Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as  IgE-mediated allergy is a hypersensitivity disease suffering from more than 25% of the population in industrialized countries. Currently, specific immunotherapy is the only allergen-specific approach for its treatment and for preventing its progression to severe manifestations , 2. The The use of hypoallergenic mutants would be a good strategy to avoid the nondesired side effects of immunotherapy. Hypoallergenic mutants have been developed for several pollens and foods allergens –18, and Peach allergy is the most prevalent plant food allergy in the Mediterranean area, and its major allergen, the LTP, Pru p 3, is the main plant food allergen in this region , 25. ThePar j 1 was developed with the T cell epitope and IgG responses not affected -thymidine (0.5 μCi/well) was added, and the incorporated radioactivity was measured by scintillation counting. Phytohemagglutinin-L from Phaseolus vulgaris  was used as a positive control. The stimulation index (SI) was calculated as the ratio between counts of antigen-stimulated cultures and counts without any activator. A SI ≥ 2 was considered a positive value.PBMCs were freshly isolated from whole blood subjected to density gradient centrifugation on Lymphoprep , as previously described . Culture4 cells per well) and their mitomycin-treated PBMCs (105 cells per well) were seeded in 96 wells (see above). They were incubated with 0.5 and 5 μg/mL of anti-HLA-DP/DQ/DR  or isotype control antibodies  for 2 h at 37°C before addition of the corresponding allergen or its mutants and testing of the proliferative response.To demonstrate the specificity of the proliferation in TCLs experiments, human leukocyte antigen (HLA) class II inhibition assays were performed using specific antibodies, as previously described . SpecifiμL of TCL supernatants, by means of matched antibody pairs according to the manufacturer's instructions . Cultures containing TCLs with mitomycin-treated PBMCs alone served as negative controls. IFNγ/IL4 ratios over 10, between 2 and 10, and less than 2 were, respectively, considered as Th1, Th0, and Th2-responses.Cytokine levels were quantified using 50 γ, F: 5′-AAATCTGTGGGGACAAGTCG-3′, R: 5′-TGAGGGTATCTGCTCCTTGG-3′; EF-1, F: 5′-CTGAACCATCCAGGCCAAAT-3′, R: 5′-GCCGTGTGGCAATCCAAT-3′; [γτ mRNA expression was normalized with endogenous control EF-1, and the relative quantification was performed using the comparative threshold cycle method (2−ΔΔCt), as described by Livak and Schmittgen [mRNA from the TCLs was isolated according to the Quiagen-RNeasy protocol  and stored at −80°C. RT-PCR was performed as previously described , 43. cDNCAAT-3′; , 45. Reahmittgen . The chaP < 0.05 were considered to be significant.Statistical analysis was performed using SPSS 17.0 and Statgraphics Centurion XVI. Wild type and mutants were compared using the Wilcoxon paired samples test, in the Ig binding ELISA assays, proliferative responses of T cell lines, cytokine levels, and gene expression. Values of Pru p 3.01 and Pru p 3.02 could be detected as a single band by both Coomassie staining and polyclonal antibodies to the wild type protein . These data suggested that B epitopes in Pru p 3.03 were altered. The decrease of Pru p 3.03-IgE binding capacity was confirmed by inhibition ELISA, using a serum pool from peach-allergic patients, and Pru p 3 wild type as solid phase . Both forms, Pru p 3.01 and Pru p 3.02, almost completely inhibited the IgE binding, being a little lower in the case of Pru p 3.02  because higher values were obtained with the mutant , indicating that these mutants have not affected these epitopes (Pru p 3.03 was decreased (P = 0.0001), with values ranging from 0 to 97% of reduction, indicating that the IgG4 epitopes were affected, at least in part.On the other hand, several patients showed low levels of specific IgG4 against the native form of Pru p 3 . In the epitopes . Nonethein vitro was confirmed by ex vivo assays, because most patients did not recognize the Pru p 3 mutant forms .The reduction in the IgE binding observed nt forms . The mutP = 0.363, P = 0.958, and P = 0.494, for Pru p 3.01, Pru p 3.02, and Pru p 3.03, resp.). So, these results suggest that the mutations did not affect the T cell epitopes. This proliferation was prevented by incubation with anti-HLA antibody in a dose-dependent manner  with more production of IL4 than IFNγ  represents the most effective treatment available toward allergies. However, in food allergy, SIT has not been practiced, mainly for the high risk to develop anaphylactic side effects for patients . A few yBy contrast, the use of hypoallergenic molecules may prevent these side effects and reduce cost, allowing us the use of higher doses of proteins and longer treatments. These features reveal them as a promising strategy to develop , 32. HypThe LTPs syndrome is an allergy mediated by members of this family present in a wide range of plant foods and some pollen . This mePru p 3.01, with four changes in the central region; Pru p 3.02, with four mutated residues in the C-terminal coil; and Pru p 3.03, including all modified residues of the other two. The three mutants retained their ability to bind blocking antibodies and to stimulate T cell proliferation. One of the mutants, Pru p 3.01, had a similar behaviour as the wild type, meaning that the four modified residues were not enough to abolish the allergenic capacity of the wild type form in a significant way. The other two, Pru p 3.02 and Pru p 3.03, showed a significant reduction in their allergenicity capacity, since they had less ability to bind IgE and less recognition by patients with peach allergy, compared to the wild type protein. The reduction in cellular response by basophil activation test in these mutants suggested that their modifications were affecting the cross-linking on the surface of the effector cells.In this work, three Pru p 3 mutant forms have been produced by site-directed mutagenesis of the essential residues for IgE binding activity , 31: PruIn immunotherapy, several studies have indicated the importance to maintain the binding to IgG blocking antibodies. Those antagonize the recognition of wild type allergens by IgE antibodies captured on the surfaces of mast cells and basophils, which triggers the allergic reaction .Pru p 3.01, Pru p 3.02, and Pru p 3.03, their ability to bind IgG1 was retained. In fact, some patients showed higher IgG1 binding to Pru p 3.02 and Pru p 3.03 than to wild type, probably due to the fact that some modified residues produced better recognition to IgG1.In the case of the mutants produced in this study, Pru p 3.01 and Pru p 3.02 rendered no differences comparing to the wild type, Pru p 3.03 showed a reduced ability to bind it. This behaviour may be explained in terms of overlapping between IgE and IgG4 epitopes, or because the loss of folding observed in this form could alter the binding to IgE and IgG4 but not to IgG1. In a recent paper [Pru p 3.03 mutant.In the case of IgG4, while nt paper , a reduc65–80 [Pru p 3.01, the mutations did not affect T epitopes, and the T cell proliferation was not affected, as predictable. By contrast, the modifications in Pru p 3.02 and Pru p 3.03 could have interfered. However, both retained their ability to stimulate the proliferation of specific T cell lines, suggesting that their modified residues did not affect T cell epitopes.Besides the loss of IgE binding and the maintaining of blocking IgG recognition, it is essential for a successful immunotherapy that the hypoallergenic mutants also retain their capacity to stimulate the immune system , preserv65–80 , 50, wasPru p 3.02 and Pru p 3.03, which could be promising tools to be used in immunotherapy for peach-allergic patients.Summarizing, in this work we have generated two hypoallergenic forms, Pru p 3.03 had the highest hypoallergenic capacity of the three mutants. However, this mutant showed a loss of its three-dimensional structure, which made it more unstable in solution. This could hinder future applications. But also, this loss can be the cause of the drop in its IgE and IgG4 binding capacity, though preserving the IgG1 epitopes. By contrast, Pru p 3.02 retained its ability to bind IgG antibodies, both IgG1 and IgG4, but exhibiting a considerable reduction of its allergenic activity, in both IgE binding capacity and patients' recognition. These characteristics make it highly recommendable for use in immunotherapy.Pru p 3.02 and Pru p 3.03, more detailed studies such as their use in animal models are necessary to test their effectiveness, the approach presented in this work may be a first step towards the implementation of a new strategy for immunotherapy.Although, for both


Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila.The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the  Drosophila, the pathway is important for many cell specification events, including patterning of the embryonic epidermis The Wnt/ß-catenin (Wnt/ß-cat) pathway is a major cell-cell signaling pathway found throughout metazoans TCF genes, while Drosophila has only one, TCF/Pangolin (TCF/Pan) A key intracellular messenger in Wnt/ß-cat signaling is ß-catenin, whose degradation is inhibited by Wnt stimulation All TCFs contain High Mobility Group (HMG) domains, which can bind DNA with sequence specificity Several TCFs increase their DNA binding specificity through a second DNA binding domain known as the C-clamp. This domain, located just C-terminal to the HMG domain, was originally discovered in “E-tail isoforms” of vertebrate TCF1 and TCF4 GLUT4myoD enhancer Drosophila, known as fly Glut4EF, which is required for proper wing position in adults In addition to the TCF1E and TCF4E isoforms, there are other C-clamp containing proteins in humans. The best characterized is known as SLC2A4 regulator (SLC2A4RG), GLUT4 enhancer factor (GLUT4EF) or Huntington’s disease binding protein 1 (HDBP1), which can bind to the promoters of Huntington’s disease gene In contrast to the HMG domain, where a structure of the domain bound to a high affinity site has been determined TCF/Pan gene containing a point mutation that specifically inhibited this inhibitory activity was compromised for activity in fly cell culture, but was able to rescue the embryonic patterning defect of TCF/Pan mutants. However, a DNA-binding mutant had no rescue activity in fly embryos. These data provide important biochemical information about the C-clamp of TCFs, and provide the first direct evidence for the importance of this domain in mediating Wnt/ß-cat target gene regulation in Drosophila embryos.In this report, we explore the physical properties and functional relevance of the C-clamp through a combination of biochemical and genetic assays. We find that the C-clamp of TCF/Pan is sufficient for binding to DNA containing a Helper site and that the C-clamp contains a zinc ion that is essential for this DNA-binding activity. Site-directed mutagenesis demonstrated that all four cysteines and a stretch of basic residues are essential for specific DNA binding, and the ability to activate W-CRMs reporters in fly cell culture. We also found that the C-clamp can bind to the HMG domain and inhibits its ability to bind to HMG site DNA. A E. coli was used to purify a His-tagged 45 amino acid fragment of TCF/Pan, containing the C-clamp. This protein can bind to a DNA probe containing a Helper site , only zinc restored high affinity binding