This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4–1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed. Collagen is the primary structural component of tissue extracellular matrix (ECM) and plays a critical role for tissue mechanical integrity and functionality , 2. CollEffects of F/T on bulk tissue properties and microstructure of collagenous biomaterials have been extensively studied. There are numerous studies showing changes in thermal and mechanical properties of collagenous tissues upon F/T –15. ChanThermodynamic destabilization of collagen by low temperatures and mechanical insult by ice formation are two alternative mechanisms that could explain previously reported F/T induced changes such as increase in fibril dimensions , 16 and Although protein cold denaturation has been previously investigated by differential scanning calorimetry (DSC) , nuclearIn this study, we address the above gaps by providing measurements of post-thaw thermal stability of collagen at multiple levels of its hierarchy using modulated temperature differential scanning calorimetry (MTDSC). Measurements were performed on collagen molecular solutions as well as on hydrogels with different collagen concentrations. Since hydrogels exhibit a fibrous mesh microstructure that is absent in molecular solution, it was possible to relate the F/T induced thermal stability changes of collagen to its distinct structures at matrix, fibril and molecular levels. In addition, morphological examination of collagen fibrils and network was performed with scanning electron microscopy to assess alterations in collagen matrices by F/T. The results were discussed to delineate the effects of ice formation and thermodynamic effects of low temperatures. The results were further analyzed by a computational model developed to relate F/T induced changes in collagen’s post-thaw thermal stability to fibril deformations.2 incubator. Afterwards, isotonic saline solution (1X DPBS) was added on top of the hydrogel to prevent dehydration and incubation was continued until the experiments next day.Rat tail collagen type-1  was purchased from the manufacturer as a concentrated stock solution. In order to study collagen in macromolecular form, the stock solution was diluted to a final collagen concentration of 3 mg/mL in 20 mM acetic acid. The resulting molecular solution of collagen remained unpolymerized when kept at room temperature for the duration of experiments. In addition, hydrogels exhibiting fibril and matrix-level structures of collagen were prepared according to manufacturer’s protocol. Appropriate amounts of 10X Dulbecco's Phosphate-Buffered Saline , 1.0 N NaOH, cell culture grade distilled water, and collagen stock solution were mixed together in a centrifuge tube on ice in the given order, resulting in a solution with neutral pH and isotonic ionic strength. This neutralized collagen solution had a final collagen concentration of either 3 or 6 mg/mL. Prepared solution was then dispensed into a chamber slide  and allowed to polymerize for 1 hour at 37°C in a COTd, was determined as the temperature attained at peak maximum. Temperature span of the transition was reported as peak width at half height, ΔT1/2, and denaturation enthalpy, ΔHd was calculated as the area under the peak. Measurements were performed 3–4 times for each treatment group and results were averaged based on standard statistical methods.Circular hydrogel specimens were isolated by a 3 mm biopsy punch and placed in DSC pans. For molecular solution experiments, 10μL of solution was directly dispensed in the DSC pan. Pans were hermetically sealed to prevent dehydration during the experiments and were loaded in a differential scanning calorimeter  which was previously calibrated with indium, water and sapphire. Measurements were taken during heating from 25 to 60°C (molecular solution) or 30 to 65°C (hydrogel) with an underlying heating rate of 1°C/min. A sinusoidal temperature modulation with amplitude of 0.75°C and period of 80 seconds was superimposed with the underlying linear temperature program. Use of temperature modulation enabled deconvolution of specific heat signal into reversing and non-reversing components . Heat abFor freezing temperature studies, samples were cooled to either -20°C or -60°C in the DSC cell at a rate of 50°C/min, kept isothermal for 2 minutes, and warmed back to 20°C with 50°C/min. MTDSC measurements were initiated within 10 minutes of thawing.In order to investigate the effects of ice formation, sample prepared in a DSC pan was placed on a temperature controlled stage  and monitored for ice nucleation while stage temperature was decreased to -15°C with a rate of 10°C/min and maintained at that temperature for 2 minutes. Small volume of the DSC sample enabled substantial supercooling allowing it to remain ice-free unless ice nucleation was deliberately induced by a liquid nitrogen cooled needle. Ice formation controlled this way enabled preparation of samples that were either supercooled and ice-nucleated, or supercooled only.Different ice formation kinetics were obtained with a procedure similar to above. Samples were first cooled to -2°C where ice was nucleated. The presence of small ice crystals in the sample was verified under a microscope. Then, the samples were cooled to -20°C with two different cooling rates at 1 or 50°C/min. These two cooling rates were observed to generate different rates of ice growth in the samples.For scanning electron microscopy, collagen samples prepared in chamber slides were placed on a directional solidification stage  and froz2SO, DMSO), hydrogels were incubated in saline solution including DMSO at concentrations ranging between 0.25 M and 1 M for 30 minutes before freezing. After 10 minutes of passive thawing at room temperature, samples were sectioned and transferred to DSC pans followed by freezing in the DSC cell as explained before. Since DMSO in the hydrogels was not unloaded after F/T, the samples prepared in this way contained DMSO during the MTDSC measurements. In order to control for possible confounding effects of DMSO in the measurements, experiments were also performed on DMSO loaded unfrozen collagen hydrogels and the results were compared with 0 M DMSO case.For experiments with cryoprotectant dimethyl sulfoxide  for 20 min followed by repeated washing in isotonic saline for 30 min. This procedure approximated the transient concentration increase of solutes during F/T. Parts of hydrogels treated with hypertonic solution were then F/T to -60°C and post-thaw denaturation was measured as before.2 critical point dryer. Through the sample preparation, the sizes of specimens unfrozen or F/T with DMSO remained unchanged while notable shrinkage was observed for the specimens frozen and thawed without DMSO. Specimens were then imaged using a scanning electron microscope . At least 3 randomly selected fields of views were imaged per sample. The fibril diameters were estimated by manually tracing the boundaries of approximately 100 fibrils per each group by ImageJ [Hydrogels were F/T on a directional solidification stage as described before and passively thawed at room temperature for 15 minutes. The samples were then immediately fixed with 2.5% glutaraldehyde and 2% formaldehyde in 1X DPBS, and stored in fixation solution at +4°C overnight. Afterwards circular sections were punched out from unfrozen and F/T regions, dehydrated against ethanol and dried in a COy ImageJ .In this study, we considered collagen thermal denaturation as a single step irreversible rate reaction based on earlier work of Miles et al. . The denN and D are the native and denatured states of collagen, respectively. Both unfrozen (UF) and frozen/thawed (F/T) collagen is assumed to go through the same reaction, but at possibly different rates.Here α is the extent of conversion from native to denatured state, k is the rate constant and f is the reaction model. Arrhenius type temperature (T) dependence for the rate constant and an nth order reaction model [For both UF and F/T, kinetics of denaturation is described by the rate law,on model  were assA is commonly referred as the pre-exponential factor and Ea is the activation energy. These together with the reaction order n, are the kinetic parameters that are to be estimated by fitting the above model to experimental data by an optimization routine developed in MATLAB®.In Freezing induced expansion of fibril was modeled by a unit cell / representative volume approach that was previously used in modeling freezing-induced tissue-level expansion . Mean coS↕, and confinement diameter, d, based on statistical mechanics modeling of a polymer in a tube [N is the number of links in the thermally labile domain of collagen , b is t 0.35 nm ) and R i 0.35 nm , 50.A, in the previously described kinetic model as:The activation entropy is related to the pre-exponential factor, perature .d2 in comparison to a reference configuration with confinement diameter d1, we obtain:By considering Eqs Therefore, reaction kinetics of an arbitrary confinement level can be predicted based on the reaction kinetics and confinement level of the reference state.d, increases resulting in a decrease in molecular confinement. The relative increase in unit cell dimensions is given by:dc and ϕ is the fibril porosity at unfrozen state. ρf and ρi are the densities of intrafibrillar fluid and ice, respectively. Porosity is calculated from geometric relationships as:dm is the collagen molecular diameter that was varied between 1.3 and 1.5 nm [d = 2dc-dm, also based on unit cell geometry.The amount of this expansion mainly depends on fibril native i.e. unfrozen porosity. Upon expansion, confinement diameter, d 1.5 nm , 52. TheEqs p-value was less than 0.05. Experimental data are reported in terms of mean ± standard deviation.Differences in treatment means were tested by one-way ANOVA. Multiple comparisons were performed by Tukey’s test. The differences were considered statistically significant when Heat absorbed by collagen during thermal denaturation is observed as an endothermic peak in a DSC thermogram . RepreseTd, determined from