The biguanide metformin is widely prescribed for Type II diabetes and has anti-neoplastic activity in laboratory models. Despite evidence that inhibition of mitochondrial respiratory complex I by metformin is the primary cause of its cell-lineage-specific actions and therapeutic effects, the molecular interaction(s) between metformin and complex I remain uncharacterized. In the present paper, we describe the effects of five pharmacologically relevant biguanides on oxidative phosphorylation in mammalian mitochondria. We report that biguanides inhibit complex I by inhibiting ubiquinone reduction (but not competitively) and, independently, stimulate reactive oxygen species production by the complex I flavin. Biguanides also inhibit mitochondrial ATP synthase, and two of them inhibit only ATP hydrolysis, not synthesis. Thus we identify biguanides as a new class of complex I and ATP synthase inhibitor. By comparing biguanide effects on isolated complex I and cultured cells, we distinguish three anti-diabetic and potentially anti-neoplastic biguanides  from two anti-malarial biguanides (cycloguanil and proguanil): the former are accumulated into mammalian mitochondria and affect oxidative phosphorylation, whereas the latter are excluded so act only on the parasite. Our mechanistic and pharmacokinetic insights are relevant to understanding and developing the role of biguanides in new and existing therapeutic applications, including cancer, diabetes and malaria. Metformin is a widely prescribed anti-diabetic, but its mode of action is disputed. It has been proposed to inhibit respiratory complex I. We show how metformin and related compounds affect both complex I and ATP synthase in mammalian mitochondria. Metformin is the most commonly prescribed drug for Type II diabetes. It decreases blood glucose by mechanisms thought to involve the activation of AMPK (AMP-activated protein kinase)  and/or i+ pool. It uses the two electrons from NADH oxidation to reduce ubiquinone to ubiquinol in the mitochondrial inner membrane, supplying respiratory complexes III and IV with electrons for the reduction of O2 to water. The energy released by the NADH:ubiquinone redox reaction is used to transport protons across the inner membrane, supporting the protonmotive force (Δp) that drives ATP synthesis by F1FO-ATP synthase. Complex I is also an important source of ROS (reactive oxygen species) production in mitochondria, with the rate of production dependent on the redox status of the NADH/NAD+ pool in the matrix  were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO50 concentrations. Per lane, 21 μg was loaded on a 3–12% Novex NativePAGE pre-cast gel (Invitrogen) and run according to the manufacturer's instructions.Octanol/PBS distribution coefficients were measured by the shake-flask method  at 32°C P<0.05, **P<0.01 and ****P<0.0001 as determined using an unpaired two-tailed Students t test. Differences between measurements with P>0.05 were considered not significant. IC50 values were determined using the standard dose–effect relationship {activity (%)=100×IC50/(IC50+[inhibitor]m); m);  with theB. taurus (bovine) heart mitochondria [50 value of 19.4±1.4 mM shows that metformin is only a weak inhibitor of complex I catalysis, in general agreement with previous data from immunoprecipitated complex I (IC50=66 mM) [P. pastoris and the bacterium E. coli was also observed, with IC50 values of 22.6±4.3 mM and 60.7±8.5 mM respectively, indicating that the biguanide-binding site is formed by the ‘core’ subunits that are common to all complexes I, not by mammalian-specific subunits [D, the octanol:PBS distribution coefficient at pH 7.5 [D and log IC50 values (50=25.8±3.9 mM) very similarly to metformin, in contrast with the results of a previous report that guanidinium has no inhibitory effect [We used complex I isolated from chondria  to test 0=66 mM) . Inhibitsubunits . Importat pH 7.5 ) reveale0 values B. Finally effect .KM and kcat values for decylubiquinone are altered by metformin we conclude that biguanides do not bind competitively in the ubiquinone-binding site. Instead, metformin is a reversible non-competitive inhibitor, that binds to complex I whether ubiquinone is bound or not. We conclude that metformin inhibits a rate-determining step that is mechanistically coupled to ubiquinone reduction. Consequently, the biguanides constitute a hitherto unknown class of complex I inhibitor, consistent with them being positively charged and relatively hydrophilic molecules that do not resemble the highly hydrophobic uncharged canonical complex I inhibitors, such as rotenone and piericidin A.To narrow down the location at which biguanides bind to complex I, we tested their effects on different steps in the catalytic cycle: NADH oxidation by the flavin mononucleotide, intramolecular electron transfer along the chain of FeS clusters, and ubiquinone reduction . First, 39, is a coupling element in the amphipathic region between the redox and proton-transfer domains; it reaches up to the hydrophilic domain from the membrane domain, pinning them together [39 can be derivatized [SMPs are robust, inverted and tightly coupled vesicles of the inner membrane of bovine heart mitochondria . NADH oxtogether ,38. In rivatized ; when suivatized .2 reactions proceed via two sequential steps in which NADH reduces the flavin and dissociates, and then the electron acceptor reacts directly  that rely on oxidation of the nucleotide-free reduced flavin [P. pastoris and E. coli  and the dose–response effects of guanidinium and metformin on H2O2 production were very similar . Stimulation of H2O2 production by complex I by guanidinium has also been described previously [2O2 production was not affected by rotenone, a canonical Q-site inhibitor, and the same effects were observed in the isolated hydrophilic domain subcomplex (Iλ) of bovine complex I [Complex I catalyses electron transfer from NADH to hydrophilic electron acceptors by two distinct mechanisms, both localized uniquely at the flavin site. The NADH:FeCN and NADH:Od flavin ,26, but,d flavin . Dose–reeviously ,42. Impoomplex I , that la2O2 production at the flavin site are the nucleotide concentration , normalized to the rate in NADH only. The potential dependence shifts in the presence of biguanide so