The estimated probabilities that reductions in the mean global population size of polar bears will be greater than 30%, 50% and 80% over three generations (35–41 years) were 0.71 (range 0.20–0.95), 0.07 (range 0–0.35) and less than 0.01 (range 0–0.02), respectively. According to IUCN Red List reduction thresholds, which provide a common measure of extinction risk across taxa, these results are consistent with listing the species as vulnerable. Our findings support the potential for large declines in polar bear numbers owing to sea-ice loss, and highlight near-term uncertainty in statistical projections as well as the sensitivity of projections to different plausible assumptions.Loss of Arctic sea ice owing to climate change is the primary threat to polar bears throughout their range. We evaluated the potential response of polar bears to sea-ice declines by (i) calculating generation length (GL) for the species, which determines the timeframe for conservation assessments; (ii) developing a standardized sea-ice metric representing important habitat; and (iii) using statistical models and computer simulation to project changes in the global population under three approaches relating polar bear abundance to sea ice. Mean GL was 11.5 years. Ice-covered days declined in all subpopulation areas during 1979–2014 (median −1.26 days year The su history . Two hav history ,8. Other history , have be history  or have  history .Figure K) is directly proportional to the availability of sea ice. Approaches 2 and 3 estimated relationships between changes in sea ice and observed changes in polar bear abundance. We evaluated projection outcomes, over three polar bear generations, relative to thresholds for threatened categories under criterion A3 of the IUCN Red List of Threatened Species . The [et al. .2.t + 1 were counted as pseudo-observations in year t. Variation in GL was evaluated, using a bootstrap procedure .Projection timeframes can incorporate biological differences across species by referencing to generation length . We estK for polar bears (ice as the number of days per year that ice area was above the midpoint (i.e. the number of ‘ice-covered’ days). Mean values of ice were projected forward, using linear models, which facilitated projections at the spatial scale of polar bear subpopulations .Satellite data of sea-ice concentration were collected between years 1979–2014 to develop an index of ice to estimates of subpopulation abundance . Approach 1 assumed a one-to-one proportional relationship between ice and N. Approaches 2 and 3 estimated linear relationships between ice and proportional changes in N, and used these relationships to predict future values of N as a function of projected ice. Approach 2 estimated a global ice–N relationship based on a maximum of two estimates of N per subpopulation, separated by at least 10 years, which were available for seven subpopulations. Approach 3 estimated a separate ice–N relationship for each polar bear ecoregion using a dataset that was similar to approach 2 but included longer time series of N available for four subpopulations. All approaches assumed that changes in N were mediated primarily through changes in K or density-independent habitat effects, and that the ratio N/K was stable relative to other factors [N were scaled to changes in MGPS, using the most recent estimate of N for each subpopulation. Based on 62 500 stochastic projections, we calculated the most likely change in MGPS over three generations. In addition, the probabilities of exceeding 0%, 30%, 50% and 80% reduction thresholds were generated following Red List guidelines [We used population projections to evaluate changes in MGPS between the years 2015 and (2015 + 3 × GL) based on three approaches relating  factors . These a factors ) for mosidelines . We perfidelines , using tidelines  to simul3.ice declined at a significance level of 0.05 in all 19 subpopulation areas during 1979–2014 .The mean subpopulation-specific estimate of GL was 11.5 years  based on 3374 observed reproductive events . Projections were performed using GL = 11.5 and 13.6 years to reflect variation in GL and approximate natural GL. We did not apply the lower fifth percentile, because harvest likely shortened several empirical estimates of GL . The metice and N . This finding reflects variability in current subpopulation status, uncertainty in estimates of N and the lack of empirical evidence for sea-ice mediated changes in global abundance over recent decades [ice–N relationship for each ecoregion. Relationships were positive at a significance level of 0.01 for the seasonal  and divergent ecoregions , reflecting observed correlations between declining sea ice and declining abundance . Relationships were not significant for the convergent  and archipelago ecoregions . Although approach 3 reflected regional variability in sea-ice dynamics and polar bear ecology, it was strongly influenced by several well-studied subpopulations and did not reflect finer-scale variation. For example, within the divergent ecoregion, multiple estimates of N were available for the declining Southern Beaufort sea subpopulation [Our analyses highlight the potential for large reductions in MGPS as climate change and sea-ice loss continue  over the decades . Approacpulation , but notOur projections  are broa


Administration of the therapeutic anti‐IgE antibody omalizumab to patients induces strong increases in IgE antibody levels.To investigate the effect of intranasal administration of major birch pollen allergen Bet v 1, omalizumab or placebo on the levels of total and allergen‐specific IgE in patients with birch pollen allergy.IL‐4/anti‐CD40‐treated PBMCs from allergic patients were studied in vitro.Based on the fact that intranasal allergen application induces rises of systemic allergen‐specific IgE, we performed a double‐blind placebo‐controlled pilot trial in which birch pollen allergic subjects were challenged intranasally with omalizumab, placebo or birch pollen allergen Bet v 1. Total and allergen‐specific IgE, IgG and basophil sensitivity were measured before and 8 weeks after challenge. For control purposes, total, allergen‐specific IgE levels and omalizumab‐IgE complexes as well as specific IgG levels were studied in subjects treated subcutaneously with either omalizumab or placebo. Effects of omalizumab on IgE production by P = .016). No relevant change in allergen‐specific and total IgE levels was observed in subjects challenged with omalizumab. Addition of omalizumab did not enhance IL‐4/anti‐CD40‐induced IgE production in vitro. Significant rises in total IgE (mean IgE before: 131.83 kU/L to mean IgE after: 505.23 kU/L) and the presence of IgE‐omalizumab complexes were observed after subcutaneous administration of omalizumab.Intranasal challenge with Bet v 1 induced increases in Bet v 1‐specific IgE levels by a median of 59.2%, and this change differed significantly from the other treatment groups (Intranasal administration of allergen induced rises of allergen‐specific IgE levels, whereas intranasal administration of omalizumab did not enhance systemic total or allergen‐specific IgE levels. BCRB‐cell receptorBSABovine serum albuminCε3Constant region 3CIConfidence intervalELISAEnzyme‐linked immunosorbent assayFCSFoetal calf serumFcεRIHigh‐affinity receptor for IgEFcRnNeonatal Fc receptorGMPGood manufacturing practiceIgEImmunoglobulin EILInterleukinISUInternational standard unitskU/LKilo units per litrekUA/LKilo units antigen per litremAbMonoclonal antibodyMFIMean fluorescence intensityPBMCPeripheral blood mononuclear cellPBSPhosphate‐buffered salineRBLRat basophilic leukaemias.c.Subcutaneous1Immunoglobulin E (IgE) is the key antibody class responsible for allergic symptoms in sensitized patients..In order to prevent IgE binding to its receptors, omalizumab—a monoclonal humanized anti‐IgE antibody that specifically binds to the constant region 3 (Cε3) of IgE—has been developed and has been shown to be effective for the treatment of a variety of allergic manifestations and in particular for severe IgE‐mediated asthma+ memory B cells. It is therefore possible that cross‐linking of the BCR on IgE+ memory cells may contribute to the strong increases in IgE levels which are observed in patients treated with omalizumab in addition to a prolongation of the half‐life of IgE within the omalizumab‐IgE immune complexes.+ B cells as well as plasma cells+B cells in the nasal mucosa. We therefore investigated the effect of intranasal administration of omalizumab, allergen or placebo on allergen‐specific and total systemic IgE. We applied in a double‐blind, placebo‐controlled clinical study omalizumab to birch pollen allergic subjects intranasally and, as positive and negative control, included allergic subjects who received intranasal allergen challenge and buffer, respectively. The effects of intranasal omalizumab and allergen exposure on total, allergen‐specific IgE and IgG levels as well as on basophil sensitivity were then analysed for a period of 8 weeks. In addition, we studied the effects of subcutaneous (s.c.) omalizumab administration in allergic subjects regarding changes in total, allergen‐specific IgE and allergen‐specific IgG levels.Omalizumab targets the Cε3 region of IgE, which is considered to be accessible also on membrane‐bound IgE in the form of B‐cell receptors (BCRs) on IgE22.1®, Novartis Pharmaceuticals Corporation, New York, NY), Bet v 1  or placebo (0.9% sodium chloride solution). One patient in the omalizumab challenge group withdrew from the study due to illness (upper respiratory tract infection) just before the first nasal challenge. At visit 1 (t1), blood samples were taken and the first nasal challenge was performed. Two more nasal challenges were performed on the consecutive 2 days. Further blood samples were taken 3 (t2), 5 (t3) and 8 (t4) weeks (±4 days) after the first challenge. The clinical investigators and study subjects were blinded regarding the administered substances. Randomization and preparation of the study drugs  were carried out by a person who was not involved in the study and had no contact with the subjects to ensure the double‐blinded nature of the study.The study was performed over 8 weeks outside of the birch pollen season (November to January) as an investigator‐initiated, randomized, double‐blind and placebo‐controlled pilot study. The study was approved by the Ethical committee of the University of Vienna and Austrian authorities and registered in the European Clinical Trials Database (EudraCT: 2012‐004193‐25) and in the ClinicalTrial.gov data base (NCT03019237). During the screening visit, 24 subjects