Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential levels of phosphorylation of many proteins and extensive changes in levels of gene expression, protein abundance, cell wall lipids, and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis, and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell.Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystem approach to determine the effects of a potent small-molecule inhibitor of the essential  Mycobacterium tuberculosis responses to inhibition of its two essential serine/threonine protein kinases. These kinases allow the bacterium to adapt to its environment by phosphorylating cellular proteins in response to extracellular signals. We identified differentially phosphorylated proteins, downstream changes in levels of specific mRNA and protein abundance, and alterations in the metabolite and lipid content of the cell. These results include changes previously linked to growth arrest and also reveal new roles for these kinases in regulating essential processes, including growth, stress responses, transport of proteins and other molecules, and the structure of the mycobacterial cell envelope. Our multisystem data identify PknA and PknB as promising targets for drug development and provide a valuable resource for future investigation of their functions.Tuberculosis is the leading killer among infectious diseases worldwide. Increasing drug resistance threatens efforts to control this epidemic; thus, new antitubercular drugs are urgently needed. We performed an integrated, multisystem analysis of  Phosphorylation-based signal transduction allows adaptation to environmental conditions by linking extracellular signals to intracellular regulatory mechanisms. In most bacteria, the dominant mechanism of phosphorylation-based transmembrane signaling is the two-component system , where lin vitro and in mice (–The success of kinase inhibitors as drugs to treat human diseases  has stim in mice –7 and onin mice –.pstP), a candidate peptidoglycan (PG) synthase (rodA) (pbpA). Phosphorylation mediated by PknA and PknB controls activity of PG synthesis regulators (–Bacillus subtilis to regulate cell growth and morphology (Mycobacterium tuberculosis through phosphorylation of the GarA regulatory protein (M. tuberculosis (–M. tuberculosis cell physiology.Targeted research has provided insights into a number of likely functions of PknA and PknB. Genes for both kinases are present in an operon that includes genes for a Ser/Thr phosphatase (e (rodA) , and a cgulators –11, and rphology , 13. Pkn protein , 15. Serrculosis –18. DespTo develop a more comprehensive view of the functions of PknA and PknB, we used a newly developed small-molecule inhibitor of these kinases together with unbiased multisystem profiling. Compared to genetic strategies that rely on protein depletion, the advantage of chemical inhibition is the rapid inhibition of enzyme activity, which allows identification of both early, direct effects on protein phosphorylation and downstream, indirect effects of blocking kinase activity. Given the potential for PknA and PknB to broadly regulate multiple cellular pathways, we performed phosphoproteomics, proteomics, transcriptomics, lipidomics, and metabolomics analyses. This approach identified changes in protein phosphorylation in response to kinase inhibition that were linked to broad downstream changes in gene expression, protein abundance, and lipid and metabolite profiles. Integration of these data identifies changes in pathways for cell growth, remodeling of the mycobacterial cell envelope, protein and small-molecule transport, and stress responses and reveals an unexpected intersection of PknA/PknB-mediated signal transduction with an essential two-component system.in vitro inhibition of PknB (inhibitor constant Ki = 0.004 μM) and PknA (Ki = 0.018 μM), its ability to inhibit growth of M. tuberculosis H37Ra [minimal inhibitory concentration (MIC) = 4.5 μM], and its selectivity for PknA and PknB relative to human kinases (Ki = >5 μM) or M. tuberculosis growth  and served as a control in all experiments.From a series of small molecules developed by Vertex Pharmaceuticals Incorporated to inhibit PknA and PknB, we selected a 5-substituted pyrimidine  based on kinases . An anal kinases  showed nM. tuberculosis growth inhibition, we measured the MIC of the active compound in a strain in which pknB expression was reduced by 80% to 90%, using clustered regularly interspaced short palindromic repeat (CRISPR) interference (pknA expression and pknB expression showed similar (4- to 8-fold) MIC shifts (data not shown). These data indicate that a major part of the growth inhibitory activity of the active compound results from inhibiting PknB and PknA, the only two essential M. tuberculosis STPKs, and strongly support the hypothesis that physiological changes in responses to the inhibitor result from inhibition of these kinases.We used this inhibitor and the inactive analogue to investigate PknA and PknB function in detail in the virulent H37Rv strain. We determined the MIC of the active compound to be 5 μM and that of the inactive analogue to be >160 μM. To test whether inhibition of PknB activity is responsible for rference . We obserference . ConsistM. tuberculosis STPKs are similar to those of PknB and PknA   , have beed genes . Express results ; in our 10.1128/mBio.02333-17.4TABLE S1 M. tuberculosis STPKs. Download TABLE S1, DOCX file, 0.04 MB.Activity of kinase inhibitor and inactive control compounds against Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We also observed significantly decreased phosphorylation of phosphopeptides from PknA and PknB, and from known PknA/PknB substrates in extracts of cells treated with the inhibitor, that indicated that PknA and PknB are primary targets of the inhibitor . In contrast, phosphorylation of peptides from PknD, which also autophosphorylates, was stable or increased and PknL phosphopeptides were not detected in any sample. Phosphorylation of phosphopeptides from PknE, PknF, and PknG was also stable or increased, and phosphopeptides from PknH, PknI, PknJ, and PknK were not detected. These data support the conclusion that major effects of the inhibitor result from inhibition of PknA and PknB and do not support the identification of PknD or PknL as targets of the kinase inhibitor.pknD and pknL deletion strains. Global gene expression in the inhibitor-treated mutants was highly correlated with the wild-type results (Pearson’s r = 0.71 for pknD and 0.78 for pknL). To the extent that kinase gene deletion and kinase inhibition have similar effects, for a gene whose expression is affected by PknD or PknL kinase activity, if the inhibitor blocks this activity, we would expect (i) that inhibitor effects on gene expression in the wild type would not be seen in the mutant treated with the inhibitor and (ii) that expression of the gene in the mutant treated with the control compound would be similar to its expression in the wild type treated with the inhibitor.We further investigated whether inhibition of PknD or PknL might account for some effects of the kinase inhibitor by performing transcriptomics analysis of inhibitor-treated 2 fold change [Log2FC], >1 or ≤1; P < 0.0001)  in the inhibitor-treated pknD and pknL mutants, respectively (n = 3) were performed in the experiments comparing mutant strains to the wild type. After excluding phage genes, expression of 27 (9.1%) and 35 (11.7%) genes (i) did not change in the inhibitor-treated pknD or pknL strains, respectively, in a manner similar to that seen with the inhibitor-treated wild-type strain and (ii) changed significantly  in the control-treated pknD and pknL strains in a manner similar to that seen with the inhibitor-treated wild type. These criteria identified genes whose expression might be affected by inhibition of PknD or PknL activity by the kinase inhibitor. Given that distinct signal transduction systems often have overlapping downstream pathways, however, this likely represents a substantial overestimate of such effects. Taken together, these several analyses strongly support the identification of PknA and PknB as primary targets of the kinase inhibitor and the supposition that inhibition of these two kinases is the main source of the changes obtained in our multisystem data.Of the 298 highly differentially expressed genes in the wild type treated with the inhibitor (log 0.0001) , 255 (86ectively . Lower c10.1128/mBio.02333-17.5TABLE S2 pknD and pknL deletion strains (D). Download TABLE S2, XLSX file, 0.6 MB.Expression ratio for all genes of RNA from samples treated with the kinase inhibitor to samples treated with the inactive control compound (A), genes with significantly increased expression and comparative gene expression from other conditions (B), genes with significantly decreased expression and comparative gene expression from other conditions (C), and comparison of highly regulated genes to their expression in Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2-fold change (Log2FC) in inhibitor-treated versus control-treated cells, with P values adjusted for multiple testing.We sought to understand kinase effects on cellular functions in a manner that would be minimally confounded by cell death pathways. Pilot studies showed growth inhibition but minimal cell death at an inhibitor concentration of 4-fold the MIC (20 μM) –0.05% Tween 80. Serial 10-fold dilutions (10−1 to 10−5) were prepared, and 10 µl of each dilution was spotted on 7H9 agar plus ADNC and incubated for 3 weeks at 37°C. Download FIG S1, DOCX file, 0.4 MB.Survival of Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Unbiased mass spectrometry (MS)-based metabolomics analysis of cell extracts detected inhibitor and control compounds in cell extracts at the earliest time point (12 h) . Ions inM. tuberculosis phosphoproteome. Analysis of these phosphorylation sites identified eight significant motifs (P < 0.01), comprising four each with Ser or Thr as the phosphorylated residue  Significant phosphorylation site motifs identified among all unique phosphopeptides. (B) Significant motifs from phosphopeptides that showed significantly decreased phosphorylation in response to the kinase inhibitor. Download Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02333-17.6TABLE S3 TABLE S3, XLSX file, 0.4 MB.List of all detected phosphopeptides (A), distribution of phosphorylated residues (B), quantified phosphopeptides (C), phosphopeptides with significantly decreased expression (D), phosphopeptides with significantly increased expression (E), and time course of quantified phosphopeptides (F). Download Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2FC values of ≤0.5 and P values of <0.001 to phosphopeptides detected in >7 of 36 samples, 68 phosphopeptides from 48 proteins showed decreased phosphorylation in response to treatment with the inhibitor (in vivo substrates of PknA and/or PknB (P < 0.0001) and significantly depleted in “intermediary metabolism and respiration” (P = 0.001)  . Thirty Significantly decreased phosphorylation  was seenP = 0.01) and depleted in “information pathway” phosphoproteins (P = 0.01). In contrast to the low proportion (12%) of Ser residues that showed decreased phosphorylation, a much higher proportion (44%) of phosphopeptides with increased phosphorylation were phosphorylated on Ser , expression of 253 