that H2O2 production is especially increased at higher potentials (when the NAD+ pool is more oxidized). Related behaviour has been observed previously for guanidinium [In vivo, the direct effects of metformin are compounded by increased reduction in the NADH pool due to inhibition of complex I catalysis, providing the possibility of more than 20-fold increases in H2O2 production from the complex I flavin site.Two conditions that control the rate of H2 access ), and th to NADH . Figure  to NADH , see fitnidinium ,44. The 2O2 production. We used the fluorescence of resorufin to follow H2O2 production by isolated rat skeletal muscle mitochondria respiring on glutamate and pyruvate (to produce NADH) in the presence of ATP and rotenone (to build ΔP), and found the rate to be stimulated by 270±34% and 360±35% in 50 and 100 mM metformin respectively. However, these extramitochondrial concentrations are very high and we are unable to control the intramitochondrial concentration or conditions, so the results are not quantitatively meaningful. Previously, incubation of isolated liver mitochondria with metformin revealed a small, but progressive, increase in H2O2 production [2O2 production in cells [2, accelerating H2O2 production by redox cycling [2O2 production, but decreases the paraquat-stimulated rate.Currently, there are few direct data by which to assess the effects of metformin on mitochondrial or cellular Hoduction , and bigoduction ; it has oduction ,47. In cin cells ; paraqua50 values for isolated complex I, on the succinate:O2 activity of complexes II + III + IV in SMPs and found that only cycloguanil had a significant effect . This complicated picture may be rationalized by either a single binding site in the interfacial region that is altered upon extraction of the enzyme from the membrane and only partially retained in the isolated F1 domain, or by the presence of more than one binding site, as in complex I.The effects of biguanides on ATP hydrolysis by Fn of ADP ,29. Figu50 for ATP hydrolysis . The cellular IC50 values are 237±13 μM and 325±25 μM for metformin, for 143B and Hep G2 cells respectively, and 3.81±1.12 μM and 3.80±0.38 μM for phenformin. By comparison with the isolated complex I IC50 values, these values equate to 60 to 80-fold accumulation of metformin, and 120-fold accumulation of phenformin. Our IC50 values are comparable with those reported for primary hepatocytes after 24 h of incubation, and considerably lower than previous values reported for Hep G2 cells [Biguanides are positively charged so they are expected to accumulate in the mitochondria of cells in response to the potential difference across the inner membrane . Each 60 mV of potential difference drives the concentration of the positive ion to 10-times higher inside the mitochondrion than outside. For a typical potential difference of 120–150 mV the concentration inside the mitochondrion could therefore be 100 to 300-times higher than outside. Plasmodium [We have found that biguanides interact with two separate sites on complex I; one site perturbs the reactivity of the flavin, the other inhibits catalysis. We have also found that biguanides inhibit ATP hydrolysis by ATP synthase, and that some biguanides inhibit only hydrolysis, not synthesis. The other three mammalian oxidative phosphorylation complexes are not significantly affected by biguanides except for a comparatively weak inhibition of complex III by cycloguanil. Dipeptidylpeptidase 4 , AMP deaasmodium , and metasmodium ,52. Receasmodium . ImportaClearly, biguanides interact with many different enzymes and some insight into this non-specificity is provided by the structural relationship between biguanides and guanidinium. At high concentrations guanidinium is a protein denaturant, but at low concentrations its trigonal-planar structure promotes a diverse set of protein interactions: it forms ion pairs with negatively charged residues and cation-π interactions with aromatic residues, is a hydrogen-bond donor, can mimic and displace the arginine side chain, and also disrupt hydrophobic interfaces ,54. In t2O2 production, as an alternative route to the activation of AMPK [Piericidin is a canonical inhibitor of complex I; it competes for the ubiquinone-binding site  and is a of AMPK ,47.1 [The site(s) at which biguanides bind to ATP synthase, and their inhibitory mechanism(s), are currently unknown, but the two antimalarial biguanides, cycloguanil and proguanil, stand out from the multitude of known ATP synthase inhibitors by their unidirectional behaviour, being inhibitors of hydrolysis but not synthesis. Except for the mitochondrial inhibitor protein of ATP hydrolysis, IF1 , we were1 , with th50 values initially presented a conundrum. Metformin is found in sera at ~2000-times lower concentration than its complex I IC50 value (~IC50/2000), whereas phenformin (the biguanide with the highest risk of lactic acidosis) is in sera at ~IC50/1000, and proguanil and cycloguanil (with no reported lactic acidosis risk) are present at ~IC50/10 and ~IC50/5000 respectively [Comparison of typical blood serum levels for the biguanides with their complex I ICectively . We haveectively ,59. The ectively , consistectively , suggestectively , is respectively .In vivo, combined plasma and mitochondrial membrane potentials of 180 mV will  accumulate metformin and phenformin by 1000-fold in the mitochondrial matrix, relative to in serum. This represents an increase from the serum concentrations of ~10 μM and ~200 nM respectively [in vivo, inhibition of oxidative phosphorylation by biguanides is heterogeneous across different tissues, varying not only with tissue drug concentrations, but also with factors that influence transport across cytoplasmic and mitochondrial membranes.Although it is not clear how much complex I inhibition is required to lower gluconeogenesis clinically, it is clear that serum levels of metformin and phenformin are much too low, themselves, to have any effect. Thus it is important to realize that accumulation of positively charged molecules in the mitochondrial matrix, in response to the protonmotive force across the inner mitochondrial membrane, is a well-described physical phenomenon that raises the intramitochondrial concentration far above the external concentration; this