between 18 and 60 years of age were assessed  (Biomay AG) or Xolair (Novartis) was diluted in sterile 0.9% sodium chloride freshly before use. Bet v 1 was applied at a concentration of 50 μg/mL as this concentration has previously been shown to lead to increase in systemic serum IgE levels after intranasal administration.2.3Betulaceae and Fagaceae), as well as mugwort, mould, cat, dog, D. pter., D. far. extract all purchased from Stallergenes .Recruitment, study population, administration of the drug as well as dosage and blood draw schedule are described in detail in Gevaert et al.2.4Total IgE and IgE specific to birch extract (t3), Bet v 1 (t215), grass (g6), olive (t9), cat (e1) and house dust mite (d1) were measured by ImmunoCAP . Allergen‐specific IgG levels were analysed using a microarray based on ImmunoCAP ISAC technology  containing more than 170 different allergen molecules2.5Allergen‐induced basophil activation was determined in heparinized blood samples obtained before and after intranasal challenge by measuring the upregulation of CD203c expression on basophils upon exposure to rBet v 1 (0.01 μg/mL), anti‐IgE (1 μg/mL) or buffer . Mean fluorescence intensities of stimulated (MFIstim) and unstimulated (MFIcontrol) cells were determined for triplicate cultures by flow cytometry, and the upregulation of CD203c expression was expressed as stimulation index (MFIstim: MFIcontrol) (SI).In addition, basophil activation tests were performed by passive loading of rat basophilic leukaemia (RBL) cells expressing the α/β/γ subunits of the human FcεRI2.6m phosphoric acid. OD levels were read at 450 nm. Measurements were performed in triplicates, and background was subtracted.ELISA plates  were coated with 1 μg/mL human anti‐omalizumab (drug/target complex) HCA238  overnight at 4°C and blocked with PBS/5% v/v BSA/0.05% v/v Tween‐20(blocking buffer). As a standard, purified monoclonal chimeric Bip 1 IgE (5 μg/mL)2.7Heparinized blood samples were obtained from 2 birch and grass pollen allergic subjects after written informed consent was given . PBMCs were isolated from heparinized blood samples of allergic patients using Ficoll density gradient separation . Cells were washed 3 times in phosphate‐buffered saline (PBS)  and then cultured in RPMI 1640 containing L‐glutamine , supplemented with 10% foetal calf serum (FCS)  and 50 μg/mL gentamicin . Cells were either left unstimulated or stimulated with IL‐4  and anti‐CD40  in the presence or absence of omalizumab (3 μg/mL) or isotype control . Supernatants were taken after 1 week of culture and frozen at −20°C until measurement of total IgE levels by ImmunoCAP . Percentage change in total IgE levels compared to supernatants from unstimulated cells was calculated.2.8P‐value of the Kruskal‐Wallis test was < .05, the two‐sided Wilcoxon rank‐sum test was calculated for pairwise group comparison. For both, the Kruskal‐Wallis test and the Wilcoxon rank‐sum test, exact P‐values were computed. To analyse the difference before and after subcutaneous administration of omalizumab, paired t tests and 95% confidence intervals (CI) for the mean difference of visits were calculated for each treatment group separately, assuming a normal distribution of the data. To assess correlation between total IgE levels and omalizumab‐IgE complexes, Pearson correlation coefficients, 95% CIs as well as exact P‐values were calculated. Due to the exploratory nature of this pilot study, no correction for multiple testing was applied.Statistical analyses were carried out with SAS 9.4.  or R 3.3.1 . For all analyses, the significance level was set to 0.05. To analyse the percentage change among the intranasal treatment groups, the Kruskal‐Wallis test was conducted. Only if the 33.1Allergic patients suffering from birch and grass pollen allergy n = 15)  or placebo (n = 6) subcutaneously3.2P = .0053, two‐sided Wilcoxon rank‐sum test Bet v 1 vs omalizumab and Bet v 1 vs placebo: P = .016)   Figure . The med) Figure .3.3P = .0463). The sensitivity of basophils loaded with sera from Bet v 1‐challenged subjects was significantly higher than that of basophils loaded with sera from omalizumab‐challenged subjects (pairwise Wilcoxon test: P = .032) .In a first set of experiments, we investigated whether loading of RBL cells expressing the human FcεRI with sera from subjects obtained 5 weeks after intranasal challenge with Bet v1 increases Bet v 1‐specific sensitivity. We found significant group differences between the intranasally challenged groups   .The analysis of sera from subjects who had received subcutaneous administration of omalizumab (n = 15) or placebo (n = 6)) Figure A. In patWhen we analysed allergen‐specific IgE levels, a fourfold to sevenfold rise in allergen‐specific IgE was observed after subcutaneous administration of omalizumab but not of placebo regardless of the nature of the tested allergen Table . InteresP = .0009, n = 15). A statistically significant correlation between serum IgE levels and the amount of IgE‐omalizumab complexes was noted; however, the corresponding confidence interval is wide due to the low sample size .Using an ELISA based on antibodies recognizing specifically IgE‐omalizumab complexes but neither noncomplexed IgE nor noncomplexed omalizumab alone, omalizumab‐IgE complexes were detected in the sera from all subjects after subcutaneous omalizumab administration. The levels of IgE‐omalizumab complexes rose significantly from 0 μg/mL before to an average of 0.104 μg/mL in subjects receiving omalizumab  with anti‐CD40 and IL‐4 in the presence or absence of omalizumab as well as an isotype control for omalizumab. Anti‐CD40 and IL‐4 stimulation induced an increase in total IgE levels in culture supernatants of both allergic subjects as previously described4+ B cells and plasma cells in the nasal mucosa.In the present study, we investigated the effect of intranasal administration of Bet v 1, omalizumab and placebo on the levels of total and allergen‐specific IgE in patients with birch pollen allergy. The pilot clinical trial performed by intranasal administration of Bet v 1 and omalizumab was based on the observation that intranasal administration of allergen to allergic patients induced a robust increase in allergen‐specific IgE antibodies, eventually by targeting IgE‐producing cells.There are several mutually not exclusive explanations why there are differences regarding allergen‐specific sensitivity when testing allergic patients basophils isolated at different time points from the patients vs testing cultured basophils. First, it is possible that the intrinsic sensitivity of the patient's basophils may vary at different time points. Second, testing of patients basophils at different time points may introduce variation. Third, experiments with patient's basophils are carried out in the presence of allergen‐specific IgG. In fact, allergen‐specific IgG increases were noted in several serum samples which may have affected allergen‐induced basophil activation Table . FinallyThe results can thus be summarized as follows: intranasal application of the major birch pollen allergen Bet v 1 led to an increase in allergen‐specific but not total systemic IgE levels, while intranasal omalizumab application had no effect on total or allergen‐specific IgE levels. Basophil sensitivity in patients did not change after challenge with the allergen despite a significant increase in Bet v 1‐specific IgE levels. In contrast, rat basophilic leukaemia cells expressing the human FcεRI which had been passively sensitized with the sera from allergen‐challenged patients showed increased allergen sensitivity after the challenge.+ B cells.Furthermore, our in vitro experiments showed that omalizumab had no effect on the secretion of IgE ex vivo using PBMCs primed with IL‐4 and anti‐CD40. In patients having undergone subcutaneous omalizumab administration, a significant rise in systemic IgE levels and the presence of IgE‐omalizumab complexes were demonstrated. In summary, these data suggest that the rise of systemic IgE levels observed in omalizumab‐treated patients is most likely due to the formation of IgE‐omalizumab complexes increasing the half‐life of IgE and not due to an increase in IgE production by IgE+ memory B cells. In the present study, both in our in vivo intranasal challenge model and in our in vitro approach using a well‐established system to induce IgE production,+ B cells and Cε germline transcription but not IgE production in vitro as we did.+ B‐cells numbers upon addition of omalizumab to the cultures. Limitations of our study include that only a limited number of patients and only 1 administration schedule were studied in vivo and regarding the in vitro experiments that the IL‐4/anti‐CD40‐switched IgE‐producing cells may not exactly mimic IgE‐BCR containing memory B cells. However, both the in vivo and the in vitro experiments did not provide evidence that omalizumab induces IgE production through activation of IgE‐producing cells.We have thus addressed two mutually nonexclusive possibilities to explain the rises of IgE levels in allergic patients undergoing therapy with the monoclonal anti‐IgE antibody omalizumab. The first possibility is based on findings that the administration of anti‐IgE antibodies into primed mice1 antibody, prolongs the serum half‐life of IgE by preventing its degradation utilizing the recycling of the complex by the neonatal receptor for IgG, FcRn.The other hypothesis for the increase in IgE upon omalizumab administration which has been proposed recently suggests that complex formation between IgE and omalizumab, a human monoclonal IgG+ memory cells causing increased IgE production but rather due to formation of nonallergenic omalizumab‐IgE complexes with prolonged serum half‐life.In summary, our results indicate that increases in IgE levels under omalizumab treatment are not due to cross‐linking of the BCR on IgERudolf Valenta has received research grants from Biomay AG, Vienna, Austria; Thermofisher, Uppsala, Sweden; Fresenius Medical Care, Bad Homburg, Germany; and Viravaxx, Vienna, Austria, and serves as a consultant for these companies. Dr. Marth reports personal fees from Novartis, personal fees from Thermofisher, personal fees from Teva, personal fees from Medmedia, non‐financial support from Boehringer Ingelheim, personal fees from Chiesi, outside the submitted work. Dr. Valenta reports grants from FWF, during the conduct of the study; grants and other from Biomay, grants and other from Viravaxx, outside the submitted work. Dr. Philippe reports grants and personal fees from Novartis, Roche, Genentech, during the conduct of the study. Dr. Lupinek reports personal fees from Thermo Fisher Scientific, outside the submitted work. Dr. Selb reports grants from Austrian Research Fund (FWF), during the conduct of the study. The other authors declare that they have no conflicts of interest.RV and VN designed and planned this study, participated in the interpretation of the findings, wrote, read and critically revised the manuscript. JED contributed to design and planning of the study, performed the data analyses, participated in the interpretation of the findings and wrote the initial manuscript and read and revised the final version. RF, RK, CL and TP performed measurements for specific and total IgE levels (CAP and CHIP measurements), read and revised the manuscript. PG and IM performed RBL assays, and KB and PV conducted basophil activation assays, read and revised the manuscript. RS, KM, RC, PZ, AM, KG assisted in planning and performing the intranasal challenge study, read and revised the manuscript. PhG kindly provided sera for analysis of subjects having received subcutaneous administration of omalizumab, read and revised the manuscript. IS and JG performed statistical analyses, read and revised the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


