematopoietic cell survival, differentiation and proliferation. IL-3 and GM-CSF exert potent survival and proliferative effects on a range of myeloid progenitor cells. Previously we have identified a novel cytoplasmic motif of the human GM-CSF/IL-3 receptor beta chain responsible for coordinating survival and proliferative responses in primary hematopoietic cells. The motif contains a serine residue (585) that is phosphorylated in response to cytokine and couples to the PI3K/AKT pathway via the 14-3-3 adaptor protein and is required for cell survival (Guthridge et al, Molecular Cell 6:99, 2000; Blood 103:820, 2004). An adjacent tyrosine residue (577) within the motif is phosphorylated via Jak2 and associates with proliferative function. We investigated the phosphorylation status of the beta chain motif in response to GM-CSF stimulation in blasts cells from 12 patients with AML, and in blood monocytes from 6 healthy volunteers. Leukemic samples consisted of >99% blasts after buffy coat separation according to cytospin morphology. All leukemic blasts and blood monocyte samples expressed alpha and beta chain by flow cytometry. The phosphorylation status of residues 577 and 585 were examined utilising beta chain phospho-specific antibodies, after stimulation of AML blasts or blood monocytes with GM-CSF and beta chain immunoprecipitation. Human monocyte preparations all showed inducible serine 585 phosphorylation and tyrosine 577 phosphorylation. In contrast AML blasts displayed strong constitutive serine 585 phosphorylation in 10 out of 12 patients. No discernible tyrosine 577 phosphorylation in the absence of cytokine was observed for the AML samples. Beta chain phosphorylation pattern was independent of FAB classification. Inhibition of Jak2 did not prevent serine 585 phosphorylation of the receptor. Constitutive Akt activation was observed in AML blasts. The data provides evidence for dysregulation of a novel GM-CSF/IL-3 receptor mediated survival pathway involving serine/threonine kinases, 14-3-3 and Akt that is active in primary AML blasts in the majority of patients. This data would suggest that dysregulated activation of serine/threonine kinases and/or phosphatases at the receptor level may contribute to leukemogenesis.

IL4 receptor polymorphism is associated with increased risk of sudden deafness in Korean population

The interleukin 4 receptor ( IL4R) polymorphism Q576R (rs 180275) has been well known to be associated with atopy and other inflammatory diseases. A single nucleotide polymorphism (SNP) A > G transition potentiates the binding specificity of the adjacent tyrosine residue. In this study we investigated the possible relationship between sudden deafness (SD) and IL4R polymorphism Q576R in 97 Korean SD patients and 613 controls using pyrosequencing method. The odds ratio (OR) for SD associated with the G vs. A allele was 2.58 [ P < 0.0001, 95% confidence interval (CI) = 1.84–3.60]. We then sub-grouped SD into Tinnitus positive (+) and Tinnitus negative (−). G allele in Tinnitus (+) is significantly associated with the development of Tinnitus (+) [ X 2 = 32.02, P < 0.0001, OR (95% CI) = 2.74 (1.91–3.93)] but not with Tinnitus (−). Taken together these results suggest that G allele could be a risk factor for SD.

Towards a Structure for a TSG-6·Hyaluronan Complex by Modeling and NMR Spectroscopy: INSIGHTS INTO OTHER MEMBERS OF THE LINK MODULE SUPERFAMILY

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-pi stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein.HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [(13)C, (15)N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.

β3 Tyrosine Phosphorylation and αvβ3-mediated Adhesion Are Required for Vav1 Association and Rho Activation in Leukocytes

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.

Crosslinking of and coupling to viral capsid proteins by tyrosine oxidation.

Cowpea mosaic virus is composed of 60 identical copies of a two-subunit protein organized in pentameric assemblies around the icosahedral 5-fold symmetry axis. Treatment of the virus with the Ni(II) complex of the tripeptide GGH and a peroxide oxidant, or irradiation in the presence of Ru(bpy)(3)(2+) and persulfate generates covalent crosslinks across the pentameric subunit boundaries, effectively stitching the subunits together. Intersubunit crosslinking was found to occur exclusively at adjacent tyrosine residues (Y52-Y103), as predicted from the X-ray crystal structure of the capsid, and to be more extensive with the photochemical ruthenium system. The Ni/GGH oxidative procedure was also used to make covalent attachments to the virion by trapping with a functionalized disulfide reagent.