to the HMG-Helper site probe  cells that had been depleted of endogenous TCF/Pan using RNA interference (RNAi) To systematically explore which regions of TCF/Pan’s C-clamp are required for activation of Wnt/ß-cat signaling, eight mutant constructs were generated. These include the four conserved cysteines, which were converted to alanines , Mutant 8 appeared to have a specific  defect in the inhibitory activity  and Mutant 5 was defective in both activities. Additional EMSA experiments confirmed these defects . Note thTo investigate the mechanism of the C-clamp inhibitory function, the ability of the HMG domain to interact with the C-clamp was tested. GST-tagged HMG domain (GST-HMG) and a His-tagged C-clamp fragment (His-C-clamp) were incubated and analyzed in a GST pulldown assay. GST-HMG binding to His-C-clamp was observed, with minimal binding by the GST negative control . In contTCF/Pan cDNAs  under the control of a Gal4-inducible promoter TCF/Pan] transgenes and a Daughterless-Gal4 driver  were crossed into genetic backgrounds containing two independent TCF/Pan mutant alleles (2TCF & 3TCF) believed to be nulls Da-Gal4 driver is active throughout the embryonic epidermis TCF/Pan transheterozygotes expressing UAS-TCF/Pan via the Da-Gal4 driver were generated via standard genetic crosses  had a weak defect in Wg/Wnt signaling in the Drosophila embryo TCF/Pan mutants have a strong segment polarity defect TCF/Pan cDNA , has been described previously The protein expression vector for The protein expression vectors for EMSA were generated by cloning the region encoding the HMG domain and the C-clamp from the pAc-TCF/Pan constructs into the XmaI and SacI restriction sites of the pET52b(+) vector (pET) (Merck Millipore). pET-HMG and pET-C-clamp were generated by cloning the respective coding regions  into the same sites. The pET vector encodes a C terminus 10xHis tag, which was used for protein purification.TCF/Pan ORF was cloned from the pAc-TCF/Pan constructs into the KpnI and XbaI restriction sites of the pUAST vector. This vector contains a C terminal V5 tag, which was used to detect the protein for Western blots.For transgenic flies, the Drosophila Schneider’s media with 10% FBS. For RNAi knockdown, cells were treated with 10 µg/1×106 cells of double-stranded RNA (dsRNA) targeting the 3′UTR of wild-type TCF/Pan. After 4 days, cells were diluted to 1×106 cells/mL and transfected with a plasmid mix containing pGL3-nkd-intE or pGL3-6xHMG, pAc-TCF/Pan (wild-type or mutant), S-188-cc-RLuc, a Renilla luciferase reporter Kc167 cells were cultured in E.coli, were used. Band signals in the gel were detected and quantified using the LI-COR Odyssey Infrared Imaging System. After background subtraction, the percentage bound was calculated as the signal in the shifted band/total signal in that lane. Signals for all mutants were then normalized to the wild-type protein.Gel shifts were carried out using the Lightshift Chemiluminescent EMSA kit (Pierce). IRDYE-700 tagged DNA probes and His-tagged recombinant TCF/Pan fragments, purified from Purified proteins in the EMSA binding buffer were incubated with 3.6 mM of 1, 10-orthophenanthroline (OPA) for 20 minutes at room temperature. The DNA probe was then added and the mixture was incubated for 20 minutes on ice, before being loaded into the gel. In the negative control, nuclease free water was used instead of OPA. For the rescue, OPA treated proteins were incubated with 100 µM of each of the salts for 10 minutes at room temperature before DNA was added.Samples of His-TCF and His-HMG proteins were sent for testing metal content using inductively coupled plasma mass spectrometry (ICP-MS), which was carried out by Dr. Ted Huston .TCF/Pan embryonic phenotype were set up as indicated in Transgenic fly lines were generated by BestGene Inc. using P-element mediated transformation. Crosses for rescue of the For the Western blots, flies were allowed to lay eggs for 4 hours and the embryos were incubated for an additional 6 hours, all at 25°C. Embryos were collected and dechorionated. They were then treated with hot SDS buffer and manually ground for 5 minutes. Samples were loaded into a gel. TCF-V5 was detected using mouse anti-V5 antibody . Tubulin was used as a loading control. The ECL kit (Pierce) was used to visualize the blots.TCF/Pan mutant alleles used were 2TCF and 3. TCF2TCF contains a base pair loss of ATT to AT leading to a frameshift at I106 and 3TCF contains a CAA to TAA mutation resulting in a stop codon at Q319 in the HMG domain 2. OPA treatment was carried out by incubating His-C-clamp in 0.363 µM OPA for 20 minutes at room temperature. GST or GST-HMG was then added and the mixture was incubated for 1 hour at 4°C with rotation prior to GST pulldown. In the negative controls for both treatments, nuclease free water was used instead of micrococcal nuclease or OPA.2 µg of His-C-clamp and 2 µg of either GST or GST-HMG were incubated for 1 hour at 4°C with rotation in 20 mM Tris-HCl (pH 7.62), 150 mM NaCl and 1% Triton X-100 binding buffer. The mixture was then incubated with Glutathione Sepharose beads  for an additional 2 hours at 4°C with rotation. After 4–5 washes with binding buffer, the sample was treated with hot SDS buffer and loaded into a gel. His-C-clamp was detected using mouse anti-His antibody . Micrococcal nuclease treatment was carried out as described previously


Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.in vitro.We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity  Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects 1% of people over age 60 and more than 5 million people worldwide. PD results primarily from the selective loss of dopaminergic neurons in the substantia nigra. Current available therapies only address the symptoms of PD, and the most effective treatment has been in use for more than 50 years. The identification of familial mutations associated with PD has presented novel targets for potential therapeutic development. Mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal familial and sporadic PD in vitro studies in vivo models in vitro and in vivoLRRK2 is a large multi-domain protein that is phylogenetically related to the Receptor Interacting Kinase branch of the tyrosine kinase-like family of the human kinome. Indeed the kinase domain does bear homology to MAP3K kinases. It contains several potential protein-protein interaction domains including N-terminal ankyrin repeats, leucine-rich repeats and a C-terminal WD40 domain. Surrounded by these domains is the catalytic core containing a GTP-binding Ras of complex protein (ROC) GTPase domain, a carboxy-terminal of Roc (COR) domain and a serine/threonine kinase domain LRRK2 is found to be phosphorylated at multiple sites throughout the protein and can be classified as autophosphorylation sites or sites of constitutive phosphorylation, which are detected on active LRRK2 expressed in cells. Multiple autophosphorylation sites on LRRK2 have been mapped in vitro LRRK2 kinase activity. These include a high-throughput screening (HTS) compatible Time-Resolved Förster Resonance Energy Transfer (TR-FRET) assay using either LRRKtide or Nictide as the substrate Several biochemical assay formats have been reported and utilized for measuring The first step for developing a LanthaScreen® TR-FRET cellular assay is to generate cells expressing substrate of interest fused to GFP which acts as the fluorescence acceptor from a terbium labeled modification-specific antibody Materials and Methods. U-2 OS cells were transduced with the indicated concentrations of BacMam LRRK2-GFP wild-type and mutants and plated in a 384-well assay plate. Cells were lysed directly in the assay plate with lysis buffer supplemented with Tb-labeled anti-LRRK2 pSer935 antibody. The presence of phosphorylated Ser935 allows the binding of the antibody to the expressed LRRK2-GFP bringing Tb in close proximity to GFP enabling a FRET to occur from Tb to GFP. The level of phosphorylation at Ser935 can then be quantitatively represented by the TR-FRET signal (the emission ratio of the acceptor GFP to the donor Tb). Shown in 50 values of 0.08 µM and 0.03 µM, respectively , a known PKA activator which increases cAMP levels.To further validate the TR-FRET cellular assay, LRRK2-IN-1 and seven other small molecule inhibitors previously reported to have inhibitory activities of LRRK2 kinase were profiled in three different cell backgrounds, U-2 OS, SH-SY5Y and HEK293T transduced with BacMam LRRK2-GFP wild-type or G2019S. The ICTo test the performance of this assay in a high-throughput screen, Tocriscreen™ Mini library of 1120 biologically active compounds which target ion channels, G-protein-coupled receptors (GPCRs), nuclear receptors and kinases were screened in the TR-FRET Ser935 phosphorylation cellular assay using BacMam LRRK2-GFP G2019S transduced SH-SY5Y cells. Each compound was tested twice at a single concentration of 20 µM in 0.1% DMSO using duplicate 384-well assay plates, to reproduce actual conditions that may be used in a larger high-throughput screen. LRRK2-IN-1 was included as a positive control on each plate and used at 10 µM. Percent inhibition of each compound is calculated and plotted in a histogram shown in 50 values for each compound in the follow-up study are summarized in columns 4 and 5 of 50 values. Several compounds such as JTC-801 and the two NSC compounds displayed steep dose-response curves  were toxic at lower concentrations than those needed to in the TR-FRET assay. The two NSC compounds also have strong red color which could potentially affect the TR-FRET readout causing steep dose-response curves.Out of the 1120 compounds, 16 compounds reduced the level of Ser935 phosphorylation by greater than 50%. Among the 16 compounds, SP600125, indirubin-3′-monoxime and ROCK inhibitor Y-27632 with previously reported LRRK2 inhibitory activity showed 88%, 67% and 63% inhibition, respectively in this screen . Both mae curves . To examin vitro activity against LRRK2 in order to make correlations between cellular and in vitro activities. We observed the previously unreported inhibitors of LRRK2, GW441756, PD407824, IKK16, exhibited in vitro IC50s of 320 nM, 320 nM and 50 nM, respectively , SB218078, BAY11-7805 and BAY11-7821 moderately reduced phosphorylation of all four residues. The observation that NFκB pathway inhibitors modulated LRRK2 kinase activity correlated with unpublished data indicating IKKepsilon and TBK1 phosphorylate LRRK2 in vitro (S. Riddle and RJ. Nichols). We therefore employed the kinase inhibitor BX-795, which was first identified as a PDK1 inhibitor, but was subsequently shown to also potently inhibit IKKepsilon and TBK1 To further confirm the TR-FRET cellular assay results, we also evaluated the effects of these compounds on the cellular phosphorylation of Ser910, Ser935, Ser955 and Ser973 by immunoprecipitation (IP) followed by Western analysis. Here, commonly used HEK293 cells inducibly expressing N-terminal GFP tagged LRRK2 G2019S 50 value for inhibitors directly acting on LRRK2. We observed that the LRRK2 A2016T/G2019S mutant rescued phosphorylation in the presence of LRRK2-IN-1 treatment as well as with GW44766, IKK16 and BX-795 even at the high concentrations utilized in these assays, revealing that the later three compounds do directly inhibit LRRK2 in cells . We provide multiple lines of evidence suggesting that LRRK2-GFP functions and behaves similarly to the previously