We found that sorafenib may act as a histone deacetylase (HDAC) inhibitor based on similar gene expression profiles. In vitro experimental analyses demonstrated that sorafenib indirectly inhibited HDAC activity in both sorafenib-sensitive and -resistant HCC cells. A cancer genomics analysis using the cBioPortal online tool showed the frequent upregulation of HDAC mRNAs. Furthermore, HCC patients with higher expressions of HDAC1 and HDAC2 had worse overall survival. Taken together, our study suggests that inhibition of HDAC by sorafenib may provide clinical benefits against HCC, and enhancement of HDAC-inhibitory activity of sorafenib may improve its therapeutic efficacy. In addition, our study also provides a novel strategy to study polypharmacology.Although sorafenib has been approved for treating hepatocellular carcinoma (HCC), clinical results are not satisfactory. Polypharmacology (one drug with multiple molecular targets) is viewed as an attractive strategy for identifying novel mechanisms of a drug and then rationally designing more-effective next-generation therapeutic agents. In this study, a polypharmacological study of sorafenib was performed by mining the next-generation Connectivity Map (CMap) database, CLUE ( Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of cancer-related deaths worldwide . Major rhttp://www.lincscloud.org/), that was recently replaced by CLUE (https://clue.io/).Polypharmacology is one drug exhibiting actions on more than one molecular target . PredictIn vitro experimental analyses validated that sorafenib indirectly inhibits HDAC activity in both sorafenib-sensitive and -resistant HCC cells. A cancer genomics analysis indicated that higher mRNA expressions of HDAC1 and HDAC2 in HCC were associated with a worse overall survival of HCC patients. Therefore, inhibition of HDAC activity is associated with the anticancer activity of sorafenib against HCC.In this study, we mined the CLUE database to discover novel actions of sorafenib. We found that inhibition of histone deacetylase (HDAC) activity is a novel function of sorafenib. https://clue.io/), the next generation CMap database, for the polypharmacology of sorafenib, we accidentally found similarities of HDAC inhibitors with sorafenib , were used. First, their sensitivities to sorafenib were determined by an MTT cell viability assay. As shown in Figure y Figure . Next, wy Figure , which iy Figure . Sorafeny Figure . Similary Figure . Therefohttp://www.cbioportal.org/) [+ for their activities [http://www.oncomine.org/) [http://www.compbio.iupui.edu/proggene/) [To gain more insights into the clinical benefits of sorafenib through its HDAC inhibition, genetic alterations, including the mutation status, copy number alterations, and mRNA expressions, were examined by accessing the cBioPortal for Cancer Genomics  , 11. Theal.org/) , 13. Claal.org/) , 13. Clativities . Herein,tivities –18 were ne.org/) . As showoggene/)  was perfTo investigate the drug-gene connectivity of sorafenib and HDAC isoforms, gene expression profiles of sorafenib-treated cells were compared to those in specific HDAC isoform-knockdown cells. As shown in Figure To investigate whether sorafenib can downregulate HDAC expression, protein levels of HDAC1, HDAC2, HDAC3, HDAC4, and HDAC6 in sorafenib-treated HepG2, HepG2-SR, and PLC5 cells were analyzed by Western blotting. As shown in Figure It is currently accepted that polypharmacology (one drug interacts with multiple targets) is a general property of small molecules. Polypharmacological studies can be applied not only for drug repurposing (new uses for old drugs) but also for identifying novel mechanisms of drugs . Gene exin vitro experimental analysis showed that sorafenib indirectly inhibited HDAC activities through downregulating protein expressions. Overexpressions of HDACs were found in tumors, leading to the epigenetic silencing of tumor-suppressor genes. Therefore, inhibition of HDACs is considered a potential strategy for treating cancers through reactivating tumor-suppressor genes [Sorafenib is a multi-kinase inhibitor that inhibits cell surface tyrosine kinase receptors (such as VEGFR and platelet-derived growth factor receptor (PDGFR)) and downstream intracellular serine/threonine kinases (such as RAF family kinases) . Our anaor genes . Our anain vitro incubation of sorafenib with nuclear cell lysates did not reduce HDAC activities, suggesting that sorafenib indirectly inhibits HDAC activities. The substrate used for the in vitro HDAC activity assay was Ac-Lys(Ac)-pNA. Theoretically, this assay can detect all HDACs that can deacetylate histone. Sorafenib selectively downregulated HDAC protein expressions; however, this seemed to incompletely explain the actions of sorafenib, because protein expressions of HDACs examined in this study were only partially reduced by sorafenib in HepG2 cells. Therefore, other mechanisms may exist. Phosphorylation of HDACs is important for their activation [The mechanism for inhibiting HDAC activities by sorafenib was not resolved in the current study. Direct tivation . For exativation –30. BecaGinkgo biloba extracts, retrieved from the Chinese herbal medicine, displayed HDAC-inhibitory activity [Ginkgo biloba extracts with sorafenib was safe and tolerable for advanced HCC patients and slightly improved their overall survival [Although sorafenib can extend the median survival time of HCC patients by about 3 months , drug reactivity . A recensurvival . Statinssurvival , 36. Oursurvival . Synergisurvival . TherefoIn summary, our study demonstrated that gene expression profiling using large-scale drug connectivity databases, such as CMap and LINCS, is highly useful for polypharmacological studies. By comparing drug connectivity profiles with sorafenib, we identified that inhibition of HDAC activities was associated with the anticancer activities of sorafenib in HCC cells. Our study provides a novel aspect of sorafenib for treating HCC and a novel strategy to study polypharmacology. However, there were still limitations in our study. For example, the mechanism(s) and the clinical benefits of the HDAC-inhibitory effect of sorafenib were not fully elucidated. These issues should be addressed in future investigations.https://clue.io/) using the “Touchstone” online tool . The “Perturbational Class” was set to “CMap Class” for searching drugs similar to sorafenib and protein kinase inhibitors. Drug connections (type: PCL) can be viewed as a heatmap. The option “Strong connections” was selected. For the connectivity analysis of sorafenib and HDAC isoform knockdown, the perturbational type was set to “Gene Knock-Down”, and a name filter was set to visualize the heatmap of HDAC isoforms.Connections of sorafenib and protein kinase inhibitors to other compounds or sorafenib to HDAC isoform knockdown were directly obtained from the CLUE database  [http://www.compbio.iupui.edu/proggene/) [A cancer genomics analysis was performed  by querying the online cBioPortal for Cancer Genomics  , 11. Thehttp://www.oncomine.org/) is a collection of cancer microarray databases with a web-based data-mining platform [p value < 0.05, and ranking of gene in the analyses > top 10%. Red signifies gene overexpression, and blue signifies gene underexpression. The intensity of the color signifies the best ranking of genes in those analyses.Oncomine  was purchased from Gibco. Dulbecco's modified Eagle medium (DMEM), L-glutamine, sodium pyruvate, non-essential amino acids (NEAAs), and an antibiotic-antimycotic  were purchased from Life Technologies. HDAC1, HDAC2, HDAC3, and HDAC4 antibodies were purchased from Cell Signaling. The GAPDH antibody was purchased form GeneTex. Horseradish peroxidase-labeled goat anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch. Sorafenib was purchased from Cayman Chemical. Dimethyl sulfoxide (DMSO) and 3--2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma. Protease and phosphatase inhibitor cocktails were purchased from Roche. Other chemicals or reagents were purchased from OneStar Biotechnology.2.HepG2 and PLC/PRF/5 (PLC5) cells were purchased from the Bioresources Collection and Research Center (BCRC), Food Industry Research and Development Institute . Sorafenib-resistant HepG2-SR cells were established by repeatedly exposing them to an increasing dose of sorafenib for several months. Cells were cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 1% L-glutamine, 1% NEAAs, and a 1% antibiotic-antimycotic solution, and incubated at 37 °C in a humidified incubator containing 5% COCell viability was examined with an MTT assay. Briefly, cells were spread on 96-well plates and treated with drugs for 72 h. Then, 0.5 mg/mL of MTT was added to each well, and cells were cultured for an additional 4 h. The blue MTT formazan precipitates were dissolved in 200 μL of DMSO. The absorbance at 570 nm was measured using a multi-well plate reader.Nuclear lysates with or without sorafenib treatment were prepared using a Nuclear/Cytosol Fractionation Kit (BioVision) according to the manufacturer's instructions. Pan-HDAC activity was measured with an HDAC Activity Colorimetric Assay Kit (BioVision). Nuclear lysates were incubated at 37°C with or without sorafenib, and the HDAC reaction was initiated by adding the Ac-Lys(Ac)-pNA substrate. After 3 h, lysine developer was added, and the mixture was incubated for another 30 min. The absorbance at 405 nm was measured using a multi-well plate reader.