the location of endothermic peak. Collagen as a macromolecule maintained its thermal stability even after F/T to -60°C  (to -60°C . Peak wito -60°C . In the  > 0.05) .Td decrease caused by ice formation was comparable to those from freeze/thaw at -20°C and -60°C. Interestingly hydrogels supercooled with the same temperature history also had a lower Td than UF. However, the difference was not statistically significant. Effects of cooling rate were also investigated by considering F/T treatments to -20°C with either 1 or 50°C/min. It was found that higher cooling rate resulted in a slightly lower Td than lower cooling rate. However, the difference between the two cases was not statistically significant.In order to distinguish between the mechanical effect of ice formation and thermodynamic effects of low temperatures, experiments were conducted on collagen hydrogels either ice nucleated or supercooled with the same temperature history. Consistent with the previous results, the hydrogels that experienced ice formation denatured at a significantly lower temperature than UF as shown in Since ice formation results in both mechanical expansion and increased concentration of solutes in collagen, we compared denaturation of collagen hydrogels before and after hypertonic solution treatment as well as F/T with and without hypertonic solution treatment. It was found that hypertonic treatment resulted in a net decrease in denaturation temperature that was significantly less then F/T alone . When hyDuring cryopreservation, cells and tissues are often loaded with cryoprotectants such as dimethyl sulfoxide (DMSO) that can help reduce freezing damage by decreasing the amount of phase-change induced expansion . To testd = 18.1 ± 4.1 nm . These filaments seemed to be a part of multiple, thick, but loosely packed bundles (d = 83.9 ± 36.0 nm) which could have originated from fibrils that have unraveled during F/T due to expansion. This was in contrast to the smooth, uniformly sized fibrils observed in unfrozen control (d = 43.3 ± 7.4 nm). The extent of this fibril level damage was alleviated by use of DMSO during freezing. The case of 0.5 M DMSO also showed mostly intact fibrils with diameters, d = 42.9 ± 9.4 nm, similar to that of unfrozen control. The distributions of fibril diameters are shown in Examination of collagen hydrogel microstructure with scanning electron microscopy revealed dramatic changes in both network and fiber level upon F/T as shown in Td by F/T by considering the freezing-induced expansion of collagen fibrils and associated relaxation of collagen molecular confinement in fibrils. First, Arrhenius parameters of denaturation were determined by kinetic analysis of denaturation endothermic peaks Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Tablen, activation energy, Ea, and the pre-exponential factor, A, are reported for selected F/T and UF cases.The best fitting values of model parameters, reaction order, (PDF)Click here for additional data file.


Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution. Summary: In contrast to textbook knowledge, the phosphorylation of translation initiation factor eIF2α is not required for UV-induced inhibition of protein synthesis, which we show in three different cell types. All types of cells activate mechanisms to regulate gene expression after exposure to environmental stress, and to a large extent, at the level of translation. These stress responses enable the cells to cope with and adapt to stressful situations such as starvation, oxidative damage, osmotic stress and DNA damage. Translation can be regulated at all stages of the process, including initiation, elongation and termination, but control of translation in eukaryotes occurs mainly at the initiation step  of the initiation factor eIF2. An early step in initiation involves the binding of the 40S ribosomal subunit to the ternary complex comprising eIF2, the initiator tRNA (Met-tRNAiMet) and GTP , three in fission yeast  and one in budding yeast (Gcn2). Many different forms of stress results in phosphorylation of eIF2α in eukaryotic cells , includiIn several different types of cells, protein synthesis is inhibited after exposure to ultraviolet (UV) light, including rat fibroblasts , mouse eThere is convincing evidence that eIF2α phosphorylation attenuates the translation of most mRNAs. However, the importance of this mechanism might have been overestimated and, in many cases, presumed to be the mechanism behind an observed inhibition of protein synthesis after stress simply because eIF2α is phosphorylated. There are a few published sets of data that indicate the presence of an inhibitory stress-induced mechanism operating independently of eIF2α, such as after oxidative stress in budding yeast  and afteIn the present work, we have investigated the role of eIF2α and the Gcn2 kinase in mediating stress-induced translational inhibition. We have directly measured the translation rate in fission yeast, budding yeast and mammalian cells after UV irradiation and shown that the inhibition of protein synthesis is largely independent of eIF2α phosphorylation in all three cell types. Our data suggest that a hitherto unrecognized mechanism regulates translation after stress.l-homopropargylglycine (HPG), which is incorporated into proteins in the place of methionine. The presence of HPG is detected by using chemoselective fluorescence-tagging by means of ‘click chemistry’ and has previously been used to label newly synthesized proteins in rat fibroblasts (Schizosaccharomyces pombe). After adding HPG to exponentially growing cells, we observed an HPG-specific signal with fluorescence microscopy that increased continuously over time. The signal intensities from HPG-containing individual cells could be conveniently quantified using flow cytometry  cells were investigated by using the same procedures as described for S. pombe cells. Because budding yeast are much less resistant to UV irradiation than fission yeast, the radiation dose was reduced to 220 J/m2. Again, UV exposure almost completely abolished protein synthesis in wild-type cells  unless starved for methionine before labelling. Such starvation has the unwanted effect of activating GCN2. Therefore, we instead employed the puromycin analogue O-propargyl-puromycin, OPP , which hporation A. GCN2−/+/+ MEFs A. Immunout cells B. These S. pombe cells that had been exposed to hydrogen peroxide (H2O2), which leads to oxidative stress and Gcn2-dependent phosphorylation of eIF2α , which prevents the initiation factor eIF4E from binding to the translation scaffold protein eIF4G, inhibiting initiation of translation . When phMany forms of stress induce cytoplasmic RNA granules in fission yeast  and in bIt is the accepted view that, after exposure to stress, phosphorylation of eIF2α leads to a global downregulation of cap-dependent translation. This model builds on the classic observations that, in cells starved for amino acids, eIF2α phosphorylation and global downregulation of translation correlate, and on the fact that phosphorylation of eIF2α prevents eIF2 rejuvenation to the active GTP-bound form. However, it is technically challenging to determine to what extent this downregulation is due to eIF2α phosphorylation or to a lack of amino acids, which in itself is expected to prevent translation.in vitro assay, protein synthesis in extracts of wild-type cells was abolished after UV irradiation of the cells, whereas no inhibition was observed in extracts from a gcn2Δ mutant  before the cells were irradiated. The strains of S. cerevisiae  were grown in minimal SD base  at 25°C with appropriate supplements and to a cell concentration of about 107/ml [equivalent to an OD (600 nm) of ∼0.45]. MEFs (either GCN+/+ or GCN2−/−) were cultivated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified environment with 5% CO2.All strains of e strain  – wild-t2A:ura4+ . Media a2A:ura4+ . Growth l-homopropargylglycine  into proteins followed by chemoselective fluorescence tagging by means of ‘click chemistry’  were collected at the indicated time points after treatment, and the cells were fixed in ice-cold 70% ethanol, washed in 0.5 ml PBS, permeabilized in 1% Triton X-100 in 0.5 ml PBS, washed and blocked with 0.5 ml 1% BSA in PBS. ‘Click chemistry’ was performed according to the manufacturer's protocol to join the amino acid alkyne with a fluorochrome azide. Incorporation was measured by using flow cytometry  and the median fluorescence intensity per cell of Alexa-Fluor-647 azide. Sytox Green and DAPI were used to stain the DNA for flow cytometry and fluorescence microscopy, respectively. Multimers of cells were excluded from the analysis as previously described . FxCycle Far Red (Life Technologies) was used to stain DNA, and multimers of cells were excluded. Samples without OPP were used as negative controls.To label newly synthesized proteins in mammalian cells, O-propargyl-puromycin  was added immediately after irradiation to a final concentration of 10 µM, and the cells were detached through trypsinisation and fixed in 70% ice-cold ethanol at different times. The cells were washed once in PBS containing 1% FBS before OPP was tagged with Alexa-Fluor-488 in a Click-iT reaction, as described by the manufacturer. Incorporated OPP was measured by using flow cytometry  after as much medium as possible had been siphoned off of the culture dishes. The medium was rapidly replaced after irradiation. Oxidative stress was applied to a culture of S. pombe cells by adding 5 mM H2O2 for 15 min, as described (The appropriate fission yeast mutants were irradiated in a suspension under continuous stirring to distribute the irradiation dose equally to all cells . The staescribed . For leuescribed . MEF celTotal cell extracts of yeast cells for immunoblotting were generated through trichloroacetic acid extraction . The extFor polysome profiling, cycloheximide was added to the culture to a final concentration of 0.1 mg/ml 30 min after UV irradiation and after 2 h of starvation for leucine. Cell extracts were applied to 10–50% sucrose gradients, centrifuged and analysed as described previously .