effect has been discussed previously for biguanides ,8. In thectively  to mitocectively . Our resPlasmodium dihydrofolate reductase [Plasmodium respiratory complex III [Plasmodium (spp.) rely on their respiratory chain (which does not contain complex I) to support pyrimidine biosynthesis, and to support the essential mitochondrial membrane potential; proguanil inhibits an alternative (respiratory-chain independent) pathway for generating the membrane potential that has been proposed to involve ATP hydrolysis by the Plasmodium ATPase [Plasmodium (spp.) appear to lack some ATPase subunits, and we have not tested proguanil on Plasmodium ATPase itself, our results suggest that proguanil may inhibit ATP hydrolysis in Plasmodium directly, thereby, in combination with atovaquone, knock out the essential Plasmodium membrane potential. Notably, mammalian cells that lack mitochondrial DNA sustain a membrane potential through electrogenic exchange of ATP and ADP, coupled to ATP hydrolysis by an incomplete version of ATP synthase [As anti-malarials, cycloguanil inhibits eductase , and proplex III . Plasmodm ATPase . Althougsynthase , demonst1) ATP hydrolysis consumes ATP and contributes to maintaining the mitochondrial membrane potential [S-nitrosothiol), has been reported to protect ischaemic heart tissue from reperfusion-induced damage [In ischaemic tissue lack of oxygen stops respiratory chain turnover and oxidative phosphorylation, and causes the mitochondrial NADH and succinate pools to become reduced. Under these conditions, the resting complex I converts into its deactive state , and (unotential . Ischaemotential , perhapsd damage . Furtherd damage , and metd damage . With and damage , and alsd damage .Pharmaco-epidemiological studies have suggested that metformin use is associated with decreased cancer risk and/or improved cancer prognosis ,5. Some


L. plantarum using reconstituted media supplemented with different levels of inulin and to select the best combination based on the modified inhibitory activity (MAU/mL) against pathogens.The present study aimed to determine the inhibitory activity of postbiotic produced by L. plantarum  using reconstituted media supplemented with different levels of Inulin  yielding 36 combinations.Postbiotics were produced by 6 strains of Pediococcus acidilactici, and E. coli. The RI11 + 0.8% Inulin, RG14 + 0.8% Inulin and RG14 + 0% Inulin had significantly (p < 0.05) higher MAU/mL against P. acidilactici than other treatments. The RI11 + 0.8% Inulin and RG14 + 0.4% Inulin had a significantly (p < 0.05) higher MAU/mL against VRE. The MAU/mL against L. monocytogenes was greater in RI11 + 1.0% Inulin, RI11 + 0.6% Inulin and RI11 + 0.8% Inulin. The combinations of RS5 + 1.0% Inulin, RS5 + 0.8% Inulin and RS5 + 0.6% Inulin had greater MAU/mL against S. enterica; whereas in E. coli, the inhibitory activity had higher activity that can only be found in RS5 + 0.8% Inulin.The combination of postbiotic and inulin had higher inhibitory activity than postbiotic alone against all indicator organisms except Combination of postbiotics and inulin which had higher optical density tends to have lower pH which corresponds to increased inhibitory activity against indicator organisms. The results of this study show that postbiotics and inulin supplementation enable to inhibit proliferation of pathogenic bacteria. The act of feeding antibiotics to livestock has been practiced for over fifty years. The modL. monocytogenes, Campylobacter, Salmonella, and pathogenic E. coli. One of the most popular disease caused by food-borne bacteria worldwide is Salmonella, which is an important pathogen found in food produced by animals. This type of pathogen usually becomes widespread by trade in non-heated food products made from animal meat. The microbial strains which show resistance to antimicrobials, usually, as a result of antimicrobial procedure in animals, cause hazardous problems for public health[Moreover, bacteria cause such problems as food poisoning and diarrhea. The bacteria considered as the main cause for food poisoning are c health.Listeria monocytogenes, Clostridium perfringens, Salmonella enterica, and Escherichia coli[Because of these consequences, there is increasing public awareness and pressure to search for alternatives to antibiotics,7. Prebichia coli-15.L. plantarum using reconstituted media supplemented with different levels of inulin (a prebiotic) and to select the best combination based on the modified inhibitory activity against pathogens and an indicator bacterium.Various studies have been conducted to test the individual efficacy of postbiotics and prebiotics separately. However, no study has been conducted using the combination of prebiotics and postbiotics. Since most postbiotics exhibit probiotic effect, there could be a synergy between a prebiotic and a postbiotic. Thus, the present study was conducted to determine the inhibitory activity of postbiotic produced by 6 strains of Lactobacillus plantarum used in this study were previously isolated from Malaysian fermented food[RG11, RG14, RI11, UL4, TL1, and RS5 as ted food,17 and kPediococcus acidilactici 4–46 was chosen as the indicator due to the fact that it is a common food spoilage bacterium in food products for both humans and animals[In this study,  animals. The preListeria monocytogenes L-MS, Salmonella enterica S-1000, Escherichia coli E-30 and Vancomysin Resistant Enterococci (VRE) are same as the postbiotic producer, except that nutrient media was used for the cultivation of VRE and S. enterica, incubated at 37°C and 30°C, respectively. E. coli was cultivated in LB broth at 37°C while L. monocyotgenes was cultivated at 30°C in Listeria Enrichment media. All the cultivation was performed under the agitation speed of 150 rpm.The reviving steps of L. plantarum RG11, RG14, RI11, UL4, TL1 and RS5 were prepared for the production of postibiotic according to their composition. They were also mixed with different levels of inulin , (w/v) before autoclaved at 118°C for 15 min.In this study, the reconstituted media of 1% (v/v) of inoculum was inoculated into the respective reconstituted media supplemented with different levels of inulin, and incubated at static condition at 30°C. The postbiotic was collected after separating