genes increased and expression of 45 genes decreased (The transcriptome sequencing (RNA-Seq) results provided the most comprehensive view of changes in ecreased . In contrpfA, rpfB, rpfC, and rpfE (rpfD was upregulated); (iii) the Mce-1 virulence-associated transport system, which plays a role in fatty acid import and affects the lipid content of the cell envelope , KEGG, and manually curated pathways identified cellular processes that were significantly affected . We obseenvelope , 32, andenvelope ; (iv) ox10.1128/mBio.02333-17.7TABLE S4 TABLE S4, XLSX file, 0.1 MB.RNA (A) and protein pathway (B) enrichment analysis. Download Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis physiology, in particular, the cell envelope, and the secretion and transport systems, which together define the interaction of the cell with the extracellular environment.Pathways with significantly increased expression included those involving (i) the SigE regulon, which is activated in response to cell envelope stress; (ii) mycobactin biosynthesis, which produces a secreted siderophore for iron acquisition; (iii) genes controlled by two copper-regulated repressors, CsoR and RicR, which derepress their regulons in response to increased copper concentrations , 35; iv, suggestTo examine the extent to which these effects are specific to kinase inhibition, we first compared our data to published transcriptome-wide analyses of gene expression in response to first-line tuberculosis drugs and to multiple stresses , 37. The38–rpf expression was decreased in hypoxia, and rpfC gene expression was decreased with rifampin treatment and in macrophages.The hypoxia and nutrient depletion data and the kinase inhibitor data showed the greatest overlap. Pathways that showed substantial overlap for those two stress conditions included those associated with translation and ATP synthesis , type I NADH dehydrogenase, and succinate dehydrogenase (hypoxia only). Mce1 locus expression was highly decreased by hypoxia and partially decreased by nutrient depletion, macrophage infection, and rifampin exposure, whereas Mce4 gene expression was decreased in hypoxia and in macrophage infection. FASII gene expression was decreased during macrophage infection, Among the pathways with increased transcription in response to kinase inhibition, the SigE regulon was upregulated under conditions of hypoxia and to a lesser extent under conditions of nutrient depletion and macrophage infection. Expression of ESX1 secretion system genes and the mycobactin biosynthetic pathway was not consistently increased under any condition. Expression of the CsoR and RicR copper-regulated operons was increased in response to hypoxia. Expression of four MazF/VapC toxin genes was increased under one other condition, and increased expression was specific to kinase inhibition for four genes. In summary, in our results, the data corresponding to transcriptomic changes that are required for growth and the hypoxia and starvation data showed substantial overlap, but many other changes were more specific to kinase inhibition  and S2C.2FC values of >0.5 or ≤0.5 and P values of <0.001, expression levels of 110 proteins were significantly decreased and those of 82 proteins were significantly increased at 48 h of exposure to the kinase inhibitor , proteins with significantly increased abundance (B), and proteins with significantly decreased abundance (C). Download Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2FC data for each detected protein and the corresponding Log2FC data for each transcript  Log2FC for all quantified proteins and the Log2FC for each corresponding mRNA. (B) Log2FC of all detected proteins for which the RNA met change criteria . (C) Log2FC of RNAs for which the corresponding protein met change criteria . In panels B and C, the red dots indicate that both the RNA and protein data met the change criteria. Download FIG S3, TIF file, 0.1 MB.Plots showing LogCopyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pathway analysis  identifim/z and retention time values (molecular features) in each experiment. Unbiased comparisons of unnamed molecular features  showed that 1,259 (10.7%) met change criteria  after 48 h of treatment with the kinase inhibitor (m/z and retention time data matched those of named lipids in MycoMap (High-performance liquid chromatography-mass spectrometry (HPLC-MS)-based lipidomics identified over 10,000 unique nhibitor . Feature MycoMap  allowed  MycoMap . Similar MycoMap , ions co MycoMap , as well MycoMap .10.1128/mBio.02333-17.9TABLE S6 TABLE S6, XLSX file, 0.03 MB.Abundance of named lipids at each time point following exposure to kinase inhibitor (A), abundance of PIMS at each time point (B), and abundance of TDM at each time point (C and D). Download Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We noted a late but significant increase in mycobactin detection , consistm/z value of 1,270.89 and a retention time of 27 min , metabolite pathway enrichment analysis results (B), TCA and methycitrate cycle metabolite abundance in antibiotic-treated samples (C), and lysine biosynthesis metabolite abundance in antibiotic-treated samples (D). Download Copyright © 2018 Carette et al.2018Carette et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis cell wall lipids (prpD (Rv1130) and prpC (Rv1131) methylcitrate cycle genes, suggesting that the increased levels of methyl(iso)citrate were indicative of an increased level of flux and, hence, of catabolism of odd chain fatty acids through the methylcitrate cycle , bu, buM. tute cycle , 50.dapD (Rv1201c), which encodes tetrahydrodipicolinate N-succinyltransferase , were interpreted to be indicative of structurally linked changes in the PG layer. We noted accumulation of (S)-2,3,4,5-tetrahydropicolinate and reduction in levels of downstream lysine and DAP intermediates. This finding led us to examine the RNA-Seq data, where we identified a significant decrease in expression of nsferase . These fh arrest  48). Th. ThdapD 213 in the DNA binding domain of the MtrA response regulator whose abundance was significantly decreased in the inhibitor-treated samples  (fbpB-Rv1885-rpfC) operon  , suggest) operon , which e at 48 h . The genrculosis . Genes fsis MtrA , 54, alssis MtrA . With thsis MtrA . Thus, o213 is in a loop immediately carboxy terminal to the DNA recognition helix of MtrA , su, su213 ifbpB promoter region that contains a known MtrA binding site  needed for de novo mycolic acid biosynthesis as well as increases in the levels of metabolites associated with mycolic acid catabolism  and FASII pathway , which demonstrated a time-dependent, statistically significant decrease in TDM levels during kinase inhibitor-induced bacteriostasis  and 0.05% Tween 80 to an optical density at 600 nm (OD600) of 0.5. Using this culture, two cultures (one in 7H9-ADN plus 0.05% Tween 80 and the other in 7H9-ADN) were inoculated at an OD600 of 0.05 after two washes in 7H9-ADN. When the Tween 80-containing culture attained an OD600 of 0.3, M. tuberculosis-laden filters were prepared by passing 1 ml of the Tween-free culture by vacuum filtration through polyvinylidene difluoride (PVDF) membranes   . The filKi of the active inhibitor and inactive analogue was determined for several M. tuberculosis STPKs . For PknD, PknE, PknF, PknH, and PknK, myelin basic protein was used as the substrate. Ki values were determined by fitting data to the Morrison tight-binding equation  in 96-well microtiter plates, performed in triplicate as previously described . For MICescribed .Triplicate filter-based cultures were harvested at each time point. The bacteria were scraped from each filter directly into TRI reagent (Molecular Resource Center), and total protein extracts were prepared as previously described . Protein18-AQ; 200-µm inner diameter by 0.5-mm length, 3-µm pore size, 120 Å) and a nano-cHiPLC column  were used. Peptides were eluted with a 60-min linear gradient from 93% buffer A (water with 0.2% formic acid) and 7% buffer B (acetonitrile with 0.2% formic acid) to 68% buffer A. The injection volume was 4 µl.Samples were dissolved in 12 µl loading buffer  and sonicated for 5 min. Samples were placed in an autosampler linked to a nanoflow HPLC system  and a Q Exactive mass spectrometer. For peptide separation, a nano-cHiPLC Trap column  data acquisition, a normalized collision energy value of 27 was used. Scans were carried out at a resolution of 35 K with an AGC target of 3 × 106 ions and a maximum injection time of 120 ms. The isolation window was set to 2 m/z. An underfill ratio of 0.5% was set and a dynamic exclusion value of 20 s applied.A Q Exactive mass spectrometer was run in positive-ion mode. Full scans were carried out at a resolution of 70 K with an automatic gain control (AGC) target of 3 × 10Data analysis was performed using Maxquant (version 1.5.2.8) and H37RV version 2 protein sequence, downloaded from PATRIC . Phospho2FC, accounting for experimental batch effect as a covariate in the linear model. P values were adjusted using the Benjamini-Hochberg method.The phosphopeptides were normalized by the sum intensity value for each sample. To determine the effects of the active inhibitor versus the control compound on phosphopeptide abundance, we adapted metagenomeSeq , which a2FC values and P values and Benjamini-Hochberg-adjusted P values for the comparison between the active inhibitor and control compound data at each time point were estimated using metagenomeSeq as described for the phosphoproteomics analysis. Protein expression enrichment for functional categories was performed on the Log2-transformed, quantile-normalized data using the Romer procedure as implemented in limma with 9,999 rotations, correcting for batch effects between experiments ; background central character, none. To identify motifs overrepresented in phosphopeptides that had significantly increased or decreased levels of phosphorylation after treatment with the inhibitor , we reran motif-x on the subset of phosphopeptides meeting these criteria. All motif-x parameters were the same as those used for the analysis of all phosphopeptides, except for the “number of occurrences” value, which was changed from 20 to 5 due to the smaller number of phosphopeptides that were significantly increased or decreased in abundance in response to the inhibitor.To identify motifs in the set of unique phosphopeptides, we used motif-x , with thTriplicate samples prepared for protein extraction as described above were used for RNA extraction. Total RNAs were extracted using a Direct-zol RNA MiniPrep kit  according to the manufacturer’s instructions. The quantity and the quality of total RNAs were determined by measuring absorbance at 260 and 280 nm using a NanoDrop instrument (Thermo Fisher Scientific). The integrity of the total RNAs was assessed by the use of a Bioanalyzer in the BCH IDDRC Molecular Genetic Core. Two micrograms of total RNAs was subjected to rRNA depletion using a Ribo-Zero rRNA removal kit . The reaction cleanup was performed using an RNeasy minikit . The cDNA library was prepared using a NEBNext Ultra Directional RNA Library Prep kit for Illumina , NEBNext multiplex oligonucleotides for Illumina (index primers set 1) , and NEBNext multiplex oligonucleotides for Illumina (index primers set 2)  according to the manufacturer’s instructions. The quantity and the quality of cDNA library were