Personalised health surveillance is infrequent or absent in occupational and environmental medicine. The shortage of functional tests in relevant cells and tissues greatly limits our understanding of environmental exposures and associated disease risk. We evaluated single cell signalling in peripheral blood mononuclear cells from 301 individuals in a cross sectional health survey 18 months after a chemical explosion of sulphorous coker gasoline. The accident created a malodourous environment leading to long-term health complaints. Multiple regression analysis revealed T-cell specific elevated phosphorylation of the stress kinase p-p38 (T180/Y182) among tobacco smokers and monocyte-specific elevated phosphorylation in employees at the explosion site. Other studies of the accident reported reduced tear film stability, and more airway obstruction and subjective health complaints among the employees at the accident site. Elevated monocyte p-p38 in the employee group was independent of such health effects, and could therefore be dependent on the sulphuric malodorous environment. The present study proposes signalling status in leukocytes as a scalable biomarker providing information about environmental exposures. Monocyte p-p38 was found to be higher in employees at the malodourous explosion site compared to people living >20 km away. The results indicate a role for single cell signalling profiling in future personalised exposure monitoring.Fixed and methanol-permeabilised peripheral blood mononuclear cells (PBMCs) from the study group  were barInitial assessment of p-p38 variation indicated no significant association between monocyte p-p38 and living within 6 km from the explosion site compared to individuals living >20 km away  . EmployeMultiple linear regression analysis with T-cell p-p38 as a dependent variable confirms no association between T-cell p-p38 and being employed or living close to the accident site compared to living >20 km away . The regPerforming the same analyses stratified by gender implied a higher level of T-cell p-p38 among male smokers compared to male non-smokers, and a higher level of monocyte p-p38 among male workers and female inhabitants as compared to their respective reference groups  and 4. TInflammatory diseases and mild infections are known to increase levels of cytokines relevant to p38 signallingReduced tear film stability, and more airway obstruction and subjective health complaints have been reported for this employee group in other studies2728in vitro experiments where exposure of epithelial cells to cigarette smoke extract (CSE) induces ROS-mediated phosphorylation of p-p38Analysis of the basal p38 (pT180/pY182) phosphorylation level indicated elevated monocyte p-p38 in individuals working close to a malodourous environment, determined in a single cell assay suitable for population studies. We also detected significantly elevated p-p38 (pT180/pY182) in T-cells in male smokers compared to male non-smokers; however, no such association was observed for females . Elevateet al. suggest that the sulphuric remains from the accident may have lead to irritation of the eyes, but that other types of pollution not related to the accident could also be involved (i.e. dust)Individuals employed at the malodorous explosion site showed significantly elevated p-p38 in monocytes but no modulation of p-p38 in T-cells when compared to individuals living >20 km away  and 2. I1/FVC ratio < 0.7) or subjective stressful life events (IES-R) in the regression models did not result in significant associations with monocyte p-p38 , this co-occurred with the persistent smell in the area during the survey28292S is common in the paper producing industry, in the processing of crude oil, in sewers and in proximity of geothermal fields404142432S, which is also an endogenous gasotransmitter, is reported to attenuate oxidative stress and p-p38 in the lungs of rats exposed to passive smoking2S alone also attenuates p-p38 in granulocytes2S does induce phosphorylation of p38 in endothelial cellsThe coker gasoline involved in the explosion accident was reported to contain large amounts of sulphurWe propose elevated phosphorylation of p38 in monocytes as a possible marker during exposure to a sulphuric malodourous environment and indicate that elevated p-p38 in T-cells could be a marker of smoking. There is a need for new biomarkers in health studies of environmental and occupational exposure1The cross sectional study is part of the health survey that was carried out 18 months after the accident described previously2627282926Of the 1,016 individuals invited to take part in the survey, 734 met for the examination . 231 ind® CPT™). Mononuclear cells were harvested after centrifugation , this was followed by washing of the cells in 0.9% NaCl. Cells for flow cytometry were fixed in 4% paraformaldehyde (PFA) for 5 minutes at room temperature (RT) prior to permeabilisation in ice cold methanol. For western blot the cells were suspended in 7% ice cold trichloroacetic acid (TCA) for protein precipitation. Samples were stored at −80 °C until transportation on dry ice to the central research lab. Health care personnel involved in the consultations also carried out the initial sample collection. The major findings of the study are cellular subset-specific for monocytes or T-cells in the same sample from the individuals, reducing the probability that sample preparation has biased the results.For the population study a blood sample was taken by venepuncture and collection in Cell Preparation Tubes with sodium citrate . An additional panel using the same surface markers and Mouse IgG1 κ Isotype control Alexa Fluor® 647  was used to determine background. The canonical surface markers CD3 (T-cells) and CD14 (monocytes) were damaged by the methanol permeabilisation during sample collection, and were replaced with CD5 and CD163, respectively. CD5 is known to co-express with CD3 ® channel was set high to increase separation between signal and background. Sufficient events were recorded to ensure a low theoretical standard error of median (σMD) for the median fluorescent intensity (MFI) . However, for 75% of the samples more cells were recorded . Gating and export of MFIs were carried out using FlowJo (TreeStar Inc). Raw value MFIs of p-p38 Alexa Fluor 647® were used in the statistical analyses.To achieve high throughput and to reduce antibody consumption, the samples from the population study were fluorescently barcoded as described by Krutzik & NolanPrecipitated protein samples from the population study were processed and analysed by SDS-PAGE and western blotting as described elsewhere®/Modular P system (Roche)).Serum hs-CRP was determined with an immuno assay (Tina-quantwww.R-project.org) to examine effects on p-p38 by the categorical predictors gender, age , body mass index (low (<25), medium (25–29.9) and high (>29.9)), smoking status (non-smoker or current smoker) and geographic groups  with Dunnett’s multiple comparison post hoc tests was used for initial assessment of variation in geographic groups in the population study. In addition, multiple linear regressions were carried out in R .