the bacterial cell by centrifugation at 10,000 × g for 15 min and used for analysis.P. acidilactici and pathogenic microorganisms; L. monocytogenes, S. enterica, VRE and E. coli using the Agar Well diffusion method[0 to 25 was conducted using 0.85% (w/v) NaCl solution. Each diluted postbiotic was inoculated at 20 μL into the corresponding well on pre-punched MRS agar plate for P. acidilactici and 100 μL into the pre-punched nutrient agar plate for L. monocytogenes, S. enterica and LB agar for E. coli while 60 μL inoculated into corresponding well on nutrient agar plate for VRE. The diameter of each well was 5.5 mm. The postbiotics were allowed to diffuse completely for 1 hr at room temperature before overlaid with 3 mL of corresponding soft agar inoculated with 1% (v/v) of P. acidilactici, L. monocytogenes, S. enterica, VRE, and E. coli, respectively. After incubation at 30°C for 24 hrs, the highest dilution factor with the clear zone’s diameter size larger than 0.1 cm of the initial diameter size was recorded. The diameter of the clear zone (mm) was measured and the modified bacteriocin activity was calculated based on the formula as shown below:The inhibitory activity of the produced postbiotics were tested against indicator microorganism, n method. A two-fOptical density measured the turbidity of a suspension which reflects cell mass or number of a bacterial culture. 1 mL of culture from each treatment group was centrifuged at 10,000 × g for 15 min. The cell pellet was washed once with 0.85% (w/v) and the optical density was determined at 600 nm using spectrophotometer . The pH of postbiotics was determined using pH meter .The factorial ANOVA was used for data analysis in this study. Data obtained for the modified bacteriocin activity (MAU/mL), inhibitory zone, pH, and optical density were subjected to generalized linear model of SAS. Duncan multiple range test was used to compare the significant difference of means.P. acidilactici than other treatments. Treatments P3.I5 (RI11 + 0.8% Inulin), P2.I3 (RG14 + 0.4% Inulin), and P2.I5 (RG14 + 0.8% Inulin) had a significantly (p < 0.05) higher MAU/mL against VRE. The MAU/mL against L. monocytogenes were greater in P3.I6 (RI11 + 1.0% Inulin), P3.I4 (RI11 + 0.6% Inulin), and P3.I5 (RI11 + 0.8% Inulin). The P6.I6 (RS5 + 1.0% Inulin), P6.I5 (RS5 + 0.8% Inulin), and P6.I4 (RS5 + 0.6% Inulin) had greater MAU/mL against S. enterica. For the E. coli, inhibitory activity was detected within only RS5, where the treatment P6.I5 (RS5 + 0.8% Inulin), P6.I1 (RS5 + 0% Inulin), and P6.I6 (RS5 + 1.0% Inulin) had higher MAU/mL activity.The modified inhibitory activity against indicator and pathogenic organisms of all the 36 combinations of postbiotics and inulin are presented in Table L. plantarum used in this study exhibited broad antimicrobial activity and had the capacity to inhibit both gram positive and gram negative pathogens. This observation corroborates the findings of Sifour et al.[L. plantarum F12 isolated from olive oil had broad inhibitory spectrum against L. monocytogenese. Similarly, Liasi et al.[L. plantarum inhibited the growth of a range of gram-positive and gram-negative microorganisms such as L. monocytogenes, E. coli, Staphylococcus aureus and Salmonella enterica. The inhibitory effect, exhibited by the postbiotics and inulin combinations which were observed by the formation of clear and distinct zones around the wells, may be due to the presence of several antimicrobial compounds such as bacteriocins or organic acids[L. plantarum is a natural antimicrobial compound capable of inhibiting the growth of pathogens at molecular and cellular levels[The postbiotics produced by the 6 strains of ur et al., who repsi et al. observedic acids. Bacteriic acids,22. Bactr levels. The pror levels.L. plantarum strains, acetic and lactic acids are produced to promote the growth of producer cells[et al.[et al.[et al.[Organic acids act as an acidifying agent, reducing the pH of surrounding and survivability of non-acid-tolerant pathogens. During the production of postbiotic by er cells,16. Higher cells,26. In aer cells. Similars[et al. and Rehml.[et al. reportedl.[et al.. Accordil.[et al., fermentl.[et al..P. acidilactici and VRE is shown in Figure P. acidilactici was 9.83 mm in RG14 (0), RG14 (0.8), RG14 (1.0), and RI1 (0.8), whereas the highest inhibitory zone against VRE was 12.16 mm in RG14 (0.4) and RI11 (0.8).The inhibitory zone of postbiotic combinations against L. monocytogenes, S. enterica, and E. coli is shown in Figure L. monocytogenes was 8.66 mm in RG11 (0), RG11 (0.2), RG11 (0.8), and RG11 (1.0), whereas the highest inhibitory zone against S. enterica was 22.66 mm in RS5 (1.0). On the other hand, in E. coli, the inhibitory activity was detected just in RS5 in which the inhibitory zone of the combination RS5 (0.8) was 7.66 mm.The inhibitory zone of postbiotic combinations against 600) and pH of various combinations of L. plantarum and inulin are shown in Table 600 between different combinations of postbiotics and inulin. The mean optical density ranges from 1.92 to 2.28. The highest optical density observed in P6.I5 (RS5 + 0.8% Inulin). In contrast, the lowest OD was observed in P5.I6 (TL1 + 1.0% Inulin). As reported by Thu et al.[L. plantarum. Choe et al.[L. plantarum tend to grow and produce various levels of metabolite which may affect the value of the OD in similar condition. However, it was observed that combinations having higher OD tend to have lower pH. It was also observed that the combinations with low pH have high inhibitory activities against different indicator organisms. This observation was in line with the report of Fooks and Gibson[The optical density , P3.I6 (RI11 + 1.0% Inulin), and P2.I5 (RG14 + 0.8% Inulin) showed a higher level of modified bacteriocin activity. The results of this study show that postbiotics and inulin supplementation enable to inhibit proliferation of pathogenic bacteria.It was evident in this study that postbiotic produced by The authors declare that they have no competing interests.FHL and LTC provided probiotic strains and method to produce postbiotic. KYK and MFO performed inhibitory tests. KYK, LTC, FHL, MFO and SAA contributed to the writing of the manuscript. All authors read and approved the final manuscript.