reported N-terminally tagged LRRK2 (GFP-LRRK2) stably expressed in HEK293 cells. First, wild-type and G2019S LRRK2-GFP displayed a diffuse cytoplasmic localization Second, LRRK2-GFP wild-type, G2019S, R1441C and D1994A showed similar Ser935 phosphorylation pattern determined by Western blot and the TR-FRET assay as reported for GFP-LRRK2 One of the common methods for detecting LRRK2 phosphorylation is immunoblotting, typically after immunoprecipation. The complexities of this methodology make it highly impractical for the processing and analysis of large sample numbers typically associated with screening experiments. The TR-FRET cellular assay reported here is in a fully homogenous format without washing, lysate transfer, or separation procedures. Cells transduced with BacMam LRRK2-GFP can be plated onto a 384-well assay plate, treated as desired and then incubated with 6 X lysis buffer containing Tb-labeled detection antibody added directly to the wells. To further simplify the procedure, we demonstrated that cells can be transduced, cryopreserved, later thawed and used directly to run the assay without the need for culturing . This inin vitro kinase assay with an IC50 of 320 nM. Interestingly, we did not observe in vivo dephosphorylation of Ser935 with the PKC inhibitors GF109203X and Ro31-8220, which were present in the Tocriscreen library and identified previously to inhibit LRRK2 autophosphorylation in vitroWe profiled 1120 compounds of the Tocriscreen™ Mini library to gain insight into the biology of Ser935 phosphorylation and demonstrate the utility of the TR-FRET cellular assay in screening. Using a maximal inhibition threshold of 50%, we observed the previously identified inhibitors of LRRK2 SP600125, indirubin-3′-monoxime and ROCK inhibitor Y-27632 induced dephosphorylation of Ser935, which increased confidence in our assays. In addition to compounds with known LRRK2 inhibitory activity, we identified several novel LRRK2 kinase inhibitors from this screen. GW441756 is a known TrkA inhibitor in vitro IC50 20 times lower than the cellular IC50, indicating that the cellular effect may be due to LRRK2 inhibition. Ro106-9920 inhibits IκB degradation and LPS induced cytokine  production in vivo kinase regulating LRRK2 phosphorylation. The inhibitor BX-795, which inhibits PDK1 as well as the TBK1 and IKKε kinases, decreased the phosphorylation of the cellular phosphorylation sites in HEK293 T-REx™ cells expressing GFP-LRRK2 G2019S, and the G2019S mutation showed decreased sensitivity to BX795 indicating it is indeed a direct inhibitor of LRRK2. BX795 treatment cannot then be utilized to rule in or out IKKε or TBK1 in their ability to phosphorylate LRRK2 in vivo. Interestingly, inhibition of LRRK2 Ser935 phosphorylation occurs in the absence of receptor stimulation and may represent perturbing a basal status of modification. Together, these data indicate that the pathways that induce NFκB activation play a role in LRRK2 phosphorylation, but this regulation is likely complex.Interestingly, we observed that 5 out of the top 16 compounds Bay 11-7085, Bay 11-7821, IKK16, Ro106-9920 and TPCA-1 intersect with the NFκB pathway. TPCA-1 and IKK16 are IKK inhibitors, however LRRK2 is inhibited by IKK16 at an in vitro and Ser935 phosphorylation in cells indicating that indeed this is potentially another direct LRRK2 kinase inhibitor. Since the LRRK2 inhibitor resistant mutant did not show decreased sensitivity to this compound, it may well be that CHK1 could act on the cellular phosphorylation sites in a LRRK2 kinase activity independent manner. In the future, it would be interesting to investigate LRRK2 activity and phosphorylation during different stages of the cell cycle, especially since there are multiple lines of evidence linking LRRK2 to cancer Inhibitors of the Cdc25 phosphatase NSC-95397 and NSC-663284 or kinase inhibitors aminopurvalanol A (CDK), SB 218078 and PD407824 (CHK1) can block cell cycle progression We also observed compounds other than kinase inhibitors that affect LRRK2 phosphorylation, but do not directly target LRRK2 enzyme itself, namely GPCR ligands such as opioid-receptor like-1 (OPRL-1) antagonist JTC801, the dopamine receptor D1 antagonist BNTX maleate, dopamine D2-like receptor ligand 3′-Fluorobenzylspiperone maleate, and an agonist of the adenosine A2 receptor CV1808. Though results are complicated for some of these compounds due to their cytotoxic effects, these compounds could indicate potential signaling pathways that also alter LRRK2 function via effects on LRRK2 phosphorylation. These represent avenues for further research.In summary, we have developed a robust high-throughput detection system for evaluating the phosphorylation of LRRK2 at Ser935 that correlates well with alternate systems. A small library was used to evaluate this screening system and hits from that screen were validated in established systems showing the reliability of the methodology. By exploiting the biology of Ser935 phosphorylation, we gained novel insights into potential pathways that intersect with LRRK2, as well as identified other possible direct LRRK2 inhibitors.ReagentsAll cell culture media and supplements, BacMam LRRK2-GFP reagents, PrestoBlue™ cell viability assay reagent, Western blotting reagents and human neural stem cells were obtained from Life Technologies . All other cell lines were purchased from ATCC . The cell lines were maintained under conditions recommended by the manufacturer. Anti-LRRK2 antibody used for Western blotting was purchased from Cell Signaling Technologies . Protease and phosphatase inhibitor cocktails and H-89 were purchased from Sigma . LRRK2-IN-1 was provided by the Michael J. Fox Foundation for Parkinson’s Research. Sunitinib was from LC Laboratories , JAK3 Inhibitor VI and Indirubin-3′-monoxime were from EMD Biosciences . H-1152, GW5074 and the Tocriscreen™ Mini library and the follow-up compounds were from Tocris . Rabbit polyclonal anti-phospho Ser910, Ser935, Ser955 and Ser973 antibodies were produced by Yenzym Antibodies  as described in U-2 OS cells are maintained in growth medium , 10 mM HEPES, 0.1 mM NEAA, 1 mM Sodium Pyruvate, and 100 U/mL Penicillin/100 µg/mL Streptomycin). HEK293T cells are maintained in DMEM with 10% dFBS, 10 mM HEPES, 0.1 mM NEAA and 100 U/mL Penicillin/100 µg/mL Streptomycin. SH-SY5Y cells are maintained in DMEM/F-12 medium supplemented with 10% dFBS and 100 U/mL Penicillin/100 µg/mL Streptomycin. HEK293 T-REx™ GFP-LRRK2 G2019S cells described in 50 values. Z′-factor values were calculated as: Z′-factor  = 1 - [(3 × stdevno virus +3 × stdevmaxvirus)/(avgmaxvirus – avgno-virus)]. The IC50 values reported in Transduced cells in the 384-well assay plate were incubated with indicated inhibitors  for 60 to 90 minutes. During this time, 6 X lysis buffer (Life Technologies) was supplemented with 30 nM Tb-labeled anti-LRRK2 pSer935 antibody and both protease and phosphatase inhibitor cocktails (Sigma). The complete 6 X lysis buffer was added to each well (5 µL/well) and the plates were incubated at room temperature in the dark for 2 hours. The plate was then read on an EnVision® multilabel plate reader  with excitation at 340 nm and emission 520 nm and 495 nm. Emission ratios of 520 nm/495 nm were calculated and plotted against the concentration of compound. A sigmoidal dose-response equation with varying slope was used to fit the data and generate IC50 value calculations were performed using XLFit4 software (IDBS) and a nonlinear regression equation for variable-slope sigmoidal dose response (model number 205). Z′-factor value was calculated as: Z′-factor  = 1 - [(3 × stdevLRRK2-IN-1+3 × stdevDMSO)/(avgDMSO – avgLRRK2-IN-1)]. The IC50 values reported in SH-SY5Y cells were transduced with 25% (v/v) BacMam LRRK2-GFP G2019S and plated  onto eight 384-well assay plates as described above. 40 nL of 10 mM Tocris library compounds in DMSO were dispensed into each well of the assay plate by the ECHO system. Equal volumes of DMSO were dispensed into the control wells. Cells were incubated with compounds for 90 to 120 min prior to cell lysis and detection as described above. The percent inhibition is calculated as: [(average emission ratios of DMSO control wells – average emission ratios of compound wells)/(average emission ratios of DMSO control wells – average emission ratios of LRRK2-IN-1 treated wells)] * 100. For the dose-response follow-up studies, fresh compound DMSO stock solutions were prepared from powder. Curve fitting and ICU-2 OS cells were transduced with BacMam LRRK2-GFP as described above. The cells were then plated on chambered coverslips  at 50% confluency and incubated for 48 hours. The growth medium was replaced with phenol-red free DMEM®  before live imaging. Cells were imaged under the same conditions for each mutant using an inverted microscope (Nikon Eclipse TE2000-S) equipped with a PlanApo 60X objective and a charge-coupled device camera (Diagnostic Instruments SPOT RT 2.3.0) using SPOT Advanced software.7 cells) were left untreated or treated with 3 µM LRRK2-IN-I for 90 min. For lysate preparation, the cells were washed once with ice-cold PBS and lysed by addition of ice-cold lysis buffer . The lysate was cleared of debris by centrifugation. Equal amount of total protein was loaded onto each lane for SDS-PAGE (3–8% Tris Acetate). The proteins were transferred onto nitrocellulose membrane using the iBLOT® dry blotting system (Life Technologies) according to manufacturer’s protocol. The membranes were blocked and incubated with a primary antibody against LRRK2 , followed by incubation with alkaline phosphatase-conjugated secondary antibody . The blot was developed by using the chromogenic BCIP/NBT substrate . Immunoprecipitation analysis: HEK293 cells were induced to express GFP-LRRK2 G2019S by inclusion of doxycycline in the culture media at 1 µg/mL. Two days post induction, cells were treated for 90 minutes in the presence of 20 µM of compound or DMSO vehicle control. Cells were rapidly lysed in ice cold lysis buffer 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (w/v) 1 mM sodium orthovanadate, 10 mM sodium beta-glycerolphosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM benzamidine and 2 mM phenylmethanesulphonylfluoride (PMSF) and one tablet each complete protease inhibitor and PhosSTOP phosphatase inhibitor (Roche) and was supplemented with 1% (v/v) Triton X-100. GFP-LRRK2 was immunoprecipitated with GFP nanotrap beads (ChromoTek). Immunecomplexes were washed twice with lysis buffer supplemented with 300 mM NaCl and once with Buffer A  2-mercaptoethanol, and 0.27 M sucrose). Equal amounts of immunoprecipitated LRRK2 were fractionated on 4–12% Novex® NuPAGE® Bis-Tris gels and proteins were transferred to nitrocellulose. Blots were probed with phosphoserine 910, 935, 955 and 973 antibodies as described previously Immunoblot analysis: BacMam LRRK2-GFP transduced U-2 OS or SH-SY5Y cells  and with 580 ng/mL LRRK2 G2019S (Life Technologies). After incubation at room temperature for 1 hour, the kinase reactions were stopped by addition of 10 µL of 20 mM EDTA and 4 nM Tb-anti-pERM (pLRRKtide) antibody in TR-FRET dilution buffer (Life Technologies). After a 30 minute incubation at room temperature, TR-FRET measurements were obtained on a BMG PHERAstar plate reader using LanthaScreen® filters and settings. A 16 point, 2-fold dilution series of each compound was tested in triplicate at each concentration and assays were also performed in triplicate.The Biochemical LRRK2 TR-FRET activity assay was performed according to the manufacture recommended assay protocol (Life Technologies). Briefly, kinase reactions were performed in 10 µL volumes in black, low-volume 384-well plates (Corning #3676), with 400 nM Fluorescein-ERM (LRRKtide) substrate and 1% DMSO , in Kinase Buffer S (Life Technologies) supplemented with 2 mM DTT. Reactions performed with ATP equal to KFigure S1C-GFP tagged LRRK2 phosphorylation at Ser935, Ser955 and Ser973. U-2 OS cells were transduced with BacMam LRRK2-GFP G2019S and left untreated (−) or treated with LRRK2-IN-1 at 10 µM (+). After cell lysis, the phosphorylation of LRRK2 was analyzed by immunoblotting with indicated antibodies.