O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability.Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the  Lf gene in some forms of cancer [Lf gene at the alternative P2 promoter leading, after translation, to a 73 kDa intracellular protein [via its role as a transcription factor. Indeed, ΔLf transactivates different target genes such as Skp1, DcpS, Bax, SelH, GTF2F2 and UBE2E1 [Lactoferrins exist as different variants due to gene polymorphisms, post-transcriptional and post-translational modifications . The twof cancer ,8. ΔLf m protein . Althoug protein . Thus, w protein . ΔLf is  protein . ΔLf exh protein ,12. ΔLf d UBE2E1 ,12–14. Ad UBE2E1 .O-GlcNAcylation is a key link between nutrient sensing and signaling. It notably regulates gene activation due to O-GlcNAc cycling on gene-specific transcription factors and components of the basal transcriptional machinery  among which iewed in ). The taeases it . Deglycoradation .Recently, we discovered that ΔLf is also modified by SUMOylation. The small ubiquitin-related modifier (SUMO) is involved in many aspects of cell function and affects pathways as diverse as DNA repair, cell cycle, transcriptional regulation, RNA processing, and cell signaling ,19. At avia additional interaction with Ubc9 [SUMOs are conjugated to lysine residues in a ΨKXE/D sequence where Ψ is a large hydrophobic amino acid residue and X represents any amino acid . This moith Ubc9 –29. Moreith Ubc9  and, as ith Ubc9 . SUMO-1 ith Ubc9 . Howeverith Ubc9  identifiith Ubc9 ,33. Receith Ubc9 .Crosstalk between the SUMOylation, ubiquitination, and acetylation pathways is crucial for the regulation of protein activity and/or stability since these modifications may have different, sometimes opposing consequences . Thus, SHere we demonstrate that the stability and transcriptional activity of ΔLf are regulated by SUMOylation, which provides a novel regulatory mechanism for controlling ΔLf function. We identified the major SUMO and acetylation acceptor sites and we evaluated the impact of the SUMOylation/ubiquitin and the SUMOylation/acetylation interplays.6 cells) using DreamFect  as described [de novo protein synthesis [HEK-293 cells ATC CRL-1573) were grown in monolayers and transfected  according to the manufacturer’s instructions with p3xFLAG-CMV10-ΔLf as template and primer pairs listed in 6 cells in 100-mm dish) were transfected with RNAiMax (Life Technologies), according to the manufacturer’s instructions, using 5 nM of siRNAs targeting Ubc9  or a scrambled control sequence (siCtrl) (Qiagen). Cells were harvested 48 h post-transfection and lysed. Cell extracts were assayed for Ubc9 content and SUMOylation levels.HEK-293 cells (2 x 10Skp1-Luc reporter vector containing a single ΔLfRE and the ΔLf-expression vector (WT), different ΔLf SUMO mutant constructs or a null vector (NV). HEK-293 cells were synchronized overnight in medium containing 1% FCS before being transfected  using DreamFect  as described in [Reporter gene assays were performed using the pGL3-S1ribed in . Reporteribed in  and exprProteins were extracted from frozen cell pellets in RIPA buffer as described . In ordeAntibodies against the 3xFLAG epitope , HA epitope , 6XHis epitope , SUMO-1 , SUMO-2/3 , Ubc9 , acetyllysine , GAPDH  were used for immunoprecipitation and/or immunoblotting. Secondary antibodies conjugated to horseradish peroxidase were purchased from GE Healthcare Life Sciences. All the antibodies were used according to the manufacturer’s instructions.ImageJ and statistical analyses were performed with PRISM 5 software . M2 densitometric values were normalized to GAPDH and expressed as DM2/DGAPDH. Means were statistically analysed using the t-test or ANOVA and differences assessed at p<0.05 (*) or p<0.01 (**). SUMO-1 and acetyllysine densitometric values were expressed as DAc/DSUMO with the WT ratio in the siCtrl condition arbitrarily set as 100%.The densitometric analysis was performed using the Quantity One v4.1 software  or In silico analysis of the ΔLf sequence with SUMOsp (http://sumosp.biocuckoo.org/) and SUMOplot (htpp://www.abgent.com/tools/sumoplot/) softwares revealed four lysine residues to be putative SUMO acceptors: K13 and K361 that are in the canonical ΨKXE/D motifs and K308 and K391 in non-canonical SUMO sequences . The other putative sites are concentrated in the central part of the protein, before the second DBD for K308, between this DBD and the PEST sequence for K361, and within the PEST sequence for K391  suggested that at least four SUMOylation sites are occupied  for WT.SUMOylation was first investigated on WT and M5S which were co-transfected with or without the SUMO-1 expression vector. An immunoprecipitation was then performed using the anti-FLAG antibody in order to specifically immunoprecipitate ΔLf or its SUMO variants. SUMOylation was then investigated using anti-SUMO-1 antibodies. The feeble amount of SUMO-conjugates  within the cells and also in cell extracts poses a significant problem for the detection of SUMOylated proteins. Therefore, we used N-Ethylmaleimide (NEM), which blocks cysteine proteases, as a SENPs inhibitor. Since NEM is not cell permeable we used it during cell lysis and immunoprecipitation steps. ΔLf-expressing cell lysates were immunoprecipitated with M2 and SUMO forms immunodetected with anti-SUMO-1 antibodies  and GAPDH  contents of HEK-293 cells were analysed following addition of CHX  with cycloheximide (CHX), a potent inhibitor of n of CHX . GAPDH wn of CHX . Densitoed cells  confirmied cells . The samed cells . Taken tSkp1 promoter [To study the physiological consequences of ΔLf SUMOylation, we next assayed the transcriptional activity of the mutants compared to wild type . SUMOylapromoter . In thispromoter ,48. We tSince SUMO modifications on the K308 site led to the highest inhibitory impact on ΔLf transcriptional activity, we next focused our attention on K308 and investigated the impact of altering its SUMO pattern. Therefore we increased SUMOylation by using the His-SUMO-1 expression vector and decreased it by performing either de-SUMOylation using recombinant SENP2 protease or knockdown using specific short interfering RNA against Ubc9 (siUbc9). Prior to performing transcriptional activity assays, we first showed that siUbc9 efficiently invalidated Ubc9 expression  leading Acetylation regulates numerous cellular processes, including the regulation of transcription ,50. AcetWe next assayed the impact of the SUMO/acetylation interplay on ΔLf-mediated transactivation . We incrIn order to confirm these results we further investigated whether an increase in the levels of acetylation may result in a reduction in the levels of ΔLf SUMOylation and conversely . TherefoO-GlcNAcylation, ubiquitination and SUMOylation are fast and efficient ways for the cell to respond to different stimuli. Transcription factors are often regulated by combinations of these different PTMs which might act as a molecular barcode [O-GlcNAcylation, phosphorylation at S10 and ubiquitination at K379 and K391 [Transient PTMs like acetylation, phosphorylation,  barcode . In thisand K391 , can alsin situ. The ΔLf SUMOylation pattern, manifested as multiple bands, is consistent with the presence of multiple SUMOylation sites on the protein. We identified five SUMOylation sites among which the K13 and K361 sites conform to the consensus sequence of ΨKXE/D. Furthermore, SUMOylation was also mapped at K308, K379 and K391, which are non-canonical sites. Among these, K13, K308 and K379 are the three SUMO hotspots.Considering the fact that, for a given protein, only a small fraction is commonly found in the SUMOylated state and that this modification is transient, it was difficult to visualize or isolate endogeneous SUMO forms. Nevertheless we were able to observe SUMO-1 isoforms of ΔLf Skp1 promoter containing one ΔLfRE. ΔLf binds to and transactivates DcpS, Skp1, Bax, SelH, GTF2F2 and UBE2E1 genes [SUMOylation of transcription factors generally antagonizes their activation potential or mediates repression, although in a few cases SUMOylation has been associated with reciprocal effects resulting in activation. Whereas SUMOylation decreases the transactivation potential of c- Jun and the androgen receptor ,53, it iE1 genes ,12–14 thSince ΔLf possesses five SUMO target sites it is difficult to determine whether each of the individual sites has a specific role. Moreover, three of them are targeted by other PTMs, rendering this study even more complex. In order to establish whether any single SUMOylation site was important for the transactivation capacity we compared the activities of mutants disabled specifically at each individual consensus SUMOylation site. The transactivation capacity of each single-site lysine mutant was similar or slightly increased compared to WT (data not shown). Since multiple sites contribute to the control of ΔLf transactivation capacity, the loss of only one SUMO site has only small effects on the activity of ΔLf suggesting either that individual sites act in a redundant manner or that SUMOylation at multiple sites is necessary. Therefore we next studied the transcriptional activity of each mutant in which only one SUMO site was preserved. The K308 mutant strongly inhibited ΔLf transcriptional activity. Moreover when SUMOylation was impaired either after Ubc9 knockdown or in the presence of increased expression of the SENP2 protease, the transcriptional activity of K308 was increased 5-fold compared to that of the K308 mutant expressed in untreated cells. This activity strongly decreased by nearly 2-fold when SUMO-1 peptides were overexpressed. These results demonstrated that SUMOylation at K308 strongly controls ΔLf activity, which may be due to the fact that the region downstream from its SUMO motif is rich in acidic residues as in NDSM. NDSM interacts twice with Ubc9, first between the consensus motif and the active site of the enzyme and also between the acidic tail of the consensus and the basic patch of Ubc9 . Thus, tin silico studies led us to discover a reverse putative consensus SIMr motif in the vicinity of the K361 SUMO site which is conserved among mammalian species Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