Genetic analyses demonstrate that the PPKs are collectively necessary for the normal light-induced phosphorylation and degradation of PIF3. PPK1 directly phosphorylates PIF3 in vitro, with a phosphosite pattern that strongly mimics the light-induced pattern in vivo. These data establish that the PPKs are directly involved in catalysing the photoactivated-phy-induced phosphorylation of PIF3 in vivo, and thereby are critical components of a transcriptionally centred signalling hub that pleiotropically regulates plant growth and development in response to multiple signalling pathways.Upon light-induced nuclear translocation, phytochrome (phy) sensory photoreceptors interact with, and induce rapid phosphorylation and consequent ubiquitin-mediated degradation of, transcription factors, called PIFs, thereby regulating target gene expression and plant development. Nevertheless, the biochemical mechanism of phy-induced PIF phosphorylation has remained ill-defined. Here we identify a family of nuclear protein kinases, designated Photoregulatory Protein Kinases ), that interact with PIF3 and phyB in a light-induced manner  et al. identify a family of nuclear protein kinases that interact with photoactivated phytochrome B and facilitate phytochrome-induced phosphorylation and degradation of PIF3.Phytochrome photoreceptors mediate degradation of PIF transcription factors in the nucleus in response to light. Here Ni  Arabidopsis)Plants constantly monitor ambient light conditions to adjust their growth and development for optimal photosynthesis and survival. The red and far-red wavelength information is perceived by the phytochrome (phy) family of sensory photoreceptors  synthesized in seedlings germinated in darkness are mainly localized in the cytoplasm in the inactive Pr conformer. Upon red-light irradiation, the photoreceptors are converted to the active Pfr conformer, and rapidly translocate into the nucleus, where they interact directly with a subfamily of basic helix-loop-helix (bHLH) transcription factors, termed PIFs . Thi2456789111213IF3 ref.  did not Arabidopsis seedlings. In vivo and in vitro functional analysis of these kinases indicates that they facilitate photoactivated-phyB-induced, multisite phosphorylation and degradation of PIF3 in the nucleus.Here, using mass spectrometry, we have identified a small family of protein kinases, designated photoregulatory protein kinases (PPKs) that rapidly associate with PIF3 and phyB in response to light-induced, intracellular phy activation in young in vivo in Arabidopsis, and found components of an LRB-Cullin3 E3 ligase complex, as well as all members of the phy family18Previously we used co-immunoprecipitation (co-IP) and mass spectrometric methods to identify proteins that interact with PIF3 in a light-dependent manner in vivo co-IPs'). The data show significantly higher levels of both PIF3:MYC and endogenous phyB co-precipitating from red-light-pulse (Rp)-treated than dark (Dk)-control seedling extracts when using PPK1:CFP as bait  and phospho-dead (A6) PIF3 mutant variants, carrying either S-to-D or S-to-A substitutions, respectively, in the five most strongly light-induced phosphosites (six residues) previously identified in vivoin vivo could reflect photoactivated-phyB stabilization of the PPK1–PIF3 interaction during active phosphorylation. Direct interaction between PPK1 and PIF3 was also demonstrated by in vitro co-IP assays using PPK1 as prey  triple mutants have normal PIF3 protein levels in dark-grown seedlings, and a relatively normal light-induced mobility-shift (phosphorylation) and degradation of PIF3 (ppk1ppk2ppk3 (ppk123) and the ppk1ppk2ppk4 (ppk124) triple mutants show a strongly lower light-induced, endogenous-PIF3 mobility shift (phosphorylation) and degradation (PPK1PPK2PPK3PPK4 (amiR-PPK1234) quadruple-knockdown mutant compared to Col WT in the light  light  light , indicatin vitro kinase assays using affinity-purified recombinant PPK1 and PIF3. The data show that PPK1 induces a strong mobility shift in wild-type PIF3, but not the PIF3 A20 variant, with S/T to A mutations in the majority of the in vivo, light-induced phosphosites of PIF3 . The data show that unlike PPK1, CK1d does not induce a strong mobility shift in PIF3  phosphorylation of PIF1 and PIF4 by the protein kinases CK2 and BIN2, respectively, may prime these PIFs for subsequent light-induced, phy-mediated phosphorylation and degradation292 kinase .et al.Arabidopsis CK2 can phosphorylate PIF1 at multiple sites in vitro, and that mutation of these sites reduces the rate of light-induced PIF1 degradation in vivo. However, because these mutations did not alter the extent of light-induced phosphorylation in vivo, and loss of function ck2 mutants enabling examination of the necessity of the CK2 family for PIF1 phosphorylation were not available, the question of the linkage of this catalytic activity to normal phy-PIF1 signalling has remained open. Our present data indicate that the PPKs are collectively necessary for light-induced, intracellular phosphorylation of PIF3, and that PPK1 has a catalytic capacity and phosphosite specificity that is very similar to the kinase(s) responsible for this in vivo phosphorylation. We propose a mechanistic model, therefore  that is necessary for the light-stimulated phosphorylation process in vivo, is represented by X in The mechanism by which photoactivated phytochrome transfers its signalling information to its signalling partner(s) in plant cells has been of central interest for decades, including the popular proposal that the phy molecule itself functions as a photoactivated S/T protein kinase, evolutionarily descended from related prokaryotic His-kinase progenitors7154568herefore , wherebyIn the broader context, there is emerging evidence that the PPKs may function in multiple related signalling pathways. Notably, it has been reported that the PPKs (MLKs) are recruited to the circadian-related evening complex in green seedlings, in a photoactivated-phyB-requiring mannerNo statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.PPK2 (At5g18190): SALK_026482; PPK3 (At3g03940): SALK_000758; PPK4 (At2g25760): SALK_017102) were obtained from ABRC. The remaining T-DNA insertion GABI-Kat line (PPK1 (At3g13670): GABI_756G08) was obtained from NASC. The higher order mutant combinations were generated by crossing these lines.Three T-DNA insertion SALK lines  line targeting all four pPIF3:GUS (ref. PPK1 cDNA was subcloned into PCR8/GW/TOPO TA cloning vector (Invitrogen) using primers specified in ppk124 mutant. To generate the pPPK1:GUS transgenic lines, the 1.9 kb PPK1 promoter region upstream of the start codon was amplified from Col genomic DNA using primers in Arabidopsis.The 35S:PIF3:MYC ref. , pPIF3:GGUS ref.  and 35SB−2 s) on growth medium as reported−2 s) or continuous far-red (3 μmol m−2 s or as indicated) for 4 days at 21 °C. Measurements were done with at least 30 seedlings with three biological repeats.Seeds were stratified for 5 days at 4 °C in darkness, and induced to germinate with 3 h white light before exposure to a terminal 5-min FRp , centrifuged and filtered through two layers of miracloth, precleared with protein A agarose beads, and then incubated with in-house rabbit anti-GFP antibodies for 1 h at 4 °C. PPK1:CFP fusion-proteins were then captured with protein A agarose beads for another hour at 4 °C, washed five times with IP buffer, transferred into a new tube before elution with boiling SDS sample buffer. The co-IP experiment was repeated three times.For co-IP using PPK1:CFP as bait, collected tissues were ground in liquid nitrogen. Proteins were then extracted into MOPS buffer  with a C-terminal HA, MYC or FLAG tag. Full-length PPK2, PPK3 and PPK4 cDNAs were cloned into the T7CFE1-CMyc vector directly using the In-Fusion HD Cloning Plus according to the manufacturer's instructions . The phyB and various PIF3 vectors were as describedin vitro co-IP assays were repeated at least two times.in vitro co-IP assays, horseradish peroxidase (HRP) conjugated antibody against HIS tag , MYC tag , HA tag  and FLAG tag  were used to detect the IP products. For co-IP assays from seedling extracts, mouse monoclonal antibodies against the MYC tag , GFP  or phyB (B1) were used for immunodetection. For immune blots from total seedling extracts, mouse monoclonal antibody against tubulin  and affinity-purified rabbit polyclonal antibody against PIF3 (ref. Immunoblots and quantification of bands were performed as describedIF3 ref. . The relPPK1 entry clone by site-directed mutagenesis (Stratagene) using primer listed in E. coli (BL21 AI). For PPK1 protein expression, 2 ml overnight-grown BL21 cells expressing the desired constructs were transferred to 500 ml fresh LB medium and grown at 30 °C until OD reached 0.7–0.8. IPTG (0.1 mM) was then added into the culture to induce the protein expression overnight at 18 °C. The induction of PIF3 protein expression was as describedg for 30 min to remove the insoluble cell debris. The supernatant was incubated with glutathione agarosebeads for 2 h at 4 °C. After washing with TBS, the GST-tagged proteins were eluted using 10 mM glutathione in TBS. Eluted protein was concentrated by ultrafiltration using Amicon centrifuge tubes (Millipore) with a 30 kD molecular weight cut-off. phyB:FLAG protein was expressed in Hela cell lysate and purified using FLAG antibody. Rat CK1d is from NEB. De-phosphorylated Casein is from Sigma.PIF3-WT and PIF3-A20 constructs, as both N-terminal GST- and C-terminal MYC-fusions as describedIn vitro protein kinase assays for phosphorylation of PIF3 by PPK1 were performed in a