Structures of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA show how the active site accommodates different acyl modifications and explain why butyryl-CoA acts as a competitive inhibitor. in vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group.Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications  To understand how Gcn5 discriminates between the different acyl-chain donors, we determined structures of the catalytic domain of human Gcn5L2 in complex with propionyl-CoA and butyryl-CoA to 2.0 and 2.1 Å resolution, respectively. These structures reveal that the active site of Gcn5 can accommodate longer acyl chains without major structural rearrangements, but that the butyryl chain would sterically clash with an incoming lysine residue. Consistent with this active-site architecture, we show that butyryl-CoA acts as a competitive inhibitor 2.2.1.et al., 2007M HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, concentrated to 9 mg ml−1, flash-frozen in liquid nitrogen and stored at −80°C.A plasmid encoding the His-tagged catalytic domain of human Gcn5L2 (hsGcn5L2) under T7 induction was obtained from Addgene (Plasmid No. 25482). The protein was expressed and purified as described previously , 100 mM HEPES pH 7.6, 50 mM NaCl and 500 µM acetyl-CoA , propionyl-CoA  or butyryl-CoA . Each reaction was incubated for 5 min at 37°C before adding acyl-CoA, and then maintained at 37°C for the remainder of the experiment. Initially, six data points were collected to find a time frame over which acyl-CoA consumption was linear with time. The reaction was quenched at the indicated time points by the addition of two volumes of quenching buffer . After all the samples had been collected, one volume of 4 mM DTNB  dissolved in 100 mM sodium phosphate pH 6.8 was added. Samples were moved to a 384-well polystyrene clear-bottom plate (Grenier Bio-One) and the absorbance at 412 nM was measured in a POLARstar Omega plate reader (BMG Labtech). Absorbances were converted to concentrations using a standard curve generated by reacting increasing concentrations of CoA  with DTNB using an extinction coefficient for 3-thio-6-nitrobenzoate (TNB) of ∊412 nm = 13 700 M−1 cm−1. Subsequent reactions were performed in triplicate and quenched after 0.5 min (acetyl-CoA), 5 min (propionyl-CoA) or 20 min (butyryl-CoA). All acylation rates were corrected by subtracting the rate of acyl-CoA consumption by Gcn5L2 in the absence of peptide.Kinetic measurements comparing rates of acetylation, propionylation and butyrylation were performed using the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) assay  and were initiated with the acyl-CoA. The absorbance at 340 nm was monitored continuously using a POLARstar Omega plate reader (BMG Labtech) for 5–20 min and converted into the molar concentration of NADH using Beer’s law, assuming ∊340 nm = 6220 M−1 cm−1. As controls, rate measurements were performed at each concentration of acyl-CoA in the absence of peptide. Each measurement was performed in triplicate, and reaction velocities in the presence of peptide were blanked by the rate of reaction in the absence of peptide. Blanked rates were normalized to enzyme concentration, plotted as a function of substrate concentration and fitted to the Michaelis–Menten equation using nonlinear least-squares regression in GraphPad Prism 5. Butyryl-CoA inhibition measurements were also performed with the enzyme-coupled assay. Reaction velocities were measured in the presence of 0.5–10 µM acetyl-CoA and 50 nM hsGcn5L2 with increasing concentrations of butyryl-CoA . Under these conditions, consumption of butyryl-CoA by hsGcn5L2 is undetectable by the same assay. Blanked rates were normalized to enzyme concentration and the resulting curves were globally fitted to a competitive-inhibition model in GraphPad Prism 5.Steady-state kinetic titrations varying the acetyl-CoA or butyryl-CoA concentration were performed with a continuous spectrophotometric assay as described previously  was mixed with each acyl-CoA to a final concentration of 1.6 mM acyl-CoA and 7.9 mg ml−1 protein. NaCl was added to a final concentration of 125 mM from a 5 M stock, and the resulting mixture was incubated on ice for 30 min. Both complexes were crystallized using hanging-drop vapor diffusion by mixing 1 µl protein–acyl-CoA complex solution with 1 µl well solution. Human Gcn5L2 bound to propionyl-CoA was crystallized in 20%(v/v) ethanol, 100 mM Tris pH 9.0. Human Gcn5L2 bound to butyryl-CoA was crystallized in 10%(v/v) 2-propanol, 3% glycerol, 100 mM HEPES pH 7.8, 11%(w/v) PEG 4000. Crystals were cryoprotected by soaking in well solution supplemented with 9% sucrose, 4% glucose, 8% ethylene glycol and 8% glycerol. Prior to data collection, crystals were flash-cooled in a liquid-nitrogen stream.Propionyl-CoA and butyryl-CoA were diluted in 20 m2.4.HKL-2000 .Diffraction data were collected using a Rigaku FR-E SuperBright X-ray generator at a wavelength of 1.54 Å and were recorded with a Saturn 944+ CCD detector. The data were processed with 2.5.5h84 (propionyl-CoA complex) and 5h86 (butyryl-CoA complex).Structures and amplitudes have been deposited in the Protein Data Bank with accession codes 3.3.1.et al., 2008Km of P/CAF for propionyl-CoA is only fourfold weaker, corresponding to a sixfold decrease in catalytic efficiency  or the extra ethyl chain for butyryl-CoA . Compared with the structure of human Gcn5L2 bound to acetyl-CoA . The conservation of the active-site geometry rules out the possibility that butyryl-CoA binding slows down Gcn5 catalytic activity by misaligning the active-site residues. The pantetheine arm and adenine moieties of coenzyme A superimpose well between all three acyl-CoA molecules; what differs is the respective position of the acetyl, propionyl and butyryl chains . Although the positions of the C2 atoms in all three acyl-CoA molecules are the same , the torsion angle formed between the S—C1 and C2—C3 bonds in propionyl-CoA is 24.5° , compared with −61° for butyryl-CoA . Whereas the C3 atom in propionyl-CoA fits within the active-site cleft of human Gcn5 , butyryl-CoA binds in an orientation that places the terminal methyl group (C4) facing the solvent, since the catalytic water molecule blocks it from occupying the Gcn5 active-site cleft . Although longer acyl chains could also bind in this orientation, we note that molecules such as crotonyl-CoA, which contain unsaturated carbon–carbon bonds that do not freely rotate, cannot adopt conformations that are compatible with this geometry.The active site of Gcn5 contains three features that facilitate transfer of the acyl chain