To identify disease markers for AICU, we investigated the single nucleotide polymorphism (SNP) of the promoter loci of high-affinity IgE receptor (FcεRIα) and CD203c expression level in Chinese patients with AICU. We studied two genotypic and allelic frequencies of rs2427827 (–344C/T) and rs2251746 (–66T/C) gene polymorphisms of FcεRIα in 20 patients with AICU, 52 subjects with airway hypersensitivity without aspirin intolerance, and 50 controls in a Chinese population. The results showed that the frequencies of two SNPs  were similar to the normal controls. The allele frequency of –344CC was significantly higher in the patients with AICU compared to those with airway sensitivity (p = 0.019). We also studied both histamine release and CD203c expression on KU812 cells to assess aspirin-induced basophil activation. We found that the activity of basophil activation of AICU was significantly higher in the patients with AICU compared to those with airway hypersensitivity without aspirin intolerance. The mean fluorescence intensity of the CD203c expression were 122.5 ± 5.2 vs. 103.3 ± 3.3 respectively, (p < 0.05), and the percentages of histamine release were 31.3% ± 7.4% vs. −24.0% ± 17.5%, (p < 0.05) respectively. Although the mean fluorescence intensity of CD203c expression and the percentage of histamine release were significantly up-regulated by aspirin, they were not affected by anti-IgE antibodies. These results suggest that a single SNP of FcεRIα (–344C>T) is less likely to develop AICU and the basophil activation activity in the sera by measuring CD203c expression can be applicable to confirm the diagnosis of AICU.There are currently no diagnostic methods  Aspirin is one of the most commonly used NSAIDs, and it is therefore important to identify disease markers and prevent the development of aspirin-induced CU (AICU).Chronic urticaria (CU) is a common allergic disease . AlthougThe pathogenesis of AICU is thought to involve two different pathways, including IgE-mediated and non-IgE mediated pathways. Urticaria, angioedema and respiratory distress are the main clinical presentations of immediate-type aspirin-induced hypersensitivity. IgE-mediated activation of basophils and mast cells has been proposed to play an important role in the pathophysiology of immediate-type drug hypersensitivity . Upon biin vitro. Since the symptoms of acute exacerbation are similar to IgE- medicated reaction and aspirin-induced acute angioedema has been case reported due to IgE [The basophil activation test has proven to be a useful tool for the assessment of immediate-type responses to allergens mediated by IgE. In addition, different activation markers (CD203c or CD63) with distinct modes of regulation have been used to evaluate the basophil response to allergens. Although flow cytometric detection of basophil activation by CD63 expression has been studied in diagnosing immediate-type allergies to various allergens such as aeroallergens , foods, hymenoptera venom, natural rubber latex and drugs , CD203c e to IgE . It is fe to IgE ,22. Howein vitro. In addition, we hypothesized that the effect of anti-IgE antibodies on IgE-mediated basophil activation may help to clarify the role of IgE in the pathogenesis of AICU.The aim of this study was to identify specific disease markers to diagnose AICU using cells and serum from patients with AICU and analyzing the promoter genes of FcεRIα and measuring basophil activation activity The general data, serum total IgE and mite-specific IgE of the patients with AICU and control groups using for analysis of CD203c and histamine release are summarized in p = 0.019)  were genotyped in 20 AICU patients and compared to 50 normal healthy controls and 52 subjects with airway hypersensitivity without aspirin intolerance. The frequencies of both polymorphisms were similar to the normal controls, however the allele frequency of –344CC was significantly higher in the patients with AICU compared to those with airway hypersensitivity without aspirin intolerance (= 0.019) . This ren = 17) and control group (n = 11) were compared by CD203c expression and histamine release. KU812 cells were incubated with aspirin and the patient’s sera in the presence or absence of anti-IgE antibody followed by the measurement of CD203c expression and histamine release. Although the MFI of CD203 expression was similar between the two groups, the expression was only significantly up-regulated by aspirin in the subjects with AICU (108.6 ± 2.8 vs. 122.5 ± 5.2) and not in control group (107.0 ± 5.1 vs. 103.3 ± 3.1). The up-regulated CD203c expression by aspirin was not affected by anti-IgE antibody  (p < 0.001) and the sensitivity and specificity were 75% and 80%, respectively. Although the histamine release was down-regulated by anti-IgE, it did not reach a statistically significant level.Differences in basophil activation activity in the sera between the patients with AICU (antibody . These r < 0.05) . For evan = 7) with and without airway allergy were compared. Basophils were incubated with aspirin and the patient’s sera in the presence or absence of anti-IgE antibody, followed by measurement of CD203c expression and histamine release. The MFI of CD203c expression was up-regulated by aspirin in both groups, however, there was no significant up-regulation in those without airway allergy. The up-regulation of MFI in both groups was not affected by anti-IgE antibody . Written informed consent was obtained from each participant before they were enrolled into this study. A total of 20 AICU subjects and 52 subjects with airway hypersensitivity without aspirin intolerance who attended the Allergy Clinic at Taichung Veterans General Hospital were recruited for this study. AICU was defined as a history of chronic urticaria for more than 6 weeks and no other known causes of chronic urticaria. The exacerbation of urticaria was proved by aspirin provocation test with 500 mg aspirin. Airway hypersensitivity was defined as having a history of recurrent nasal stuffiness, nasal itching, sneezing, rhinorrhea, and/or asthma as defined by Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines . Normal Because mite allergens commonly affect allergic patients, total IgE and specific IgE to mite concentrations were measured using a UniCAP system  as a background value.A set of primers was designed for the amplification and sequencing based on GenBank sequences. The forward and reverse primer sequences for the FcɛRIα promoter region were 5'-AAgAAAAgCgTTggTAgCTCTggTg-3' and 5'-ATCTTCTTCATggACTCCTggTgC-3', respectively. Sequence variants were verified using chromatograms on an ABI 3730 DNA analyzer . Two SNPs in the promoter region were identified at –344 (rs2427827) and –66 (rs2251746).The sera from 19 patients with AICU and 12 aspirin-tolerant control subjects were collected for basophil histamine release assay. The patients were instructed to avoid the use of systemic antiallergenic drugs (including antihistamine and systemic corticosteroid drugs) for at least 24 h before blood sampling. We excluded those subjects with pre-medications, there were only 17 out of 19 patient with AICU and 11 out of 12 subjects in control group were performed histamine releasing assay.6 cells/mL using trypan blue. Passive sensitization of the KU812 cells with sera from the patients or controls (1/5 the volume of subjects’ sera) was performed for 4 h at 37 °C. Anti-IgE antibody  was added to detect the role of IgE-mediated basophile activation in the patients with AICU. The anti-IgE antibody selectively binds to free human IgE in the blood and to the membrane-bound form of IgE (mIgE) on the surface of mIgE-expressing B lymphocytes. Before the passive sensitization, the anti-IgE antibody was added and mixed with the sera at a concentration of 100 μg/mL for 30 min. After incubation with the sera, the sensitized cells were then incubated with 100 μg/mL of aspirin  for 30 min at 37 °C, and with 1 μg/mL of A23187  for total release histamine, and then the supernatant was collected and reacted with O-phthalaldehyde  for 7 min. The reaction was stopped by adding H2SO4 (0.04 M). The histamine released into the supernatant was measured by a fluorescence spectrophotometer, and the percentage of histamine release was calculated by the following formula: (stimulated released histamine—Spontaneously released histamine)/ × 100%.KU812 cells  were resuspended in medium RPMI-1640 by adjusting to 1 × 106 cells/mL using trypan blue. Passive sensitization of KU812 cells with sera from the patients or controls (1/10 the volume of subjects’ sera) was performed for 4 h at 37 °C. Anti-IgE antibody  was added to detect the role of IgE-mediated basophile activation in the patients with AICU. Before the passive sensitization, the anti-IgE antibody was added and mixed with the sera at a concentration of 100 μg/mL for 30 min. After incubation with the sera, the sensitized cells were then incubated with 100 μg/mL of aspirin  for 30 min at 37 °C, and with 1 μg/mL of A23187 (Sigma) for maximum CD203c expression. The cells was collected and suspended in PBS, and then reacted with FITC-conjugated anti-CD203c mAb  for 30 min at 4 °C. All of the samples were centrifuged  and analyzed within 2 h on a FACScan flow cytometer . At least 10,000 basophilsper tube were acquired, and the expression of CD203c was analyzed on the gated cell population. The results were given as mean fluorescence intensity (MFI) of basophil expression.The sera from the same donors for the basophil histamine release assay were collected. KU812 cells were resuspended in medium RPMI-1640 by adjusting to 1 × 102 tests. Differences in genotype frequency between the patients and controls were also analyzed using χ2 tests. Differences in histamine release between the patients with AICU and those who were not allergic to aspirin were examined using the Mann–Whitney U test. A p value of 0.05 or less was regarded as indicating significance. Statistical comparisons of the CD203c expression between the patients and controls were performed using paired t-tests. Sensitivity, specificity and area under the curves (AUC) were calculated for KU812 cells using in expression of CD203c and basophil histamine releasing assay.Statistical analysis was performed using SPSS software . The genotype frequencies of each SNP were examined for significant departures from Hardy-Weinberg equilibrium using χin vivo using various forms of oral aspirin have been used to diagnose AICU for many years, it is not practical due to unpredictable severe systemic reactions. This study demonstrates that patients with a single SNP of FcεRIα (–344C>T) are less likely to develop AICU, and that CD203c expression by basophil activation activity in sera can confirm the diagnosis of AICU. These in vitro disease markers may be a useful tool for assessing aspirin hypersensitivity in patients with chronic urticaria.Patients may not be aware of aspirin intolerance and the association between the use of NSAIDs and AICU exacerbations. Although provocation


