While constantly rising, the prevalence of allergies is globally one of the highest among chronic diseases. Current treatments of allergic diseases include the application of anti-histamines, immunotherapy, steroids, and anti-immunoglobulin E (IgE) antibodies. Here we report mammalian cells engineered with a synthetic signaling cascade able to monitor extracellular pathophysiological levels of interleukin 4 and interleukin 13, two main cytokines orchestrating allergic inflammation. Upon activation of transgenic cells by these cytokines, designed ankyrin repeat protein (DARPin) E2_79, a non-immunogenic protein binding human IgE, is secreted in a precisely controlled and reversible manner. Using human whole blood cell culturing, we demonstrate that the mammalian dual T helper 2 cytokine sensor produces sufficient levels of DARPin E2_79 to dampen histamine release in allergic subjects exposed to allergens. Hence, therapeutic gene networks monitoring disease-associated cytokines coupled with in situ production, secretion and systemic delivery of immunomodulatory biologics may foster advances in the treatment of allergies. The standard treatment for an allergic response is anti-histamines, steroids and anti-IgE antibodies. Here the authors present a genetic circuit that senses IL-4 and IL-13 and responses with DARPin production to bind IgE. In the early phase of an allergic reaction, allergens are recognized by professional antigen-presenting cells (APCs)5. These cells are able to take up and display fragments of allergens on major histocompatibility complex (MHC) class II molecules, which they carry on their cell surface6. Naive T cells can recognize the fragments via T-cell receptors, which leads to their differentiation into effector T helper 2 (TH2) cells7. These specialized effector cells produce and release several cytokines, including interleukin 4 (IL-4). IL-4 serves as an autocrine growth and differentiation factor8 and is responsible for the class switching of B cells to immunoglobulin E (IgE) synthesis9. Mast cells and basophil granulocytes bear high-affinity receptors for IgE (FcεRI) and bind free IgE in blood or tissue10. Pre-formed IgE–FcεRI complexes permit a rapid response by mast cells and basophil granulocytes: the allergens directly bind to the IgE on the surface, thereby promoting the aggregation of the IgE–FcεRI receptor complexes and triggering the intracellular inflammatory cascade12. Subsequently, an immediate release of soluble mediators promotes the allergic inflammation. These mediators consist of pre-formed and newly synthesized compounds including histamine, cytokines, chemokines and leukotrienes13. Among the cytokines released after FcεRI aggregation, interleukin 13 (IL-13) plays a major role in the development of atopic asthmatic disease15. IL-13, along with epidermal, neural, vascular and fibroblast growth factors, drives the production of mucus and is responsible for the remodeling of airway walls16. Elevated amounts of IL-13 can cause excessive production of mucus thereby narrowing the airways and increasing the typical asthmatic symptoms18. Once the early phase of the allergic reaction has developed into a chronic allergic inflammation, the production of IL-4 and IL-13 occurs in a positive feedback loop, which results in an increase in IgE levels19.Allergies are reactions of the immune system toward foreign substances that do not cause immunogenicity under healthy conditions. The development of allergies is often inherited20. Histamine is produced by mast cells and basophil granulocytes and is responsible for the main symptoms of allergic diseases22. Allergen-specific immunotherapy, another form of treatment, consists of the desensitization of the immune system by administration of appropriate concentrations of allergen extracts23. Of all these approaches, the direct targeting of the IgE molecule appears to be the most promising because IgE plays a pivotal role in allergic disease and the amount of IgE in the serum correlates with the disease severity25. Recently, a humanized monoclonal antibody, quilizumab, was designed to neutralize IgE-expressing B cells, thereby depleting the net amount of IgE in the serum26. Although the therapy was able to reduce serum total and allergen-specific IgE by 30–40 %, it was not able to reduce the asthma exacerbations, lung function, or patient-reported symptoms27. Another murine humanized anti-IgE antibody, omalizumab, has been developed and is capable of binding IgE in the serum of allergic patients28. However, the administration of very high doses of omalizumab is required in patients because of the high sensitivity of mast cells and basophil granulocytes to IgE. Thus, the major drawbacks of this therapy include the high costs and the restriction of the therapy to a small group of patients with severe asthma29. Recently, a novel IgE-binding protein (DARPin E2_79) was developed. DARPin E2_79 is a so-called designed ankyrin repeat protein (DARPin), a small variant derived from a very broad class of naturally occurring ankyrin repeat proteins31. Due to its high specificity to IgE, DARPin E2_79 is capable of inhibiting the formation of IgE–FcεRI receptor complexes32. At high concentrations, DARPin E2_79 has also been shown to disrupt previously formed complexes33 and thus prevents the onset of an allergic reaction.Various attempts have been made to develop potent treatments against allergies. Many options include either the neutralization of histamine or the prevention of its release35. Hence, the development of implantable cell-based synthetic cytokine sensor-effector devices may represent an important therapeutic advancement compared to conventional treatment strategies that may help to improve the quality of life of allergic patients.Here, we report genetically engineered human cells able to produce IgE inhibitors at a precise time and in a dose-dependent manner to prevent the development of allergic inflammatory responses. The effective and accurate production of IgE inhibitors that is initiated by the two cytokines IL-4 and IL-13 dampens the histamine release in whole blood cell cultures. The use of such mammalian designer cells defines a major advance in the prevention, treatment and cure of beginning allergic reactions and helps alleviating allergic rashes, allergic symptoms and preventing daily administration of medicationsH2 cytokine sensor (DCS) device is based on a common IL-13 receptor (IL-13R) that is shared by the allergy-associated cytokines IL-4 and IL-13 41. To enable optimal signal detection and high sensitivity of the DCS, the co-transfection of the constructs pCH23 (PhCMV-IL13RA1-pA) and pCH24 (PhCMV-IL4RA-pA) encoding for the IL-13 receptor subunits combined with pLS16 (PhCMV-STAT6-pA) for the constitutive STAT6 gene expression was necessary  enabled the controlled production and secretion of the human placental secreted alkaline phosphatase (SEAP) in response to input cytokines. After optimization of the plasmid ratios in HEK-293 cells, the step-by-step addition of individual DCS components showed considerable improvement in signal detection  displayed very low leakiness  and reporter plasmid pLS12 (PSTAT6/cEBP-SEAP-pA), the SEAP activity in the presence of high input levels suggests that HEK-293 cells express the IL-13Rα1 and IL-4Rα receptor chains endogenously  is essential for the functionality of the DCS device  was replaced by the IgE inhibitor DARPin E2_79. Initially, the sequence of the IgE-binding protein was optimized for its production in Escherichia coli33. The sequence of the protein used for this work has therefore been codon-optimized for human protein translation and has been equipped with a human secretion signal (SSSEAP) to ensure its correct production and secretion by mammalian cells, respectively. The DARPin E2_79-expressing construct pCH26 (PSTAT6/cEBP-SSSEAP-His-DARPin E2_79-pA) was cloned by replacing the SEAP gene of pLS12 (PSTAT6/cEBP-SEAP-pA) with the human codon-optimized and secretion-engineered DARPin E2_79 sequence. As expected, the DCS-transgenic cells secreted the IgE inhibitor protein DARPin E2_79 in a dose-dependent manner into the supernatant of engineered cells when exposed to varying concentrations of IL-4 and/or IL-13 -lysine-alginate capsules to protect the cells from the attack of human immune cells contained in the whole blood. To ensure the correct performance of the encapsulated cells, the dose-dependent DARPin E2_79 expression -lysine-alginate capsules were shown here to be permeable for the cytokines IL-4 and IL-13, both necessary to trigger the DARPin E2_79 production of the encapsulated cells. Furthermore, the dose-dependent DARPin E2_79 expression 33. Also, the half-maximal disruptive concentration of the IgE-FcεRI complexes shows that µM concentrations are required to disrupt the IgE-receptor interaction (ID50 1.57 × 10−6M)44.Because the high-affinity interaction of DARPin E2_79 with IgE is critical to hinder the formation of a stable IgE-FcεRI receptor complex, it was essential to show that the DARPin E2_79 produced in mammalian cells has similar binding properties as the reported 45 enabled the quantification of released histamine via the production of SEAP -lysine-alginate capsules . The cytokine concentrations correlated with DARPin E2_79 production and total IgE levels. The cytokine and total IgE levels remained also low for the non-allergic group. Calculations of the DARPin E2_79 over IgE ratios , reduced the histamine release of the basophil granulocytes significantly  and IL-4 receptors  as well as STAT6  was run in parallel in the whole-blood samples of the same donors. The addition of allergen extract triggered histamine release by basophil granulocytes  although the cytokines levels in the blood samples where somewhat lower than in Fig. −1, showing that the optimized DCS device was able to make up for the lower cytokine concentration in the blood. Furthermore, the