determined by Bioanalyzer analysis.2FC values, P values, and Benjamini-Hochberg-adjusted P values comparing active inhibitor data to control data, treating batch effects as a covariate.RNA-Seq was performed at the Dana Farber Cancer Institute Center for Cancer Computational Biology (CCCB) core facility on an Illumina NextSeq 500 sequencer set to obtain 75-base single reads (SR75). RNA-Seq data were aligned using Spliced Transcripts Alignment to a Reference (STAR) software  to the Hhttp://networks.systemsbiology.net/mtb/). Gene ontology (GO) and KEGG Pathway functional categories were downloaded from PATRIC  ultra-high-performance column with electrospray TOF MS detection in the negative mode, using 1-propanol and cyclohexane as the eluting mobile phase in solvent B, water and methanol in initial solvent A, and 2 mM ammonium acetate in both solvents. For higher levels of phosphatidylmannosides, up to the hexamannoside, a gradient of solvent A (1:19 water/methanol) and solvent B (9:1 1-propanol/cyclohexane) was run as follows: the initial eluent was 100% solvent B, and, after 4 min, the ratio was linearly increased to 100% solvent B at 14 min and held at 100% solvent B until min 18. To equilibrate for the next run, solvent A was run at 100% for 20 min.For targeted analysis of very polar lipids, including phosphatidylinositol mannosides, a reversed-phase method was developed using an Agilent Poroshell 120  and solvent B (7:3 1-propanol/cyclohexane) as described above. Chloroform (0.25 ml) was added to the lipid extract to improve lipid solubility followed by addition of solvent C to make a final concentration of 1 mg/ml. Runs started with 80% solvent B until min 2 with a linear increase to 100% solvent B at min 6, continuing at 100% solvent B until min 20, with a linear decrease to 80% solvent B at min 20 to min 22.Triplicate samples were harvested at serial time points as described above. Metabolite extraction and metabolomics analysis were performed as previously described .mtrA was obtained by PCR using M. tuberculosis H37Rv chromosomal DNA as the template and primers XC202 and XC203. The amplicon contained NcoI and HindIII restriction sites for cloning into pET28a to express an N-terminally His6-tagged MtrA. Mutations Y102C, T213A, and T213D were introduced by the use of a QuikChange XL site-directed mutagenesis kit  and, respectively, primers XC208 and XC209, primers XC210 and XC211, and primers XC212 and XC213. All alleles were confirmed by sequencing of the entire gene.Wild-type 6-MtrA proteins were produced in E. coli BL21 (DE3) pLysS  as previously described (MRW) in degrees per square centimeter per decimole.Hisescribed . The valfbpB promoter region (PfbpB) was analyzed by EMSA using a Cy3-labeled PfbpB probe sequence  and XC220 to XC221, respectively. The reaction buffer consisted of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM KCl, 10 mM dithiothreitol (DTT), 5 µM EDTA, 5% glycerol, 0.05% NP-40, 2 µg poly(dI) ⋅ poly(dC), 200 fmol probe, and 100 pmol MtrA (probe/protein molar ratio of 1:500). The samples were incubated for 30 min at room temperature and resolved by 7.5% TG native gel electrophoresis . The gel was scanned using a Kodak Image Station 4000MM system.Binding of the protein to the sequence . The proGSE110508. Proteomics data are in PXD008968. Lipidomics data are provided in Transcriptomics data are in


The observational items compatible with disorganization in the original NOMAS were divided into three groups: cluster 2 , cluster 3 , and cluster 4  and further divided into incoordination-positive (cluster 4) and incoordination-negative groups (clusters 2 and 3). Premature infants in the incoordination-positive group  showed a median transition time of 22 days (range: 4–121 days) which was longer than that in the incoordination-negative group . Univariate linear regression analysis revealed that the presence of incoordination among disorganized sucking patterns (NOMAS cluster 4 vs. clusters 2 and 3), birth weight, total parenteral nutrition (TPN) duration, non-invasive positive pressure ventilation duration, the presence of moderate to severe bronchopulmonary dysplasia, pulmonary hypertension, sepsis, small for gestational age (SGA), and necrotizing enterocolitis are associated with the transition time to FOF. In a multivariate linear regression analysis, the variables revealed to be associated with the transition time were TPN duration, SGA, and the presence of stress signals (incoordination-positive group) among disorganized sucking patterns. When selecting premature infants to be treated with swallowing therapy, it is reasonable to pay more attention to the incoordination-positive group described in the NOMAS, that is, premature infants with stress signals to shorten the time to attain FOF.Several treatments have been proposed to shorten the time to the attainment of full oral feeding (FOF) for premature infants, but there are only a few evaluation methods useful in estimating predictors of this period. We investigated whether specific items within the disorganized sucking patterns described by the Neonatal Oral-Motor Assessment Scale (NOMAS) could estimate the time to FOF in preterm infants with feeding difficulty. Preterm infants diagnosed with a disorganized sucking pattern in the NOMAS evaluation before 50 weeks of postmenstrual age were included. Video recordings of at least 2 min of oral feeding were further analyzed retrospectively by two assessors and the premature infants who exhibited disorganized sucking patterns ( Achieving full oral feeding (FOF) early in preterm infants can shorten hospitalization time, reduce hospital costs, and enable greater interaction between the mother and child –3. StratThe normal sucking pattern is the coordination of suck, swallow, and respiration (SSR), which means that the jaw and tongue make rhythmic movements. The term “feeding difficulty” is used to express the problem of swallowing in the broad sense, including all states that do not allow FOF. If the abnormality of oromotor function is thought to be the cause of feeding difficulty, the evaluation is often performed to distinguish between the disorganized sucking pattern and the dysfunctional sucking pattern to interpret feeding difficulty –13. The The Neonatal Oral-Motor Assessment Scale (NOMAS) is a method of visual observation that is used to assess non-nutritive and nutritive sucking in an infant from birth to 48 weeks of postmenstrual age (PMA) , 12. TheThe observational items compatible with disorganization in the original NOMAS were divided into three groups according to the presence of arrhythmical, unable to sustain, and the incoordination items : clusterObservational findings of the “incoordination of SSR that results in stress signals” in the NOMAS include nasal flaring, head turning, and extraneous movements of the body or limbs during sucking, as the description in the study by Palmer et al. . The NOMIn this study, we investigated whether the stress signals during bottle feeding were associated with a longer time to FOF in preterm infants who showed feeding difficulty at the beginning of the oral feeding.Between May 2014 and March 2017, 148 infants in Neonatal Intensive Care Unit (NICU) were referred for consultation to the Division of Pediatric Rehabilitation for feeding difficulty during the transition period from enteral tube feeding to oral feeding. Regarding all infants who were referred, a video recording was done for over 2 min, which was a necessary preparation for the NOMAS evaluation , 16. InfAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical approval was obtained from the Seoul National University Hospital Institutional Review Board (No. 1705-122-855).The following parameters were investigated by retrospective medical records analysis: birth weight (BW), gestational age (GA) at birth, sex, Apgar score at 1 and 5 min after birth, total parenteral nutrition (TPN) duration, and PMA at the time of the NOMAS assessment Table . We alsoRegarding all infants who were referred, a video recording of bottle feeding was done for over 2 min, which was a necessary preparation for the NOMAS evaluation. The caloric density of breast milk was variable and known to be about 57–65 kcal/100 ml . PrematuFindings from the 28 items in the NOMAS were categorized into five clusters according to the suggestions of the Dutch NOMAS working group. An arrhythmic sucking pattern in isolation was classified as cluster 2. The NOMAS cluster 3 was defined as adding the “unable to sustain” item to cluster 2, which was defined by at least one of the following findings: (1) the infant stops sucking completely in the first 2 min of nutritive sucking; (2) the pause is longer than the burst; or (3) the bursts are shorter than three sucks. Cluster 4 refers to the addition of at least one of the “incoordination” items to cluster 2 or 3. Incoordination items defined by the Dutch group include stress signals, such as head bobbing, extraneous movements of the body or limbs during sucking, choking, gagging, coughing, yelping, and grunting . DisorgaThe primary outcome was the transition time from the initiation of oral feeding (IOF) to FOF according to the presence of the incoordination item among the disorganized sucking patterns. FOF was defined as the ability to feed three or more times only by bottle feeding, which corresponds to the time of removing the nasogastric tube .U test. For the analysis of the categorical variables, the chi-square test or Fisher’s exact test was performed. A p-value < 0.05 was considered statistically significant. Cohen’s kappa was obtained for the interrater reliability of the two evaluators in the cluster level and the presence of the incoordination item.We analyzed the data from the presence of the stress signals  among disorganized sucking patterns in terms of transition time to FOF and baseline characteristics. The continuous variables, including transition time, were compared between the two groups (cluster 4 vs. clusters 2 and 3) using the Mann–Whitney p < 0.05, removal condition p > 0.15). Analyses were performed using the IBM SPSS 20 software .Univariate linear regression analysis was performed with the transition time to FOF as the dependent variable. Afterward, the multivariate linear regression was carried out through a stepwise selection . Disagreement occurred between clusters 3 and 4 (three infants) and clusters 2 and 3 (three infants). The reliability of the two evaluators on the presence of the incoordination items (cluster 4 vs. cluster 2 or 3) was higher with the Cohen’s κ of 0.933, with only three disagreements occurring.n = 6) or because the NOMAS assessment point was more than 72 h after oral feeding initiation (n = 12). Of these 111 preterm infants, 109 preterm infants belonged to the disorganization group and were included in our study: incoordination-negative group (clusters 2 and 3) and incoordination-positive group  , and 5 infants with oromotor dysfunction  were excluded because the PMA at the time of the NOMAS assessment exceeded 50 weeks, or because GA exceeded 37 weeks. Of the remaining 129 NOMAS evaluation records, 18 cases were further excluded because the infants received postnatal surgery resulting in the interruption of oral feeding  among the disorganized sucking patterns are summarized in Table The median [range] transition time to FOF of 109 preterm infants was 9 