reaction mixture containing 50 mM Tris pH 7.5, 160 mM NaCl, 5 mM ATP, 10 mM MgCl2, 2 mM DTT, protease inhibitor cocktail from Roche, 0.5 μg PPK1 and/or 5 μg PIF3 in 10 μl. Reactions were stopped after incubation at 30 °C for 1 h unless specified. PIF3 or total phosphorylated proteins were detected by western blot analysis using anti-MYC antibody or pIMAGO , respectively. For assays comparing PPK1 with CK1d, we used 0.5 μg PPK1 or CK1d, 2 μg PIF3 or 0.5 μg Casein in a total of 10 μl reaction volume. For in vitro assays with phyB, 0.25 μg of PPK1, 2 μg of PIF3 and 0.4 μg of phyB were used in a 10 μl reaction volume. The kinase assay buffer used for testing the effect of phyB was similar to the above buffer except that the concentration of NaCl was reduced to 100 mM. All in vitro kinase assays were repeated at least two times.in vitro kinase reactions using wild-type and mutant PPK1, in-solution Trypsin or AspN digestion (enzyme to substrate ratio at 1:20) was carried out overnight after reduction and alkylation. Peptides were cleaned by ziptip. Data were first acquired in the Data Dependent Acquisition (DDA) mode and searched against the TAIR database (http://www.arabidopsis.org/) from December 2010, concatenated with sequence randomized version of each protein  using Protein Prospector with parameters the same as describedFor mass spectrometric analysis of PIF3 phosphorylation, following −1 Xgluc). Data from biological triplicates of two independent transgenic lines were collected, and representative images are shown for each transgene.Histochemical GUS staining assays were performed on 2-day-old seedlings as describedA full-length PPK1 cDNA in an entry vector (G11831 from ABRC) was cloned into a pEG202 gateway vector to generate the lexA BD:PPK1 construct. A full-length PIF3 entry vectorAgrobacterium tumefaciens GV3101. Nicotiana benthamiana plants were grown in long day conditions (16 h white light (120 μmol m–2 s)/8 h dark) at 22 °C. Agroinfiltration was performed as described in ref. 600 of 0.1 for each culture. 35S:p19 (ref. −2 s) and kept in the dark. Infiltrated Nicotiana leaves were tested 3 days post-inoculation. The abaxial epidermis of test leaves was peeled, and images were taken using a Leica DM4000 B microscope fitted with a YFP filter. At least five leaves from different plants were examined for each construct. For signal quantification, samples were examined with a × 40 magnitude objective and nuclei with a fluorescent signal were counted in five microscopic fields per construct. The number of nuclei displaying fluorescence in each field was expressed as a percentage of the total number of nuclei visible in that field (Full-length PPK1 cDNA in a gateway entry vector (G11831 from ABRC) was cloned into the pEarleyGate 104 vector to generate the 35S:YFP:PPK1 vector. For BiFC assays, the vector system described in ref. p19 ref.  was co-iat field .ppk124) transgenic line were performed by using an Axiovert 200 microscope  with excitation and detection of the CFP fluorophore. Images were recorded with a digital Axiocam camera (Zeiss) and processed for optimal presentation using the Adobe Photoshop software package.Epifluorescence microscopy analysis of PPK1:CFP in the hypocotyl cell of a PPK1:CFP .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Table


Phosphorylation is the most abundant post-translational modification, regulating several aspects of protein and cell function. Quantitative phosphoproteomics approaches have expanded the scope of phosphorylation analysis enabling the quantification of changes in thousands of phosphorylation sites simultaneously in two or more conditions. These approaches offer a global view of the impact of cellular perturbations such as extracellular stimuli or gene ablation in intracellular signaling networks. Such great potential also brings on a new challenge: to identify, among the thousands of phosphorylations found in global phosphoproteomics studies, the small subset of site-specific phosphorylations expected to be functionally relevant. This review focus on updating and integrating findings on T lymphocyte signaling generated using global phosphoproteomics approaches, drawing attention on the biological relevance of the obtained data. The signaling cascades triggered in response to both extracellular and intracellular stimuli are often controlled by post-translational modifications (PTMs). Protein phosphorylation is the most abundant and best characterized PTM in eukaryotic cells. Phosphorylation involves the covalent attachment of an inorganic phosphate group to amino acid residues, and it is a reversible process determined by the balanced activity of kinases and phosphatases , 2. Site32P radioactive labeling, and antibody-based techniques such as western blotting, immunohistochemistry, ELISA, or flow cytometry. These approaches have generated invaluable knowledge on signaling cascades over the past years, but they do not offer a comprehensible, global view of the phosphoproteomic profile. More often than not, these approaches cannot identify the exact phosphorylation site. In our effort to understand intracellular communication, signaling components have been isolated and cascades have been “linearized.” However, the road from stimuli to changes in cellular functions is more complex. For example, a single stimulus often triggers simultaneously different pathways that synergize and/or antagonize in the regulation of specific cellular functions. In addition, signaling cascades often activate intrinsic positive and negative feedback mechanisms. The complexity of the phosphorylation networks is further increased by the fact that a particular protein can hold different phosphorylation sites, which may be targeted by different kinases and phosphatases. These regulatory mechanisms expand protein function, allowing a particular protein to control different activities and at the same time, to integrate different signals into coherent cellular responses. Thus, the identification and quantification of the vast amount of phosphorylation events that occur in a cell require unbiased techniques to complement traditional biochemical approaches used to study kinases and substrates on an individual basis. In this context, a recent breakthrough is the application of mass-spectrometry (MS) based quantitative phosphoproteomics to study signaling networks. This experimental approach has expanded the scope of phosphorylation analysis, enabling the simultaneous identification and quantification of changes in thousands of phosphorylation sites between two or more conditions. This powerful approach provides a global and unbiased view of the impact of natural or experimental perturbations such as gene ablation or extracellular stimuli. This great potential comes together with a new challenge: to identify, among the thousands of phosphorylations found in global phosphoproteomics studies, the small subset of site-specific phosphorylations that will be functionally relevant for the control of a specific cellular response. Starting in the last decade, an increasingly large number of studies have applied these techniques to interrogate signaling pathways in lymphocytes. Here we recapitulate how knowledge on T lymphocyte signaling has been lighted up using global phosphoproteomic approaches.The study of site-specific phosphorylations and kinase-substrate networks has been traditionally approached using two-dimensional gels, 2)  signaling pathway  Figure . Thus, p pathway , 15, imm pathway . A caveaFollowing p-peptide or p-protein enrichment, samples are subjected to analysis using reversed-phase liquid chromatography coupled to tandem MS. Due to the stochastic nature of peptide sampling by MS, different methods have been developed to achieve maximal accuracy in the quantification of changes in site-specific phosphorylations produced as a result of cell perturbations. The most commonly used methods for comparison and accurate quantification of p-peptides rely on the use of differential isotopic or isobaric labeling. Using these methods, proteins or peptides from different conditions are labeled using compounds with near identical chemical properties yet each one containing a unique isotopic composition, resulting in slightly different masses that can be distinguish by MS. Subsequently, samples from different conditions can be combined and simultaneously analyzed by MS. Stable isotope labeling strategies such as SILAC, dimethyl labeling, isobaric tagging reagents such as isobaric tags for relative and absolute quantitation (iTraq) or tandem mass tagging (TMT) labeling are often used in phosphoproteomics workflows to achieve maximal accuracy in the quantification Figure . The choTo summarize, phosphoproteomic workflows and MS analysis yields a global view of cellular phosphoproteomes with accurate quantification of changes in thousands of p-sites between two or more conditions. These changes require validation using alternative techniques , often antibody-based techniques such as western blotting using anti-phospho-specific antibodies. Finally, the challenge is to identify and characterize, among the whole phosphoproteome, the site-specific phosphorylations that are relevant for cellular functions. Some examples of experimental strategies followed by different authors to determine functional relevance of the data reported in phosphoproteomic studies can be found in Figure The recognition of peptide/major histocompatibility complexes (p/MHCs) by the TCR on the surface of antigen-presenting cells is a key event during the adaptive immune response. The initial events of TCR signaling