(TIF)Click here for additional data file.Figure S2Dose response curves of hit compounds in the TR-FRET cellular assay for LRRK2 Ser935 phosphorylation. 25% BacMam LRRK2-GFP G2019S transduced SH-SY5Y cells were incubated with indicated concentrations of indicated compounds for 90 min prior to the TR-FRET detection with Tb-anti-LRRK2 pSer935 antibody. The % inhibition is calculated as described in Materials and Methods and plotted against the concentrations of the compounds.(TIF)Click here for additional data file.Figure S3Dose response curve of LRRK2-IN-1 in the TR-FRET cellular assay using cryopreserved pretransduced SH-SY5Y cells. SH-SY5Y cells were transduced with 25% BacMam LRRK2-GFP G2019S and then cryopreserved. Cryopreserved cells were thawed, resuspended in assay medium and plated onto 384-well assay plate for 20 hours. Cells were incubated with indicated concentrations of LRRK2-IN-1 for 90 min prior to the TR-FRET detection with Tb-anti-LRRK2 pSer935 antibody. The raw emission ratios are plotted against the concentration of LRRK2. The IC50 using cryopreserved cells is about 40 nM, similar to the one generated using freshly transduced cells.(TIF)Click here for additional data file.


Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src.c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6  Three SFKs, Src, Yes and Fyn, are ubiquitously expressed, whereas the others, including Lyn, Lck, Hck and Blk, are mainly expressed in non-adherent cells of the haematopoietic system. SFKs play key roles in cell proliferation, survival, adhesion, morphology and motility The Src family proteins are characterized by four highly conserved Src homology (SH) domains termed SH1 to SH4 Drosophila to human in sequence comparison, but not found in Caenorhabditis elegansThe human trihydrophobin 1 (TH1) gene is the homolog of Drosophila TH1, which was originally identified during positional cloning of mei-41 Recently, we reported that TH1 served as a negative regulator of A-Raf and PAK1, inhibiting MEK/ERK signaling pathway in vivo and in vitro, which led to lowered association with A-Raf and PAK. Dephosphorylated form of TH1 (TH1-Y6F) inhibited c-Src mediated MAPK/ERK pathway activation, whereas phosphorylated form of TH1 did not inhibit. Moreover, c-Src driven cell migration was attenuated by TH1-Y6F over-expression. Induced tyrosine phosphorylation of TH1 was found by EGF and estrogen treatments. In conclusion, we proposed a mechanism for comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src.In this study, we identified TH1 as an interaction partner of c-Src by immunoprecipitation and GST-pull down assay. Further investigation revealed that c-Src directly phosphorylated TH1 at Y6 in vitro and in vivo.In previous study, we demonstrated that TH1 served as a regulatory protein in MAPK/ERK pathway It has been demonstrated that c-Src stays in a close, inactive state through auto-inhibition without stimuli, switches to an open state upon stimulation, including serum and EGF bona fide substrate of c-Src, we performed in cell phosphorylation assay. COS-7 cells were transfected with Myc-TH1 and HA-c-Src or pcDNA3 empty vector, precipitated by anti-Myc monoclonal antibody, immunoblotted with an anti-phosphotyrosine antibody (4G10). As shown in in vitro  to predict possible phosphorylation sites within TH1 and got 4 potential phosphorylation sites: Y5, Y6, Y175 and Y431. Based on this, TH1 point mutants including single mutants  and a double mutant (Y6/431F), generated by replacing Tyrosine with Phenylalanine, were transfected into COS-7 cells along with c-Src (Y527F). After precipitation, phosphorylation of TH1 mutants was measured by the anti-phosphotyrosine antibody (4G10). in vitro. Taken together, these data indicate that c-Src phosphorylates TH1 at Tyr-6.To determine the phosphorylation site of TH1, we constructed 3 Myc-tagged fragments of TH1: FL, full length (residue 1–581); F1, N-terminal domain (residue 1–300) and F2, C-terminal domain (residue 301–581). Next, we transfected HA-c-Src(Y527F) into COS-7 cells with Myc-tagged TH1 fragments, precipitated with anti-Myc antibody and detected phosphorylation of TH1 with anti-phosphotyrosine antibody. As shown in To investigate biological impact of TH1 phosphorylation, we constructed two Y6 mutant derivatives, Y6E (replacing Tyrosine with Glutamic acid) to mimic constitutive phosphorylation and Y6F to abolish phosphorylation. Our previous study demonstrated that TH1 played an inhibitory role in MAPK/ERK signaling It has been demonstrated that constitutive active c-Src triggers sustained activation of ERK signaling Our previous study showed that TH1 interacted with A-Raf and PAK1. Through interaction, TH1 inhibited their kinase activities and attenuated MAPK/ERK signaling We next determined whether Tyr-6 phosphorylation of TH1 would impact its stability. As shown in To address whether c-Src (Y527F) driven TH1 lowering is mediated through decreased TH1 stability, we treated HeLa cells with cycloheximide (CHX), an inhibitor of protein synthesis, investigated the time course of TH1 degradation. Ectopic expression of TH1 was reported to affect cell adhesion dynamics, which plays a critical role in cell migration guidance To investigate the regulation of TH1 phosphorylation, we transfected GFP-tagged TH1 expression vector into HEK293T cells, serum starved for 18 h, and then stimulated with serum or serum plus EGF for 15 min. After stimulation, tyrosine phosphorylation of TH1 was measured. Interestingly, EGF plus serum induced tyrosine phosphorylation of TH1 significantly, whereas serum alone was not sufficient to induce tyrosine phosphorylation of TH1 . MeanwhiThe c-Src/Ras/ERK signaling pathway plays key role in the regulation of cell migration, which is orchestrated at multi-levels Our previous studies have demonstrated that TH1 serves as an interaction partner and a negative regulator of A-Raf and PAK1, inhibiting MAPK/ERK signaling transduction In addition to MAPK/ERK pathway, c-Src also regulates AKT and JNK pathways. c-Src activates PI3-K and Akt in response to GDNF Our previous results indicated that c-Src enhanced cell migration through phosphorylation of TH1. Tyr-6 phosphorylation of TH1 led to decreased inhibition on cell migration, while TH1-Y6F, the dephosphorylated form of TH1, shows increased inhibition on c-Src mediated cell migration. The cell migration is an integrated process that requires the coordinated regulation of various molecules, including kinases, phosphatases, scaffold proteins and cell skeleton proteins et al. demonstrated that phosphorylation of p27 by c-Src impaired its Cdk2 inhibitory action and reduced its steady-state binding to Cyclin E-Cdk2 to facilitate Cyclin E-Cdk2-dependent p27 proteolysis, serving as an alternative way for c-Src promoting cell cycle progression Previous study by Chu et al., unpublished data), suggesting possible role of c-Src in transcription elongation regulation.In addition to its role in negative regulation of MAPK signaling, TH1 is an integral subunit of the human negative elongation factor (NELF) complex In summary, the present study demonstrates that c-Src interacts with and phosphorylates trihydrophobin 1. The Tyr-6 phosphorylation of TH1 decreases its inhibition on MAPK/ERK signaling. Moreover, Tyr-6 phosphorylation of TH1 enhances c-Src mediated cell migration. Thus, phosphorylation of TH1 by c-Src may serve as an alternative way for c-Src promoting MAPK/ERK signaling and cell migration.Leupeptin, aprotinin, phenylmethylsulfonylfluoride, PP2, 17β-estradiol were purchased from Sigma-Aldrich Inc . Antibodies against ERK1/2, p-ERK 1/2 (Thr-202/Tyr-204), PAK1, GST and HA were purchased from Santa Cruz Biotechnology . Antibodies against c-Src and phospho-Src (Tyr-416) were purchased from Cell Signaling Technology . Anti-Phosphotyrosine (4G10) antibody was purchased from Millipore . Antibodies against paxillin were purchased from Abcam. Antibodies against Myc and pcDNA3.1 vector were purchased from Invitrogen . Protein G-agarose and anti-GFP antibody were products of Roche Applied Science . TH1 antiserum was described in previous studies 2. MCF-7 breast cancer cells were cultured in 10% FBS–DMEM and supplemented with 0.01 mg/ml insulin, and A549 lung cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS. Cell transfection was carried out using Lipofectamine 2000  according to manufacture's instructions.HEK293T, HeLa, COS-7, MCF-7, and A549 cell lines were obtained from the Institute of Cell Biology Academic Sinica . HEK293T, HeLa, and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in 5% CO5′- TCAGAACCAAGGTTCTTTCAGCTGC-3′ and Reverse: 5′- TCTGCAGCTGAAAGAACCTTGGTTC-3′, Y175F Forward: 5′-ATTTCTGACGCAGGGTTCCAGGGGG-3′ and Reverse: 5′- CTGGTGATCTCCCCCTGGAACCCTG-3′, Y5F Forward: 5′- ATGGACGAGGACTTCTACGGGAGCGCGG-3′ and Reverse: 5′- GAGGATCCGTTCACCATGATGAAG -3′,Y6F Forward: 5′-ATGGACGAGGACTACTTCGGGAGCGCGG-3′ and Reverse: 5′- GAGGATCCGTTCACCATGATGAAG -3′,Y5/6F Forward: 5′- ATGGACGAGGACTTCTTCGGGAGCGCGG-3′ and Reverse: 5′- GAGGATCCGTTCACCATGATGAAG -3′, The double mutant TH1 Y6/431F was generated by sequentially using the single mutant TH1 Y431F as Template. All the constructs were confirmed by sequencing.Myc-tagged full length TH1 in pcDNA3.1 vector and pEGFP-N3 vector containing a GFP tag were described in previous studies 2, 60 mM beta-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml phenylmethylsulfonyl fluoride) for 1.5 h at 4°C. The whole cell lysates were incubated first with indicated antibody at 4°C for 1 h and then with protein G-agarose beads at 4°C overnight. The bound proteins were eluted by boiling in SDS sample buffer and detected by western blot assay.Immunoprecipitation assay was performed as previously described Western blot experiments were performed as previously described Cells were processed for immunofluorescence assay as described previously Escherichia coli containing the corresponding pGEX-4T-1 constructs, purified by Glutathione-Sepharose 4B beads according to the manufacture's instruction . As for GST-pull down assay, purified GST-tagged proteins (5 µg) were incubated with HeLa cell lysates, precipitated by glutathione-Sepharose 4B beads. The unbound proteins were removed by washing the beads three times with IP lysis buffer, whereas retained proteins were resolved by SDS-PAGE and analyzed by Western blot.The recombinant GST-tagged wild type TH1 and mutants were expressed by isopropyl-thiogalactoside (IPTG) induction of Thirty-six hours after transfection, cells were trypsinized and washed three times with ice-cold PBS. Cell pellets were suspended in RIPA buffer  with additional 1% SDS and boiled for 10 min to disrupt protein–protein interaction. The lysates were further diluted with ten volumes of RIPA buffer, sonicated on ice, clarified by centrifugation, pre-cleared with Protein-G agarose for 45 min at 4°C and immunoprecipitated with indicated antibody. The immunoprecipitated proteins together with whole cell lysates (40 µg) were analyzed by western blots using anti-Phosphotyrosine (4G10) antibody.2, 100 µM ATP) for 30 minutes at 30°C. The reaction was stopped by adding SDS sample buffer and boiling for 5 min. Samples were subjected to SDS-PAGE on 8% gels and transferred onto PVDF membranes for western blot using anti-Phosphotyrosine (4G10) antibody.Purified GST, GST-TH1 or GST-TH1 mutant (5 µg) was incubated with recombinant human c-Src  in 30 µl kinase buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 2.5 mM DTT, 0.01% Triton X-100, 10 mM MnCl6 cells in serum-free medium were added to the upper chamber and 500 µl of the appropriate medium with 10% FBS was added to the lower chamber. The Transwell was incubated for 18 h at 37°C. Non-migratory cells on the upper membrane surface were removed with a cotton swab, and the migratory cells on the undersurface of the membrane were fixed and stained with 0.1% crystal violet for 20 min at room temperature. Photographs of 6 random regions were taken, and the number of cells was counted to calculate the average number of migrated cells.Transwell assay was performed as described previously


Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions. Hepadnaviruses, or hepatitis B viruses, are hepatotropic DNA viruses, consisting of an enveloped icosahedral capsid containing an approximately 3 kb DNA genome in a partially double-stranded, relaxed circular form (RC DNA). The virus carries its own polymerase enzyme, a reverse transcriptase, that converts the packaged pregenomic RNA (pgRNA) to RC DNA through reverse transcription inside the capsid The CTD is highly basic, rich in arginines, but also contains multiple sites of serine/threonine (S/T) phosphorylation For both HBV and DHBV, the core protein has been detected in both the cytoplasm and the nucleus It is still unclear how the state of phosphorylation regulates core protein function in RNA packaging, DNA synthesis, nuclear import, and potentially additional steps in the viral life cycle. One possibility is that the core protein exerts its multiple roles by interacting dynamically with distinct viral or host factors at different stages of viral replication, in a CTD phosphorylation state-dependent fashion. We have now indeed identified cellular proteins that interacted preferentially with the unphosphorylated CTD and could potentially modulate viral replication.We previously reported that during DHBV reverse transcription, phosphorylation of the DHBc CTD is required for first strand DNA synthesis while its dephosphorylation is required for second strand maturation 35S]cysteine/methionine and [32P]orthophosphate metabolic labeling showed that the proteins were expressed in both cell lines and that they were indeed phosphorylated as expected , with WT sequences or phosphorylation sites substituted with A or D Fig. 1E.To test for host factor interaction, we transfected HEK293T cells with the GST-DCC fusion protein expression constructs and performed GST pulldown experiments. We observed no detectable co-purifying bands in any of the fusion proteins containing only the phosphodomain, either as WT or phosphomutants  Fig. 1 w5B, lane 5). Barely detectable B23 interaction could be seen in GST co-purifications performed with 196 WT and 196-DDDDDD . The use of NP-40 or Triton X-100 in the lysis buffer did not make any difference in these interactions.To confirm the interaction between GST-DCC fusion proteins and B23 or I2PP2A, we performed coimmunoprecipitation experiments using 3X-Flag-tagged B23 or Flag- and HA- double-tagged I2PP2A. Additionally, since the 229 constructs, in contrast to the 196 constructs, did not show any host factor interaction, we constructed another fusion protein, containing only the linker region from 196–228 Fig. 1D,, lane 14 vs. 15). I2PP2A interaction could also be detected with 196-SSAAAA  translation system and asked if B23 or I2PP2A purified from HEK293T cells could coimmunoprecipitate the in vitro-translated proteins. Attempts to detect interactions between full-length core protein expressed in HEK293T cells and B23 or I2PP2A were unsuccessful, likely due to the relatively low expression levels (compared to the fusion proteins) and the rapid assembly of full-length core proteins to capsids in cells. The in vitro-translated full-length DHBc containing the AAAAAA substitutions indeed interacted with both B23 and I2PP2A strongly , expressed in RRL, with either B23 or I2PP2A . Interaction was also seen between the HCC141-AAA mutant and I2PP2A (middle panel). Also similar to DHBc observations, interaction between HCC141-EEE, representing the phosphorylated CTD, and B23 was much weaker (top panel) and HCC141-EEE and I2PP2A interaction was undetectable (middle panel). No interaction was detectable between the WT HBc CTD and either B23 or I2PP2A in these experiments  and representing the hypo- or non-phosphorylated CTD showed strong interaction with B23  and I2PP2A . These interactions were identified by using GST-fusion proteins containing the isolated CTDs of DHBc and HBc and were confirmed for the full-length DHBc. Although the DHBc binding site for both host factors was localized to the linker region (196–228) upstream of the CTD phosphodomain, for both the isolated CTD and the full-length core protein, host factor interactions were nevertheless strongly modulated by the phosphorylation state of the downstream CTD phosphodomain.panel I), and CTD phosphorylation, by partially neutralizing the overall positive charge of the CTD, could weaken the host interactions (panel II). It is also possible that the phosphorylated CTD could fold back to interact with the positively charged linker and thus preclude the linker-host interactions, if negatively charged patches are formed in the phosphodomain as a result of phosphorylation (panel III). HBc CTD-B23 and -I2PP2A interactions were similarly phosphorylation state-dependent. The HBc linker region is much shorter (9 amino acids) (panel I). Introduction of negative charges next to the basic clusters by phosphorylation of the neighboring serine residues would decrease their positive charges and weaken the interactions with B23 and I2PP2A (panel II). However, as discussed below, electrostatic interaction alone is unlikely to mediate the observed CTD-B23 or I2PP2A interaction.There have been a few reports on cellular factors that interact with HBc, including a 46-kD serine kinase o acids) Fig. 8 aE. coli did not exhibit any detectable interactions with eukaryotic proteins. The fusion protein with WT sequences was not phosphorylated in bacteria, so it might have been predicted that the WT fusion protein made in E. coli would behave similarly to 196-AAAAAA. The lack of any interaction by the bacterially-expressed fusion proteins, regardless of the phosphodomain sequence, was possibly due to their misfolding, which may have masked the host protein binding sites when they were expressed in E. coli. This result further suggests that electrostatic interaction alone is unlikely to be sufficient to mediate the CTD-B23 or I2PP2A interaction. Similarly, B23 and I2PP2A have been reported to interact with the basic capsid proteins from the West Nile Virus GST-DCC fusion proteins that were purified from The linker region where B23 and I2PP2A bind is thought to be responsible for the non-specific RNA/DNA binding activity of DHBc, and together with the viral reverse transcriptase, is essential for packaging specifically the viral pgRNA The DHBc linker region also harbors a nuclear localization signal (NLS), a cluster of basic residues Fig. 1D,Efforts to directly determine the role of B23 and I2PP2A in core functions and in viral replication in vivo have so far been complicated by the multifunctionality of B23 and I2PP2A, which affect multiple cellular functions including transcription, translation, chromatin remodeling, and cell signaling - expresses a core-defective DHBV genome and pcDNA-Dcore expresses wild type (WT) DHBc pCMV-DHBV/CHBc CTD (HCC) coding sequences from 141 to 183 (WT and phosphorylation site mutants S-to-A or S-to-glutamic acid (E) at the three major SP sites: S155, S162, and S170) were generated by PCR amplification and subcloned to pEBG A rabbit polyclonal antibody against DHBc was generously provided by Dr. William Mason HEK293T (human embryonic kidney) cells were obtained from Dr. Zhijun Luo  GST-fusion proteins were purified from bacteria as previously described 35S-metabolic labeling, three days post-transfection, 60 mm dishes of HEK293T cells were washed twice with 4 ml cysteine/methionine-free DMEM (Invitrogen). Cells were incubated in 0.5 ml cysteine/methionine-free DMEM supplemented with 10% dialyzed FBS (HyClone) for 1 hour to deplete cellular stores of cysteine and methionine. One mCi [35S]cysteine/methionine Express Protein Labeling Mix  was added to the cells which were then incubated for an additional 6 hours. GST-tagged proteins were purified as described above.For 32P]orthophosphate metabolic labeling procedure was carried out as described 32P]orthophosphate . GST-tagged proteins were purified as described above.The methionine-labeled using the TnT in vitro rabbit reticulocyte lysate (RRL) transcription and translation coupled kit according to manufacturer's recommendations (Promega). Where indicated, translation products were treated with 1 U calf intestinal alkaline phosphatase  per µl translation for 16 hours at 37°C. DNase I  and RNase A (100 µg/ml) were then added to the reactions, after which the translation reactions were incubated at room temperature for 1 hour with gentle agitation. Precipitates were removed and each reaction was evenly divided into three aliquots for binding to the I2PP2A-Flag-HA resin, 3X-Flag-B23 resin or untransfected control resin, as prepared above from HEK293T cells. Unbound proteins were removed after collection of the resin by centrifugation. Beads were washed extensively in Lysis Buffer containing 0.1% NP-40. Bound proteins were eluted by boiling in SDS sample buffer and resolved by SDS-PAGE. The gels were stained with Imperial Protein Stain (Pierce) to visualize antibody chains and to ensure samples had been loaded equally. 35S-labeled full-length DHBc or GST-DCC fusion proteins were visualized by autoradiography.Full-length DHBc or GST-DCC fusion proteins were expressed and [Viral DNA was isolated from transfected cell lysates as previously described