DARPin E2_79 concentrations produced by the improved variant of the DCS device were enough to abolish the histamine release for blood donors no. 4, 5, and 6, after 24 h  are perceived by the DCS device, thereby adjusting the DARPin E2_79 concentration. Usage of the DCS device during a season with high pollen occurrence due to warm and dry temperatures . For the treatment of such disorders, the DCS device should ideally be implanted prior or during the sensitization phase to prevent the onset of an allergy. This may also prevent the development of late allergic phases of the disease and decrease the risk of recurrent allergic inflammations becoming chronic.DCS-containing designer cells may develop into an alternative to established drug-based treatment of allergies. However, there are major challenges toward clinical translation. For example, the DCS device is difficult to validate in an in vivo context, the precision of the sense-and-response dynamics may turn out to be suboptimal and the treatment of acute manifestations of the allergic disease will be complicated. Nevertheless, the cell-based DCS strategy holds the promise for life-long protection of patients, in particular those with a genetic predisposition for allergies -derived pancreatic progenitor cells could be reprogrammed into glucose-sensitive insulin-secreting beta-like cells63. Similarly, this provides the unique opportunity of rewiring patient-derived stem cells to achieve personalized implants capable of protecting allergic patients reliably and durably. As the DCS device is programmed to interfere with the onset of perennial allergic and asthmatic reactions as well as attenuate ongoing allergic reactions, it is important to note that the treatment of other serious medical conditions such as life-threatening anaphylactic reactions are beyond the scope of the DCS device.As stated in the literature, the IgE-binding domains are efficiently blocked by the DARPin E2_79 molecules78. We show here for the first time that DARPin E2_79 can be successfully produced in mammalian cells without loss of functionality. Besides, due to their production in mammalian cells, the molecules are free of toxic contaminants such as bacterial endotoxins and are particularly safe for the use in patients. Using a biparatopic IgE-targeting approach, the disruptive potency of DARPin E2_79 has been improved by approximately 100-fold47. The production of novel generations of IgE-binding DARPins is very encouraging, especially if they are used in a precisely controlled manner.DARPin technology is relatively recent, and the construction of new generations of DARPins is very promising, bearing a vast development potential for IgE-binding DARPinshCMV-IL13RA1-pA) and pCH24 (PhCMV-IL4RA-pA) for constitutive expression of the IL-13 receptor subunits, pLS16 (PhCMV-STAT6-pA), encoding constitutive expression of STAT6 to enable the synthetic signaling cascade and pCH26  and secretion-engineered (SSSEAP) DARPin E2_79.Comprehensive design and construction details for all expression vectors are provided in Supplementary Table 79) and human fibrosarcoma cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FCS  and a 1% penicillin/streptomycin solution . Chinese hamster ovary cells  were cultivated in ChoMaster HTS  supplemented with 5% FCS and a 1% penicillin/streptomycin solution. FreeStyle 293-F cells  were cultured in FreeStyle™ 293 Expression Medium . All of the cell types were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cell numbers and viability were quantified using an electric field multichannel cell-counting device . The cells were transfected using an optimized PEI transfection protocol. The cells were seeded 16 h before transfection at 3 × 104 per well of a 48-well plate. The transfection was performed upon the addition of 0.4 µg plasmid DNA and 1.2 µL PEI  per well to FCS-free DMEM for 15 min at 22 °C before it was added dropwise to the cells. For the SEAP output, 0.0125 µg of pCH23 (PhCMV-IL13RA1-pA) and pCH24 (PhCMV-IL4RA-pA), 0.150 µg of pLS12 (PSTAT6/cEBP-SEAP-pA) and 0.05 µg of pLS16 (PhCMV-STAT6-pA) were used. For the pre-optimized version of the DCS tested in a set of different human and rodent cell lines with SEAP output, 0.05 µg of pCH23 (PhCMV-IL13RA1-pA) and pCH24 (PhCMV-IL4RA-pA), 0.150 µg of pLS12 (PSTAT6/cEBP-SEAP-pA) and 0.05 µg of pLS16 (PhCMV-STAT6-pA) were used. For regulated DARPin E2_79 production, 0.0125 µg of both pCH23 (PhCMV-IL13RA1-pA) and pCH24 (PhCMV-IL4RA-pA), 0.05 µg of pCH26 (PSTAT6/cEBP-SSSEAP-His-Tag-DARPin E2_79-pA) and 0.05 µg of pLS16 (PhCMV-STAT6-pA) were used. For the increased expression of DARPin E2_79 with increased basal levels, 0.025 µg of both pCH23 (PhCMV-IL13RA1-pA) and pCH24 (PhCMV-IL4RA-pA), 0.1 µg of pCH26 (PSTAT6/cEBP-SSSEAP-His-DARPin E2_79-pA) and 0.1 µg of pLS16 (PhCMV-STAT6-pA) were used. After 6 h, the medium was replaced by either standard cultivation medium or medium supplemented with the indicated amounts of recombinant proteins, including human recombinant interleukin 4  and human recombinant interleukin 13 . Unless stated otherwise, the transgene expression was profiled after 48 h. For the expression and secretion of DARPin E2_79 in FreeStyle 293-F cells, 2 µg mL−1 of pCH15 and 6 µL of PEI per 1 × 106 cells were mixed with FreeStyle 293 Expression Medium for 15 min at 22 °C before it was added to the cells. The cell culture supernatant was collected after 4 days.Human embryonic kidney cells , human cervical carcinoma cells , immortalized human mesenchymal stem cells  diethanolamine, pH 9.8). After the addition of 20 µL of 120 mM para-nitrophenylphosphate  diluted in 1x SEAP assay buffer, the time-dependent increase in light absorbance was profiled at 405 nm for 30 min using a GeniosPro multi-well reader . DARPin E2_79 profiling: To assess the production of DARPin E2_79, human IgE  was immobilized at 3 µg mL−1 on an ELISA plate . A mouse anti-His-tag secondary antibody  was added to bind DARPin E2_79. A third sheep anti-mouse HRP-tagged antibody  was used to detect DARPin E2_79. Cytokine measurement: The IL-4 and IL-13 levels in the blood samples were assessed using an IL-4- and IL-13-specific ELISA  according to the manufacturer’s instructions. IgE quantification: To assess the total IgE level in the serum, goat α-human IgE  was immobilized at 2.5 µg mL−1 overnight at 4 °C on an ELISA plate . The serum was diluted 1:1 with Mabtech Ready-to-use diluent for ELISA . An anti-human Ig kappa light-chain FITC  was then added to detect the bound IgE. Quantification of IgE-DARPin E2_79 complexes: To assess the IgE-DARPin E2_79 complexes in the serum, goat anti-human IgE  was immobilized at 5 µg mL−1 overnight at 4 °C on an ELISA plate . A mouse anti-His-tag secondary antibody  was added to bind DARPin E2_79. A third sheep anti-mouse HRP-tagged antibody  was used to detect DARPin E2_79. Histamine release from human basophil granulocytes: The release of histamine from human basophil granulocytes was quantified as previously described45. In brief, 200 µL of whole blood containing the microencapsulated DCS device were mixed with the indicated concentrations of allergen with an adapted protocol using DARPin E2_79, anti-human IgE, the allergens and fMLF diluted in PBS instead of degranulation buffer. The histamine release assay was performed in an incubator at 37 °C in a humidified 5% CO2-containing atmosphere for 30 min. Thereafter, the cells were centrifuged for 10 min at 700 × g at 4 °C. The resulting serum was either directly used for subsequent assays or was stored at −20 °C.SEAP levels: The production of human placental secreted alkaline phosphatase was quantified in the cell culture supernatant as described previouslyhCMV-SSSEAP-His-DARPin E2_79-pA) was incubated overnight at 4 °C with equilibrated Ni-NTA agarose . After centrifugation of the agarose, the supernatant was discarded and washing steps were performed with NPI-20 wash buffer . The elution of DARPin E2_79 was performed using NPI-250 elution buffer . Finally, the DARPin E2_79 fraction was concentrated by ultrafiltration using concentrator columns  and the elution buffer was successively replaced by PBS. The final protein concentration was determined using a Bradford assay  and Coomassie blue gel staining.The purification of His-tagged DARPin E2_79 was performed under native conditions. In brief, a cell culture supernatant of FreeStyle 293-F cells transfected with the DARPin E2_79 expression vector pCH15 . For the disruption of the IgE-FcεRI complex, IgE was added to each well at a 1 µg mL−1 concentration, incubated for 30 min at room temperature and subsequently washed. Increasing concentrations of DARPin E2_79 were added , incubated for 2 h and removed by washing. Then, an anti-human Ig kappa light-chain FITC  was added for IgE detection.To assess the binding of DARPin E2_79 to IgE, human recombinant FcεRI  was immobilized at 2 µg mL35. Briefly, coherent alginate-poly-(l-lysine)-alginate beads (400 µm diameter) were formed using an Inotech Encapsulator Research Unit IE 50R  set to the following parameters: 0.2 mM nozzle with a vibration frequency of 1025 Hz, 25-mL syringe operated at a flow rate of 410 units and 1.12 kV for the bead dispersion. 