are mediated by cytosolic tyrosine kinases and adaptors that couple antigen recognition to a network of serine–threonine kinases that propagate the signal from the cell membrane to the nucleus, promoting the transcriptional changes that support T cell differentiation and acquisition of effector functions . SeveralThe TCR signaling cascade sets off phosphorylation events on tyrosine residues that have been characterized during the past 20 years . Using pex vivo isolated T cells from two different mouse strains, the B6g7 and the non-obese diabetes (NOD) mice, as a strategy to find molecules responsible for hyperreactive T cells in NOD mice , which is susceptible to inhibition using an analog of the kinase inhibitor 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolopyrimidine  Ser557 residue as a PKD substrate. Remarkably, this study reported the generation of a “knockin” mouse bearing a phosphorylation-deficient SHP1 variant (S557A mutation), to demonstrate that SHP1-Ser557 phosphorylation is required for optimal T cell differentiation in the thymus  to transmit the signal originated at the cell surface to the cytosol. Local changes in the concentration of DAG are sensed by serine/threonine kinases of the protein kinase C (PKCs) and the PKDs families . The sige thymus .An interesting yet unsolved question in the T lymphocyte signaling field is whether effector T cells use unique signaling components that contribute to the distinct effector T cell response signaling. Quantitative phosphoproteomics is a powerful tool to provide insights into this question. For example, while effector T cell immune synapses (IS) recruit in CD28 and PKCθ, regulatory T cells (Tregs) IS are enriched in CTLA4 instead of CD28, lacking PKCθ . The conT cell activation depends on TCR signaling pathways triggered by antigen recognition. In addition, T cell differentiation and the acquisition of effector functions are strongly influenced by the cytokine milieu generated during the inflammatory response. Secreted mediators such as cytokines, chemokines, and growth factors are critical intercellular messengers that mediate the generation of effector T cells. In this context, IL-2 is the main controller of CD4 and CD8 clonal expansion and differentiation . In addiThe IL-2 signaling pathway has been also examined in primary CTLs. Upon stimulation of murine CTLs with IL-2 for 15 min, phosphorylation was induced in more than 700 p-sites and reduced on 220 p-sites, revealing the mRNA binding protein YBX1-Ser100 as a novel component of the IL-2 signaling pathway . In thisNef and Tat viral transcripts. As a consequence, SRRM2 depletion causes a change in the ratio of spliced forms, decreasing virion release. This is an example of how studying a process such as virus entry using global phosphoproteomics has unexpectedly lead to the identification of cellular proteins involved in the splicing of viral transcripts. Moreover, the authors found other 239 HIV-responsive phosphorylations on 175 different proteins, each of them with a potential role in HIV infection that merits additional investigation. The analysis of virus entry using global phosphoproteomics has been also applied to lytic gammaherpesvirus (The binding of the chemokine CXCL12 (SDF1) to its receptor CXCR4 has been implicated in HIV-1 infection and cancer metastases. This signaling pathway was explored in a phosphoproteomic study using the T lymphoblast cell line CEM. This study confirmed previously known SDF1-regulated p-sites in core T cell kinases such as Akt and Erk2, and also revealed novel SDF1-responsive p-sites in proteins such as Stathmin . Comparipesvirus  and inflpesvirus .In addition to analysis of virus entry, phosphoproteomics has been recently used to study HIV cell–cell spread . ChangesPhosphoproteomics is being used to understand the role of phosphorylation networks on T cell activation, but it can also offer insights into the alterations of regulatory mechanisms that underlie the transformation processes causing lymphomas, leukemia, and other lymphocyte-mediated diseases such as inflammatory and autoimmune conditions. Although this approach has been exploited so far to study mostly B cell tumors , some stIn vitro kinase assays demonstrated that PKM2 is a substrate of ALK. Moreover, the combination of pharmacological inhibition and phosphorylation mutants of PKM2 showed that ALK-mediated phosphorylation of PKM2-Tyr105 inhibits PKM2 activity, thus promoting glycolysis, Warburg metabolism, and tumor progression. Thus, the blockage of PKM2 phosphorylation represents a potential mechanism to control cell proliferation in ALK-mediated lymphomas. Interestingly, increased PKM2 phosphorylation on Ser37 residue, which induces the expression of genes that promote the Warburg metabolism, was detected in a phosphoproteomic study in response to IL-23 stimulation in the Kit225 human T cell line  is often driven by a constitutively active tyrosine kinase, the nucleophosmin-anaplastic lymphoma kinase (ALK). To evaluate the therapeutic potential of inhibiting ALK to treat ALCL, the effect of an ALK inhibitor was analyzed in ALCL-derived cells using phosphoproteomics . Among tell line . Hence, In addition to leukemia and lymphomas, aberrant T lymphocyte function underlies pathological conditions such as multiple sclerosis. Phosphoproteomic analysis of multiple sclerosis brain lesions detected sphingosine 1-phosphate receptor (S1P1) phosphorylation on Ser351 . S1P orcIn addition to phosphorylation, other PTMs also participate in signaling processes to control protein and cell function. The cellular scenario becomes even more complicated because the different PTMs crosstalk. As an example, phosphorylation can control the ability of proteins to undergo additional PTMs of different types . The advUbiquitination consist in the addition of ubiquitin to a Lys residue of substrate protein. The enrichment strategy for “ubiquitome” profiling more often used is based on immunoprecipitation using an antibody that recognizes the glycine–glycine bound to the modified Lys-residue (K-ε-GG), generated after trypsin digestion of ubiquitinated proteins. Ubiquitinated sites are subsequently identified by MS  to target proteins at the ε-amino group of Lys residues, regulating protein function, and activity. His-tagged versions of SUMO have been expressed in HEK293T cells, allowing the enrichment of SUMOylated proteins using affinity chromatography. Then, samples are digested and immunoprecipitated with monoclonal antibody that specifically recognize SUMO remnants left after tryptic digestion of SUMOylated proteins. This experimental approach has led to the identification of 954 SUMO3-modified Lys residues, 86% of the sites previously unknown  addition (O-GlcNAcylation) is particular type of protein glycosylation on Ser/Thr residues, involved in diverse cellular metabolic and signaling pathways including T cell self-renewal and malignant transformation (13C6-glucose, produces 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. In combination with O-GlcNAc peptide enrichment using phenylboronic acid solid phase extraction cartridge and MS identification, 105 O-GlcNAc and their dynamics have been determined on HeLa cells following this method (O-linked β-ormation . As a tos method . To summThe overall conclusion of the work recapitulated here is that phosphoproteomic approaches have fulfilled their potential. They have been successfully applied in the identification of novel components of signaling networks, previously unknown kinase substrates and novel biological functions controlled by site-specific phosphorylation. These experimental approaches have also shed light into unexpected feedback mechanisms. In addition, already generated datasets will keep on generating new discoveries, since hundreds of site-specific phosphorylations on key molecules for T lymphocytes are waiting for further study of novel functions and regulatory mechanisms. From the initial work based on studies in cell lines, phosphoproteomics is now being successfully applied to more relevant experimental settings such as virus infection or analysis of tissue samples from T cell-mediated inflammatory diseases. Remarkably, this knowledge is unique to phosphoproteomics, since it will not have been generated using other experimental approaches.Phosphoproteomic analysis is ready to be taken into complicated experimental settings. An interesting yet unsolved question in the T lymphocyte signaling field is if effector T cells use unique components that contribute to the distinct outcome of TCR signaling in the different effector subtypes. The robustness of quantitative phosphoproteomic analysis can be used to examine TCR signaling in the different types of effector T lymphocytes in order to identify unique signaling events that can be used to manipulate the function of specific T cell subpopulations. In addition to novel components of signaling cascades, parameters such as stoichiometry of site-specific phosphorylations, abundance of signaling molecules, or even detailed kinetics on a particular phosphorylation event can be extracted from phosphoproteomics datasets. These parameters are often estimated from experimental systems, but phosphoproteomics datasets offer empirical data that can implement systems biology and mathematical modeling of signaling networks to make predictions and generate new hypothesis-driven experiments . These sAll listed authors have made substantial contributions to the conception of the work, drafted the manuscript, and approved it for publication.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.