days 1–121 days]. As shown in Figure 1 days. AR value of the final model was 0.699, and the included variables were TPN duration, SGA, and the presence of incoordination items (NOMAS cluster 4 vs. clusters 2 and 3) , showed a longer transit time to FOF than did those of the incoordination-negative group (clusters 2 and 3).An interesting finding is that patients with SSR coordination reached early FOF quicker than did those with incoordination. For successful oral feeding, premature infants should achieve maturity with developments in the central and enteric nervous system-mediated reflexes involved in the activation, control, coordination, and adaptation of oromotor, aerodigestive, and peristaltic functions –22. In cThe stress signals observed in the premature infants may be due to incomplete maturation of the aerodigestive system. Jadcherla et al. reported that swallow-integrated esophageal motility is an important factor in distinguishing between a primary oral feeder and a chronic tube feeder . SwallowAlthough there might be a relationship between the incoordination of the SSR and the NOMAS cluster 4 , the meaning of these two items is different. Therefore, the results of this study could be interpreted as follows: the stress signals in the NOMAS during nutritive sucking in preterm infants are more associated with a longer transition time. Therefore, if sucking patterns in premature infants are compatible with the NOMAS cluster 4 , active oromotor facilitation and swallowing training, such as applying pacifier, non-nutritive oral-motor stimulation, massage therapy, and direct tactile stimulation on specific oral structures, could be considered as supplemental oral sensory and motor experiences, and as a result, shorten the transition time to FOF. Also, giving the infant time for maturation could be considered.Neonatal Oral-Motor Assessment Scale defines incoordination as an observational finding, while Lau et al. objectively evaluate these findings through a catheter with a pressure sensor . During Lau and Smith  also evaPrevious studies have reported that GA, BW, and medical conditions, such as BPD, cardiac, gastrointestinal, and neurological conditions were factors which could affect the time to reach FOF , 27–29. Bingham et al. reported that the NOMAS was a poor predictor  and noteJadcherla et al.  reportedThere have been inconsistent results from studies regarding the implication of feeding difficulty on the neurodevelopmental outcomes in preterm infants –34. ThisThere are some limitations to this study. Since instrumental evaluation was not included and measurements were only made through video recordings, it is possible that the process of sucking was not objectively evaluated. If manometry, pulse oximetry, and plethysmography had been added through a multidisciplinary approach, a more integrated interpretation might have been possible. This study examined the time to reach FOF from the IOF but did not evaluate the long-term feeding performance or neurodevelopment outcome of preterm infants. In this study, the stress signals of the incoordination-positive group included head bobbing, extraneous movements of the body or limbs, choking, gagging, coughing, yelping, and grunting. However, each stress signal is thought to be different. For future studies, incoordination symptoms could be further classified into different subcategories and investigated for their relative implications upon oral feeding and development.The stress signals reflecting incoordination of SSR among disorganized sucking pattern are associated with the transition time to FOF in preterm infants with feeding difficulties. When selecting premature infants to be treated with swallowing therapy, it is reasonable to perform treatment to shorten the time to attain FOF with an incoordination-positive group in the NOMAS, that is, premature infants with stress signals.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical approval was obtained from the Seoul National University Hospital Institutional Review Board (IRB) No. 1705-122-855.YY: acquisition of data; analysis and interpretation of data; analysis and interpretation of data; and critical revision of manuscript. B-MO: acquisition of data; study concept and design. SS: acquisition of data; study concept and design. JS: acquisition of data. E-KK: acquisition of data; study concept and design. H-IS: study concept and design; acquisition of data; analysis and interpretation of data; study supervision; and critical revision of manuscript for intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


East Asia experienced a record-breaking cold event during the 2015/16 boreal winter, with pronounced impacts on livelihood in the region. We find that this large-scale cold spell can be attributed to the concurrent super El Niño event in the tropical Pacific. Our analysis reveals that all super El Niño winters  were accompanied by a rapid sub-seasonal North Atlantic Oscillation (NAO)/Arctic Oscillation (AO) phase reversal from a positive to a negative state during early January, which was largely caused by the interaction of these super El Niño events with the subtropical jet annual cycle. The NAO/AO phase transition leads to a rapidly strengthened Siberian High, which favors southward intrusions of cold air to East Asia and thus causes severe local cooling. Similar cold spells can also be detected over Northern Europe associated with the fast sub-seasonal NAO/AO phase reversal. Due to the weaker amplitude of the ENSO forcing, these sub-seasonal atmospheric responses cannot be detected for moderate El Niño events. The super El Niño associated sub-seasonal signal of the East Asian and Northern Europe wintertime temperature responses carries important implications for future predictability of regional extreme events. Despite a long-term global warming trend, Eurasia has been suffering from more frequent extreme cold surges in recent years4, which often results in heavy snowfall and freezing precipitation with tremendous impacts on human societal and economic activities5. During the 2015/16 boreal winter, East Asia first experienced remarkably warmer than normal surface air temperatures (SAT) in the early winter season. However, an extreme cold spell swept subsequently across East Asia during mid-to-late January 2016  on seasonal-to-interannual climate variability in East Asia and Europe14. An anomalous low-level anticyclone is evident over the western North Pacific (WNP) during El Niño winter seasons, which results in the transport of more moist and warm air masses northward to East Asia and thus warmer and wetter than normal winter conditions13. Additionally, El Niño tends to cause a negative NAO phase, which results in a colder and drier than normal climate during the late winter season in Europe8. Moreover, the ENSO phase is also closely related to the interannual variability of cold surges. For instance, more frequent cold surges occur in East Asia during El Niño winters and less during La Niña winters, due to the modulation of the ENSO associated short-wave train across the North Pacific15. However, few studies investigated possible effects of El Niño on the strong sub-seasonal winter climate variations. Especially, the question whether the concurrent super El Niño was responsible for the strong cooling event during the 2015/16 winter is not answered. Here, we explore a possible dynamical linkage between El Niño and strong wintertime SAT cold surge events over East Asia and Northern Europe. Our main finding is that super El Niño winters are generally accompanied by a pronounced North Atlantic Oscillation (NAO)/Arctic Oscillation (AO) phase reversal from a positive to a negative state in early January (not observed during moderate El Niño winters) that gives rise to the severe cooling in both East Asia and Northern Europe in the subsequent mid-to-end January period.During the 2015/16 boreal winter, we also witnessed a super El Niño event (by the Niño 3.4 measure) in the tropical Pacific, with comparable amplitude to the well-known 1982/83 and 1997/98 extreme El Niño events13, this relationship exhibits a large degree of uncertainty. For instance, no prominent warm DJF-mean temperature anomalies were observed in East Asia during two super El Niño events (1982/83 and 2015/16)  from 1979 to 2015 Fig. . Conspic16) Fig. . During 16) Fig. . It can th . The severe cold anomalies superimposed on the lowest climatological SAT can therefore potentially lead to catastrophic consequences.Next we utilize a composite analysis for all (both super and moderate) El Niño events to investigate their possible impacts on East Asian sub-seasonal SAT variations. East Asia shows only a very weak and spatially confined warm SAT anomaly in the early winter  and cold (P2: January 12th to 25th) periods are defined based on the sub-seasonal phase transition of the SAT indices as shown in Fig. A scientific question that remains to be answered is why the super El Niño events are accompanied by a severe sub-seasonal SAT phase reversal over East Asia and Northern Europe. To address this question, we first show the anomalous atmospheric circulations for the anomalous warm (P1: early winter) and the following anomalous cold (P2: middle winter) periods Fig. . Here, t19. Earlier studies have shown that the NAO is a dominant factor controlling East Asian and Northern European cold surge events23. In a winter season coinciding with a positive NAO phase, the intense polar vortex and strengthened mid-latitude winds tend to confine the cold polar air to high latitudes. In contrast, during a winter with a negative NAO phase, the weak polar vortex and weakened mid-latitude winds more easily allow southward intrusions of cold polar air southward into mid-latitude regions26. Over Northern Europe, the negative NAO phase is usually concurrent with weakened Atlantic westerlies, which inhibits warm air transport from the Atlantic Ocean towards Europe. Thus, the NAO phase transition seemingly acts as a bridge to connect super El Niño events and the rapid sub-seasonal transition of winter SAT.During the anomalous warm period (P1) of the super El Niño winter composite, an anomalous low-level anticyclone over the western North Pacific (WNP) and a pronounced positive NAO/AO pattern in the North Atlantic region are evident, which tend to transport more water vapor and warm air from the adjacent ocean towards land, respectively. One can also identify a significantly weakened Siberian High and East Asian trough Fig. . In cont28. This sub-seasonally varying ENSO-NAO relationship could be responsible for the weak ENSO-NAO correlation during the boreal winter season mentioned in previous studies30. Here, this sub-seasonal atmospheric reversal is further confirmed by the super El Niño composite zonal-mean sea level pressure (SLP) response over the North Atlantic region  boundary conditions from 1979 to 2008, a period that includes two super El Niño (1982/83 and 1997/98) and seven moderate El Niño events . The composite zonal-mean SLP sub-seasonal variability over the North Atlantic is displayed for the two super and seven moderate El Niño winters . This timing inconsistency may be ascribed to intrinsic model biases and deficiencies. We also examine the anomalous Northern Hemisphere SAT difference after and before the NAO phase transition date during the composite super El Niño winter in the