New multifunctional drugs that target multiple disease-relevant networks offer a novel approach to the prevention and treatment of many diseases. New synthetic oleanane triterpenoids (SO), such as CDDO  and its derivatives, are multifunctional compounds originally developed for the prevention and treatment of inflammation and oxidative stress. However, the protein binding partners and mechanisms of action of these SO are not yet fully understood. Here we characterize the putative target profile of one SO, CDDO-Imidazolide (CDDO-Im), by combining affinity purification with mass spectroscopic proteomic analysis to identify 577 candidate binding proteins in whole cells. This SO pharmaco-interactome consists of a diverse but interconnected set of signaling networks; bioinformatic analysis of the protein interactome identified canonical signaling pathways targeted by the SO, including retinoic acid receptor (RAR), estrogen receptor (ER), insulin receptor (IR), janus kinase/signal transducers and activators of transcription (JAK/STAT), and phosphatase and tensin homolog (PTEN). Pull-down studies then further validated a subset of the putative targets. In addition, we now show for the first time that the mammalian target of rapamycin (mTOR) is a direct target of CDDO-Im. We also show that CDDO-Im blocks insulin-induced activation of this pathway by binding to mTOR and inhibiting its kinase activity. Our basic studies confirm that the SO, CDDO-Im, acts on a protein network to elicit its pharmacological activity. Chronic diseases are now the prime cause of most pain and suffering in American society, as well as the largest financial burden in the health care system in vitro and in vivo models. The SO, CDDO-methyl ester  is currently in late-stage clinical development for treatment of chronic kidney disease (diabetic nephropathy) Over the past decade a series of synthetic triterpenoids based on the natural triterpenoid, oleanolic acid (OA) has been synthesized In the development of second generation OA derivatives, two electrophilic Michael acceptor sites were incorporated in the A and C rings of OA to increase the reactivity of OA toward cellular targets in vivoA few biologically important targets of SO have already been discovered. Activation of the KEAP1/Nrf2 pathway has been shown to be critical for many of the activities of both CDDO-Me and its close relative, CDDO-Imidazolide (CDDO-Im) CDDO-Im, TP-154 and TP-304 have been described previously 2. In some cases cells were plated and treated in culture media containing 1% horse serum. To measure proliferation, an MTT assay was performed as described previously HEK293 and PC-3 cells were from ATCC, and maintained in DMEM (HEK293) or RPMI (PC-3), with 10% FBS and in 5% CO3VO4), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 µmol/L leupeptin, and 5 µg/ml aprotinin). Protein concentration in centrifuged lysates was determined by Bradford assay. Lysates were immunoblotted as described previously 4, pH 7.2, 0.3% CHAPS (to preserve the mTOR-Raptor interaction), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM beta-glycerophosphate, 1 mM Na3VO4). Clarified lysate supernatants were incubated with anti-RAPTOR polyclonal antibodies for 2 h at 4°C, followed by incubation with protein A Sepharose beads for 2 h. Immunoprecipitates were washed three times in lysis buffer B, and resuspended in 50 µl of Laemmli loading buffer followed by immunoblotting with mTOR antibodies For Western blots, HEK293 cells were washed twice with cold PBS and suspended in RIPA buffer . Centrifuged lysates were incubated with 50 µl of MyOne™ Streptavidin T1 paramagnetic Dynabeads®  for 1 h. Dynabead®-biotinylated triterpenoid protein-complexes were isolated magnetically and washed a total of five times with buffer A. An additional sample was treated with TP-304 and washed with a more stringent wash buffer  Triton X-100, 300 mM sodium actate, 0.1% (v/v) SDS) to disrupt protein complexes and remove non-specific binding proteins. Complexes were boiled in Laemmli loading buffer and separated on 4–20% gradient, SDS-PAGE gels followed by staining overnight in a solution of 50% MeOH, 40% H18-AQ particle filled  fritless-tip microcapillary columns   using an HPLC-driven (Agilent 1200 capLC) split-flow system designed to maintain 1200–1500 psi at the head of the analytical column. The resultant peptide eluate was directed into an LTQ-Orbitrap high performance mass spectrometer operating in a data-dependent sequencing acquisition mode across a 60-minute reverse-phase gradient (5% acetonitrile / 0.1% formic acid to 30% acetonitrile / 0.1% formic acid). The collected tandem mass spectra were data-searched using the SEQUEST algorithm Coomassie-stained gels were divided into 8 regions and digested with trypsin overnight at room temperature. The next day, peptides were extracted, dried, and analyzed by nanoscale microcapillary LC-MS/MS as described. Samples were loaded using a FAMOS autosampler  onto a custom manufactured reverse-phase, Reprosil-Pur 3 µm, 200 Å Cwww.Ingenuity.com).For Go annotation, UniProt IDs were submitted to GORetriever To identify candidate cellular targets of SO, biotinylated SO (btSO) conjugates have been synthesized. The pharmacological activity of these btSO was initially measured here by their ability to inhibit cellular proliferation . BiotinyUtilizing TP-304 and TP-154 as probes, we developed and optimized protocols to treat cells in culture for proteomic analysis. Because of the electrophilic nature of the SO and their ability to bind to redox active targets, it was important to ensure that the biotinylated probes interact with protein binding partners in their natural environment where the context-dependent nucleophilicity of -SH groups in cysteine residues is preserved. Following treatment with TP-304 and TP-154, both at 4 µM, for 1 h, HEK293 cells were lysed, and lysates were incubated with Neutravidin-coated Dynabeads to affinity purify protein targets . AffinitThe putative SO targets were annotated using gene ontology (GO) identifiers. For GO annotation, UniProt IDs were submitted to GORetriever and categorized in GO_slim categories using CateGOrizer. GO annotation based on molecular function showed that catalytic activity, protein kinase activity, transcription regulator activity, kinase activity and enzyme regulator activity represented 75% of putative TP-304 targets . GO cateThus we analyzed TP-304 targets using the Ingenuity software program to identify specific canonical pathways within the TP-304 binding partner dataset. This analysis identified a large number of canonical pathways targeted byTP-304 with very high significance . The topNext we determined the validity of the putative SO binding partners identified in the above proteomics studies. We used methods previously employed to identify IKK as an SO target. HEK293 cells were treated with 4 µM TP-304 for 1 h, followed by lysis and pull-down of TP-304-protein binding partners with Neutravidin-coated agarose beads. Following a series of washes to eliminate nonspecific binding, the remaining bound proteins were eluted and separated by SDS-PAGE followed by Western blotting. This was performed for a subset of proteins specifically identified above , 3 by LCThe SO can bind to nucleophilic Cys residues on binding partners through Michael addition. Therefore, we sought to confirm that such binding had a functional effect on some of the binding partners identified above. We interrogated the Ingenuity-assigned canonical pathways to identify specific SO binding partners which appear in multiple pathways, and reasoned that proteins that appear in multiple canonical pathways may represent critical SO target nodes. Thus, components of the PI3K/AKT/mTOR pathway were highly enriched in our canonical dataset. We used the highly potent CDDO analog, CDDO-Im for these studies and found that it was a potent inhibitor of the phosphorylation of insulin-stimulated ribosomal S6 (RS6) at serine 235/236 (S235/236). As can be seen in Thus, based on the observed enrichment for this pathway from our proteomics experiments and the observed inhibition of phosphorylation of both RS6 and 4E-BP1, we then investigated the effects of CDDO-Im on this pathway in greater detail in a hierarchical manner, beginning with RS6 and 4E-BP1 and working back up the pathway to determine at which level the CDDO-Im is inhibitory. Since P70S6 induces RS6 phosphorylation we looked at the insulin-induced activation of this important kinase. As expected, insulin rapidly induced phosphorylation of threonine 389 (T389) on P70S6K . HoweverTo examine the effects on mTOR in more detail we conducted an in-vitro mTOR activation assay which examines the phosphorylation levels of serine 2481 of mTOR In order to exclude the possibility that the CDDO-Im was inactivating the pathway upstream of mTOR, and in turn blocking its activation we examined the activity of the upstream activating kinases, PI3K and AKT, by looking at phosphorylation levels of their substrates, AKT and GSK3β, respectively. As expected, we found that insulin rapidly phosphorylated AKT at S473 . This efTo determine if this inhibition of the mTOR signaling pathway was idiosyncratic to HEK293 cells we examined the effect of CDDO-Im in the prostate cell line, PC-3. PC-3 cells have constitutive activation of the PI3K/AKT/mTOR signaling axis due to mutations in the lipid phosphatase, PTEN, which dephosphorylates PI3K. In PC3 cells CDDO-Im inhibited the phosphorylation of RS6 S235/236 and 4E-BP1 S65 , indicatUtilizing a novel, chemical proteomics approach, we have identified 577 proteins which interact with the biotinylated SO, TP-304, in cell culture. While some of these proteins had previously been identified, to date, this is the first report of a comprehensive and system-wide analysis of SO binding partners. As expected, a large number of new binding partners were identified, forming a large interconnected network of proteins that interact with SO. Many of the canonical pathways previously implicated as being targeted by SO, such as NFκB, JAK/STAT, PTEN and AMPK were found in this analysis In addition to these networks identified in silico, we have functionally validated the mTOR pathway as a target for CDDO-Im. Previous studies have suggested that this pathway may be inhibited by very high concentrations of the SO, CDDO-Me The SO are extremely potent inhibitors of cellular proliferation in vivo models. Currently the development of network-based, multi-target drugs similar in function to the SO is gaining increased interest in the field of drug development One caveat of our proteomics-based target search is its qualitative nature, which lacks quantitative information about the SO-target interaction. Binding in such assays is a function of both protein/target abundance and affinity; therefore, highly abundant-low affinity proteins will co-purify with low abundance high-affinity proteins, making it difficult to infer relative affinities of the SO for the identified targets. However, multi-functional drugs typically bind to targets with lower affinity than a single target drug. Previous studies have suggested that low-affinity, multi-target drugs have a lower prevalence and a reduced range of side-effects than high-affinity, single-target drugs while also being more efficacious e.g. the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib, are, in fact more promiscuous than originally thought In summary, technological advances in the integration of proteomics, pharmacology and molecular biology now provide the means to map drugs and targets at a systems level. Indeed, it has become evident that many drugs thought to specifically target a single protein, Figure S1Complete list of protein TP-304 binding proteins identified by LC-MS/MS. The collected tandem mass spectra were data-searched using the SEQUEST algorithm, filtered to less than 1% false discovery rate using the target-decoy strategy as described in the (PDF)Click here for additional data file.Figure S2High resolution, scalable image of the network map depicted in (TIF)Click here for additional data file.Figure S3High resolution, scalable image of the sub-network map depicted in (TIF)Click here for additional data file.