Dependent on their cellular localization, Protein Kinase D (PKD) enzymes regulate different processes including Golgi transport, cell signaling and response to oxidative stress. The localization of PKD within cells is mediated by interaction with different lipid or protein binding partners. With the example of PKD2, we here show that phosphorylation events can also contribute to localization of subcellular pools of this kinase. Specifically, in the present study, we show that tyrosine phosphorylation of PKD2 at residue Y87 defines its localization to the focal adhesions and leads to activation. This phosphorylation occurs downstream of RhoA signaling and is mediated via Src. Moreover, mutation of this residue blocks PKD2’s interaction with Focal Adhesion Kinase (FAK). The presence and regulation of PKD2 at focal adhesions identifies a novel function for this kinase as a modulator of cell adhesion and migration. PKDs often have redundant functions, but recently, cellular responses where individual isoforms have unique targets were described. For example, PKD1 expression has been shown to block breast cancer cell migration and invasion in vitro and in vivo and to block matrix-metalloproteinase (MMP) expression5. In contrast to this, the two other isoforms promote these processes7, and unlike PKD1, PKD2 induces cell invasion by regulating MMP expression and secretion9. In addition to its effects on cell motility, PKD2 also has been implicated in enhancing tumor cell proliferation and tumor growth11. Beyond its functions in cancer, PKD2, unlike the two other isoforms, also has been shown to have a critical role in T-cell antigen receptor signaling in mature T-cells; and in PKD2 deficient mice, loss of PKD2 is associated with enlarged lymph nodes and spleen13.Protein kinase D 2 (PKD2), along with PKD1 and PKD3 constitute a family of serine/threonine kinases implicated in a wide variety of biological processes such as epithelial to mesenchymal transition, cell migration, proliferation, survival and angiogenesis14. Moreover, its localization to other cellular sites such as the Golgi has been reported8. To date, there are no reports linking PKD2 to focal adhesion (FA) function, except one study indirectly showing that PKD (as detected with a pan-antibody that picks up PKD1 and PKD2), as well as cortactin and FA-localized paxillin can be isolated from invadopodia of breast cancer cells15.In the absence of stimulation, PKD2 is mostly resident in the cytoplasm, but in response to receptor-mediated activation can translocate to the plasma membrane16. FAs undergo constant flux, and their formation and dissolution is indispensable during cell adhesion and migration16. The FA complex is formed by a multitude of proteins, including adapters such as p130Cas and Paxillin, and the kinases focal adhesion kinase (FAK) and non-receptor tyrosine kinase Src17. FAK can autophosphorylate at Y397, which leads to binding and activation of Src. After activation, Src then phosphorylates a variety of FA proteins including FAK and p130Cas19. The pivotal role of Src in the remodeling processes that contribute to the dynamic nature of FAs becomes evident after its inhibition, which results in a loss of integrin-mediated adhesions20 and FA turnover during cell migration21.In the present study, we show that tyrosine-phosphorylated PKD2 is localized at the focal adhesions. FAs are integrin-based macromolecular structures that link the actin cytoskeletal network within cells to matrix componentsIn this report, we define PKD2 as a new target for Src at the focal adhesions. We describe phosphorylation at Y87 as a defining characteristic of PKD2 localization to the focal adhesions. We further show that RhoA functions upstream of Src in mediating this phosphorylation, and that inhibition of Y87 phosphorylation by RhoA/Src impairs cell adhesion and migration.22. In order to study the role of tyrosine phosphorylation of PKD2 at this residue, HeLa cells, as well as NMuMG and MDA-MB-231 cells were deemed as an ideal systems, because they express only PKD2 and PKD3 , but not Rac1 (Rac1.CA) or Cdc42 (Cdc42.CA), can induce phosphorylation of PKD2 at Y87  that is shared by PKD1 and PKD2, but not PKD3  within the Y-G-M/L-Y motif can be phosphorylated downstream of oxidative stress22, we here show that in PKD2 the corresponding tyrosine residue (Y87) is phosphorylated downstream of RhoA and Src , possibly due to the minimal difference in its amino-acid sequence (pY87-G-L-Y). Therefore, we conclude that Y87 phosphorylation is important for PKD2 localization to the FAs, which brings the kinase into proximity to another, yet unidentified, nPKC that mediates activation loop phosphorylation.Tyrosine phosphorylation of PKD2 at Y87 also contributes to its activation, since a PKD2.Y87F mutant was less phosphorylated in its activation loop, and showed less autophosphorylation activity, both direct measures for PKD activity Fig. . Activat36. In addition, Src can induce adhesion turnover which also contributes to cell migration21.PKD2 regulation at Y87 at the FA seems to contribute to cell adhesion Fig. , as well37, and depletion of PIP5Klγ leads to severe attachment and cytoskeletal defects in cells38. PIP5Klγ regulates PI4,5P2 levels, and such alterations regulate proper FA function and FA dynamics40. The protrusive forces that regulate cell spreading, as well as dynamic regulation of FAs, are also linked to actin cytoskeletal reorganization42. Cofilin/ADF is a key factor for actin re-organization events that is regulated by its phosphorylation state44. PKD2 has been shown to regulate the phosphorylation status of cofilin by activating PAK445 and decreasing activity of SSH1L23. However, if this occurs at FA and if this is dependent on Y87 phosphorylation remains elusive.Now that we have identified RhoA-Src-pY87-PKD2 signaling as a mechanism to target PKD2 to the FAs, prospective targets for PKD2 in this pathway still need to be identified in future studies. Possible substrates for PKD2 at the FAs could include FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ), which previously has been shown to be a target for PKD1In summary, we identify focal adhesions as previously undescribed sites of PKD2 localization. This localization is characterized by its phosphorylation at Y87, which is mediated by Src downstream of RhoA. Attenuation of this phosphorylation antagonizes cell adhesion and cell migration Fig. .22. Secondary HRP-linked antibodies were from Millipore. Secondary antibodies (Alexa Fluor 546 F(ab’) fragment of goat anti-rabbit IgG (H + L), Alexa Fluor 488 F(ab’)2 fragment of goat-anti-rabbit IgG, Alexa Fluor 568 F(ab’)2 fragment of goat-anti-mouse IgG) were from Invitrogen . Alexa Fluor 633-phalloidin and Rhodamine-phalloidin were from Invitrogen. Zenon Alexa Flour  labeling kits were from Thermo Fisher Scientific . For transient transfection of HeLa cells, TransIT-HeLa Monster  was used. Recombinant human PKD2 (cat # 14–506) and Src (cat # 14–326) were from Millipore. Fibronectin was from Sigma-Aldrich, PP2 was from Millipore, and Rho Inhibitor I was from Cytoskeleton Inc . All other chemicals were from Thermo Fisher Scientific.Cell lines were obtained from ATTC  and cultured in DMEM supplemented with 10% FBS (plus 10 µg/ml insulin for NMuMG). Primary antibodies are described in detail in Supplemental Table 24. RFP-LifeAct was from ibidi . GFP-tagged PKD2 was from Dr. T. Seufferlein . PKD2.Y87F mutations were introduced by site-directed mutagenesis  using 5′-TTCCCTGAGTGTGGCTTCTTCGGCCTTTACGACAAGATC-3′ and 5′-GATCTTGTCGTAAAGGCCGAAGAAGCCACACTCAGGGAA-3′ as primers. Non-targeting (scr) shRNA (SHC001), shRNAs targeting PKD2  and PKD2-shRNA#2, NM_016457.x-1335s1c1) as well as shRNAs targeting Src (Src-shRNA#1 NM_198291.1-1579s1c1 and Src-shRNA#2 NM_198291.1-648s1c1) were from Sigma-Aldrich.The expression plasmids for constitutively-active Src (Src.Y527F), pEBG-RhoA.CA, Rac1.CA, Cdc42.CA and FLAG-tagged PKD2 have been described before2O at 70 °C for 10 min. 5x buffer, 40 U RNAsin , 200 µM dNTP (NEB), 10 mM DTT, 300 U Superscript II reverse transcriptase were added to a total volume of 20 µl. The reaction was carried out at 45 °C for 60 minutes and then heat-inactivated at 95 °C for 5 minutes. Resulting cDNA was subjected to PCR analysis using specific primer sets. Primers were: 5′-TTCTTCCACCTCAGGTCATC-3′ and 5′-TGCCAGAGCACATAACGAAG-3′ for a 600 bp fragment of human PKD1, 5′-CAACCCACACTGCTTTGAGA-3′ and 5′-CACACAGCTTCACCTGAGGA-3′ for a 698 bp fragment of PKD2; and 5′-TCATTGACAAACTGCGCTTC-3′ and 5′-GTACATGATCACGCCCACTG-3′ for a 485 bp fragment of PKD3. Reaction conditions for the PCR reactions were: 1 min annealing at 55 °C, 1 min amplification at 72 °C, with 25 cycles.Cellular RNA was isolated using RNA-Bee  according to the manufacturer’s instructions and mRNA was transcribed into cDNA using Superscript II (Invitrogen). For the transcription reaction 1 µg Oligo dT(18) primer  and 1 µg RNA were incubated in a total volume of 10 µl H2HPO4, 1.5 mM KH2PO4 pH 7.2) and lysed with Buffer A  containing Protease Inhibitor Cocktail . After 30 minutes of incubation on ice, lysates were centrifuged  and protein concentration was determined. Proteins were immunoprecipitated by incubation with a specific antibody (2 μg) for 1 hour, after which samples were incubated for a further 30 minutes with Protein G Sepharose . Immunecomplexes were washed 3 times with TBS , and resolved in 20 μl TBS and 2x Laemmli buffer. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes and visualized by immunostaining.Cells were washed 2 times with ice-cold PBS  in 40 µl of kinase buffer  supplemented with 100 μM ATP. The reaction was incubated for 30 minutes at room temperature and was stopped by addition of 2x Laemmli buffer.The TM PermaFluorTM Aqueous Mounting Medium (Thermo Fisher Scientific). Samples were evaluated using either a LSM 880 or a LSM 510META confocal laser scanning microscope , or an IX81 DSU Spinning Disc Confocal .Cells were transfected or stimulated and seeded in ibidi 6 channel μ-slides (ibidi) or on coverslips Figs. , C, as i6 cells/coverslip. Cells were imaged at the indicated times and counted manually to determine the number of cells attached.Cells were transfected or infected as indicated and then reseeded onto fibronectin-coated glass coverslips (10 μg/ml) at a density of 0.02 × 103. Briefly, cells were harvested, washed once with media containing 1% bovine serum albumin (BSA) and re-suspended in media containing 0.1% BSA. Then 100,000 cells were seeded per transwell insert. NIH-3T3 conditioned medium served as a chemoattractant in the lower chamber. Remaining cells were used to control the expression of genes of interest by Western blot. After 16 hours, cells on top of the transwell insert were removed and cells that had migrated to the lower surface of the filters were fixed in 4% paraformaldehyde and counted.Transwell migration assays were performed as previously describedt-test using Graph Pad software, and p < 0.05 was considered statistically significant.Data are presented as mean ± SEM. P values were acquired with the student’s All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Data