HIRAM model simulations  simulations. The Geophysical Fluid Dynamics Laboratory global High Resolution Atmospheric Model  is utilized to examine whether the seasonally dependent NAO response during super El Niño winters can be simulated. The experiment is forced with Atmospheric Model Inter-comparison Project (AMIP)-styleers Fig. . The moders Fig.  despite ons Fig. . Remarkaons Fig.  nor the th, exhibiting a similar timing as in the observations. Meanwhile, prominent SAT cooling signals are also detected, with 1.1 °C and 2.4 °C SAT decreases after the NAO phase reversal in East Asia and Northern Europe, respectively (not shown). These results demonstrate a convincing dynamical linkage between super El Niño events, the rapid sub-seasonal phase reversal signals of the NAO, and East Asian and Northern Europe SAT.We further conduct two sets of AGCM experiments using the GFDL Atmospheric Model, version 2.1 (AM2.1) (see the Methods for details) to investigate whether the sub-seasonal SAT anomaly and NAO phase evolution are mainly related to super El Niño-SST over the tropical Pacific. The simulated SLP response over the North Atlantic to super El Niño forcing also realistically reproduces the observed NAO phase reversal signal from positive to negative during the winter season Fig. . The NAO34, a modulation by the delayed tropical North Atlantic (TNA) SST warming35, or variations of the tropospheric subtropical jet waveguide36. Next we investigate these three mechanisms individually. Neither a significant stratospheric downward propagation nor a significant delayed TNA SST warming signal can be detected  and NOAA high-resolution blended analysis of Optimum Interpolation SST version 238 (OISST v2) respectively. The 2-metre (T2m) temperatures are utilized to represent the SAT. The monthly and daily SAT and the associated atmospheric circulation variables used to investigate the processes of wintertime cooling events are obtained from the European Centre for Medium-Range Weather Forecasts (ECMWF) Reanalysis (ERA)-Interim dataset39. The ERSST v4, OISST v2 and ERA-Interim data are derived from January 1979 to February 2016 and are globally at 2.0° × 2.0°, 0.25° × 0.25° and 1.5° × 1.5° grid resolutions, respectively.The monthly and daily SST data are obtained from the National Oceanic and Atmospheric Administration (NOAA) Extended Reconstructed SST version 435 is defined as an area average of the SST anomalies over 0°–25°N, 80°W–0°. The NAO index is defined by the difference of normalized SLP anomaly between 35°N and 65°N over the North Atlantic sector 40. An AO index is calculated by subtracting zonal mean SLP at 65°N from zonal mean SLP at 35°N41. The daily evolution of SAT during December and January is the focus of this study since the rapid temperature transitions appear in early January. El Niño events are selected based on a threshold of 0.5 °C for the Niño 3.4  SST anomaly. We therefore identified 11 El Niño winters . Among them, three events  are classified as super El Niño events since their corresponding Niño 3.4 SST anomalies reach 2 °C during their mature phases. The remaining El Niño events are referred to as moderate El Niño events. All statistical significance tests were performed using the two-tailed Student’s t test and the effective degrees of freedom were determined by considering the autocorrelation of the respective time series.Anomalies for all variables were computed as the deviation from the long-time climatological mean. A 5-day moving average filter is performed to exclude the high frequency variability during the boreal winter season when analyzing the daily data. We also used 7-day and 9-day moving average filters and the qualitative results remain unchanged (not shown). We defined an East Asian SAT index as the SAT anomalies averaged over the region of 25°–55°N and 105°–130°E to approximately represent the variability of East Asian SAT. Additionally, a Northern Europe SAT index is calculated as the average of the T2m anomalies over the region of 60°–80°N and 20°W–30°E. The TNA SST index42. We utilize an ensemble average of three simulations with perturbed initial conditions for better reliability.To verify the connection between super El Niño and regional wintertime cooling events, the AMIP simulations of GFDL HiRAM are used. The model uses a finite-volume dynamical core on a cubed sphere grid with each face consisting of a 180 by 180 grid, with 32 levels in the vertical dimension. The model’s physics parameterizations are similar to GFDL’s AM2.1 model, with modifications deemed appropriate for the increased resolution43. These experiments are referred to as the “CTRL” (control), “Super El Niño” run, etc. The first set of experiments is forced with a global annual cycle SST while the others have the same annual cycle but with specific El Niño SST anomalies (from the developing year June to decaying year February) added in the tropical Pacific  (see Supplementary Table Moreover, to further confirm the relationship between El Niño forcing and sub-seasonal NAO phase reversal, five sets of experiments are performed based on the GFDL AM2.1, with a horizontal resolution of 2.5° × longitude 2° latitude with specified SST boundary conditionsSupplementary Information


This study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA).Kic), quenching constant (KFQ) and binding constant (Kitc).The interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/The combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA. BTEblack tea extractsCcatechinDSCdifferential scanning calorimetryECepicatechinECGepicatechingallateEGCepigallocatechinEGCGepigallocatechingallateFQfluorescence quenchingGTEgreen tea extractsITCisothermal titration calorimetryKiccompetitive inhibition constantKiuuncompetitve inhibition constantKFQfluorescence quenching constantKitcbinding constantOTEoolong tea extractsPPAporcine pancreatic α‐amylaseTAtannic acidTFtheaflavinTF1theaflavin‐3’‐gallateTF2theaflavin‐3, 3’‐digallate1Dietary phenolic compounds have been suggested as potential alternatives to medicines for controlling and treating type II diabetes, a chronic disease caused by reduced insulin sensitivity, as they have inhibitory effects on α‐amylase 50), kinetics of inhibition, fluorescence quenching (FQ) and docking analysis 50 indicates the strength of inhibition, i.e. lower IC50 value means higher inhibitory activity. From the kinetics of inhibition, the inhibition type can be identified, and competitive inhibition constant (Kic) and uncompetitive inhibition constant (Kiu) are obtained KFQ) indicating the binding affinity of polyphenols to the enzyme is obtained from the quenching effects of polyphenols on the intrinsic fluorescence of tryptophan side chains within the structure of α‐amylase KFQ and 1/Kic needs further study, as both constants suggest the binding of polyphenols with the enzyme Previously, the interactions between polyphenols and α‐amylase have been characterized by determination of the half inhibition concentration  can be applied to monitor phase and conformational transitions through measurement of specific heat capacity as a function of temperature for a sample 50, Kic and Kiu) of PPA by a range of tea polyphenols were systematically studied Kic, KFQ and Kitc are compared and correlated to analyze the relationships between PPA inhibition and binding behavior. The role of the galloyl moiety in binding of catechins and theaflavins with PPA is highlighted.In our previous study, the kinetics of inhibition (IC22.1Aqueous extracts of various teas (TE), green (GTE), black (BTE) and oolong (OTE) were prepared as described 2.2Kic and uncompetitve inhibition constant, Kiu, Dixon (1) and Cornish–Bowden (2) equations were applied as follows v is the initial reaction velocity determined in the experiment, Vmax is the maximum initial reaction velocity, a is the concentration of starch, mK is the Michaelis constant, i is the concentration of inhibitor, Kic is the competitive inhibition constant and Kiu is the uncompetitive inhibition constant.The kinetics of inhibition of PPA by TEs and tea polyphenols were as reported in our previous research 2.3® spectrofluorimeter  according to a previously reported method ex) set as 282 nm and the emission λem recorded from 300 to 500 nm. Both the slit widths were 10 nm.Fluorescence spectra of PPA in the absence and presence of TEs and pure polyphenols were recorded by using a Shimadzu0F and F are the fluorescence intensity in the absence and presence of the quencher (TE or tea polyphenol), respectively; qk is the bimolecular quenching constant, τ0 is the lifetime of the fluorophore. For α‐amylase, the τ0 is 2.97 ns Q] is the concentration of the quencher; KFQ is the fluorescence quenching constant.Fluorescence quenching is described by the Stern–Volmer equation:0/FF against [Q] describes an upward curve, concave towards the y axis. Commonly, the upward curvature indicates that there are several mechanisms responsible for the quenching effects on fluorophores in protein, or it suggests the existence of a ‘sphere of action’, i.e. apparent static quenching Usually, a linear Stern–Volmer plot indicates that there is a single class of fluorophore in the protein interacting with the quencher in the same way and that only one quenching mechanism (dynamic or static) takes place. However, positive deviations for the equation are frequently observed when the quenching extent is large. In this case, the plot of 2.4® Instrument, New Castle, DE). Solutions of eight individual phenolic compounds (60 mg/mL), concentrations of the three TEs  and 47 mg/mL PPA were prepared in PBS (20% DMSO) buffer. The DSC procedure was performed according to a previous method 13 with some modifications: 50 μL of PPA and 50 μL of each polyphenol (or TE) solution were mixed thoroughly in a 1.5 mL microcentrifuge tube. After incubation for 30 min at 4°C, 15 μL of the mixture was pipetted into a TA® Tzero pan and tightly sealed with a Tzero Hermetic lid. 15 mg of each sample solution was loaded. The control was PPA with PBS (20% DMSO) buffer. Thermograms were recorded from 10 to 100°C with 5°C/min heating rate, using an empty pan as the reference. The enthalpy values (∆H) were calculated based on the mass of protein in DSC sample pans (J/ g protein).The thermostability of the PPA control and that bound with TEs and pure phenolic compounds was studied by use of a differential scanning calorimeter  was used to determine enthalpy changes associated with polyphenol–PPA interactions at 298 K. In a typical ITC experiment, a polyphenol solution in the injection syringe was titrated into 1.175 mg/mL PPA solution in the sample cell of the calorimeter with stirring at 1000 rpm. All the solutions were prepared in PBS buffer. Each polyphenol solution  was titrated as a sequence of 20 injections. The volume of each injection was 2 μL and the duration of each was 4 s. The time delay between the injections was 150 s. Control experiments included the titration of polyphenol solutions into PBS buffer, the titration of PBS buffer into PPA solution and the titration of PBS buffer into PBS buffer. The latter two control experiments resulted in no measurable enthalpy changes; therefore, they were not considered in the data analysis −1) against time (min). Then, the data was integrated peak‐by‐peak and normalized using MicroCal Origin  to obtain a plot of corrected enthalpy