200 µL of microencapsulated cells  and 300 µL of RPMI-1640 Medium  were added to 500 µL of whole blood. Controls for constitutive production of DARPin E2_79 were run with a mock plasmid (pColaDuet-1). The plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The plates were prepared for each time point . For the plasma collection, the plates were centrifuged for 10 min at 700 × g at 4 °C. The plasma samples were stored at −80 °C.The encapsulation of HEK-293 cells was performed as previously described45. In brief, 5 × 104 HSDSEAP32 cells were seeded into each well of a 96-well plate containing 105 µL of standard cultivation medium. After 12 h, 15 µL of human serum derived from histamine release assays was added, and the cells were incubated for an additional 24 h. The supernatant was collected and either directly used for analytical assays or stored at −20 °C until further SEAP quantification.The cell-based allergy test was performed as previously describedThe basophil activation test was performed with the Flow CAST Basophil Activation Test Kit . Blood samples of allergic donors were analyzed after 24 h according to the manufacturer’s instructions.The quantification of the IgE–FcεRI complex dissociation is based on the Flow CAST Basophil Activation Test Kit. Whole-blood samples were incubated overnight with increasing concentrations of DARPin E2_79 . A staining reagent containing anti-Human CD193 (CCR3) PE  and anti-Human Ig kappa light-chain FITC  was added to the blood samples according to the manufacturer’s instructions instead of the Flow CAST Basophil Activation Test Kit Staining Reagent. Samples were measured after 24 h according to the manufacturer’s instructions.−145.Crude allergen extracts, including including Betula alba pollen , Corylis avellana pollen , Cynodon dactylon pollen , Phleum pratense , Lolium perenne pollen , Dermatophagoides pteronyssinus allergen , were dissolved in degranulation buffer  , 110mM sodium chloride , 5mM potassium chloride  and 0.01% (v/v) thimerosal ; pH 7.4), and each mixture was stirred at 300 r.p.m. at 4 °C for 16 h. Subsequently, each allergen mixture was centrifuged, the supernatant was filter-sterilized and the total protein concentration was measured using the Bradford assay. The allergen solutions were stored at −20 °C at a concentration of 1 mg mLPeripheral blood was drawn from allergic and non-allergic donors after obtaining their informed consent. The study was approved by the Ethics Committee of ETH Zürich (no. EK 2012-N-42).All data and materials are available upon request.Supplementary Information


The BT is complex and confounded by the younger closely timed and compositionally similar Penifiler Tephra (PT). Two of the deposits found in the ice are in Greenland Interstadial 1e (GI‐1e) and an older deposit is found in Greenland Stadial 2.1 (GS‐2.1). Until now, the BT was confined to GI‐1‐equivalent lacustrine sequences in the British Isles, Sweden and Germany, and our discovery in Greenland ice extends its distribution and geochemical composition. However, the two cryptotephras that fall within GI‐1e ice cannot be separated on the basis of geochemistry and are dated to 14358 ± 177 a b2k and 14252 ± 173 a b2k, just 106 ± 3 years apart. The older deposit is consistent with BT age estimates derived from Scottish sites, while the younger deposit overlaps with both BT and PT age estimates. We suggest that either the BT in Northern European terrestrial sequences represents an amalgamation of tephra from both of the GI‐1e events identified in the ice‐cores or that it relates to just one of the ice‐core events. A firm correlation cannot be established at present due to their strong geochemical similarities. The older tephra horizon, found within all three ice‐cores and dated to 17326 ± 319 a b2k, can be correlated to a known layer within marine sediment cores from the North Iceland Shelf . Despite showing similarities to the BT, this deposit can be distinguished on the basis of lower CaO and TiO AMSaccelerated mass spectrometryCFAcontinuous flow analysisEPMAelectron probe micro‐analysisGIGreenland interstadialLA‐ICP‐MSlaser ablation inductively‐coupled plasma mass spectrometryMCEmaximum counting errorNANorth AtlanticREErare earth elementSCsimilarity coefficientTAtotal alkaliTAUtephra analysis unitWDwavelength dispersiveWDSwavelength dispersive spectrometryXRFX‐ray fluorescenceet al., et al., et al., et al., et al., Tephrochronology has long been established as a tool that exploits ash deposits with unique geochemical fingerprints to precisely correlate a diverse range of palaeoarchives from widely separated localities  region. The BT and PT are close in age and composition and, as a result, present problems for correlation purposes  and Penifiler (PT) cryptotephras in the Greenland ice cores. Both are distinguishable from other Lateglacial cryptotephras by low FeO and TiO, et al.  in early , by Jarvis , derived from mollusc shell material located 4.5 cm above the tephra layer . Stratigraphic and chronological evidence therefore indicates that the marine layers were deposited in GS‐2.1‐equivalent glacial sediments and this discrepancy between terrestrial and marine‐based radiocarbon age estimates was first noted by Lowe , MD99‐2275 (3679 cm) and MD99‐2271 (806–808 cm) from the North Iceland shelf have been discovered by Eiríksson et al. , Sønderget al.  and Gudm  and uncertainty is based on a maximum counting error (MCE) of ambiguous layers, equivalent to 2σ, where cumulative errors increase with depth .Three Greenland ice‐cores  were used to search for the BT, PT and the older Borrobol‐type tephras between GS‐2.1 and GI‐1. The timing of Greenland interstadials (GI) and stadials (GS) and ages presented in this study have been defined by Greenland Ice Core Chronology 2005 (GICC05)  is the preferred method for major element characterization of individual tephra grains and requires flat exposed sections through grains for electron bombardment and X‐ray generation  with the NIST 612 reference glass used for calibration, taking concentrations from Pearce et al.  at the Department of Geography and Earth Sciences, Aberystwyth University. Here a Coherent GeoLas ArF 193 nm Excimer LA system was operated with a fluence of 10 J cme et al. . A fract  ranges between 7.69 and 8.55 wt%, the SiO2 values range between 75.90 and 77.40 wt% and the FeO and TiO2 contents are between 1.20 and 1.83 wt% and 0.08 and 0.19 wt%, respectively . Lind et al. , so with respect to the ice‐core age estimates, the older ice deposit GRIP 1734 m (14 308 ± 177 a BP) is consistent only with the BT . Statistical investigation of the dataset shows compositional similarity between these two layers, with SC and D2 values of 0.974 and 1.614, respectively  shows Sr decreasing with CaO, consistent with a possible genetic link between them by feldspar extraction. Additionally, almost all the other incompatible elements  increase from the ice‐core layers to BO521. This suggests the relationship between these samples is related to an eruption from a compositionally zoned or stratified magma chamber, with the more evolved upper part of the magma body producing the BO521 deposit, and later erupted (less evolved) magma from deeper in the magma body travelling to Greenland to be deposited as GRIP 1727.75 m/NGRIP 1582.75 m or GRIP 1734 m. Deposits from the younger ice‐core layer GRIP 1727.75 m/NGRIP 1582.75 m overlap with BO521 in terms of their Sr and Y concentrations   sediments. The best (reservoir‐corrected) age estimate for this marine isochron is 17 179–16 754 cal a BP Jarvis,  and is cca. 106 years apart in GI‐1e ice, the correlation issues that plague the BT remain. Without any diagnostic geochemical features, pinpointing a correlation to either the BT or the PT in terrestrial sequences is limited. Our new trace element data show some tentative and subtle differences but require further exploration to robustly assess their use for discrimination purposes. Furthermore, while the older ice‐core deposit  is consistent only with the calibrated age range of the BT, the younger deposit  overlaps with the age ranges of both the BT and the PT layers. Although a firm correlation is precluded, we discuss various possibilities below that will require testing in future work.Three cryptotephra deposits with a Borrobol‐type composition have been identified for the first time in Greenland ice spanning GS‐2.1 (one horizon) and GI‐1e (two horizons), but Borrobol‐type deposits were absent from GI‐1d ice. For the two compositionally identical events, et al. , whereas other sites (e.g. Abernethy Forest) may only preserve one of these ice‐core deposits Fig. . The abivs. TiO2 content (Fig. et al., The tephra identified in GS‐2.1 is simpler in terms of its wider application as an ice‐marine tie‐point. This is the oldest known deposit with a Borrobol‐type composition, but we demonstrate that it can be separated from the BT and PT on the basis of CaO ent Fig. C. This cent Fig.  (e.g. RaAdopting a contiguous ice‐core sampling approach has provided further insight into the complexity of the Borrobol Tephra. Two cryptotephra deposits detected in GI‐1e ice probably equate to the BT found in terrestrial records but a firm correlation is precluded given the indistinguishable composition and closely timed deposition of the BT and PT. In this study trace element compositions show possible but tentative signs that may prove fruitful for future discrimination purposes. If these deposits are to be used as valuable marker deposits, further work is urgently required in this area. As yet there are no trace element analyses from terrestrial records that contain both the PT and the BT, and this is essential if differences are to be observed between these deposits. Re‐analysis of BT and PT major element signatures with improved microprobe operating conditions may also prove beneficial to tease out any subtle differences that may be obscured by analytical noise. Furthermore, ultra‐high‐resolution sampling of Scottish Lateglacial sequences together with high‐precision chronologies may prove beneficial to unpick the diffuse tephra profiles associated with the BT. Lastly, the GS‐2.1‐RHY horizon identified in three ice cores illustrates the value of marine‐ice tie‐points in an interval plagued by dating uncertainties and highlights its potential to assess marine reservoir offsets for the North Iceland Shelf.Supporting information relating to this article can be accessed via the publisher's website.Table S1. Major oxide concentrations and secondary standards.Click here for additional data file.Table S2. Trace element data.Click here for additional data file.


Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X‐ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1–TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB. Mtb Decaprenylphosphoryl‐β‐d‐ribofuranose 2‐oxidase (DprE1) is a flavin adenine dinucleotide (FAD)‐dependent enzyme, which together with decaprenylphosphoryl‐d‐2‐ketoerythropentose reductase (DprE2), converts decaprenylphosphoryl‐beta‐d‐ribose (DPR) to decaprenylphosphoryl‐beta‐d‐arabinofuranose (DPA), an essential cell wall component.dprE1 results in a strong bactericidal effect in vitro.Mtb, further validating DprE1 as an attractive anti‐tuberculosis (TB) drug target.dase DprE is a flaWe previously reported the identification of TCA1 from a cell‐based phenotypic screen, and demonstrated it is a DprE1 inhibitor.Mtb, and selectively targeting DprE1 over CYP2C9.Here we report the X‐ray crystal structures of WT DprE1, a TCA1‐resistant DprE1 mutant, and CYP2C9 in complex with TCA1 analogs. Along with structure activity relationship studies, the mechanism of resistance by the mutant DprE1 enzyme and the tight correlation between DprE1 and CYP2C9 inhibition was elucidated. We further generated analogs with excellent in vitro and in vivo activities against In the DprE1–TCA1 structure, TCA1 showed a planar conformation and tight fit in the DprE1 active site. The benzothiazole core is oriented parallel to the FAD isoalloxazine ring and the thiophene carboxamide resides in small hydrophobic pocket. Besides substantial hydrophobic interactions, multiple hydrogen bonding interactions are formed between TCA1 and residues Lys418, His132, and Ser228 . Guided by the structure, our medicinal chemistry effort focused on the benzothiazole core (red), thiophene carboxamide (green) and the acylcarbamate (blue) of TCA1  compared to TCA1 (48±14 nm) . Only a fluoro substituent on the thiophene afforded an analog (TCA481) that is 5‐fold more potent than TCA1 based on MIC . Replacement of thiophene with other heterocycles or adding bulky substitutions resulted in reduced activity against m, IC50=370±133 nm)   and the mutant strain . To elucidate the molecular basis of the resistance mechanism, the structure of DprE1 Y314C bound with TCA1 was determined to 2.2 Å , suggesting a low risk of cardiotoxicity. In addition, we observed no genetic toxicity in a mini‐Ames mutagenicity test. TCA007 showed no inhibition of four major CYPs, 1A2, 3A4, 2D6, 2C19, but a strong inhibitory effect for CYP2C9 (SI methods).A typical treatment of TB lasts for more than six months, and therefore, the safety profile of anti‐TB drugs is of great importance. TCA007 is not cytotoxic against Vero and HepG2 cell lines at up to a concentration of 100 μThe potent inhibition of P450 2C9 could reflect oxygenation of thiophene moiety of TCA analogs to an electrophilic S‐oxide or epoxidePhe100, Leu102, and Phe114 form a hydrophobic pocket together with Phe476 where the carbamate group of TCA007 is bound  for TCA582 and TCA711 were 33 and 1515, respectively, which, compared with TCA007 (ratio=1.8), represents a significant improvement in selectivity   Figure  D. The iIn summary, we demonstrate the mode of inhibition of DprE1 by the thiophene scaffold via enzymatic and structural studies. The SAR studies not only helped us to optimize the potency of the inhibitors but revealed an unconventional resistant mechanism of DprE1. The optimized lead compound TCA007 showed excellent in vitro and in vivo activities, PK properties, and low risk of toxicity profile except for the strong inhibition of CYP2C9. Guided by the structures of CYP2C9 in complex with TCA1 analogs, an optimized molecule, TCA711, was generated with differential inhibitory activities against DprE1 and CYP2C9, which paved the road for further development of a clinical candidate to treat TB.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


A sequence conserved in both AP180 and CALM (LDSSLA[S/N]LVGNLGI) was found to be the major interaction site and mutation caused a deficit in clathrin assembly, which is the first example of a mutation having this effect. In contrast, single or double mutation of DL(L/F) motifs in full length AP180 had no significant effect on clathrin binding, despite higher clathrin affinity for isolated peptides containing these motifs. We conclude that the novel clathrin interaction sites identified here in CALM and AP180 have a major role in how these proteins interface with clathrin. This work advances the case that AP180 and CALM are required to use a combination of standard clathrin N-terminal domain binding motifs and the sequence identified here for optimal binding and assembling clathrin.The clathrin heavy chain N-terminal domain interacts with endocytic adapter proteins via clathrin binding motifs to assemble clathrin triskelia into cages. However, the precise mechanism of clathrin assembly is not yet known. Clathrin assembly protein AP180 has more clathrin binding motifs than any other endocytic protein and has a major role in the assembly of the clathrin coat during synaptic vesicle biogenesis. We now demonstrate that some of the previously identified binding motifs in AP180 may be non-functional and that a non-conventional clathrin binding sequence has a major influence on AP180 function. The related protein, clathrin assembly lymphoid myeloid leukemia protein (CALM), has fewer clathrin binding motifs and functions ubiquitously in clathrin-mediated endocytosis. The C-terminal ~16 kDa sub-domain in AP180, which has relatively high similarity with CALM, was shown in earlier work to have an unexplained role in clathrin binding. We identified the specific sequences in this sub-domain that bind to clathrin. Evidence for a role for these sequences in promoting clathrin binding was examined using  An early event in clathrin-mediated endocytosis (CME) is the formation of a clathrin-coated pit at the plasma membrane which requires clathrin binding to endocytic adaptor proteins ,2. Clathin vitro was shown to be directly proportional to the number of CBMs , anti-VAMP2 and anti-AP180 were from Abcam. Anti-CALM and anti-β-Actin was from Sigma. Anti-GFP was from Roche. All horseradish peroxidase-conjugated secondary antibodies were from DAKO. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), Trypsin-EDTA and Alexa Fluor 594 labelled transferrin were from Invitrogen. Paraformaldehyde was from Scharlau Chemie. All reagents were of analytical grade unless specified otherwise.Arrays of 15mer peptides were synthesised directly on cellulose membrane. The PepSpots arrays were synthesised by JPT Technologies. The N-termini of the peptides were acetylated to mimic an internal sequence. Each 15mer is separated from the membrane by an Ala linker at the C-terminus of the 15mer sequence. The 15mer peptides from the ADΔCLAP of AP180 and CALM were designed to ensure an overlap of 12 out of 15 amino acid residues for adjacent peptides on the array, which enables a minimum 3 or more amino acid residues that could be part of a short linear binding motif. Some additional out-of-sequence 15mers were used to allow comparison between imperfectly-aligned AP180 and CALM sequences  and human CALM isoform 3 . CALM isoform 3 was selected since it has the longest ADΔCLAP. CALM isoform 1 was used for all other experiments. The membrane was rinsed in methanol initially for 5 min to solubilise hydrophobic peptides. Then, the membrane was washed 3 x 10 min with Tris buffered saline spiked with Tween 20 , and blocked for 16 h at 4°C with agitation in 3% bovine serum albumin (BSA)/T-TBS. The membrane was incubated with purified clathrin from bovine brain  was de novo synthesised and cloned into pGEX-6P-1 (using BamH1 and Not1) and pEGFP-C1  by GenScript. AP180 and CALM mutants were generated by a commercial service (GenScript). Mouse AP180 isoform 2 Site 1 mutant consisted of L750A + L754A + L757A + L761A + I763A. Mouse AP180 isoform 2 Site 2 mutant consisted of L887A + L890A + I892A + F895A + L896A. Human CALM isoform 1 Site 1 mutant consisted of L540A + L544A + L547A + L551A + I553A. Human CALM isoform 1 Site 2 mutant consisted of I649A + F651A + M652A. Plasmid sequences were confirmed by a commercial sequencing service  after transformation. Transformations were performed in JM109 competent Escherichia coli cells (50 μl) with 25 ng DNA. A single colony containing transformed DNA was selected. Cultures were grown in Lysogeny Broth media containing 100 μg/ml ampicillin. Glutathione S-transferase (GST) fusion protein expression was induced with the addition of 1 mM IPTG and grown for a further 4 h, then pelleted at 10,000 rpm for 15 min at room temperature (~23°C) in a Beckman Coulter JLA-10.500 fixed angle rotor. Each protein pellet from 500 ml of culture was resuspended in 40 ml salt-Tris-EDTA buffer , to which was added 1 complete EDTA-free protease inhibitor cocktail tablet. The solution was then made up to 1% Triton X-100, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 mM benzamidine and 5 μg/ml pepstatin A. Lysozyme was added to a final concentration of 0.1 mg/ml, and incubated on rotation at 4°C for 30 min. The pellets were subjected to two freeze-thaw cycles and probe sonication. The resulting lysate was centrifuged at 20,000 rpm for 20 min in a Beckman Coulter JA-25.50 fixed angle rotor. The supernatant was collected and stored on ice. Glutathione-Sepharose beads were prepared by washing in 0.1% Triton X-100/PBS three times, and incubated with the same solution for 30 min with rotation at 4°C. A 1 ml packed volume of beads was added to the collected lysate. The beads were incubated with the lysate for 1 h with rotation at 4°C. The beads were washed with 40 ml of ice cold buffers in the following order: 1) 1 wash with PBS; 2) 2 washes with 1.5 M NaCl in PBS; 3) 1 wash with 0.1% Triton X-100; 4) 3 washes with PBS. A final 2 ml of 50% slurry of approximately 1 mg/ml suspension of immobilised fusion protein was produced.Mouse AP180 isoform 2 was cloned into pGEX-6P-1, as described previously . Human CBeads with bound GST fusion protein were added to a spin-column  and were washed with 4 x 500 μl ice cold PBS before addition of synaptosome lysate (11–13 mg/ml). Lysate was prepared from P2 synaptosomes prepared from rat brain as described previously . All aniEscherichia coli, as above. The beads were transferred to a 10 ml Bio-Rad Poly-Prep Chromatography Column. This column was washed with 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM DTT supplemented with 0.01% Triton X-100 and then with the same solution containing 25 mM reduced glutathione at 4°C. The liquid was eluted and dialysed (membrane MW cut-off 12–14 kDa) against 1 L of TBS with 1 mM DTT for 20 h at 4°C with a buffer change at 4 h. The dialysed protease was collected, diluted with an equal volume of glycerol before being aliquoted and stored at -80°C.GST-tagged human rhinovirus 3C protease was expressed in GST-AP180 fusion proteins beads were washed in 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Triton X-100 and 50 mM Tris-HCl pH 7.0. The recombinant human rhinovirus 3C protease was added (10 μl protease/200 μl packed bead volume) and the spin-column was incubated and rotated at 4°C for 16 h. The cleaved protein was eluted, aliquoted and stored at -80°C. The eluted AP180 solution was dialysed against a 1 L solution of 0.5 M Tris pH 8 / 1 mM DTT for 20 h at 4°C with a buffer change at 4 h. Frozen pellets (drops from a glass Pasteur pipette into liquid nitrogen) were prepared immediately after dialysis and stored at -80°C.Purified clathrin and AP180 protein stocks were