Protein kinase-mediated phosphorylation is among the most important post-translational modifications. However, few phosphorylation sites have been experimentally identified for most species, making it difficult to determine the degree to which phosphorylation sites are conserved. The goal of this study was to use computational methods to characterize the conservation of human phosphorylation sites in a wide variety of eukaryotes. Using experimentally-determined human sites as input, homologous phosphorylation sites were predicted in all 432 eukaryotes for which complete proteomes were available. For each pair of species, we calculated phosphorylation site conservation as the number of phosphorylation sites found in both species divided by the number found in at least one of the two species. A clustering of the species based on this conservation measure was concordant with phylogenies based on traditional genomic measures. For a subset of the 432 species, phosphorylation site conservation was compared to conservation of both protein kinases and proteins in general. Protein kinases exhibited the highest degree of conservation, while general proteins were less conserved and phosphorylation sites were least conserved. Although preliminary, these data tentatively suggest that variation in phosphorylation sites may play a larger role in explaining phenotypic differences among organisms than differences in the complements of protein kinases or general proteins. Arabidopsis thaliana genome encodes more than 1000 [Protein kinases play a critical role in regulating cellular processes by catalyzing the phosphorylation of amino acid residues , which may activate, deactivate, or change the activity of the target protein , 2. Mosthan 1000 . The comGiven their functional importance, as well as their integral role in determining the phenotype of a given organism, several studies have been devoted to characterizing the evolution and conservation of phosphorylation sites. For example, Rinschen et al. discovered that cross-species comparisons of phosphorylation site data can provide insight into the architecture of specific signaling systems , while KIn another study, Jimenez et al. found that phosphorylation sites are less well-conserved in different organisms than would be expected given their functional significance , althougA. thaliana), our knowledge of phosphorylation sites in other species ranges from sparse  to almost nonexistent  [While the studies cited above have examined the evolution and conservation of phosphorylation sites in a limited number of organisms and/or for a limited number of protein kinases, to our knowledge there has not yet been a study characterizing the general conservation of human phosphorylation sites in a wide variety of species. Whereas protein kinases can be readily identified using sequence and structural homology, allowing the kinomes of different species to be characterized and compared relatively easily , 16, sevoneybee) . Third, oneybee) –20.In this study, a methodology that addresses these challenges is used to characterize the degree of conservation of human phosphorylation sites among a wide variety of species. Phosphorylation site conservation is then compared to the conservation of both protein kinases and proteins in general. Given that fewer genomic changes are required for the gain or loss of a phosphorylation site as compared to the gain or loss of a protein kinase, we hypothesize that phosphorylation sites will be less conserved than protein kinases.n = 146306) were gathered from the online databases PhosphoSitePlus [PF and the subset consisting only of sites with low-throughput references as PL. Complete eukaryotic proteomes (n = 432) were downloaded from UniProt [Experimentally-determined human phosphorylation sites  = 1 if the proteome of species S contains phosphorylation site i; otherwise, fi(S) = 0. Additionally, define the functions Ei and Bi as follows:Ei is equal to 1 if either SA or SB (or both) contains phosphorylation site i. Bi is equal to 1 only if both SA and SB contain phosphorylation site i. Finally, we define C, which represents the number of phosphorylation sites present in both SA and SB divided by the number present in at least one of SA and SB (expressed as a percentage). Stated differently, the values C represent the percentage of phosphorylation sites that are present in at least one of SA and SB that are present in both. Mathematically,p is the number of phosphorylation sites examined. Greater values of C indicate greater conservation, and vice versa.The degree of phosphorylation site conservation between each pair of species fi’s using experimental data. Thus, we calculated the fi’s by employing the online tool DAPPLE [RRKIPQCSQLQEDVD, which spans residues 46–60 in the full protein sequence. A BLAST search against the bovine proteome using this peptide as a query gives RRKIPQGSQLQEDVD as the best match, which is found in the bovine protein with accession number Q5EA18. This peptide comprises residues 48–62 of the full protein, and the phosphorylated residue is S55. Given that there is only one mismatch between the two sequences (C→G in position 7 of the peptides), it is likely that residue S55 in the bovine protein Q5EA18 is a phosphorylation site.As the number of experimentally-determined phosphorylation sites in many species is limited, it is difficult to accurately calculate the l DAPPLE  to predil DAPPLE , 21, 22.fi’s were calculated from the DAPPLE output as follows. Let Qi represent the 15-mer peptide corresponding to known human phosphorylation site i, and Hi, S denote its best match in species S. We set fi(S) = 1 if the number of sequence differences between Qi and Hi, S was less than or equal to a threshold T; otherwise, fi(S) = 0. To select an appropriate value for T, we used previously-published data concerning how the number of sequence differences between a query 15-mer and its best match in the target proteome affects the likelihood that the best match is a known phosphorylation site [fi, an alternative function gi was defined in which gi(S) = 1 if the number of non-conservative sequence differences between Qi and Hi, S is less than or equal to T, and 0 otherwise. A substitution was considered non-conservative if the entry for that substitution in the BLOSUM62 substitution matrix was less than zero. The usage of fi or gi is explained further at the end of this section.The ion site . In addiT, the values C were calculated for each pair of species SA and SB, and the results were compiled into a table (similarity matrix). As the number of species was 432, the number of possible pairs of species was D = 100−C was also calculated for each pair, and the R function hclust was used to perform hierarchical clustering using these distances. Average linkage was used as the linkage method. The function hc2Newick from the ctc R package was used to convert the hierarchical clustering to Newick format. The Newick file was visualized using TreeGraph 2 [After selecting an appropriate value for eGraph 2 .fi(S) or gi(S) was used to determine if species S contains phosphorylation site i, and whether the set of phosphorylation sites used was PF or PL. Specifically, Method 1 used fi and PF, Method 2 used fi and PL, Method 3 used gi and PF, and Method 4 used gi and PL.Four different variations of the above procedure were performed. The differences between each variation relate to whether C derived using Method 1 compared to those derived using Methods 2, 3, and 4, the following procedure was used. The methods were compared both in terms of the degree of difference  and the direction of difference . Let C1, C2, C3, and C4 denote the value of C obtained using Methods 1, 2, 3, and 4, respectively. To determine the degree of difference between Method 1 and Method 2, the value |C1−C2| was computed for all possible pairs , generating a list of 93096 values, with each value representing the degree of difference between the two methods for one pair of species. The mean, median, standard deviation, and range of these differences were then calculated, which together describe the overall degree to which the two methods differ in calculating phosphorylation site conservation. The degree of difference between Method 1 and Method 3 was then determined by calculating the values |C1−C3| and then determining the aforementioned statistical parameters, and similarly for Method 1 versus Method 4. To compute the directionality of the difference between Method 1 and Method 2, the same procedure as above was used except without taking the