change per mole of injection (∆H kJ mol−1) against molar ratio of polyphenol to PPA (or weight ratio of TEs to PPA). The data obtained were fitted using a single‐site binding model. The equation for this binding model is iQ is the total heat released after injection i, 0V is the volume of the calorimeter cell, [M] is the total concentration of PPA, [P] is the total concentration of polyphenols, n is the molar ratios of interacting species, ∆H is the enthalpies, Kitc is the equilibrium binding constants.A GE2.6t‐test at the 95% significance level (P < 0.05). The ITC data were analyzed to obtain the binding parameters and fit standard deviation using MicroCal Origin software.The data in this study are expressed as the means of duplicates and analyzed through one‐way analysis of variance (ANOVA) using SPSS 18.0 Statistics . The mean values were evaluated by Dunnett's 33.150 values, which are shown together with values of Kic and Kiu in Supporting Information Table S1 as reported previously In our previous study 3.2λex = 282 nm with addition of three TEs;  and eight pure phenolic compounds . In all cases a decrease in the fluorescence intensity, albeit to different extents, was observed from the quenching. Notably, there was a red‐shift of the maximum λem for PPA with three TEs, EGCG, ECG, TA and TF2, while no significant shift was observed for EC, TF1, TF or EGC. The Stern–Volmer plots presented for EC, TF1, TF and EGC showed a linear character, and the plots for TEs, EGCG, ECG, TA and TF2 had an upward curvature, concave toward the y axis  for TEs and pure polyphenols are summarized in Table KFQ values for TEs and pure polyphenols are GTE>BTE>OTE and TF2>TA≈TF1>TF>ECG>EGCG>EC. Both these orders are similar to those of the inhibitory activities of these compounds against PPA evaluated by IC50 values (Supporting Information Table S1).The fluorescence intensity of PPA before and after addition of tea polyphenols was determined to investigate the interactions between them. Figure 3.3dT) at 67.54°C and denaturation enthalpy (∆H) of 26.91 J/g protein . The lowest dT of PPA bound with BTE and OTE were also observed at their respective highest concentrations and determined as 64.21 and 64.40°C, respectively. Generally, the ∆H of PPA bound with TEs was lowered as well. A lower ∆H demonstrates that a smaller amount of energy is required to unfold the PPA molecule. Together with the decreased dT by TEs, this indicates that PPA molecules bound with TEs were less thermally stable than non‐bound PPA. For pure phenolic compounds,the dT of PPA bound with TA, ECG, EGCG and TF2 decreased by 5.55, 2.39, 2.21 and 1.80°C, respectively, while TF, TF1, EGC and EC had no significant effects on the dT of PPA. Consistent with the changing tendency of dT, the ∆H of PPA bound with TA, ECG, ECCG and TF2 decreased to 18.40, 21.32, 20.39 and 23.15 J/g protein, respectively. However, the ∆H values for TF, TF1, ECG and EC did not change significantly.DSC was applied to assess interactions with PPA in terms of thermal stability. The effects of TEs and pure polyphenols on DSC characteristics of PPA are presented in Fig. 3.4ITC is a powerful technique to analyze the thermodynamics of binding of polyphenols with proteins Kitc (11600 M−1) and ∆H (−8790 J mol−1), indicating the strongest binding with PPA and the greatest amount of heat released during the binding process. Similarly, EGCG was shown to have the weakest interaction with PPA with the lowest values of Kitc (482 M−1) and ∆H (−988 J mol−1). In addition, TA showed the lowest, and ECG showed the highest stoichiometry (n), i.e. the polyphenol to PPA ratio.The single‐site binding model that assumes a single set of multiple binding sites 4The difference in fluorescence quenching activities of three TEs indicates that the changes in the micro‐environment of Trp residues caused by interactions of TEs and PPA may depend on the phenolic constituents in TEs. In comparison with the data of PPA inhibition, it was found that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity.qk, which reflects the efficiency of quenching or the availability of quenchers to fluorophores, can be used to determine if the quenching results from complex formation between proteins and quenchers. The qk is close to 1 × 1010 M−1 s−1 for the typical dynamic mechanism (collision‐controlled quenching) qkvalues of EC, TF1 and TF are 9‐150‐fold higher than that given in Table KFQ values  between the two compounds Kic, represents the dissociation constant of the PPA–polyphenol complex; therefore, the reciprocal of Kic (1/Kic) indicates the association constant of polyphenols with PPA. As discussed above, quenching constant, KFQ, indicates the binding affinity of a quencher to protein; therefore, if the quencher binding is related to enzyme inhibition, there should be a relationship between KFQ and 1/Kic. Based on this concept, the correlation between KFQ and 1/Kic is shown in Fig. KFQ = 13.97·1/Kic + 1517.1, R2 = 0.9590), suggesting that lower Kic corresponds to higher KFQ. Hence, KFQ and 1/Kic obtained through fluorescence quenching and inhibition kinetics methods, respectively, may be combined to characterize the binding of polyphenols with PPA. FQ reflects the change of micro‐environment in the vicinity of Trp residues caused by both collisional encounters and complex formation between Trp and quenchers. In addition, other amino acid residues near Trp may also affect the fluorescence of a fluorophore Kic definitely reflects the formation of enzyme–inhibitor complex KFQ against 1/Kic) is much higher than 1. Previous studies also suggest that KFQ values can be much higher than the inhibition constants The competitive inhibition constant, λem of PPA by TEs, EGCG, ECG, TA and TF2  if the group is buried within a native protein, while it may be red‐shifted (longer wavelength) when the protein is unfolded λem. As shown in Table λem at its highest concentration. This was the largest shift for all the pure polyphenols investigated and was followed by ECG (9 nm), EGCG (6 nm) and TF2 (4 nm). This indicates that the order of the extent of potential structural unfolding for PPA upon binding with the four polyphenols was TA>ECG>EGCG>TF2. The denaturation process for a protein usually takes place in two steps. One is reversible, arising from the protein unfolding process. In this step, there is a partial loss of activity for the protein due to the disruption of intramolecular non‐covalent interactions λem), also made PPA most vulnerable to heat during DSC . Also, the Td of PPA bound with ECG, EGCG, and TF2 corresponded to the extent of structural unfolding caused by binding of the three polyphenols, i.e. lower Td corresponded to a higher red‐shift of maximum λem. In addition, the Td and ∆H of PPA incubated with EGC, TF1, TF and EC remained similar to the PPA control, indicating that these four polyphenols did not change the thermostability of PPA. This is because they induced hardly any structural unfolding for PPA, since there was no significant red‐shift of maximum λem for PPA interacting with these polyphenols. Therefore, as no structural unfolding occurred, the thermostability of PPA did not change. A similar relationship of red‐shift of maximum λem and Td (∆H) was also observed for PPA bound with three TEs, in which the higher mass ratio of TEs to protein caused lower thermostability of PPA. This is because higher concentration of TEs induced more extensive structural folding of the PPA molecule , indicating that the equilibrium of competitive inhibition (suggested by 1/Kic) actually resulted from the binding of polyphenols with specific sites on the enzyme (suggested by Kitc). Therefore, the constants obtained through the inhibition kinetics and ITC show a consistent relationship, and the two methods may be combined to analyze the binding of polyphenols with α‐amylase.The kinetics of inhibition of PPA indicates that TA, EGCG and TF2 were competitive inhibitors of PPA, specifically binding with the active site of the enzyme ∆H and Kitc values of the three compounds were in the order of TF2>TF1>TF. Therefore, for catechin and theaflavin families, the introduction of galloyl groups in the molecular structures could enhance the binding of these polyphenols with PPA. Additionally, TA, with ten galloyl groups in its molecular structure had a high binding affinity to PPA . This finding is consistent with our previous results that galloyl substitution plays an important role in association of catechins and theaflavins with PPA, as deduced from the kinetics of inhibition of PPA by tea polyphenols n), i.e. the polyphenol to PPA ratio n, 4), indicating that the fewest TA molecules are needed to saturate the binding with PPA. In addition, as the number of galloyl moieties decreased in the theaflavin molecules, n increased . The changing tendencies of n for both ECG to EC and EGCG to EGC were the same as that for theaflavins. Through molecular docking analysis it appeared that the galloyl moiety interacts with PPA, not only through hydrogen bonds between its three hydroxyl groups and the catalytic amino acid side‐chains , but also through hydrophobic π‐π (aromatic‐aromatic) interactions with the active site of the enzyme (Trp59) Very little corrected exothermic heat was observed for the titration of EC and EGC into PPA Fig. G and H, KFQ and Kitc demonstrate the binding of polyphenols with PPA, it is of interest to analyze the correlation between the two constants in order to evaluate the accuracy of each method and the rationality of combining fluorescence quenching and ITC. As shown in Fig. KFQ and Kitc . Because both constants directly reflected the interactions between polyphenols and PPA, the correlation coefficient was close to 1. This also indicates that the binding constants obtained through fluorescence quenching and ITC are comparable and that analysis of the interactions between polyphenols and enzyme by combination of the two methods is feasible. In addition, the fact that KFQ and Kitc are quantitatively similar suggests that tea polyphenols binding to PPA manly include the interactions with the tryptophan and with the vicinity of tryptophan at the active sites of the enzyme.As both 5λem in FQ and the decreased thermostability of PPA in DSC. Through ITC analysis and inhibition kinetics, it was shown that the equilibrium of competitive inhibition resulted from the binding of polyphenols with specific sites on the enzyme. There were positive and linear correlations among 1/Kic, KFQ and Kitc, indicating that all three constants reflect the binding affinity of polyphenols to α‐amylase. The galloyl moiety was shown to be an important substituent group in the binding of catechins and theaflavins with α‐amylase and thus in inhibiting the catalytic activity of the enzyme; therefore, galloyl substitution should be considered in the extraction and synthesis of pharmaceutical ingredients for type II diabetes.Overall, our data show that tea polyphenols which had higher inhibitory effects on PPA showed higher fluorescence quenching effects on the enzyme. Our study shows, for the first time, the potential structural unfolding of PPA upon binding of polyphenols with the enzyme as indicated by the red‐shift of maximum Supplementary informationClick here for additional data file.Table S1. Kinetics of PPA inhibition by TEs and pure phenolic compounds [6].Click here for additional data file.