dialysed at 4°C into 10 mM Tris pH 8.0 (with 2 mM DTT added to the clathrin solution). The concentration of each protein was determined by spectrophotometric measurement of absorbance at 280 nm. The purified clathrin stock was 3.75 μM and was used directly. The AP180 and CALM proteins were diluted to a working stock of 10 μM in 10 mM Tris pH 8.0. A light scattering assay  was perf2. Confluent COS-7 cells were resuspended and diluted in DMEM/10% FBS and seeded in 12 well plates (containing glass coverslips coated with poly-D-lysine) at ~80,000 cells/well, and left in the incubator for 24 h. DNA (1 μg) and Fugene transfection reagent (3 μl) were mixed in 500 μl DMEM/10% FBS. The mixture was left to stand at room temperature for 20 min. The DNA-Fugene complexes were added drop wise to each well (500 μl/well) and the dishes gently swirled. The dishes were returned to the incubator for 3 h, and then topped up with 1 ml DMEM/10% FBS per well and returned to the incubator. Transfected cells were used in Tfn uptake assays 24 h post-transfection. Each plasmid was transfected in two wells for each independent experiment.DMEM was supplemented with 10% FBS and used to maintain COS-7 cells at 37°C in a humidified atmosphere of 5% COThe cells were serum starved in DMEM without FBS for 4 h in the incubator. Tfn conjugated to Alexa Fluor 594 (5 μg/μl) was added and the cells were placed back in the incubator for 10 min. Tfn containing medium was immediately aspirated, washed 3 x with warm PBS (after 5 min incubations in PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature, followed by 3 more PBS washes. Coverslips were mounted on slides spotted with DABCO to stain the nuclei and help the coverslip stick to the slide; the coverslip was sealed around the edges with clear nail polish to avoid drying out of the fixed cells, and the slides were stored in the dark at 4°C until imaging. The cells were visualised using an Olympus IX81 motorized inverted microscope (Hamamatsu Photonics) and images captured in three channels: red for Tfn (exposure 800 ms) and green for AP180 or CALM (exposure 200 ms). Metamorph software was used to calculate average intensity (AI) of Tfn uptake per cell, with the transfected cells normalised to non-transfected control cells (a minimum of 30 cells quantified per condition):The average and SEM from three independent cell cultures were calculated using Prism 5 (GraphPad Software). Statistical analysis was done using a one-way ANOVA followed by Tukey’s multiple comparison test. GFP-tagged WT and mutant expression of AP180 and CALM was determined by lysis, SDS-PAGE and Western blotting with anti-GFP . Anti-β-actin  was used to demonstrate equal protein content in the lysates.n = 1 is typical [The specific sequences within the AP180 and CALM ADΔCLAP  which bi typical . Howeverin vivo [The relative binding of clathrin to the AP180 CBMs has not been systematically examined. Therefore, these peptides are not only important positive controls, but provide novel insight on the relative binding strength of AP180 CBMs. The 11 peptides from the AP180 CLAP domain  containein vivo . Also, ain vivo . Thus, tThe overlay assay of the ADΔCLAP peptides revealed two corresponding sequences in AP180 and CALM that bound clathrin above the background level. The AP180 and CALM ADΔCLAP peptides were approximately aligned in The second site that bound clathrin, hereafter “Site 2”, was located in the extreme C-terminus of both CALM and AP180 . The levThe C-termini of yeast AP180 and epsin homologs, YAP180-1 and Ent1p, contain clathrin box motifs where the C-terminal carboxyl group suffices for the last polar group . We compc.f. peptides c.f. peptides c.f. peptides To determine the amino acid residues in Site 1 and Site 2 important for clathrin binding, we used peptides with alanine substitutions at specific residues in the sequences. Conserved, acidic and hydrophobic residues were targeted, since these residues occur in known CBMs and these sequences are likely to have a conserved function. Substitution of all the conserved Leu/Ile residues with Ala in CALM iso3 490-LDSSLANLVGNLGIG-504 prevented almost all clathrin binding  were substituted for Ala in AP180 Site 2 peptide 882-PAKDPLADLNIKDFL-896 and this was sufficient to block clathrin binding . When thP < 0.001 for all three mutants, comparing to the wild type, n = 4). The combined mutation, Site 1&2, further reduced clathrin binding for CALM (P < 0.001). The AP180 Site 1&2 mutant was not significantly reduced compared to S1. However, both AP180 and CALM S1&2 mutants did not bind clathrin significantly different to the non-specific binding of GST alone, whereas Site 1 alone was significantly different to GST alone for both AP180 (P < 0.01) and CALM (P < 0.001). The more pronounced effect of Site mutation for CALM could be due to an overall lack of CBMs compared to AP180. We conclude that Site 1 was the major ADΔCLAP binding site and that, at least for CALM, both Site 1 and 2 contributed to efficient clathrin extraction.The conserved Leu/Ile residues in AP180 and CALM Site 1 and the crucial hydrophobic residues identified above for each Site 2 sequence were mutated See . Note thin vitro clathrin assembly assay was used to measure the effect of AP180 ADΔCLAP mutation on clathrin cage assembly. GST-tags were cleaved from the bacterially expressed AP180 sequences and the untagged proteins were purified . Likewise, mutation of a DLF motif (L/F) site mutation would have a similar magnitude effect on clathrin binding, which has not been tested in full length AP180, until now. We mutated CBM sites in AP180 and compared the ability of GST-AP180 WT and mutants to extract clathrin from synaptosome lysate , similarWe mutated five Leu/Ile residues within Site 1 without regard for a potential degenerate three-residue D(L/I)(L/F) CBM within this sequence (750-LDSSLASLVGNLGI-763). The most likely candidate for a three-residue CBM is the 756-SLV-758 sequence, since it has adjacent hydrophobic residues and Ser is occasionally found instead of Asp at the first position in longer CBMs ,7. Furthin vitro and the existence of a multitude of CBMs suggested that the AP180 CLAP domain has the required functional elements to form a clathrin coat on nascent synaptic vesicles. However, these D(L/I)(L/F) CBMs were not systematically tested for clathrin binding function, prior to this work. A peptide array overlay assay allowed the first examination of the relative binding of clathrin to each AP180 CLAP domain CBM. It was found that not all CBMs on AP180 are equal. Three of eleven CBMs bound relatively low levels of clathrin indicating they might not be functional. Since two previously studied AP180 CBMs did not change their localised structure whether free in solution or bound to the clathrin NTD [in situ. If an arbitrary binding affinity threshold is applied, it is likely AP180 has only eight functional CBMs in the CLAP domain. How many CBMs does AP180 require? Contact between AP180 and multiple clathrin triskelia is required for assembly. AP180 has excess CBMs for this requirement and there is the assumption that abundant CBMs allow the formation of relatively small synaptic vesicles. The CALM CLAP has not been as thoroughly surveyed for non-canonical CBMs. The exact function of abundant CBMs within adapter proteins may become known as further mechanistic requirements for assembly, and the stages of coat maturation, are discovered.Since CME relies on either AP180 or CALM for the formation of a clathrin coat, these two proteins are essential for regulated vesicle biogenesis. Here, we have partially determined why AP180 and CALM have sequence similarity in the C-terminal ~16 kDa and how this sequence affects clathrin binding and assembly. The ability of AP180 to assemble clathrin hrin NTD , our resThe overlay assay identified two novel sequences in the ADΔCLAP of AP180 and CALM that bound clathrin. The results of the overlay assay were confirmed via binding, assembly and Tfn uptake assays. The first sequence, Site 1, is the major site. Site 2 is a minor site that was not confirmed as functional in all experiment types. Site 1 is conserved in AP180 and CALM and bound approximately half as much as the weaker CBMs from the CLAP domain. Site 2, which was not conserved but positioned at the extreme C-termini of each adapter, had much weaker binding and would not be considered a functional binding site if the same threshold, as above for a CBM, was applied. Clathrin binding to Site 1 was dependent on Leu/Ile only. The experiment using 756-SLV-758 to AAA mutation in AP180 confirmed that Site 1 was not harbouring a degenerate DL(L/F) type CBM. In contrast, Site 2 in AP180 was dependent on both hydrophobic and acidic residues, indicating  similarity to classical CBMs. Site 2 in CALM was dependent on hydrophobic residues and had no acidic residues. These data demonstrate that Site 1 is a novel clathrin binding sequence, which allows the possibility that it binds to a non-NTD site on clathrin.in vitro clathrin assembly. It is counterintuitive that a relatively weak clathrin binding sequence could have such a strong effect. The lack of measurable effect of mutated AP180 DL(L/F) sites in a pull-down experiment exemplified that Site 1 has a surprisingly large influence on clathrin binding, which was not predicted by the relatively low affinity of clathrin for isolated Site 1 peptides. These data suggest the proper function of the CLAP domain CBMs, which are undoubtedly essential for assembly, also requires the ADΔCLAP Site 1. Recent modelling of the role of adapter proteins in clathrin assembly [Site 1 mutation was sufficient to ablate the clathrin assembly function of AP180 and abolish the dominant negative Tfn uptake phenotype of overexpressed WT AP180 or CALM. This is the first time that mutation of a clathrin binding site has affected assembly  may prov1-3ΦX2-3ΦXΦ, where Φ is most often Leu, is conserved within AP180 and CALM [c.f. Figures C and D in c.f. AP180 747–761 to CALM iso3 487–501 and AP180 753–767 to either CALM iso3 491–505 or 494–508, c.f. peptide It is important to note that Site 1 overlaps with a nuclear export signal (NES) in CALM ,27. The and CALM –28. An Nand CALM . The seqSite 2 in AP180 and CALM both have a di-hydrophobic residue motif at their C-termini. Extreme C-termini CBMs exist in yeast AP180 and epsin (YAP180-1 and Ent1p). The clathrin binding ability of these yeast extreme C-termini was compared to the termini of mammalian AP180, CALM and epsin 1. All mammalian C-termini were qualitatively weaker than the yeast epsin C-terminus. This data suggests an evolutionary gulf between the function of yeast and mammalian C-termini that may not have been entirely ablated. Site 2 in both AP180 and CALM did not have a consistent effect on clathrin binding in the experiment types presented here and so is less likely to be a functional binding site.In conclusion, we have identified a novel clathrin binding site in the ADΔCLAP of AP180 and CALM. Thus, CALM now has at least two identified clathrin binding sites (including the lone DLL motif). The evidence here, and the original observation that the isolated ADΔCLAP sub-domain binds clathrin cages, suggests that Site 1 increases clathrin-AP180/CALM avidity, or provides an ideal pathway to regulated clathrin cage nucleation, and is essential for efficient cage assembly. This study has updated the role of the previously identified CBMs in AP180 and provided a sequence-specific explanation of clathrin-ADΔCLAP interaction.S1 File(PDF)Click here for additional data file.