To set the cut-off values of IgG4, IgE, and the IgE/IgG4 ratio for predicting positive results in oral challenges, receiver operating characteristic curves were plotted and the area under the curves (AUC) were calculated.The hen’s egg oral food challenge test was performed in 105 sensitized children without atopic dermatitis, and the titers of egg white-specific immunoglobulin G4, and IgE/IgG4 for the prediction of positive results were 0.609, 0.724, and 0.847, respectively. Thus, the IgE/IgG4 ratio generated significantly higher specificity, sensitivity, positive predictive value (%), and negative predictive value (%) than the individual IgE and IgG4. The negative predictive value of the IgE/IgG4 ratio was 90% at a value of 1.Sixty-four of 105 oral challenges with whole eggs were assessed as positive. The AUC for IgE, IgG4 ratio is important for predicting reactivity to egg during food challenges.We have demonstrated that the egg white-specific serum IgE/IgG Hen’s egg is one of the common food in children, and is the most common immediate-type food allergy in Japanese children . The pro4 antibodies is commonly identified in atopic patients but their role remains unclear  × 100). The higher the response, the more specific IgG4 is present in the specimen. To evaluate the test results, the responses from patient samples were directly compared with a reference serum run in parallel. ImmunoCAP Specific IgG4 Calibrators were used for total IgG4 determination, and values are expressed in μg/L. In the ImmunoCAP Specific IgG4 assay, these calibrators were used for the determination of specific IgG4 antibodies, and values are expressed in mgA/L, where A represents antigen-specific antibodies. Values above the limit of quantitation represent a progressive increase in the concentration of antigen-specific IgG4 antibodies. With a sample dilution of 1:100, IgG4 antibody levels up to 30 mgA/L were measured. The crossreactivity of the enzyme-labeled anti-IgG4 with IgG1, IgG2, IgG3, IgA, IgM, and IgE was <0.5%.Serum samples were obtained from all subjects on the day of the food challenge test and immediately processed for quantification of egg white-specific IgE antibody titers using the UniCAP SystemTM , according to the manufacturer's instructions. Egg white-specific IgGAll egg-food challenges were open challenges, performed in hospital settings and supervised by physicians. Clinical features to react hen’s egg were investigated for clinical purposes via an open challenge test as described in the Japanese food allergy management guideline 2008  or afterThe Mann–Whitney test, the χ2 test (2-tailed), or the Kruskal-Wallis rank test was used to test differences between groups. A p-value of 0.05 was considered significant. Performance characteristics  were calculated for various cut-off values, including the optimal cut-off values proposed by the receiver operating characteristics (ROC) plots. Statistical analysis was carried out using SAS System V8.2 . The box plot and whisker plots were generated in the study because the data did not show normal distribution.Oral food challenges using egg white were performed in 105 children sensitized to hen’s eggs. Forty-five children were negative, while 60 were assessed positive: 25 children reacted under loading doses of <0.8 g of hen’s egg, 13 at 1.6 g, 11 at 3.2 g, and 11 at 6.4 g. No child reacted at doses of either 12.8 g or 25.6 g.Patient characteristics are presented in Table 4 antibodies were 2.17 mgA/L  and 0.29 mgA/L (0.095-0.715 mgA/L) in the NFC and PFC groups, respectively  was 4.83 , whereas it was 46.71 (18.05-131.04) in the NFC group  in the NFC group (45 children) and 14.45 kUA/L (6.93-30.9 kUA/L) in the PFC group  reacted at less than 0.8 g of eggs, the B group (n = 13) at 1.6 g, the C group (n = 11) at 3.2 g, and the D group (n = 11) at 6.4 g. The E group was negative. The IgE/IgG5 reacted4, IgE, and the IgE/IgG4 ratio for predicting positive results in oral challenges, ROCs were plotted and the AUCs were calculated. The AUC for serum egg-specific IgE was 0.697; for the estimated cut-off of 8.9 IU/mL, values of 68.3% and 68.9% were obtained for the sensitivity and specificity of the assay, respectively . All 4 parameters exhibited relatively high IgE/IgG4 ratios compared to IgE and IgG4 in the 2 groups.The patients were divided into 2 groups, over 5 years of age and under 4 years of age, and their ROC curves were compared. The area under the ROC curves (AUCs) for egg-specific IgE, IgG4 antibodies as well as IgE antibodies to hen’s egg white is of value in the prediction of reactivity to hen’s egg during food challenges, and therefore helpful in the confirmation of food tolerance in egg-sensitized children. The titers of food-specific IgE antibodies have already been reported to be useful in the prediction and possible diagnosis of IgE-mediated food allergy; several studies have proposed “cut-off” values of antigen-specific serum IgE or weal diameter sizes for the diagnosis of food allergy, to optimize the use of food challenge tests [In recent years, it has been highly recommended that unclear cases of food allergy should be confirmed by standardized food challenges, such as the DBPCFC, for definite diagnosis although it carries a risk of severe adverse events, such as anaphylaxis ,20. In tge tests ,22. Sampge tests -25. Compge tests ,24. Thes4 antibodies during treatment [4 antibodies act as blocking antibodies by competing with IgE for allergen binding to IgE receptor-expressing cells, such as mast cells and basophils [4 antibodies at the level of antigen-presenting cells has also been detected in vitro[4 antibodies is up-regulated by IL-4 produced from activated Th2 cells [4 production [4 antibodies in the absence of IgE antibodies [Recent studies on immunotherapy with inhalant allergens suggest a protective role of IgGreatment ,27. It hasophils , and com in vitro. The proh2 cells . Howeveroduction . Anothertibodies .4 in natural tolerance development in food allergy. Stapel et al. have shown that food-specific IgG4 does not indicate food allergy or intolerance, but rather indicates immunological tolerance, linked to the activity of regulatory T cells [4 levels were the highest in atopic subjects who were tolerant to cow’s milk, whereas Shek et al. [4 response in children allergic to cow’s milk compared to atopic patients without suspected food allergy. Ahrens et al. [4 levels between tolerant and allergic children. The discrepancy between our study and Ahrens’s result may be due to the following reasons: (1) the age of the study population, (2) the challenged materials, and (3) the procedure of the challenge test, (4) the exclusion of atopic dermatitis patients. The main reason for the discrepancy may be the age of the population. The mean age in our study was 5 years, although it shows 2 years of age of its study. We also observed a lower level of AUC for hen’s egg white-specific IgG4 in the under 5 years of age group than in the over 5 years of age group. Children younger than 2 years of age have less ability to produce IgG4[4 in food allergy is related to tolerance or repeated exposure to food ingestion. In our study, children demonstrating a lower ratio of IgE/IgG4 to hen’s egg  were more likely to be able to eat eggs. The cut-off level of IgE/IgG4 for more than 90% of the NPVs was 1, and that for 20% of the post-test probability values was 13.5, based on the calculation of negative likelihood ratios (0.2). This means that 90% and 80% of patients who showed cut-off levels of IgE/IgG4 that were less than 1 and 13.5, respectively, were able to eat hen’s eggs.Several reports have described the role of specific IgG T cells . Ruiter  T cells  have repk et al.  have dess et al.  reportedduce IgG4. Thus, i4 ratio to egg white is a more useful parameter for predicting the outcome of oral challenge with eggs than IgE alone in patients without atopic dermatitis or particularly in older children. Further study will be needed to evaluate the role of egg white-specific IgG4 in oral tolerance induction in hen’s egg allergy.Our study demonstrated for the first time that the IgE/IgGThe authors declare that they have no competing interests.SO and ST conceived the study, designed the study and wrote the paper. SK and YH participated in its design and writing. NK and TS helped to edit the manuscript. KK helped to collect data for analysis. All authors read and approved the final manuscript.