to lysine: an active-site glutamate that functions as a general base  were generated based on the structure of Tetrahymena Gcn5 (ttGcn5) bound to a bisubstrate analog consisting of CoA covalently linked to a histone peptide , which is a reasonable angle of attack for a carbonyl group by a nucleophile . By contrast, the terminal methyl of butyryl-CoA projects into the channel occupied by the lysine . The Gcn5L2 active site cannot accommodate butyryl-CoA without ejecting the catalytic water molecule, so the butyryl chain sterically clashes with the incoming lysine. This explains why Gcn5L2 is a poor butyryltransferase, as the acyl chain, catalytic water molecule  and incoming lysine  cannot all fit into its active site.Since Gcn5 uses a direct-transfer mechanism to catalyze lysine acetylation , we wondered whether it might act as a competitive inhibitor versus acetyl-CoA. To test this idea, we measured acetylation rates as a function of acetyl-CoA concentration in the presence of increasing concentrations of butyryl-CoA. As shown in Fig. 5a), Gcn5 is robustly acetylates histone peptides under conditions where butyrylation is nearly undetectable. Fitting our initial velocity measurements to the Michaelis–Menten equation, we determine a Km for acetyl-CoA of 0.91 ± 0.09 µM , which is comparable to previously reported Km values for yeast Gcn5, human Gcn5 and human P/CAF  of 5.6 ± 0.7 µM from our global fit to a competitive-inhibition model . Taken together with our structural findings , these data indicate that butyryl-CoA competitively inhibits acetylation by Gcn5 by binding to the free form of the enzyme and preventing acyl-chain transfer.Our results suggest that a naturally occurring acyl-CoA molecule, such as butyryl-CoA, could inhibit Gcn5 activity by binding to the enzyme in a way that prevents lysine from entering its active site. Since butyryl-CoA is a poor substrate for Gcn5 Fig. 1 but is sµM Fig. 5a, whichel Fig. 5b. Taken4.d), which prevents the peptide substrate from accessing the active-site cleft of Gcn5. We further show that butyryl-CoA is a competitive inhibitor versus acetyl-CoA for human Gcn5 , raising the question as to whether fluctuating levels of acyl-CoA molecules in cells may regulate the activity of Gcn5.We have determined crystal structures that describe how Gcn5 accommodates propionyl-CoA in its active site and provide a structural mechanism that explains our biochemical data showing that human Gcn5 discriminates between different acyl-CoA molecules. Since unsaturated acyl chains greater than three C atoms in length (propyl groups) cannot fit into the active site of Gcn5, butyryl-CoA binds in a conformation that is incompatible with catalysis. The butyryl-CoA C3 and C4 atoms occupy the channel for the incoming lysine Fig. 3d, whichn5 Fig. 5c, raisiin vivo , acetyl-CoA availability may regulate the activity of human Gcn5  of 6.7 and 0.44 µM, respectively. Combined with the observation that free CoA is present at roughly equimolar concentrations to acetyl-CoA in cells (Gao et al., 2007et al., 2014et al., 2011et al., 2015et al., 2012et al., 2002in vivo and provide a clue as to how other CoA-based scaffolds may be exploited to design future generations of acetyltransferase inhibitors.Other CoA-based molecules have been implicated as acetyltransferase inhibitors human Gcn5L2, bound to propionyl-CoA, 5h84PDB reference: bound to butyryl-CoA, 5h86PDB reference:


Forced expression of β-TrCP increased the polyubiquitination and decreased the stability of CREB-H-ΔTC, whereas knockdown of β-TrCP had the opposite effect. An evolutionarily conserved sequence, SDSGIS, was identified in CREB-H-ΔTC, which functioned as the β-TrCP-binding motif. CREB-H-ΔTC lacking this motif was stabilized and resistant to β-TrCP-induced polyubiquitination. This motif was a phosphodegron and its phosphorylation was required for β-TrCP recognition. Furthermore, two inhibitory phosphorylation sites close to the phosphodegron were identified. Taken together, our work revealed a new intracellular signaling pathway that controls ubiquitination and degradation of the active form of CREB-H transcription factor.CREB-H is an endoplasmic reticulum-resident bZIP transcription factor which critically regulates lipid homeostasis and gluconeogenesis in the liver. CREB-H is proteolytically activated by regulated intramembrane proteolysis to generate a C-terminally truncated form known as CREB-H-ΔTC, which translocates to the nucleus to activate target gene expression. CREB-H-ΔTC is a fast turnover protein but the mechanism governing its destruction was not well understood. In this study, we report on β-TrCP-dependent ubiquitination and proteasomal degradation of CREB-H-ΔTC. The degradation of CREB-H-ΔTC was mediated by lysine 48-linked polyubiquitination and could be inhibited by proteasome inhibitor. CREB-H-ΔTC physically interacted with β-TrCP, a substrate recognition subunit of the SCF CREB-H, also known as cAMP responsive element-binding protein 3-like 3 (CREB3L3), is an endoplasmic reticulum (ER) membrane-bound transcription factor which is mainly expressed in the liver and small intestine1467CREB-H is a multifunctional protein which regulates lipid metabolism, iron homeostasis, gluconeogenesis, cell secretion, cell growth and acute phase response45712131512171216104511The ubiquitin-proteasome system is one major pathway for intracellular protein degradation2427293032β-TrCP. We also identified two degron-proximal inhibitory phosphorylation sites in CREB-H-ΔTC. When our study was completed, an independent group also reported SCFβ-TrCP-mediated degradation of CREB-H-ΔTCDegradation of membrane-bound CREB-H is thought to be mediated through the ER-associated protein degradation (ERAD) pathwayCREB-H is known to be stabilized by proteasome inhibition and degraded through the ERAD pathway45To verify the polyubiquitination status of CREB-H-ΔTC and the type of ubiquitin linkage, we compared the ubiquitination patterns of CREB-H-ΔTC immunoprecipitated from HEK293T cells overexpressing wild-type and mutant ubiquitin. The ubiquitination smear of CREB-H-ΔTC was evident when wild-type ubiquitin but not its lysine-free mutant K0 was overexpressed . These dBecause CREB3L1 and CREB3L2 are degraded by SCF29β-TrCP is a bona fide E3 ubiquitin ligase responsible for CREB-H-ΔTC degradation, several lines of experiments were performed. First, we explored the interaction between CREB-H-ΔTC and SCFβ-TrCP by reciprocal co-immunoprecipitation. Detection of β-TrCP in the CREB-H-ΔTC immunoprecipitate and vice versa indicated the interaction between CREB-H-ΔTC and β-TrCP , we introduced