The stone fish is an under-utilized sea cucumber with many nutritional and ethno-medicinal values. This study aimed to establish the conditions for its optimum hydrolysis with bromelain to generate angiotensin I-converting enzyme (ACE)-inhibitory hydrolysates. Response surface methodology (RSM) based on a central composite design was used to model and optimize the degree of hydrolysis (DH) and ACE-inhibitory activity. Process conditions including pH (4–7), temperature (40–70 °C), enzyme/substrate (E/S) ratio (0.5%–2%) and time (30–360 min) were used. A pH of 7.0, temperature of 40 °C, E/S ratio of 2% and time of 240 min were determined using a response surface model as the optimum levels to obtain the maximum ACE-inhibitory activity of 84.26% at 44.59% degree of hydrolysis. Hence, RSM can serve as an effective approach in the design of experiments to improve the antihypertensive effect of stone fish hydrolysates, which can thus be used as a value-added ingredient for various applications in the functional foods industries. Marine invertebrates are naturally endowed with valuable proteins that can be exploited in the production of functional hydrolysates and their constituent’s biopeptides. Many peptides exhibiting one or more biological activities including those for the production of antimicrobial, antioxidant, antithrombotic, anti-Alzheimer, opiate and antihypertensive activity have been reported . The angMost commercially-available synthetic ACE inhibitors, such as enalapril, captopril, and lisinopril, have been reported to effectively reduce blood pressure by blocking the formation of angiotensin II from angiotensin I . HoweverTherefore, development of food protein hydrolysates with an ACE-inhibitory effect is considered to be a safe alternative to the use of the synthetic ACE inhibitors in the management of hypertension .Protein hydrolysates and peptides with ACE-inhibitory activities are the most studied among the different classes of food biopeptides . They ocActinopyga lecanora) was hydrolyzed by six different proteases whereas the activity decreased with trypsin, papain, pepsin, and flavourzyme generated hydrolysates [Thus, the choice of an appropriate enzyme is critical for obtaining inhibitory hydrolysates with strong activity against ACE. As previously reported, alcalase and bromelain-generated hydrolysates yielded the highest ACE-inhibitory activity when stone fish  using bromelain to determine the best conditions to produce stone fish hydrolysates with maximum inhibitory effects on ACE. It is hoped that improving the target activity will expand the utilization of The proximate composition analyzed for the powdered stone fish sample is given in p < 0.05 and estimated regression coefficients for the DH and ACE-inhibitory-activity quadratic models (Y1 and Y2), respectively. Statistical parameters including coefficient of determination (R2) and F-test probability have also been indicated. The R2 values of Y1 and Y2 were 98.50% and 98.70%, respectively.The actual and predicted responses for the degree of hydrolysis  and ACE-inhibitory activity (Y2) of stone fish hydrolysates under the combined effects of the different hydrolysis conditions pH (X1), temperature (X2), E/S ratio (X3) and time (X4) are presented in p < 0.05) and interaction (p < 0.05) effects of pH (X1), temperature (X2), E/S ratio (X3) and time (X4) on DH, whereas temperature had no significant (p < 0.05) effect in the quadratic region of the model for DH (The results for the degree of hydrolysis of stone fish hydrolysates (SHs) are shown in l for DH . The DH l for DH .Such increase in DH with an increase in hydrolysis time and E/S ratio has been reported previously, ,16 and wAs shown in p < 0.05) and interaction (p < 0.05) effects of pH (X1), temperature (X2), E/S (X3) and time (X4) on ACE-inhibitory activity, while only time (X4) had a significant (p < 0.05) effect in the quadratic region of the model for ACE-inhibitory activity  of protein hydrolysates was reported to be influenced by the protein unfolding and denaturation as well as the appearance of carbohydrates and other non-protein components ,23. Accodrolysis . Proteindrolysis ,24.Moreover, the fat binding capacity of SHs was 6.25 ± 0.01 mL/g protein. High oil/fat binding capacity is an important quality of protein hydrolysates for use in the confectionery and meat industries. These include the use of hydrolysates to bridge the fat and water content in sausages , bulk deThe results of CCD are given in t test and p values.The individual and combined influence of the independent factors on DH and ACE inhibition were identified using student’s 2) based on the effects of the process variables and their mode of interactions. The higher R2 values obtained indicated a high level of reliability which means the model is well-fitted to the experimental data and that 97.74% and 98.14%, respectively, of the variability in the observed DH and ACE-inhibitory-activity responses can be explained by the model.The extent of variability in the observed response was measured using the coefficient of determination (Rp > 0.05) lack-of-fit values of 0.076 for DH and 0.771 for the ACE-inhibitory activity further verified the significance of each model and its fitness for the experimental data.The non-significant (A. lecanora) were locally supplied from Kedah and Langkawi breeding centers in Malaysia. The internal organs were immediately removed, and samples were washed and transported in iced bags to the laboratory. The samples were rewashed, packed in plastic bags and stored at −80 °C until use. Prior to analysis, the samples were freeze dried and grounded with a Waring® blender , sieved through 600 µm wire gauze and kept at −40 °C until analysis. Bromelain from pineapple stem tissue, 100 gr ) was obtained from Acros Organics . Angiotensin converting enzyme (≥2.0 units/mg protein) and N-Hippuryl-His-Leu hydrate-powder, (≥98%) were purchased from Sigma–Aldrich . Dithiothreitol and O-phthaldialdehyde (OPA), 97% were obtained from Sigma–Aldrich . All other chemicals used for this study were analytical grade and obtained from Acros organics , Fisher Scientific , J.T Baker  and Merck KGaA .Fresh samples of stone fish  was subjected to dialysis for 24 h in a 12–14 kDa molecular mass cut-off dialysis tube (4 h against deionized water at room temperature and 20 h against reaction buffer at 4 °C). After dialysis, the sample was then mixed with 50 mL of the buffer  under different hydrolysis conditions according to the central composite design using RSM by MINITAB software Version 16.0 . The mixThe protein concentration of the stone fish hydrolysates was determined following a modified Lowry method using a micro Bicinchoninic Acid (BCA) protein Assay kit obtained from Sigma–Aldrich.The DH was determined based on OPA spectrophotometric assay according to Nielsen, Petersen, & Dambmann  and Li eO-phthaldialdehyde 97% was solubilized in 4 mL ethanol and added to the solution above by rinsing with deionized water. A weight of 0.176 g dithiothreitol (DTT) was dissolved with deionized water and added into the solution above. The final volume of the solution was then adjusted to 200 mL using deionized water. Then, 3 mL of the OPA reagent was added into 400 μL aliquot of stone fish hydrolysates and 400 μL of deionized water (blank), respectively, and gently vortexed for 5 s. The tubes were allowed to stand for 120 s at 25 °C and the absorbance was spectrophotometrically read at 340 nm.The OPA working reagent was freshly prepared as follows: 7.620 g disodium tetraborate decahydrate and 0.20 g sodium dodecyl sulfate were completely dispersed in 150 mL deionized water. 0.160 g tL is the amount of free amino groups released following hydrolysis at a time t, L0 is the amount of free amino groups in the original stone fish sample before enzyme addition and maxL is the total amount of free amino groups in the original stone fish sample obtained following acid hydrolysis with 6 M HCl at 110 °C for 24 h.The free amino group content of the samples was calculated as serine amino equivalent from a standard curve constructed using L-serine. The DH of the SHs was then calculated using the following equation:sA is hippuric acid absorbance when ACE, substrate captopril and stone fish hydrolysates are all present; cA is hippuric acid absorbance when only ACE and substrate captopril are present, and bA is hippuric acid absorbance when only substrate captopril is present. The percentage for ACE-inhibitory activity was obtained as the mean value of triplicate determinations.The inhibitory effect of the stone fish hydrolysates on ACE was determined as described by Jimsheena & Gowda , with mog for 10 min. The protein content was then quantified by micro BCA protein assay and expressed as the percentage of total soluble protein according to the following:The solubility of SHs was determined in accordance with the method of Tsumura et al. , with sog for 25 min. The supernatant volume was measured and the fat absorption capacity (FAC) was calculated as volume of oil (mL) absorbed per gram of stone fish hydrolysates.The ability to absorb fat by SHs was assessed following the description of Shahidi & Han , with mog for another 30 min. The supernatant was removed and the centrifuge tube was weighed together with the sediment. The WHC was then estimated as grams of water absorbed per gram of stone fish hydrolysates.The water-holding capacity was determined as explained by Beuchat , with mofV is the whipped volume (mL) and iV the volume (mL) before whipping. The analysis was conducted in triplicate and the FS was determined as the percentage of foam volume that remains after 3 min as shown below:sV is the volume (mL) after standing and iV is the volume (mL) before whipping.The foaming capacity (FC) and foam stability (FS) of SHs were assessed according to Sathe & Salunkhe  method, The software Minitab Version 16.0 was used for experimental design, data analysis and model-building. Four process hydrolysis variables, including pH (X1), temperature (X2), E/S ratio (X3) and time (X4), were recruited at three levels for each variable . A centr3 full factorial designs, were generated. Randomization was made to reduce the effects of variation on the experimental responses due to external factors that cannot be explained by the model. The experimental values of the model were fitted using the following second order polynomial equation or its reduced form obtained from the regression coefficient:Y is the response or dependent variable (ACE-inhibitory activity and DH) and β0 is the constant of the model. i, βiiβ and ijβ are the model’s regression coefficients. iX and jX are the levels of the process or independent variables  and represent their linear, quadratic and interaction effects on the responses. The response due to the independent variables was estimated using Minitab software version 16.0. The significance of β-coefficients of each process variable was determined using a student’s t test, and ANOVA was applied to assess the significance and fitness of the model, as well as the effects of the process variables on the responses. The accuracy of the reduced final model was further ascertained by comparing the experimental values with the fitted values predicted by the response regression equation.The experiments were conducted based on the design presented in Three-dimensional (3D) response surface plots were used to show the pattern of relationship between the responses and the levels of each of the process variables being studied. Response optimizer was employed to establish the actual optimum levels of the four process variables that yielded the maximum ACE-inhibitory activity of the stone fish hydrolysates. Under these conditions, confirmatory tests were performed for statistical validation of the experimental procedures.In this study, we have successfully established the hydrolysis condition of stone fish with bromelain using RSM. The temperature of 40 °C, E/S ratio at 2%, pH of 7 and time of 4 h were identified as optimal for the hydrolysis of stone fish to generate hydrolysates with strong ACE-inhibitory potential. The experimental data of 84.26% for ACE-inhibitory activity and 44.59% for DH obtained under these conditions were not statistically different from the predicted values of 84.42% and 46.26% for ACE-inhibitory activity and DH as calculated by RSM, within a 95% confidence interval, thus indicating a good fit of the model to the experimental data.The optimization process involved the whole peptide content of the hydrolysates. This is because it is more efficient, inexpensive, and preserves the nutritional value of the protein. Thus, the ACE-inhibitory effect of the hydrolysates is the net inhibitory effect of the different peptides present.The stone fish derived ACE-inhibitory hydrolysates are likely to exhibit no life-threatening side effects. Hence, they could have various applications as functional ingredients in the formulation of foods and beverages for the control of blood pressure to reduce the risks of cardiovascular diseases in hypertensive individuals.