AMP-activated protein kinase appears to have evolved in single-celled eukaryotes as an adenine nucleotide sensor that maintains energy homeostasis at the cellular level. However, during evolution of more complex multicellular organisms, the system has adapted to interact with hormones so that it also plays a key role in balancing energy intake and expenditure at the whole body level. Although discovered via its ability to phosphorylate and inactivate two enzymes involved in lipid synthesis, i.e., acetyl-CoA carboxylase and HMG-CoA reductase , 9, 10, AMPK appears to exist universally as heterotrimeric complexes comprised of catalytic α-subunits and regulatory β- and γ-subunits , and welSaccharomyces cerevisiae (SNF1 and SNF4) were cloned in the 1980s  . In the rk cycle . In the darkness . Thus Snstresses , 21. TakAs with many other protein kinases, AMPK and its orthologs are only significantly active when phosphorylated at a conserved threonine within the kinase domain by upstream kinases ; this thVia these two effects, AMP binding causes a sensitive switch to the active, phosphorylated form of AMPK. This activation is further amplified by allosteric activation, which is triggered only by AMP . The phy1) promotion of Thr172 phosphorylation; 2) inhibition of Thr172 dephosphorylation; 3) allosteric activation. Only the second effect is mimicked by ADP, although all three are antagonized by ATP  increases in response to glucose starvation and correlates with large increases in cellular ADP-to-ATP and AMP-to-ATP ratios , first demonstrated 10 years earlier .Treatment of cells with agents that increase intracellular Ca earlier , was phy earlier , 34, 74  earlier . Increas neurons , activat neurons , and treS. cerevisiae strain that lacked all three kinases upstream of the yeast AMPK ortholog . This appeared to reduce the rate of subsequent Thr172 phosphorylation and activation by LKB1 in a cell-free assay, whereas prior insulin treatment also reduced Thr172 phosphorylation during ischemia in perfused rat hearts . This meA full discussion of downstream targets for AMPK are beyond the scope of this article, and readers are referred to other reviews –25, 53; AMPK also switches off almost all biosynthetic pathways, including synthesis of fatty acids, sterols, triglycerides, phospholipids, glycogen, ribosomal RNA, and proteins. These pathways are often downregulated via multiple mechanisms, involving both phosphorylation of key enzymes in the pathway and longer-term effects on gene expression. In the case of protein synthesis, the major action of AMPK is to inhibit the mechanistic target-of-rapamycin complex-1 (mTORC1), a central signaling node that integrates signals arising from growth factors, nutrient availability, and energy status, and promotes cell growth when circumstances are appropriate. mTORC1 promotes cell growth by phosphorylating ribosomal protein S6 kinase-1 (S6K1), by stimulating translation of proteins critical for growth  and transcription of ribosomal RNA, and by phosphorylating eIF4E binding protein-1 (4E-BP1), relieving its inhibitory effects on translational initiation . InhibitRecently, O'Malley's group reported that steroid receptor co-activator-2 (SRC-2) was phosphorylated by AMPK and provided evidence that this promoted transcription of mRNA encoding the bile salt export pump, BSEP, in hepatocytes . Since tGiven that it evolved in simple single-celled eukaryotes, the original primary function of AMPK appears to have been to regulate energy homeostasis at a cell-autonomous level. However, during the evolution of multicellular eukaryotes, hormones involved in regulating whole-body energy balance have also developed the ability to regulate AMPK. This final section of the review will focus on the effects of these hormones, whose effects are summarized in 3 and consequent release of intracellular Ca2+ in the presynaptic neuron. The released Ca2+ then triggers phosphorylation of AMPK via CaMKKβ  that allows continued Ca2+-dependent release of the neurotransmitter glutamate onto the NPY/AgRP neurons , even after ghrelin release has stopped , ghrelin increases and leptin and insulin decrease. This will increase AMPK activity in the presynaptic neurons, causing increased activity of NPY/AgRP neurons and decreased activity of POMC neurons, driving a powerful orexigenic output . The conAs well as repressing appetite in leptin-sensitive individuals, inhibition of AMPK by leptin in the hypothalamus also causes activation of sympathetic nerves leading to the periphery. One effect of this is to activate AMPK-α2 and stimulate fatty acid oxidation in skeletal muscle, thus increasing whole body energy expenditure, prolonging the more transient direct effects of leptin on muscle . These fAlthough secreted by adipocytes, adiponectin paradoxically displays high plasma levels in lean individuals and low levels in obese individuals, suggesting that it is released when fat stores are low. Adiponectin binds to two related receptors, AdipoRI and AdipoRII , and binThe thyroid hormone tri-iodothyronine (T3) binds to nuclear receptors that regulate transcription and has effects on almost all cells. However, a major effect of T3 on whole-body energy balance appears to involve inhibition of AMPK in neurons of the ventromedial hypothalamus . Like th2+-CaMKK pathway and the ability of insulin, and perhaps leptin, to inhibit AMPK activation by promoting phosphorylation of the ST loop.It is seems likely that the appearance of the AMPK system was a very early event in evolution of eukaryotes, providing a mechanism for the host cell to interact with and cooperate with its new intracellular partner, the mitochondrion. As more complex multicellular eukaryotes developed, this ancestral role as a cell-autonomous energy sensor was retained and even appears to have become more sensitive. Thus the mammalian system is regulated by AMPK by a very sensitive tripartite mechanism, whereas its budding yeast ortholog lacks at least one of these mechanisms  and also appears to be regulated by ADP rather than AMP. In addition, new systems of regulation have been added on top of these ancestral mechanisms, which allowed AMPK to play new roles in regulating energy balance at the whole body level. Although these additional mechanisms are still being elucidated, they include the ability of the starvation hormone ghrelin to activate AMPK by the Ca


Omalizumab, a humanized monoclonal anti-IgE antibody has the potential to alter allergen processing. Recently, it has been postulated the assessment of PHA-stimulated adenosine triphosphate (ATP) activity as maker of CD4+ T cells activity in peripheral blood cells. We present the case report of a 35-year-old woman with a history of chronic idiopathic urticaria and angioedema of 8 years of development with poor response to treatment. The patient was partially controlled with cyclosporine at doses of 100 mg/12 h. However, she was still developing hives daily. Finally treatment with omalizumab was started at dose of 300 mg every 2 weeks. The patient experienced a decrease in urticarial lesions 2 days after starting therapy. We also evaluated the effects of omalizumab therapy on the activity of peripheral blood CD4+ T cells from the patient, in order to determine the potential modification of anti-IgE therapy on the process of antigen presentation-recognition. Activity of CD4+ cells by ATP release was clearly increased demonstrating an enlarged CD4 activity. Omalizumab may be useful in the treatment of severe chronic urticaria. ATP activity of peripheral blood CD4+ T cells might be a non-subjective method to assess Omalizumab activity. We have read the interesting manuscript recently published in Clinical and Molecular Allergy entitled "Down regulation of the high-affinity IgE receptor associated with successful treatment of chronic idiopathic urticaria with omalizumab" . The stuHowever, anti-IgE appears to provide a therapeutic option for cases of many allergic diseases and conditions in which IgE plays a significant role. Although, the potential use of omalizumab in other IgE-mediated conditions is being investigated ,4 and trWe would like to present a 35-year-old woman with findings of rhinoconjunctivitis and episodic asthma by mite sensitization from childhood, severe chronic urticaria and angioedema since November 1999 with normal initial study conducted in 2000 . Poor control was obtained with conventional treatments . Subsequently, the patient consulted several specialists (dermatologists) without success and was re-evaluated by Allergology during hospitalization caused by severe urticaria angioedema exacerbation coincident with an episode of retinal detachment. In previous years the urticaria and angioedema had not changed and she still had symptoms daily. Only in 2004 during pregnancy and subsequent breastfeeding showed a slight improvement in their symptoms.A new study was done with normality of all the tests, including complement proteins study again. Then, we tried different treatments with antihistaminics, doxepin and corticosteroids. In April 2005, we began cyclosporine at doses of 200 mg per day with good response initially. Despite of oral contraception method the patient had a spontaneous miscarriage in that year. During the next 4 years the minimal doses of cyclosporine were of 100 mg per day and the last 2 years with daily cutaneus lesions. The pacient had exacerbations after walking, exposure to cold, premenstrual phase and the laboral absenteeism were important. Due to the poor control obtained previously, we decided to initiate Omalizumab therapy in 2008 with 300 mg every 2 weeks, based on weight and IgE level (178.0 UI/ml). Dramatic relief was obtained within 72 hours. The patient discontinued by own decision all medication with no exacerbation. Two weeks later, she had not injuries and did not take any medication. We began to gradually increase the intervals between doses. Currently we give 300 mg every 6 weeks and the patient remains asymptomatic without any side effects. Further, we tried to extend it to 8 weeks, but resulting with small hives in patient's extremities.® CD4, Dynal Biotech A.S.A., Oslo, Norway) were added to immunoselect CD4 cells from both the stimulated and non-stimulated cells. After washing the selected CD4 cells on a magnet tray, a hypotonic basic solution as lysis reagent was added to release intracellular ATP. During incubation, increased ATP synthesis occurs within the cells that respond to PHA. The ATP result was measured by luminescence (562 nm). Serum obtained was stored and total IgE  of every sample were determined in the same immunoassay with no significant differences among samples. However, T cell activation was significantly increased from basal (365 ng/ml ATP-moderate response) to first point, 2 weeks after first injection (593 ng/ml-strong response). That activation was maintained during following 18 weeks  were obtained and used in fresh for an immune cell function assay to detect T cell activation . Briefly, PBMC were incubated 18-h either in the absence of stimulant to assess basal activity or with specific stimulant for T cells (phytohemagglutinin-PHA). Magnetic beads coated with mouse monoclonal anti-human CD4 (DynabeadsThe mechanism of action of omalizumab, an anti-IgE monoclonal antibody, in urticaria  is unknoThe authors declare that they have no competing interests.ISM & JIS studied the case report and wrote the initial draft of the manuscript. AF & YB performed every single lab assay. for in vivo tests. RG was responsible for the Drug Allergy Section and for safety of administration with Omalizumab. VM & YB conceived the idea and are responsible of the final version of the manuscript. All authors approved the final version of the manuscript.