absolute value −C2 was computed for each  instead of |C1−C2|), and similarly for Method 1 versus Method 3 and Method 1 versus Method 4.To determine how the values of Homo sapiens, Pan troglodytes, Canis lupus familiaris, Mus musculus, and Rattus norvegicus; insects: Drosophila melanogaster, Apis mellifera, and Anopheles gambiae; fish: Danio rerio and Tetraodon nigroviridis; plants: Arabidopsis thaliana, Oryza sativa, and Ricinus communis; birds: Gallus gallus; arachnids: Ixodes scapularis; nematodes: Caenorhabditis elegans; others (single-celled organisms of different lineages): Plasmodium falciparum, Saccharomyces cerevisiae, Chlamydomonas reinhardtii, and Trypanosoma vivax). A list of orthologous groups was obtained from OrthoMCL-DB, where the entry for each group contained a list of the proteins from each species (if any) that were members of that group. Groups for which at least one of the member proteins contained at least one of the terms “tyrosine kinase”, “protein kinase”, or “serine/threonine kinase” were included in the analysis. The degree of protein kinase conservation was assessed using the same general framework as was used for determining phosphorylation site conservation. Specifically, fi(S) = 1 if there existed a protein kinase from species S in orthologous group i, and fi(S) = 0 otherwise. All of the other functions were defined analogously; in particular, C represented the proportion of orthologous groups containing a protein kinase from at least one of SA or SB that contained a protein kinase from both.To determine the degree to which protein kinases are conserved in different species, protein orthology data were used. As we are not aware of an orthologue database that includes all (or even most) of the 432 species whose proteomes were downloaded from UniProt, twenty species were selected that were present in the orthologue database OrthoMCL-DB . These sTo assess the general degree of protein conservation among the 20 species selected, the same procedure as described in the previous section was used, except all orthologous groups from OrthoMCL-DB were used, rather than just those containing proteins annotated as protein kinases.T  was determined between each pair of species SA and SB for which a complete proteome was available (432 species). C represents the percentage of phosphorylation sites present in at least one of SA and SB that were present in both. As described earlier, we performed four different variations of our procedure for determining the C values. These variations were denoted Method 1, Method 2, Method 3, and Method 4. The differences between the four methods relate to the dataset of human phosphorylation sites  and in how phosphorylation site conservation was measured . The reason that Methods 2 and 4 used the subset of human phosphorylation site data verified using low-throughput techniques (rather than the full dataset of human phosphorylation sites) is that it has been suggested that a sizeable portion of sites identified only using discovery-mode mass-spectrometry experiments are non-functional and do not play a role in cellular signaling [The degree of phosphorylation site conservation ignaling , 29, andignaling . Thus, rC when using Method 1 for the 20 species described in the Materials and Methods section; The upper triangle of D for the 20-species subset, and was compared to the taxonomy for those species as given by the National Center for Biotechnology Information (NCBI) [D for all 432 species is given in A dendrogram created using hierarchical clustering was generated using the distances n (NCBI)  Taxonomyn (NCBI)  to compun (NCBI) . The calC values was 21.8 for Ser sites, 31.7 for Thr sites, and 28.7 for Tyr sites  for Ser, Thr, and Tyr sites, respectively. C for each species  for each phosphorylated residue.Interestingly, the degree of phosphorylation site conservation appeared to differ depending on the residue being phosphorylated. Specifically, the average of all the H. sapiens and P. troglodytes were found in both  = 94), whereas C = 21 for H. sapiens and D. melanogaster. The pair of species with the lowest degree of similarity included H. sapiens and the parasite T. vivax, with C = 2.Figs C determined using Method 1 and the values obtained using Method 2, |C1−C2| was calculated for each pair , where C1 represents the value of C calculated using Method 1 and C2 represents the value of C calculated using Method 2. Let E represent the list of differences for all pairs . Some simple descriptive statistics  were then calculated for the values in E. The same procedure was then used to characterize how the values of C differ between Method 1 and Method 3, and between Method 1 and Method 4. The results of this analysis are given in C can range between 0 and 100, this represents a mean difference of approximately 3 percentage points. The mean differences when comparing Method 1 with Method 3 and when comparing Method 1 with Method 4 were slightly higher . The standard deviations of the differences were also quite small, ranging from 1.9 (Method 1 versus Method 2) to 3.0 (Method 1 versus Method 4). This suggests that, while the choice of method for calculating the C values makes some difference, it does not markedly change the measured degree of phosphorylation site conservation among the different species.To determine the degree of difference between the values of C values. This was done using a simple modification of the above procedure: to compare Method 1 with Method 2, C1−C2 was calculated for each instead of |C1−C2|, and similarly for Method 1 versus Method 3 and Method 1 versus Method 4. The results of this analysis are also shown in The four methods were also compared in order to measure the direction of differences between the Interestingly, the mean conservation among species was greater when using Method 1 (which used the full dataset of known human phosphorylation sites) than when using Method 2 . This is inconsistent with some prior findings, which have suggested that phosphorylation sites with unknown functions are less likely to be conserved , 10. An Determining phosphorylation site conservation by counting only non-conservative substitutions rather than all substitutions had a relatively small effect on the measured levels of phosphorylation site conservation  obtained for all 432 species when using Method 2, Method 3, and Method 4, respectively; similarly, H. sapiens and M. musculus, C = 88 for phosphorylation sites and C = 99 for protein kinases. The difference in conservation appeared to heighten at greater evolutionary distances; for instance, for H. sapiens and I. scapularis, C = 20 for phosphorylation sites versus C = 65 for protein kinases. However, there were a few exceptions to this trend, typically when one species in a pair was a plant. For the pair A. thaliana and A. gambiae, for example, C = 27 for both phosphorylation sites and protein kinases.The degree of protein kinase conservation for the 20 species described earlier is shown in the lower triangle of H. sapiens and M. musculus, C = 95 for general proteins, compared to 88 for phosphorylation sites and 99 for protein kinases. This trend appeared to extend to more distantly-related species; for instance, C = 40 for general proteins between H. sapiens and I. scapularis, compared to C = 20 and C = 65 for phosphorylation sites and protein kinases, respectively. As before, pairs that included a plant were often exceptions to this pattern; for instance, C = 26 for A. thaliana and A. gambiae versus 27 for both phosphorylation sites and protein kinases.The degree of conservation of proteins in general among the 20 species is shown in All eukaryotic species—even those that are closely related—exhibit substantial phenotypic differences. While some of these differences can be explained by the presence/absence of, or sequence variation within, specific genes and their corresponding proteins, regulatory mechanisms  also play a significant role. Phosphorylation is the most widespread post-translational modification in eukaryotes, and is integral to the control of almost every cellular signaling process. Thus, phosphorylation patterns dramatically affect the phenotype of a given organism. There are two major mechanisms by which phosphorylation-mediated regulatory pathways may be modified: the gain or loss of phosphorylation sites, and the gain or loss of protein kinases . We hypothesized that phosphorylation sites would be less conserved than protein kinases because less genomic modification is required for their gain or loss.The data presented here support this hypothesis. For a given pair of species, the percentage of phosphorylation sites found in at least one of the two species that were found in both was generally less than the percentage of protein kinases found in at least one of the two species that were found in both  Tables. While there exists a generally accepted taxonomy for eukaryotes, there is no definitive method for ascertaining phylogenetic relationships. Methods using genetic information, while widely used and accepted, can nonetheless exhibit significant biases . Nonethehuman phosphorylation sites, and does not capture the conservation of phosphorylation sites that are not found in human. It has been found that some species contain entire classes of protein kinases that are absent from other species; for example, around a third of the protein kinases in the proteome of P. falciparum belong to a class of protein kinases called FIKK , which appear to be largely absent from other eukaryotic kinomes [Although the results presented here provide considerable insight into the conservation of human phosphorylation sites in different species, certain limitations should be noted. First, the existence or non-existence of a given non-human site was predicted using sequence homology to known human sites. While sequence homology is well-established as a powerful predictor of structure and function, the fact that the sequence surrounding a known phosphorylation site in one organism is conserved in a second organism does not guarantee that the site is actually phosphorylated in the second organism. Phosphorylation site prediction is a difficult problem, and the accuracy of even the best predictors is limited –20. Howe kinomes . The con kinomes  estimateIn summary, we would like to emphasize that, given the predictive nature of this study, additional research—preferably using experimentally-determined phosphorylation sites—is required to more clearly characterize phosphorylation site conservation among species, and to define the relative importance of phosphorylation site evolution and protein kinase evolution in influencing phenotype. As more and more phosphorylation sites are discovered in different organisms using mass spectrometry and other biological techniques, our ability to accurately characterize phosphorylation site conservation will continue to improve.