Melastomataceae, Fabaceae, Rubiaceae and Euphorbiaceae) had significantly lower leaf chlorophyll content in the polluted areas compared to the unpolluted areas. This suggests that these families are more sensitive to petroleum pollution. The polluted site is dominated by Melastomataceae and Rubiaceae, suggesting that these plant families are particularly competitive in the presence of pollution. This study provides evidence of a decrease of plant diversity and richness caused by petroleum pollution and of a plant family-specific response of leaf chlorophyll content to petroleum pollution in the Ecuadorian Amazon using information from field spectroscopy and radiative transfer modelling.In recent decades petroleum pollution in the tropical rainforest has caused significant environmental damage in vast areas of the Amazon region. At present the extent of this damage is not entirely clear. Little is known about the specific impacts of petroleum pollution on tropical vegetation. In a field expedition to the Ecuadorian Amazon over 1100 leaf samples were collected from tropical trees in polluted and unpolluted sites. Plant families were identified for 739 of the leaf samples and compared between sites. Plant biodiversity indices show a reduction of the plant biodiversity when the site was affected by petroleum pollution. In addition, reflectance and transmittance were measured with a field spectroradiometer for every leaf sample and leaf chlorophyll content was estimated using reflectance model inversion with the radiative tranfer model PROSPECT. Four of the 15 plant families that are most representative of the ecoregion ( The Amazon rainforest plays an essential role in and is deeply influenced by ecological, climate and biogeochemical processes on Earth –4. In thGeologically, the Amazon region is a sedimentary basin containing vast underground petroleum reservoirs from which large amounts of oil and gas have been extracted in recent decades. During the petroleum production from oil wells, crude oil and formation water are extracted together and then separated by a chemical process. Crude oil is transported by pipelines to storage and distribution centres. Due to the potentially toxic nature of formation water, a common practice of the petroleum industry today is to re-inject it into its original geological formation. In this process accidental or deliberate oil spills from pipelines and facilities have occurred, leading to the release of large amounts of crude oil into the environment.In Ecuador, the majority of petroluem reservoirs are located in the Amazon rainforest region and have been exploited since 1967. During the first decades of petroleum production, billions of gallons of toxic formation water were disposed into unlined open pits without treatment or environmental monitoring. Over the years, the toxic waste has seeped into the natural environment causing water, soil and vegetation pollution. In addition, accidental and deliberate oil spills from the pipeline networks have resulted in approximately 16.8 million gallons of crude oil leaking into the environment –14. Fig Despite the capacity of vegetation for fast acclimation and certain stress tolerance mechanisms, vegetation usually responds to sudden short-term or long-term stressors with reduced cell activity and reduced plant growth or even plant mortality. The stress factors vary in their intensity and duration which can cause damage to plants . DiffereNoomen et al.  found thLeaf chlorophyll content is sensitive to hydrocarbon pollution, therefore this study assesses the chlorophyll content of leaf samples collected from several plant families in polluted and unpolluted sites of the Amazon rainforest in Ecuador. Large sample sizes are required to allow a plant family-level comparison, but are only achievable with a rapid and cost-effective method to estimate leaf chlorophyll content. Field spectroscopy and radiative transfer model inversion provide a physical understanding of the light transmission and reflectance in plant canopies and can be used to estimate leaf chlorophyll content with a much higher sample throughput than wet chemistry extraction.The hypothesis that vegetation stress by petroleum pollution leads to reduced leaf chlorophyll content in plant families of the Amazon rainforest in Ecuador is tested here.This study addresses the following research questions:Is the diversity of plant community reduced in areas affected by petroleum pollution?Is there a measurable effect of petroleum pollution on leaf chlorophyll content in tropical forest?Is the effect specific to individual plant families?The three study sites are located in the lowland evergreen tropical rainforest of the Amazon region of Ecuador. Sites 1 and 2 are secondary forests, with Site 1 containing open oil pits that were created to dump crude oil into the environment. In Site 2, there is no evidence of any petroleum pollution. Permissions to sample in these sites were granted by the petroleum company operating in this region (Andes Petroleum Ecuador Ltd). Site 3 is a pristine primary rainforest inside the protected Yasuni National Park and is considered our control site. Appropriate permissions to sample vegetation and soils were granted by the Environmental Ministry of Ecuador.In order to define a representative and comparable sampled area between secondary and pristine forests, area size was considered. We kept an equivalent sample size between secondary and pristine forests. The sample areas in the secondary forest plots represents 48% of the total area while the plots in the pristine forest represent 52% of the total area  Many traits, including physiological ones, are conserved within plant clades . A largeAlthough the unequal sampling of the three areas somewhat hampered the biodiversity analyses, it is the distinct strength of the approach used that it allows for a reasonable interpretation of the data despite an unequal sampling effort . Trends This multi-pronged approach was used as the different measures represent different aspects of diversity. Observed family richness has the disadvantage of being difficult to interpret when only unequal samples are available. Rarefaction, however, allows for such comparisons in several ways. First, by smoothing the accumulation curves and providing confidence intervals it allows for the statistical comparison of family richness, at least for the sampling effort of the least sampled site. Secondly, by extrapolating the family accumulation curves of sites with smaller samples or by using mathematical estimators, such as the Chao 1 index, it is possible to compare expected richness at the level of the sample with the highest effort. Of the diversity indexes, Fisher´s alpha index is the least biased one if the sample size is uneven. The Exponential Shannon Index in turn provides a family equivalence value biased towards the rarer taxa, and the Inverse Simpson Index provides a family equivalence value biased towards the more common taxa . FamilyField spectroscopy measurements of the leaf samples were taken with an ASD FieldSpec HandHeld-2 spectroradiometer . This instrument measures in the wavelength range from 325 nm to 1075 nm at a sampling interval of 1 nm. The spectroradiometer was attached to a plant probe with an internal 4.05-W halogen light source and a leaf clip that includes a rotating head with both white and black reference panels . These sRadiative transfer models simulate the interaction of light with plant leaves based on the relationship between spectral reflectance and trasmittance of vegetation and the biochemical and biophysical properties of the leaves. The LIBERTY model (Leaf Incorporating Biochemistry Exhibiting Reflectance and Transmittance Yields) is a model for conifer (particularly pine) needles , while tCab)  was applied to the reflectance and double-transmittance data , 44. ThiCab) see .Soil analysis results show increased levels of Total Petroleum Hydrocarbons (TPH) of nearly 9000 mg/kg in the secondary forest affected by petroleum pollution (Site 1), confirming that the open petroleum pits are still active sources of pollution. Soil samples from Site 2 (secondary forest unpolluted) and Site 3 (pristine forest) had <200 mg/kg TPH in the soil because they are indeed unpolluted .From the 1134 leaf samples collected during the fieldwork, 739 samples (65%) were identified at plant family level and used in this study. The distribution of plant families across the three study sites is shown in Melastomataceae, Fabaceae, Bombaceae, Rubiaceae, Myristiaceae, Moraceae, Euphorbiaceae, Meliaceae, Cluseaceae, Lauraceae, Flacourtiaceae, Violaceae, Burseraceae, Lecythidaceae and Cecropiaceae.The cumulative percentage shows that the 15 most frequently occurring plant families represent 73.5% of the samples collected in the three sites. Those plant families are: Clusiaceae are present in the pristine forest. The plots in the secondary forests are less diverse.The occurrence of each plant family across sites and heterogeneous family composition depending on the forest type is illustrated . Of the The species richness estimates are not statistically significantly different between sites. In fact, estimates for the polluted secondary forest site are close to those of the pristine forest and those of the unpolluted secondary forest site are lowest ; Table 2Chlorophyll content is an indicator of plant stress (see Section 1). Here we compare chlorophyll content between plant families in the study sites. We test the assumption that plant families growing in the polluted site (Site 1) show stress symptoms caused by the petroleum pollution. The vertical gradient of chlorophyll content across the forest layers exhibits significantly lower levels in the understory . This isAn ANOVA test was conducted at 99.9% confidence level (p < 0.001), followed by a post-hoc pairwise comparison using the type I error adjustment method of Holm and TukeyHSD (honest significant difference) see . The ANOFinally, Tukey’s HSD two-factor pairwise comparisons of leaf chlorophyll content across sites and canopy layers revealed highly significant differences (p < 0.001) in the understory compared to the other canopy layers. These differences are consistent with the reduced photosynthetic activity in the understory caused by the vertical light gradient in the forest. These findings present scientific evidence that vegetation growing in areas affected by pollution has significantly lower levels of chlorophyll content due of vegetation stress.Melastomataceae, Fabaceae, Rubiaceae and Euphorbiaceae.To identify the plant families that are affected by petroleum pollution, leaf chlorophyll content at the polluted Site 1 and the pristine forest control Site 3 were compared. The ecological conservation status of these two forests is considerably different, therefore plant physiology is likely to show stress symptoms caused by the pollution in Site 1. From the 15 more representative plant families, four families exhibit significantly lower levels of chlorophyll content in the polluted site: The high biodiversity of tropical forest and the remoteness of the study area presented challenges when selecting the sites and defining the sampling procedure. Careful consideration was given to the selection of Sites 1 and 2. Both of them are disturbed forest that has been exposed to selective logging, agricultural activities, petroleum industry impacts and secondary forest regrowth over the last 20 years following diminishing human influence. These sites are located near to each other and share the same environmental conditions including soil type, weather, forest type and anthropogenic influence. The only differenes between them is that Site 1 has been permanently affected by petroleum pollution over the last decades. This may be the cause of some shifts in plant family diversity and biochemical composition of the vegetation. The pristine forest represents a tropical forest under natural conditions and was considered as our control site.Although results based on species richness are inconclusive, pollution leads to a reduction of the diversity of the plant community by most diversity measures used. Species richness measures are probably affected by overall densities of individuals at the study sites. Although the area sampled at Sites 1 and 2 are similar, the densities at Site 2 are considerably lower. If the sampling had been extended until a similar number of individuals had been reached, the shape of the rarefied species accumulation curve may have been quite different. The shallow curve of the polluted secondary forest site is due to a lower evenness, but it also means it takes longer to stabilize to an asymptote, hence the high species richness estimates due to extrapolation and the Chao 1 index.Melastomataceae and Rubiaceae, suggesting that plants from these families are particularly competitive in the presence of pollution although the leaf chlorophyll assessment of both plant families showed significantly lower values.Diversity indexes reveal stronger trends in the data than the species richness indices. As expected, pristine forest has the highest diversity, followed by unpolluted secondary forest. This result is due to a greater evenness of abundances there than in the polluted secondary forest site. Most notable at the polluted site is the dominance of the The Morisita-Horn similarity measures seem to be influenced primarily by the more abundant families in the samples and the exclusive communality of families between two sites  compared to the secondary forest non-polluted site (Site 2) and pristine forest (Site 3). These results together with the analysis at three vertical canopy levels lead to the conclusion that plant stress caused by petroleum pollution can be differentiated from the natural photosynthetic gradient of the forest canopy.Melastomataceae, Fabaceae, Rubiaceae and Euphorbiaceae). These four families make a strong contribution to the forest structure of each site, especially at the polluted site which represents 53% of the total samples  compared to the non-polluted sites (Site 2 and 3). Chlorophyll content at Site 2 (secondary non-polluted forest) and Site 3 (pristine forest) were not significantly different from each other which suggest normal photosynthetic activity. Hence, vegetation stress is only observed in the forest exposed to hydrocarbons.Melastomataceae, Fabaceae, Rubiaceae and Euphorbiaceae) are sensitive to petroleum pollution while the other eleven families showed no significant effects. Most notable at the polluted site is the dominance of the Melastomataceae and Rubiaceae, suggesting that plants from these families are superior competitors in the presence of pollution.We conclude that photosynthetic activity at the oil polluted Site 1 is reduced due to the hydrocarbon impacts on the photosynthetic system. The detailed analysis at plant family level found that four of the 15 representative plant families at the three study sites Click here for additional data file.S1 FigError bars represent the 95% confidence intervals and suggest that the diversity of site 3 is significantly different from that of site 1, but that the diversity of site 2 is not significantly different from those of the other sites .(DOCX)Click here for additional data file.S2 FigError bars represent the 95% confidence intervals and suggest that the diversity of site 1 is significantly different from those of sites 2 and 3, but that the diversity of site 2 is not significantly different from that of site 3 .(DOCX)Click here for additional data file.S3 FigError bars represent the 95% confidence intervals and suggest that the diversity of site 1 is significantly different from those of sites 2 and 3, but that the diversity of site 2 is not significantly different from that of site 3 .(DOCX)Click here for additional data file.