Efforts to select the appropriate number of implants in adolescent idiopathic scoliosis (AIS) instrumentation are hampered by a lack of biomechanical studies. The objective was to biomechanically evaluate screw density at different regions in the curve for AIS correction to test the hypothesis that alternative screw patterns do not compromise anticipated correction in AIS when using a segmental translation technique.Instrumentation simulations were computationally performed for 10 AIS cases. We simulated simultaneous concave and convex segmental translation for a reference screw pattern  and four alternative patterns; screws were dropped respectively on convex or concave side at alternate levels or at the periapical levels (21 to 25% fewer screws). Predicted deformity correction and screw forces were compared.p < 0.0003). There was no statistical bone-screw force difference between convex and concave alternate dropouts and between convex and concave periapical dropouts (p > 0.28). Alternate dropout screw forces were higher than periapical dropouts (p < 0.05).Final simulated Cobb angle differences with the alternative screw patterns varied between 1° to 5° (39 simulations) and 8° (1 simulation) compared to the reference maximal density screw pattern. Thoracic kyphosis and apical vertebral rotation were within 2° of the reference screw pattern. Screw forces were 76 ± 43 N, 96 ± 58 N, 90 ± 54 N, 82 ± 33 N, and 79 ± 42 N, respectively, for the reference screw pattern and screw dropouts at convex alternate levels, concave alternate levels, convex periapical levels, and concave periapical levels. Bone-screw forces for the alternative patterns were higher than the reference pattern (Using a simultaneous segmental translation technique, deformity correction can be achieved with 23% fewer screws than maximal density screw pattern, but resulted in 25% higher bone-screw forces. Screw dropouts could be either on the convex side or on the concave side at alternate levels or at periapical levels. Periapical screw dropouts may more likely result in lower bone-screw force increase than alternate level screw dropouts. Pedicle screw fixation has become the state-of-the-art instrumentation for the surgical treatment of adolescent idiopathic scoliosis (AIS), resulting in better deformity correction and lower revision surgery rates compared to hybrid or hook-rod constructs –3. HowevScrew density is defined as the number of screws per level fused. There may be multiple clinical and biomechanical factors in determining screw density and distribution. Certain screw types and distributions are required in order to perform specific correction maneuvers, such as apical vertebral derotation and segmental vertebral derotation . The effA structured literature review revealed that the mean reported screw density varies from 1.04 to 2.0, whereas the average curve corrections only varied from 64 to 70% , 9. HighUntil recently, studies of screw density have been underpowered, included hybrid constructs, or based on retrospective review of clinical data. Further, there is a lack of biomechanical data guiding screw number and placement. Thus, practice is mostly based on individual preferred technique, and scientific progress is limited by the inability to test alternative screw patterns on a given patient. In contrast to studies based on clinical data analysis, biomechanical studies using computerized patient-specific models allow the assessment and comparison of variable screw numbers and patterns with different correction techniques simulated for the same case. The objective of this study was to use computerized patient-specific spine models to biomechanically evaluate screw dropouts at different regions in the curve for AIS correction to test the hypothesis that alternative screw patterns do not compromise anticipated correction in AIS when using segmental translation as the primary correction technique.Numerical simulations of posterior spinal instrumentations were performed using computerized patient-specific biomechanical models of 10 AIS patients in order to assess the effect of screw density on curve correction and bone-screw forces. With the institutional review board approval, the cases were randomly selected from AIS patients having undergone instrumented spinal fusion at our university hospital center during the last 6 years. Clinical indices are provided in Table Three-dimension (3D) spine geometry of the selected cases was built using calibrated preoperative coronal and lateral radiographs and 3D multi-view reconstruction techniques . The proVertebrae from T1 through L5 and the pelvis were modeled as rigid parts which were connected with multiple flexible elements respectively representing the biomechanical effect of the intervertebral disc, anterior longitudinal ligament (ALL), posterior longitudinal ligament (PLL), ligamentum flavum (LF), intertransverse ligament (ITL), facet joint capsule (FC), and interspinous ligament (ISL) combined with supraspinous ligament (SSL). Six translational springs were used to respectively represent (1) ALL, (2) PLL, (3) LF, (4) left ITL, (5) right ITL, and (6) the combined effect of ISL and SSL. The biomechanical behavior of the facet joints is more complex compared to the other intervertebral ligamentous elements; they were respectively represented with a six-dimensional general spring . A primaThe six translational springs were modeled as cable-like elements on the computer-aided engineering platform, Adams/View, Version MD Adams 2010 . Their stiffness in compression were set to null and those in traction were adapted to the reported experiment results on cadaveric specimens, e.g., 23.6 N/mm (ALL), 24.9 N/mm (PLL), 32.6 N/mm (LF), 12.9 N/mm (ITL), and 32.1 N/mm (ISL combined with SSL) –23 for TBased on the actual instrumented fusion levels of the selected cases and the well-accepted alternative screw densities , five scThe computed geometric indices from the reconstructed preoperative spine models and the simulated instrumented spine models are presented in Table p < 0.0003). Alternate dropout screw forces were higher than periapical dropouts (p < 0.05). There was no statistical bone-screw force difference between convex and concave alternate dropouts (p > 0.28). Although there was no statistical bone-screw force difference between convex and concave periapical dropouts (p > 0.25), the convex periapical screw dropouts had less impact on bone-screw force vector pattern in some of the cases in both the coronal and the sagittal plane, i.e., bone-screw force vector pattern was the closest to the reference screw pattern. Bone-screw force vectors for a representative case are provided in Fig. Average bone-screw force was computed for each simulation and the results are presented in Fig. s p < 0.0. There wComparing alternative screw patterns respectively with the maximal density reference screw pattern, differences in the final simulated MT Cobb angles, thoracic kyphosis, and apical vertebral rotation did not exceed 5° for all except one case. These differences are within the accepted systematic error found in clinical Cobb angle measurements . The meaAlternative screw patterns with fewer screws resulted in higher overall bone-screw forces. Previous studies showed that higher density screw patterns had usually higher bone-screw forces due to the overconstraining effect . The difIn summary, with simultaneous two-rod segmental translation using polyaxial pedicle screws with dorsal height adjustability, there was no statistical difference between convex side screw dropouts and concave side screw dropouts. Periapical screw dropouts may more likely result in lower bone-screw force increase than alternate level screw dropouts. The number of vertebrae spanned by the major curve or the sharpness of the curve affects the bone-screw forces; screw dropouts should be reduced within short structural curves with high local curvature. Screw density and distribution should be determined by taking into account the local geometric characteristics of the curve in both the sagittal and coronal planes and the shape of the rods in addition to the curve type, deformity magnitude, and spinal stiffness.Different correction techniques and instrumentation construct designs may have important roles in curve correction and bone-screw forces. Since the simulated correction technique was simultaneous two-rod segmental translation using polyaxial pedicle screws with dorsal height adjustability, findings in this study may not be directly applied to other techniques, types of screws, and construct designs. However, based on the fundamental laws of mechanics, for an equivalent curve correction, the overall effective corrective forces should be at an equivalent level independent of correction techniques and construct designs. Findings in this study provided therefore useful data on the overall effect of screw density and distribution. Knowledge of potential bone-screw forces in AIS instrumentation using alternative computer-simulated screw constructs can help surgeons select the best possible screw configuration specific for the patient. All bone-screw forces may not contribute to the actual curve correction due to the high mechanic complexity of the instrumented spine. Parts of the bone-screw forces are the “true corrective forces,” which are necessary and sufficient to achieve the desired correction and the rest are overconstraining forces which are induced when forcing to ensure proper rod seating and locking at all pedicle screws as required by the construct design . The effThis study is limited by the available experimental data to calibrate and describe the biomechanical properties of the scoliotic spine model. However, the modeling technique has been adapted to make the best of the available calibration data to meet the needs of this study. Some simplifications were made, such as modeling the vertebral bodies as rigid parts, limiting the model solving in the quasistatic domain, and approximating the intervertebral connection with limited number of elastic elements. Since the focus was on the overall comparative curve correction and bone-screw forces, these simplifications and approximations were considered as adequate for this study. To establish baseline data for screw density and distribution, studies through simulations using computerized biomechanical models should be combined with prospective clinical studies and biomechanical experiments. The computerized model will be refined and better calibrated using the more comprehensive clinical and experimental results and then used to perform more extensive studies which may not be possible in a clinical and experimental context.Deformity correction can be achieved with 23% fewer screws than maximal density screw pattern, in which screw dropouts could be either on the convex side or on the concave side at alternate levels or at periapical levels. Using fewer screws resulted in higher average bone-screw forces. Findings in this study provided preliminary data on the effect of screw density and distribution. Further studies should be conducted on more screw densities and distributions using different correction techniques and different instrumentation in order to acquire comprehensive biomechanical knowledge to assist in individualized surgical treatment for AIS.


Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The  Tat substrates have N-terminal signal peptides containing a conserved twin-arginine motif and are transported across the membrane in a folded state driven by the protonmotive force –3.in vivo some TatA constitutively associates with this complex, most likely in an equimolar ratio with TatB and TatC B/r, Δ(argF-lac)U169, −λ, e14-, flhD5301, Δ(fruK-yeiR)725(fruA25), relA1, rpsL150(StrR), rbsR22, Δ(fimB-fimE)632(::IS1), deoC1— (HRP conjugate) was purchased , and an HRP-conjugated goat anti-rabbit antibody  was used as secondary antibody for the TatA, TatB and TatC antisera. Cross-reacting bands were visualized after incubation with Clarity™ Western ECL Blotting Substrate (BioRad) using a CCCD camera .For analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using Tris-glycine gels . In totaescribed ,31. Two 4.3.ymol . Multimers were built using TatA–TatC/TatB–TatC disulfide cross-links as unambiguous constraints for docking using HADDOCK [Molecular modelling was carried out as described previously . All ima HADDOCK . In all 3, while the heterononameric systems comprised approximately 115 000 atoms, with box dimensions in the region of 100 × 100 × 100 Å3. The systems were equilibrated for 1 ns with the protein restrained before three repeats of 100 ns of unrestrained atomistic MDS, for each configuration of the molecular system (see below), using the Gromos53a6 force field [All MDS were performed using GROMACS v. 5.1.2 . The Marce field . Molecul−5 using the Parrinello–Rahman barostat [All simulations were executed at 37°C, with protein, lipids and solvent separately coupled to an external bath, using the velocity-rescale thermostat . Pressurbarostat . All bonbarostat . Electrobarostat , while a