Immunoblotting and amperometry were performed in dorsal striatal slices from wild-type (WT) and TAAR1 knockout (KO) mice. T1AM increased TH phosphorylation at both Ser19 and Ser40, actions that should promote functional activity of TH. Indeed, HPLC data revealed higher rates of L-dihydroxyphenylalanine (DOPA) accumulation in WT animals treated with T1AM after the administration of a DOPA decarboxylase inhibitor. These effects were abolished both in TAAR1 KO mice and by the TAAR1 antagonist, EPPTB. Further, they were specific inasmuch as Ser845 phosphorylation of the post-synaptic GluA1 AMPAR subunit was unaffected. The effects of T1AM on TH phosphorylation at both Ser19 (CamKII-targeted), and Ser40 (PKA-phosphorylated) were inhibited by KN-92 and H-89, inhibitors of CamKII and PKA respectively. Conversely, there was no effect of an EPAC analog, 8-CPT-2Me-cAMP, on TH phosphorylation. In line with these data, T1AM increased evoked striatal dopamine release in TAAR1 WT mice, an action blunted in TAAR1 KO mice and by EPPTB. Mass spectrometry imaging revealed no endogenous T1AM in the brain, but detected T1AM in several brain areas upon systemic administration in both WT and TAAR1 KO mice. In contrast to T1AM, tyramine decreased the phosphorylation of Ser40-TH, while increasing Ser845-GluA1 phosphorylation, actions that were not blocked in TAAR1 KO mice. Likewise, β-PEA reduced Ser40-TH and tended to promote Ser845-GluA1 phosphorylation. The D1 receptor antagonist SCH23390 blocked tyramine-induced Ser845-GluA1 phosphorylation, but had no effect on tyramine- or β-PEA-induced Ser40-TH phosphorylation. In conclusion, by intracellular cascades involving CaMKII and PKA, T1AM, but not tyramine and β-PEA, acts via TAAR1 to promote the phosphorylation and functional activity of TH in the dorsal striatum, supporting a modulatory influence on dopamine transmission.The trace amine-associated receptor 1 (TAAR1) is expressed by dopaminergic neurons, but the precise influence of trace amines upon their functional activity remains to be fully characterized. Here, we examined the regulation of tyrosine hydroxylase (TH) by tyramine and beta-phenylethylamine (β-PEA) compared to 3-iodothyronamine (T Classical trace amines (TAs), including tyramine, beta-phenylethylamine (β-PEA), tryptamine and octopamine, have been implicated in a number of neuropsychiatric disorders associated with monoaminergic dysfunction, including schizophrenia, major depression, and Parkinson’s disease . TAs are1AM), which is a derivative of thyroid hormone , has been found to be an endogenous agonist at TAAR1  which were found to be activated by TAs . These rat TAAR1 . The redtivation . In the tivation . Howevertivation . The dettivation .s proteins, but its signaling also involves the G protein independent β-arrestin2/Akt/Glucogen Synthase Kinase-3β (GSK-3β) pathway -4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB) is a selective TAAR1 antagonist  in TAAR1 KO mice rendered unilaterally dopamine denervated by 6-hydroxydopamine injections in the median forebrain bundle , which regulates DA synthesis  receptor subunit GluA1, which plays a crucial role in regulating transmission and plasticity at excitatory synapses in striatum. Finally, mass spectrometry imaging was used to detect T1AM at baseline and upon systemic administration.Here we extended the studies of TAs, thyronamines and TAAR1 on dopamine neurotransmission in the dorsal striatum. Special emphasis was put on Tsmitters . Remarkaon of T4 . We usedynthesis , along wad libitum.The experiments were approved by the local ethical committee at Karolinska Institute (N351/08) and conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Adult male WT and TAAR1 KO mice on a C57Bl6 background were used . They we2/5% CO2) artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 2.4 CaCl2, 10 glucose and 26 NaHCO3, pH 7.4. Coronal slices (300 μm thick) were prepared using a Leica vibratome . Dorsal striata were dissected from the slices in ice-cold aCSF buffer. Each slice was placed in a polypropylene incubation tube with 2 ml fresh aCSF buffer. The slices were preincubated at 30°C under constant oxygenation (95% O2/5% CO2) for 60 min with a change of buffer after 30 min. The buffer was then replaced with fresh aCSF and slices were treated with tyramine , T1AM , β-PEA , SCH23390 , EPPTB , KN-92 , H-89  and 8-CPT-2Me-cAMP , alone or in combination. The higher doses of all compounds exceeds by far the IC50 or Kd values for their respective target, but it is known that much higher concentrations are needed to exert actions in brain slices when compared to cell culture systems . Immunoblotting was performed on the membranes using P-Ser19-TH , P-Ser31-TH (Millipore), P-Ser40-TH (Millipore), P-Ser845-GluA1 (UBI), and antibodies, which are not phosphorylation state-specific to estimate total levels of TH (Millipore) and GluA1 (UBI). The antibody binding was detected by incubation with goat anti-mouse or anti-rabbit horseradish peroxidase-linked IgG (1:6000–8000 dilution) and detected using ECL immunoblotting detection reagents .Immunoblotting was performed as described earlier . Frozen 1AM (10 μM) or tyramine (100 μM), and then for 15 min with T1AM or tyramine along with the L-amino acid decarboxylase inhibitor NSD-1015 . After the removal of the solutions, tissue slices were frozen and sonicated  in 100 μL perchloric acid (0.1 mM). The pellets were resuspended in 100 μl 1% sodium dodecyl sulfate and the protein content was determined. The level of L-DOPA in the supernatant was determined using HPLC coupled to an electrochemical detection system with a refrigerated microsampling unit . The HPLC apparatus comprised an HPLC pump  that kept a constant flow of 0.2 mL/min of the mobile phase  and a pressure of ∼0.5 bar on a reverse-phase ion pair C-18 column prepacked with Biophase ODS 5 μm particles . L-DOPA was detected with an amperometric detector  and a glassy carbon electrode set at 0.75 V. The limit of detection was ∼10 nM.Dorsal striatal slices were incubated for 5 min with TSagittal striatal brain slices were prepared and maintained as above. Amperometric detection of DA release was performed as described earlier . Carbon 1AM  and killed by decapitation 30 or 60 min post-dose. All brains were immediately removed, snap frozen, and stored at -80°C until further analysis. The frozen brain tissues were cut using a cryostat-microtome  at a thickness of 14 μm, thaw-mounted onto conductive indium tin oxide (ITO) glass slides , and stored at -80°C. Sections were dried gently under a flow of nitrogen and desiccated at room temperature for 15 min, after which they were imaged optically using a photo scanner (Epson perfection V500). The samples were then coated with derivatization reagents, 2, 4-diphenylpyrylium tetrafluoroborate (DPP-TFB). Stock solution of DPP-TFB (8 mg in 1.2 ml MeOH) was prepared and diluted in 6 mL of 70% methanol containing 3.5 μL of trimethylamine. An automated pneumatic sprayer  was used to spray DPP-TFB solution over the tissue sections. The nozzle temperature was set at 80°C and the reagent was sprayed for 30 passes over the tissue sections at a linear velocity of 110 cm/min with a flow rate of about 80 μL/min. Samples were then incubated for 15 min (dried by nitrogen flow every 5 min) in a chamber saturated with vapor from a 50% methanol solution. MALDI-MSI experiment was performed using a MALDI-TOF/TOF  mass spectrometer with a Smartbeam II 2 kHz laser in positive ion mode. The laser power was optimized at the start of each run and then held constant during the MALDI-MSI experiment.Adult male WT and TAAR1 KO mice were injected with saline or Tpost hoc test. Data from Amperometry were acquired and analyzed with the pClamp 9 or pClamp 10 software (Axon Instruments). Data are expressed as % of the baseline response measured for each slice during the 5–10 min preceding start of perfusion with T1AM. Statistical significance of the results was assessed by using Student’s t-test for paired observations (comparisons with baseline within single groups) or one-way ANOVA multiple comparison test followed by Newman–Keuls post hoc test since the samples from WT and KO mice were loaded in separated gels. The numbers of individual replicates are shown in the graphs, while p-values, degrees of freedom and F values are detailed in the results part.Autoradiograms from western blotting experiments were digitized using a Dia-Scanner (Epson Perfection 4870 PHOTO). Optical density values were measured using NIH Scion Image for Windows . Biochemical data were analyzed using one-way ANOVAs followed by Newman–Keuls 1AM on the phosphorylation of TH and GluA1 in striatal slices from TAAR1 receptor WT and KO mice. To study effects of compounds in the both genotypes, their individual baseline was set at 100%. One way ANOVA analysis showed that tyramine caused a significant change of P-Ser40-TH in both WT  and KO mice . Post hoc test showed that the lower (1 and 10 μM) concentrations of tyramine did not have any significant effect, whereas 100 μM tyramine significantly reduced phosphorylation of P-Ser40-TH in both groups of mice, suggesting an effect independent of TAAR1 . Meanwhile, there were no effects at the same concentration of tyramine on P-Ser19-TH  and P-Ser31-TH  in neither WT nor KO mice . One way ANOVA analysis in T1AM treated mice revealed that only in the WT but not in the KO mice, the drug affected significantly the levels of P-Ser19  and P-Ser40-TH . Post hoc test showed that 10 μM T1AM enhanced P-Ser19 and P-Ser40-TH in the striatum of WT but not TAAR1 KO mice . The effects of T1AM were biphasic with a further increase in drug concentrations resulting in less phosphorylation of TH. There was no effect of T1AM on P-Ser31-TH both in WT  and KO mice  . Tyramine altered significantly the phosphorylation of P-Ser845-GluA1 in WT  and KO mice  . In contrast, T1AM had no effect on P-Ser845-GluA1 in striatal slices from either TAAR1 WT  or KO mice  . These data suggest that tyramine and T1AM act differently on pre- and post-synaptic striatal targets.We first studied the dose responses of the tyramine and T1 receptor antagonist SCH23390 (5 μM). As shown in Figure 2, we found that β-PEA, like tyramine, decreased P-Ser40-TH  while not significantly affecting P-Ser19-TH  or P-Ser31-TH . The effects of tyramine and β-PEA on P-Ser40-TH were not affected by SCH23390. On the other hand, tyramine significantly enhanced P-Ser845-GluA1 , an effect that was reversed to baseline by D1 receptor blockade using SCH23390. Likewise, β-PEA tended to increase P-Ser845-GluA1, but this effect did not reach significance. The data of β-PEA was further to confirm that classical TAs and T1AM act differently.To further study the effect of a high dose of endogenous TAs, we incubated striatal slices from WT mice with the tyramine (100 μM), β-PEA (100 μM) alone or with the DF: 6.856; p = 0.0061) . Post hoc test showed that T1AM (10 μM), but not tyramine (100 μM), induced L-DOPA accumulation in the presence of a DOPA decarboxylase inhibitor in WT mice. No effects were detected in TAAR1 KO mice , indicative of a TAAR1-mediated mechanism of action of T1AM.There was also a baseline increase of TH activity in TAAR1 KO mice as compared to WT mice . To stud1AM-induced phosphorylation of TH, we combined the T1AM with KN-92 or H-89, inhibitors of CamKII and protein kinase A (PKA), respectively. The effects of T1AM on both P-Ser19  and P-Ser40-TH  could be significantly inhibited by either KN-92 or H-89 . Since TAAR1 is a Gs-coupled receptor and generates cAMP, we also examined the effects of 8-CPT-2Me-cAMP, an EPAC (Exchange Protein directly Activated by cAMP) activator, on TH phosphorylation. 