them in the background of CREB-H-ΔTC-A4 . Fast-miAbove we demonstrated a stabilizing effect of the A4 mutation on CREB-H-ΔTC , lane 6.41218To further verify the role of β-TrCP in the regulation of PEPCK gene expression, the two β-TrCP-targeting siRNAs were used to deplete endogenous β-TrCP expression in HepG2 cells. The knockdown effect was highly specific to β-TrCP and the siRNAs had no influence on the expression of CREB-H mRNA . By contβ-TrCP ubiquitin ligase mediates the ubiquitination and degradation of CREB-H-ΔTC, the active form of liver-enriched transcription factor CREB-H. An evolutionarily conserved β-TrCP phosphodegron with the SDSGIS motif was identified and characterized. In addition, the degron-proximal S73 and S77 were identified as the inhibitory phosphorylation sites for β-TrCP-dependent ubiquitination and degradation. Based on our findings, we propose a model for regulation of CREB-H-ΔTC activity GxxS(+4)xxxS(+8)xxS(+11) motif, in which the β-TrCP degron is bolded. In this case, priming phosphorylation at the serine residue at +11 position is required for subsequent phosphorylation at serine residues at +8, +4 and 0 positions39(−3)TDS(0)GϕS(+3) sequence. Here S(−3) served as a priming phosphorylation site for S(0) and S(+3)(−14)xxxS(−10)xxxS(−6)xxxS(−2)DS(0)GϕS(+3) sequence, S(−14)xxxS(−10)xxxS(−6) plausibly functions as the priming phosphorylation signal for subsequent S(−2)DS(0)GϕS(+3) phosphorylation. Another possibility is that CREB-H-ΔTC, like CREB, contains a kinase-inducible domain(−14)xxxS(−10)xxxS(−6)xxxS(−2)DS(0)GϕS(+3)xxxxS(+8)xxxxT(+13) region to regulate CREB-H-ΔTC activity and stability.The relationship between the β-TrCP phosphodegron and its proximal region warrants further investigations. One possibility is that phosphorylation of the degron-proximal region “primes” or sensitizes degron phosphorylation. Such “priming phosphorylation” has been found in many other β-TrCP substrates3940(0)GϕS(+3), is very similar to but does not exactly match the canonical β-TrCP-binding motif DS(0)GϕxS(+4). However, several substrates without the x residue, such as RV-NSP1, YAP and Nrf2, have been identified, indicating that the x residue is not essential4041(0)GϕS(+3) always prefers phosphorylatable residues . Experimentally, we also demonstrated the requirement of S85 for the binding of CREB-H-ΔTC to β-TrCP. Thus, we propose the phosphorylatable residue at −2 position should be important for the binding of the SDSGϕS motif to β-TrCP. In other words, phosphorylation at −2 position might be “priming” degron phosphorylation at other sites. Alternatively, phosphorylation at −2 position could provide a negative charge to increase the binding of the SDSGϕS motif to β-TrCP. Previous studies have shown that negatively charged residue is most critical to β-TrCP bindingThe phosphodegron of CREB-H-ΔTC, DSWe have found that CREB3L4 contains the same β-TrCP recognizing sequence (SDSGIS) as CREB-H. In addition, the SDSDGS motif in CREB3L1 and the SDSEGS motif in CREB3L2 are very similar to the motif found in CREB-H. Plausibly, β-TrCP-mediated degradation is a common mechanism shared by most of these proteins. CREB3L1 and CREB3L2 are the substrates of Fbxw7. Both Fbxw7 and β-TrCP are F-box proteins of the SCF E3 ubiquitin ligase and they share many similar properties. Fbxw7 recognizes an S/TxxxS/T motif, in which S/T are phosphorylatedThe expression and activity of CREB-H are regulated at multiple levels including proteolytic activation, phosphorylation, ubiquitination and degradation. Most of the results are obtained with CREB-H-ΔTC in our study and in the other work29Expression plasmids pcDNA3.1-V5-CREB-H and pcDNA3.1-V5-CREB-H-ΔTC for human CREB-H and CREB-H-ΔTC have been previously described4515254www.agilent.com/genomics/qcpd; Agilent).Plasmid FLAG-β-TrCP-ΔFbox and plasmids expressing various CREB-H-ΔTC mutants were generated by site-directed mutagenesis using reagents supplied by Agilent. Mutagenic primers were designed using the web-based QuikChange Primer Design Program (Primary antibodies used in this study included mouse anti-V5 (Invitrogen), rabbit anti-FLAG , mouse anti-FLAG , rabbit anti-MYC , rabbit anti-glyceraldehyde-3-phosphatase dehydrogenase , rabbit anti-β-TrCP  and rabbit anti-β-actin (Sigma-Aldrich).Human hepatoma cell line Hep3B and human embryonic kidney cell lines HEK293 and HEK293T were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum (Life Technologies). Human hepatoma cell line HepG2 was grown in Eagle’s Minimum Essential Medium (Life Technologies) supplemented with 10% fetal bovine serum. Transfection of cells was performed as previously described56For proteasome inhibition, transfected cells were treated with 10 μM MG132  for 6 hours before harvest. For protein turnover assay, 200 μM cycloheximide (Sigma-Aldrich) was added into transfected cells 5 hours before harvest.Forty-eight hours after transfection, cells were harvested in NP40 buffer  supplemented with protease inhibitor cocktails (Roche). Protein concentration of cell lysates was determined by Bradford method (BioRad). Protein samples were separated by SDS-PAGE, electroblotted onto polyvinylidene difluoride membranes (Millipore), incubated with primary and secondary antibodies, and visualized by enhanced chemiluminescence (Amersham).Co-immunoprecipitation was carried out as describedPhosphatase assay was performed as describedin vivo polyubiquitination, cells were co-transfected with the MYC-ubiquitin plasmid in combination with the wide-type or mutant V5-CREB-H-ΔTC plasmids. Forty-eight hours after transfection, total protein was collected, immunoprecipitated with anti-V5 antibody, resolved with SDS-PAGE and immunoblotted with anti-MYC antibody.For Cells were harvested 36 hours after transfection for dual luciferase assay as previously described4RNA knockdown experiments were performed as described60Total RNA was extracted from cells using the RNAiso Plus reagent (Takara) according to the manufacturer’s instructions and then incubated with DNase I (Ambion) to remove remaining genomic DNA. Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis reagents (Roche). Real-time quantitative PCR was conducted in the presence of SYBR Premix Ex Taq (Takara) with the StepOnePlus real-time PCR system (Applied Biosystems). The mRNA level of β-tubulin was determined as an internal control and the results were analyzed using StepOne Software v2.3 (Applied Biosystems). Primer sequences used are listed in How to cite this article: Cheng, Y. et al. β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H. Sci. Rep.6, 23938; doi: 10.1038/srep23938 (2016).