Protonation of two adjacent tyrosine residues influences the reduction of cytochrome c by diphenylacetaldehyde: a possible mechanism to select the reducer agent of heme iron

We have shown that diphenlacetaldehyde (DPAA) is able to promote mitochondrial ΔΨ disruption accompanied by damage in mitochondrial DNA, lipids, and proteins [Almeida, A. M.; Bechara, E. J. H.; Vercesi, A. E.; Nantes, I. L. Free Radic. Biol. Med. 27:744–747; 1999]. In this work, DPAA was used as a model of carbonyl reagent for cytochrome c. The results suggest that DPAA is a redox cytochrome c modifier. Conversion of Fe(III) to Fe(II) cytochrome c promoted by DPAA is pH dependent. The second-order rate determined for heme iron reduction ( k 2) is 698 M −1 s −1 and this process occurs with an activation energy of 8.5 ± 0.8 kcal/mol. Analysis of the pH profile suggests the presence of two ionizable cytochrome c groups (p K a1 = 8.9 and p K a2 = 11.4) related to the electron transfer from DPAA to heme iron. The heats of ionization of the two prototropic groups, p K a1 (Δ H ion = 6.5 kcal/mol, Δ S ion = −29.0 cal/mol·K), and p K a2 (Δ H ion = 5.0 kcal/mol, Δ S ion = −24.0 cal/mol·K), suggest involvement of two tyrosine residues, probably Y67 and Y74, related to DPAA-promoted heme iron reduction. The cytochrome c chemical modification by iodination of tyrosine groups significantly decreased the reduction rate promoted by DPAA, and shifted the pH opt value from 10.0 to 9.25. The cytochrome c-promoted DPAA electron abstraction quickly produces the expected enol-derived radical, as indicated by 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) spin trapping EPR measurements. This radical reacts with molecular oxygen, producing a peroxyl intermediate radical that, via a putative dioxetane intermediate, promotes formation of benzophenone as the main final product of this reaction, detected by high-performance liquid chromatography coupled with tandem mass spectrometry.

The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor.

Atopic diseases are very common, and atopy has a strong genetic predisposition. Using single-strand conformation polymorphism analysis and DNA sequencing, we searched for mutations in the a subunit of the interleukin-4 receptor that would predispose persons to atopy. We examined the prevalence of the alleles among patients with allergic inflammatory disorders and among 50 prospectively recruited adults. Subjects with atopy were identified on the basis of an elevated serum IgE level (> or = 95 IU per milliliter) or a positive radioimmunosorbent test in response to standard inhalant allergens. The signaling function of mutant interleukin-4 receptor a was examined by flow cytometry, binding assays, and immunoblotting. A novel interleukin-4 receptor alpha allele was identified in which guanine was substituted for adenine at nucleotide 1902, causing a change from glutamine to arginine at position 576 (R576) in the cytoplasmic domain of the interleukin-4 receptor alpha protein. The R576 allele was common among patients with allergic inflammatory disorders (found in 3 of 3 patients with the hyper-IgE syndrome and 4 of 7 patients with severe atopic dermatitis) and among the 50 prospectively recruited adults (found in 13 of 20 subjects with atopy and 5 of 30 without atopy; P=0.001; relative risk of atopy among those with a mutant allele, 9.3). The R576 allele was associated with higher levels of expression of CD23 by interleukin-4 than the wild-type allele. This enhanced signaling was associated with a change in the binding specificity of the adjacent tyrosine residue at position 575 to signal-transducing molecules. The R576 allele of interleukin-4 receptor alpha is strongly associated with atopy. This mutation may predispose persons to allergic diseases by altering the signaling function of the receptor.

Mechanistic Studies Relevant to Bromouridine‐Enhanced Nucleoprotein Photocrosslinking: Possible Involvement of an Excited Tyrosine Residue of the Protein

AbstractThe results of mechanistic studies on formation of uridine (U) and N‐acetyl‐in‐(5‐uridinyl)tyrosine N‐ethylam‐ide (2) from irradiation of aqueous, pH 7 solutions of bromouridine (BrU) and N‐acetyltyrosine JV‐ethylamide (1) are reported. Solutions were irradiated with monochromatic laser emission at 266, 308 and 325 nm. Quantum yield measurements as a function of excitation wavelength suggest that both products result from excitation of the tyrosine derivative followed by electron transfer to BrU, possibly with intermediacy of the hydrated electron. The BrU radical anion ejects bromide to form the uri‐dinyl radical, which then abstracts a hydrogen atom from 1 or adds to the aromatic ring of 1. Formation of adduct 2 is a model for photocrosslinking of nucleic acids bearing the bromouracil chromophore to adjacent tyrosine residues of proteins in nucleoprotein complexes. The value of 325 nm excitation in photocrosslinking, where the tyrosine chromophore is more competitive for photons, was demonstrated with an RNA bound to the MS2 bacteriophage coat protein; more than a 60% increase in the yield of photocrosslinking relative to that obtained with 308 nm excitation was achieved.

Formation of dityrosine cross-links in proteins by oxidation of tyrosine residues

1. 1.|Enzymic oxidation of proteins with peroxidase and hydrogenperoxide at a basic pH value leads to an oxidative phenolic coupling of adjacent tyrosine residues forming cross-linked proteins. 2. 2.|Dityrosine (3,3′-bityrosine) was identified as the cross-link in oxidised proteins by thin-layer chromatography, amino acid analysis and fluorescence measurements. 3. 3.|Gel filtration experiments with oxidised insulin showed that the cross-linkage is predominantly intermolecular. 4. 4.|In tetranitromethane treated proteins, dityrosine could be identified after hydrolysis.