8-CPT-2Me-cAMP alone tended to increase TH phosphorylation, but did not interact with T1AM .To further study intracellular signaling cascades underlying the T1AM on stimulation-evoked release of DA from DA-containing fibers present in sagittal striatal slices, as shown in Figure 5. We found that bath application of T1AM (10 μM) significantly increased the amplitude of evoked DA release measured with carbon fiber electrodes coupled to amperometry in dorsal striatal brain slices from WT mice, and that this effect was significantly reduced in TAAR1 KO mice. In presence of the TAAR1 antagonist EPPTB (10 nM), the effect of T1AM on evoked DA release in WT mice was significantly reduced, but not completely blocked . EPPTB had no effect on evoked DA release by itself (data not shown). Similar to the results as above, T1AM significantly increased P-Ser40-TH . This effect was blocked when T1AM was combined with EPPTB, suggesting a mechanism of action mediated via TAAR1.We evaluated the effect of T1AM was derivatized by 2, 4 diphenyl pyranylium and detected by MALDI-MSI. No clear endogenous signal of T1AM was found in uninjected sections neither from WT nor TAAR1 KO mice. However, widespread signals corresponding to derivatized T1AM was detected in mice intraperitoneally administered with T1AM (20 mg/kg). The concentration of the drug appeared higher after 30 min compared to 60 min post-dose .T1AM enhance phosphorylation and activity of TH along with evoked DA release in dorsal striatum, while not significantly affecting the phosphorylation of post-synaptic AMPA receptor GluA1 subunits. The effects on TH phosphorylation observed following T1AM administration were abolished in TAAR1 KO mice, while the effects on evoked DA release were attenuated in TAAR1 KO mice and following TAAR1 blockade, supporting a role of TAAR1 as a partial mediator of these effects. We can conclude that T1AM acts through TAAR1 to enhance the production of dopamine. This de novo dopamine creation heightens the synaptic dopamine content and raises extracellular dopamine. In contrast, tyramine and β-PEA reduced TH phosphorylation via a mechanism independent of TAAR1.Our experiments demonstrate that the thyronamine T19, Ser31, and Ser40 have been identified as the functionally most important sites of TH phosphorylation /Ca++/NFAT pathways . PP-1 inhibits the phosphorylation of CamKII, which activates phosphorylation of Ser19-TH. CamKII can also inhibit protein phosphatase 2A which activates phosphorylation of Ser40-TH. The regulation of the suggested signal transduction pathways could explain how KN-92 and H-89, inhibitors of CamKII and PKA respectively, both can block the effect of T1AM. 8-CPT-2Me-cAMP, the EPAC analog, had no influence on TH phosphorylation. In summary, this higher phosphorylation rate leads to the accumulation of DOPA.Alterations of DA synthesis are regulated via phosphorylation of TH, the rate-limiting enzyme in the synthesis of catecholamines , and Serrylation . Phosphod by PKA . TH phosrylation . Notablynsations . An incrpathways . Here we is MAPK . As a correstin2 . In orde1AM, under the present conditions, the endogenous TAs tyramine and β-PEA appeared to elicit mainly non-TAAR1 dependent effects. Tyramine, at a high dose, reduced phosphorylation of Ser40-TH, but increased phosphorylation of the post-synaptic AMPA receptor GluA1 subunit in striatal slices from both WT and TAAR1 KO mice. Similarly, another endogenous TA, β-PEA also reduced phosphorylation of Ser40-TH and tended to increase phosphorylation of Ser845-GluA1 in slices from WT mice. The quantification of GluA1 phosphorylation on Ser845, help us to evaluate the status of GluA1 in the post-synaptic membrane of corticostriatal and thalamostriatal synapses, which are the main classes of glutamatergic synapse in the striatum (1 positive or D2 positive) are activated. Nevertheless, it is shown that D1 agonists and D2 antagonists induce robust increases in GluA1 phosphorylation while D2 agonists and D1 antagonists have no effect  blunted the para-chloroamphetamine (PCA) induced dopamine release in both structures  is implicated in the modulation of histaminergic neurotransmission and might likewise interact with dopaminergic pathways: this remains to be clarified (In this study we incubated the slices in a Tred T1AM . Using Mboxylase , which iboxylase . Apart flarified .1AM on dorsal striatal TH phosphorylation, activity and evoked dopamine release. No comparable alterations were found after application of tyramine and β-PEA. This simultaneous augmentation in TH phosphorylation and striatal dopamine release after the administration of T1AM indicates that this thyronamine favors dopamine synthesis and subsequent secretion through TAAR1. Conversely, TAs act in a TAAR1 independent manner to influence dopamine secretion resulting in feedback inhibition of TH. This study further indicates that the modulatory properties of TAAR1 may differ depending on the identity of the ligand in question, the extracellular milieu, basal levels of monoamines, neuronal circuitry, and the cellular localization of TAAR1, which are mutually regulated by interactions with D2 receptors and DAT, and by the available signaling transduction systems. Further elucidation of the complex pattern of influence of TAAR1 upon monoaminergic and other pathways controlling mood, motor function and cognition may lead to the elaboration of urgently-need, novel strategies for improving the treatment of depression, schizophrenia, Parkinson’s disease, and other neuropsychiatric disorders (This study demonstrates that TAAR1 mediates the effects of Tisorders .Participated in research design: XZ, KC, and PS. Collected the samples and conducted the experiments: XZ, MS, MP, AN, and TY. Performed the data analysis and discussed the data: XZ, IM, AA, TY, JK, PEA, MJM, KC, and PS. Contributed to the writing of the manuscript and to revising it critically for scientific discussions: XZ, IM, AA, MJM, KC, and PS. All authors approved the final version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fbx15. Here, we investigated the crosstalk between MAPK inhibition and the ubiquitin–proteasomal degradation of P-gp. Proteasome inhibitors or knockdown of FBXO15 and/or UBE2R1 cancelled MEK inhibitor-induced P-gp downregulation. RSK1 phosphorylated Thr162 on UBE2R1 but did not phosphorylate FBXO15. MEK and RSK inhibitors increased UBE2R1-WT but not UBE2R1-T162D and -T162A expression. UBE2R1-T162D showed higher self-ubiquitination and destabilisation than UBE2R1-WT and -T162A. Unlike UBE2R1-WT and -T162A, UBE2R1-T162D did not induce P-gp ubiquitination. UBE2R1-WT or -T162A downregulated P-gp expression and upregulated rhodamine 123 level and sensitivity to vincristine and doxorubicin. However, UBE2R1-T162D did not confer any change in P-gp expression, rhodamine 123 accumulation and sensitivity to the drugs. These results suggest that RSK1 protects P-gp against ubiquitination by reducing UBE2R1 stability.P-glycoprotein (P-gp) is a critical determinant of multidrug resistance in cancer. We previously reported that MAPK inhibition downregulates P-gp expression and that P-gp undergoes ubiquitin–proteasomal degradation regulated by UBE2R1 and SCF Receptors of tyrosine kinase, such as epidermal growth factor receptor (EGFR) or vascular endothelial growth factor receptor (VEGFR), are activated by ligand-dependent self-phosphorylation, which subsequently activates the MAPK pathway by phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinases (ERKs) and p90 ribosomal S6 kinases (RSKs)1416171819MEKs, ERKs and RSKs lowered P-gp expression by promoting its degradationFbx15, which consists of S-phase kinase-associated protein 1 (SKP1), Cullin 1 and F-box only protein 15 (FBXO15), and then ubiquitinated by the ubiquitin-conjugating enzyme E2 R1 2324In our previous studies, MEK inhibitors or siRNAs for FBXO15 and/or siUBE2R1 for 60 h followed by treatment with trametinib  in the presence of 32P-labeled ATP . Tagless UBE2R1-WT, -T79A, -T162A and -T185A were incubated with active His6-RSK1 in the presence of 32P-labeled ATP  and protein phosphatase 2A, regulatory subunit B, gamma (PPP2R3C) downregulates P-gp expression3031KIP1 and promotes their proteasomal degradation33KIP1 are negative regulators of cell cycle progression at the G2/M transition and at both G1 and G2/M phases, respectively1735KIP1. BI-D1870 has been reported to arrest HCT 116 and oral squamous cell carcinoma cell lines at G2/M37UBE2R1 also ubiquitinates WEE1 and p27®, developed by Japan Tobacco, Inc. and GlaxoSmithKline) and bortezomib  have already been approved and clinically used for melanomas harbouring BRAF V600 mutations and refractory multiple myelomas, respectively39404142P-gp is expressed in normal cells and tissues, including colon, kidney, liver, small intestine, and the blood–brain and blood–placenta barriers10More importantly, our data show that trametinib downregulates the expression of P-gp by promoting its degradation through the upregulation of UBE2R1, resulting in the downregulation of P-gp-mediated resistance to the anticancer agents vincristine and doxorubicin . TrametiOverall, this study should be beneficial not only for understanding the molecular mechanism of P-gp expression but also for predicting the side effects of drugs used in combination with trametinib or bortezomib and for overcoming P-gp-mediated chemoresistance to anticancer agents.6-ERK1 and His6-RSK1 were purchased from Merck Millipore .MG132, CHX, doxorubicin, cyclosporine A and recombinant GST-ERK1 were purchased from Sigma-Aldrich . U0126 and bortezomib were obtained from Cell Signaling Technologies  and Takeda Pharmaceutical Company Ltd. , respectively. Trametinib and BI-D1870 were from Selleck Chemicals . Recombinant HisFBXO15- or CDC34/UBE2R1-targetting siRNAs were from GE Healthcare UK Ltd. .Non-silencing control siRNA was purchased from Qiagen , and XbaI and NotI, followed by ligation of each cDNA to pQCXIP or pGEX-6P-3 vectors. Substitution of each amino acid in UBE2R1 was performed using QuikChange II Site-Directed Mutagenesis Kit , in accordance with the manufacturer’s instructions. All other plasmids had also been generated previouslypQCXIP-UBE2R1, pGEX-UBE2R1 and pGEX-FBXO15 were generated by the digestion of pcDNA3.1-HA-UBE2R1 or -FBXO15, which had been generated previouslyFuGENE HD transfection reagent  and Lipofectamine 2000 transfection reagent  were used for the transfection of cells with plasmids and siRNAs, respectively, in accordance with the manufacturers’ instructions.UBE2R1-WT, -T162A or -T162D, respectively. Similarly, 620/mock, 620/WT, 620/TA and 620/TD cells were established based on SW620-14 cells. All cell lines including human embryonic kidney HEK293 cells were maintained in DMEM supplemented with 7% FBS and 50 μg/mL kanamycin at 37 °C in 5% CO2.SW620-14 cells had been previously isolated from human colorectal cancer SW620 cellsFlow cyctometric analyses and cell growth inhibition assays were performed as described previously2122Immunoprecipitation and immunoblotting were performed as described previously2122E. coli with Glutathione Sepharose 4B  was incubated with BSA or GST-ERK1, His6-ERK1 or His6-RSK1 in kinase reaction buffer  for 30 min at 30 °C in the presence of 40 μCi [γ-32P]ATP. The reaction was stopped by incubation for 3 min on ice followed by the addition of 5 × Laemmli sample buffer and boiling for 3 min at 95 °C. The proteins were subjected to SDS-PAGE, and the incorporated radioactivity was visualised by autoradiography using an image reader FLA-7000 . To confirm that the proteins were present at equal levels, the gel was subjected to silver staining.GST-tagged UBE2R1 or FBXO15 purified from How to cite this article: Katayama, K. et al. RSK1 protects P-glycoprotein/ABCB1 against ubiquitin-proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1. Sci. Rep. 6, 36134; doi: 10.1038/srep36134 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.