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their  Here we tested whether IBPs are also highly expressed in situ, as can be expected based on their presumed critical role for survival in this habitat; and explored the phylogenetic diversity of IBP transcripts in natural sea ice communities.Sea ice constitutes one of the largest ecosystems in the world, accommodating a thriving community of microorganisms ranging from viruses to small metazoans . Many ofWe first analysed the abundance of eukaryotic IBP transcripts in five microbial sea ice communities sampled from both polar regions. Sanger sequenced metatranscriptomes  were estTranscripts encoding IBPs and, for comparison, reference genes responsible for core cellular functions  were retrieved from the datasets based on sequence similarity . All metSample ANT-B2 differs considerably from the other metatranscriptomes containing an order of magnitude more IBP transcripts and higher transcript ratios of IBPs to photosynthetic reference genes . However, we are not able to explain these differences either by physical properties of the sea ice , filter size  or by sphttp://pfam.xfam.org/) domains. Eighty-nine percent of the IBP transcripts from the metatranscriptomes were placed into two clades, which we refer to as ‘diatom' and ‘microalgae and copepod' clades (de novo phylogeny calculated by using all environmental reads longer than 150 amino acids in addition to the PfamA reference set revealed a diversity of IBP sequences especially in the ‘microalgae and copepod' clade, which were not closely related to the terminal nodes (We next analysed the diversity of the environmental type 1 IBP transcripts by phylogenetic placement upon a backbone tree  construc' clades . Unlike ' clades . This mial nodes . This shal nodes , as wellal nodes . Howeveral nodes . Neverthal nodes , a lowerLaboratory studies of IBP transcript  and prot


Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. Protein Tyrosine Phosphatase 1B (PTP1B) is an intracellular protein which is widely expressed in the body including the brain, liver, muscles, and adipose tissue, and which is up-regulated in obesity, type 2 diabetes and breast cancer12575R(S/T), where the cysteine (C) is a conserved active site that is essential for enzyme catalysisHowever, there are some outstanding challenges in PTP1B-based small-molecule therapeutics. First, it is difficult to achieve inhibition selectivity against PTP1B by targeting the active site. PTP1B is a member of the Protein Tyrosine Phosphatase (PTP) family, which contains more than 100 members. Most PTPs have a consensus active loop signature (H/V)C(X)Due to the challenges of the active site targeted inhibitors mentioned above, an alternative drug design strategy has been proposed to develop inhibitors targeting the PTP1B allosteric site instead1519in vitro2122et al. showed that lupeol can inhibit PTP1B with a high potency (IC50 = 5.6 μM), and acts as a non-competitive inhibitor of PTP1BRecently, the lupane triterpenes, lupeol, lupenone, betulin and betulinic acid , have beThe PTP1B crystal structure (PDB id: 1T49) used in this study was the open, inactive conformation containing 282 amino acids, in which the α7 was not resolved (labelled as PTP1B282). Considering that there was no crystal structure of PTP1B with α7 available in the inactive state, and also the important role of α7 in allosteric inhibition, homology modelling with Modeller 9v8 was carried out to construct the missing helix based on the active conformation of PTP1B (PDB id: 1PTY) (labelled as PTP1B299)Docking simulations were carried out with Autodock Vina (version 1.1.2)+. The salt (NaCl) concentration was set to 0.15 mol/L. Simulations were carried out using NAMD 2.92 over 10 ns. The simulations were continued for 30 ns without any restraints. For the molecular dynamics simulations of the docked complexes, a harmonic restraint on the backbone atoms was applied with a decreasing force constant from 32.0 to 1.0 kcal/mol/Å2 over 3 ns followed by 20 or 100 ns equilibrium simulations  was applied to calculate binding free energy3134Trajectory snapshots were saved every picosecond  and analysed using either the the molecular visualisation program VMD 1.9pNPP Protein Phosphatase Assay kit . The recombinant human TCPTP protein (ab42575) was purchased from Abcam Inc . Lupeol, betulin, and betulinic acid were purchased from Sigma-Aldrich . Lupenone was purchased from Faces Biochemical Co Ltd . Briefly, TCPTP was incubated in different concentrations of compounds for 10 min in assay buffer at 25 °C, and then the reaction was initiated by adding the pNPP reagent and stopped by NaOH after 30 min at 25 °C. All readings were calibrated on the negative control wells without enzymes. The TCPTP inhibition potency of the lupane triterpenes was determined by IC50, calculated using the GraphPad Prism 5 program . In addition, both the inhibition of TCPTP and PTP1B  were performed with the control drug compound 3 , a reported allosteric PTP1B inhibitor. The PTP1B assay process was the same as the TCPTP assay mentioned above.The inhibition of TCPTP was determined by using the EnzoLyte Colorimetric pNPP Protein Phosphatase Assay kit is described above. The reaction mixture consisted of six different concentrations of pNPP  and was used as a PTP1B substrate with different concentrations of lupeol, betulin, and betulinic acid. The Lupeol group was used as a control group. The absorbance at 405 nm was detected by a spectraMAX 384 microplate spectrophotometer twice every minute for a total of 15 min. Michaelis-Menten constant (Km) and maximum velocity (Vmax) of PTP1B were determined via Lineweaver-Burk plots using the GraphPad Prism 5 program .The PTP1B-catalysed hydrolyses in the presence of betulin and betulinic acid were assayed at 25 °C, and the 2. Prior to treatment, the cell culture medium was replaced with DMEM containing 1% penicillin/streptomycin for 4 h. Murine TNFα was obtained from Sigma-Aldrich . Lupeol and betulinic acid were dissolved in Dimethyl sulfoxide (DMSO), then diluted in sterile water, and mixed with serum free cell culture medium. The DMSO concentration was controlled below 0.15%. The final concentrations of lupeol and betulinic acid in the cell culture medium were 28 μM  and 7.5 μM , respectively.Mouse hypothalamic cell line (mHypoE-46) neurons were grown in monolayer in Dulbecco’s modified Eagle medium (DMEM)  with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin. They were maintained at 37 °C with 5% COThe method was described in our previous study with modificationspNPP Protein Phosphatase Assay kit , and resuspended in the same buffer. Samples were analysed for the phosphatase activity using the same pNPP assay kit mentioned above.Based on the instructions of the Pierce Classic IP Kit  with minor modificationspost-hoc Tukey-Kramer honestly significant difference (HSD) test.Data from the western blot analysis and immunoprecipitation assays were analyzed using the SPSS 19 statistical package . One-way analysis of variance (ANOVA) was applied followed by the The homology model of PTP1B in the inactive state with compound 2  binding free energy will be verified with rigorous free energy calculations.We further examined the component contributions to the binding affinities. As expected, for both ligands, the van der Waals interactions made the dominant contributions, because the allosteric site was characterised of the hydrophobic interactions with the hydrophobic skeleton of lupane triterpenes. The difference of van der Waals components between lupeol and betulinic acid to PTP1B299 was −0.6 kcal/mol . The dif1 and R2 , or the 50 of compound 3 on PTP1B was 9.4 μM  and betulinic acid  were administrated to the mHypoE-46 cell line for 10 h after treatment of TNFα for 8 h. The cells were collected for western blot and immunoprecipitation analysis. The Western blot results showed that lupeol and betulinic acid slightly decreased PTP1B protein expression 42, which are in Phase II and Phase I testing, respectively18. Recently, allosteric inhibition has become a promising alternative strategy to develop selective PTP1B inhibitors. The current study firstly predicts that lupane triterpenes bind to the PTP1B allosteric site by the application of molecular docking and molecular dynamics simulations. Enzymatic assays and neuronal cell cultures were used to demonstrate that lupane triterpenes are potential selective and allosteric inhibitors that target PTP1B. PTP1B allosteric inhibitor is featured by displaying a selective inhibitory effect on PTP1B over TCPTP. In an addition to these results, we also performed blind docking of lupane triterpenes targeting TCPTP  and compPrevious structural and biochemical studies have unveiled the important role of α7 as a regulatory helix in the PTP1B conformational transitionin vitro.Lupane triterpenes are of great interest to traditional medicine46474950 data in bo for betulinic acid .