Thus, effective strategies for the quantitative determination of phosphorylation degrees for low abundant proteins, for which antibodies are not available, are required in order to evaluate the functional regulation of proteins attributed to phosphorylation. In this study, we used the heart β1-adrenergic receptor (Adrb1) as a model protein and developed FLAG-Adrb1 knock-in mice, in which the FLAG tag was inserted at the N-terminus of Adrb1. The phosphorylation sites and levels of Adrb1 in the heart were elucidated by immuno-affinity purification followed by quantitative mass spectrometry analysis using ion intensity ratio of the phosphorylated peptide versus corresponding unphosphorylated peptide. The phosphorylation levels at Ser274 and Ser462 of Adrb1 were approximately 0.25 and 0.0023. This effective strategy should be useful for not only analyzing site-specific phosphorylation levels of target proteins, but also quantifying the expression levels of proteins of interest when appropriate antibodies are not available.Protein function is mainly modulated by dynamic reversible or irreversible post-translational modifications. Among them, the identification of protein phosphorylation sites and changes in phosphorylation levels  To overcome these difficulties, we have established a strategy to analyze protein phosphorylation levels at specific sites in vivo using the GPCR β1-adrenergic receptor (Adrb1) as a model protein for which all commercially available antibodies do not work.Phosphorylation is the most extensively studied post-translational modification (PTM) regulating protein activity and plays a pivotal role in cellular signaling events and metabolic processes. In particular, G-protein coupled receptors (GPCRs) are phosphorylated at multiple sites, acting as a barcode for regulating signaling in a tissue-specific manner through a combination of phosphorylation sites ,2. GPCRsin vivo rather than the expression level and the distribution of total protein.Adrb1 is predominantly expressed in the heart and plays a critical role in the regulation of heart rate and the force of myocardial contraction . It is ain vivo, a knock-in (KI) mouse of Adrb1 fused with FLAG-tag (FLAG-Adrb1 KI mouse) would potentially be an effective tool. In a KI model, the target gene is inserted into a specific locus in the genome via homologous recombination resulting in the natural expression pattern and level , sonicated, and added with NP-40 at a final concentration of 1%. The lysate was mixed by inverting the tube several times every 10 min for 30 min at room temperature (RT) and ultracentrifuged for 30 min at 200,000× g. The recovered proteins were then subjected to IP.Isolated mouse heart was cut into small pieces with scissors in ice cold homogenization buffer [20 mM Tris-HCl (pH 7.4)] containing phosphatase and protease inhibitor cocktails  and homogenized using a physcotron homogenizer  for 30 s. The homogenate was centrifuged at a speed of 2000× FLAG-Adrb1 was purified using an anti-FLAG M2 affinity gel (Sigma-Aldrich). Briefly, to the membrane protein lysate prepared from FLAG-Adrb1 KI mouse heart , 40 µL of M2 affinity gel equilibrated with lysis buffer was added and rotated for 16 h at 4 °C. The resin was washed three times with lysis buffer and eluted with 40 µL of 0.3% sodium dodecyl sulfate (SDS) and 40 µL of 0.15% SDS.Immunoprecipitates were diluted with Milli-Q water and added with 1 µL of 1.5 M Tris-HCl (pH 8.8) followed by reduction with 5 mM Tris (2-carboxyethyl)phosphine hydrochloride. The reaction mixture was digested with 0.5 µg of Lys-C  for 2 h at RT, followed by 1 µg of trypsin  for 16 h at RT. Protein digests were alkylated with 20 mM iodoacetoamide and subjected to strong cation exchange (SCX) column chromatography.2H (Solvent A). The peptides were eluted with 30% ACN containing 350 mM NH4HCO2 at pH 3 (Solvent B) using a gradient from 0% to 10% B for 20 min, 10% to 20% B for 10 min, 20% to 40% B for 5 min, and 40% to 80% B for 5 min, followed by 80% to 100% B for 1 min at a flow rate of 0.1 mL/min.SCX chromatographic fractionation was performed using a Shimazu HPLC system with a polysulfoethyl A SCX column . Phosphorylated peptides were loaded on the column equilibrated with 80% acetonitrile (ACN) containing 0.1% HCOFractionated peptides were analyzed by online nano liquid chromatography (LC) using an Easy nLC1000 System  coupled to a Fusion Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a trap column  and resolved on an analytical column  at 300 nL/min over 45 min. The mass spectrometer was operated collecting MS spectra in the Orbitrap mass analyzer at a resolution of 120,000 and data-dependent higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) spectra in the ion trap with normalized collision energy of 30%. Peak areas in the extracted ion chromatogram (XIC) were calculated using Xcalibur software .m/z 465 and 540. The resolution was set to 500,000, and MS/MS was performed in a data-dependent acquisition scheme by selecting the target precursor ions at m/z 484.748 and 524.721 corresponding to unphosphorylated (QGFSSESKV) and phosphorylated peptides (QGFSpSESKV), respectively.When analyzing SCX fractions containing tryptic peptides extending from 458 to 466 on Adrb1 (QGFSSESKV), the MS scan ranges were set between All MS raw files were processed to identify and quantify peptides with Proteome Discoverer 1.4 (Thermo Fisher Scientific) using Mascot  against the Uniprot mouse protein database. The mass tolerance of the precursor and fragment were set to 10 ppm and 0.45 Da. Up to one missed trypsin cleavage was allowed.For Mascot search, the following parameters were set: carbamidomethylation of cysteine was selected as a fixed modification; oxidation of methionine, and the phosphorylation of serine, threonine, and tyrosine were selected as variable modifications. The phosphorylation site localization of identified phosphorylated peptides was performed by phosphoRS algorithm 3.1 implemented in Proteome Discoverer. A site localization probability of at least 0.75 was used as the threshold. A false discovery rate of 0.01 was applied to peptide identification.t-test was performed to compare the mRNA expression for mAdrb1 or Signal sequence of FLAG (Sig S-Flag) between wilt type (WT) and KI mice. p < 0.05 was considered statistically significant.Welch’s Adrb1 CDS into the Adrb1 locus  signal sequence [Adrb1 mRNA levels.Taking these points into consideration, a mouse model where a protein of interest is fused with an epitope tag such as FLAG or hemagglutinin is a powerful tool for analyzing the phosphorylation states of target proteins ,26. Thersequence . In the 2 purification [2 purification, the Adrb1 tryptic peptides could not be detected in an analysis of total proteins derived from a single intact heart. In particular, in the case of Adrb1, it was difficult to identify the specific band of FLAG-Adrb1 in WB analysis using anti-FLAG antibody even after immune-affinity purification (data not shown). On the contrary, the target peptide fragment was assigned with certainty in the MS analysis. To examine phosphorylation levels at specific sites on Adrb1 in KI mouse hearts, we made use of proteomic technology, which combined the specificity of antibody-based purification with the high sensitivity of MS analysis. As a result, six tryptic peptide fragments were identified, and three were detected together with their phosphorylated peptides from the membrane protein fraction in the KI mouse heart . All thefication . On the As for the phosphorylation sites, phosphorylations at Ser274 and S462, which previously were not detected in the perfused mouse heart without the stimulation of ligand , were obin vivo. Next to clarifying the relative stoichiometry of phosphorylated forms in Adrb1, the determination of the absolute phosphorylation site occupancy which determines the nature of the signaling response is an important aspect and the issue in the future. This KI mouse can also be utilized to analyze the expression of the receptor without radioligands by quantifying the unphosphorylated peptide in MS analysis. Furthermore, this mouse line may be an effective model for investigating the physiological functions of Adrb1 in vivo, such as distribution, trafficking and characterization. In conclusion, we have established an effective strategy for quantitatively analyzing protein phosphorylation levels at specific sites in vivo.As for the quantitative analysis of phosphorylation, the phosphorylation level at each site was determined reproducibly except for Ser462 whose ion intensity was extremely low compared with the other sites. These data demonstrate that FLAG-Adrb1 KI mouse will be applicable for analyzing the stoichiometry of Adrb1 phosphorylation