They matched variation in striping patterns at 16 sites across its geographical range to a suite of environmental variables as well as tsetse fly (glossinid) distribution and lion (Panthera leo) presence. They found that greater intensities of intraspecific striping were associated with warmer temperatures and high precipitation.The functional significance of the extraordinary black and white stripes of zebras is still mysterious but now an active field of research. Four major hypotheses have been put forward: stripes are an antipredator defence operating through crypsis  or confuet al.  investigEquus africanus) which has leg stripes but no body stripes, and the unstriped species of equids , and 20 of their subspecies, and compared several aspects of the four hypotheses [Crocuta crocuta), social interactions, tsetse fly distribution, tabanid distribution using both temperature and humidity ranges as a proxy for abundance, and temperature isoclines to assess the cooling hypothesis. First we ran univariate tests on individual factors to identify the hypotheses with the strongest predictive ability, then subsequently used AICc model selection procedures to pit the most promising predictors against each other—a powerful statistical protocol for testing multiple hypotheses. Further, our study was corrected for phylogenetic relatedness between the species and subspecies, allowing us to draw conclusions about correlated evolution between body striping and environmental selective pressures. Our study provided strong evidence that our proxy for tabanid fly parasitism was essentially perfectly correlated with the presence of stripes across species across almost every area of the body, and especially for leg stripes, the level at which biting flies prefer to land on hosts.Last year we published a similar but phylogenetically controlled analysis using all seven species of equids, namely the three striped zebra species, the African wild ass  and constant annual temperatures (BIO3) which they found were associated with striping measures in plains zebras. In short, the studies find that striping is associated with warm humid conditions both inter- and intraspecifically.The studies additionally concur in finding aspects of striping being related to temperature and moisture. We found many aspects of striping  are associated with temperatures lying between 15°C and 30°C and humidity between 30 and 85% (a proxy for tabanid abundance) when these conditions persist for a minimum of six to seven months. Note, such warm humid conditions are congruent with Larison E. africanus that are under heavy tabanid pressure, body stripes might stem from genetic correlation with leg stripes since more species have leg stripes than body stripes.Where the studies differ is in their interpretation of these results. Larison and co-workers focus on their temperature rather than on their rainfall association and discuss how temperature is correlated with striping measures in plains zebras although they are agnostic as to mechanism. While acknowledging that stripes might set up convection currents over the torso  and so cN=57 individuals), impalas Aepyceros melampus 34.2°C (N=49), buffalo Syncerus caffer 34.1°C (N=35) and giraffe Giraffa camelopardalis 33.3°C (N=27) [Caro and others suggest that striping is associated with tabanid and possibly other biting fly annoyance. There is experimental evidence that tabanids eschew landing on white , stripedC (N=27) .Interestingly, Larison and her co-workers suggest that trypanosomes and other diseases carried by tsetse flies may have a compromised ability to develop at colder temperatures, reminding readers that the distribution of biting flies may have less relevance than the distribution of diseases carried by those flies. They concede that their temperature-striping associations could reflect favourable conditions for infectious diseases carried by biting flies that adversely affect zebras: ‘We suggest that temperature may influence trypanosome prevalence in tsetse flies and as a consequence help explain variation in striping’.Caro and others tested an additional hypothesis interspecifically that the Larison group did not test intraspecifically. This was striping being associated with mean and maximum group sizes as a proxy for social interactions, for which we found no evidence.While it is often expedient to call attention to differences in findings between studies, in the interests of publication and publicity, it is often more constructive in the long run to focus on similarities. Here, we suggest that the only two multifactorial studies of this problem , conductThe studies' joint handicap, as both sets of authors explicitly recognize, is that we are hampered by lack of understanding of the extent to which temperature and humidity predict fly annoyance, the specific problems posed by biting flies, the extent to which biting fly distributions and the diseases that they can carry elide, and the mechanism by which stripes prevent flies from landing on zebra pelage mediated through disruption of outline, modulation of brightness or of polarized light , or conf


Background: Gdown1 is a substoichiometric subunit of RNA polymerase II that blocks termination by TTF2 and the function of TFIIF during initiation and elongation.Results: Domains of Gdown1 responsible for inhibiting TTF2 and TFIIF as well as a controlling phosphorylation site are identified.Conclusion: Gdown1 can be regulated by phosphorylation of Ser-270.Significance: Our results reveal a mechanism that allows termination of all polymerases during mitosis. in vitro transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified. In vitro, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using  Regulation of Pol IId-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor DSIF, the negative elongation factor NELF, Gdown1, and the Gdown1 negative accessory factor GNAF. DSIF is required for the P-TEFb inhibitor (DRB) to convey a negative effect on elongation  of Gdown1 is required to block TTF2 and the C-terminal domain (CTD) is responsible for blocking TFIIF. We also identified a phosphorylation event that reduces the affinity of Gdown1 with Pol II, thereby allowing TFIIF and TTF2 to function on elongation complexes. These results provide mechanistic insights toward the function and regulation of Gdown1 during mitosis and perhaps during mRNA production.Full-length human Gdown1 in the pET100 directional TOPO vector (Invitrogen) was described previously . Gdown1 m A/U/GTP spiked with [α-32P]CTP. After stopping the reaction with EDTA the elongation complexes were washed with 1.6 m salt to remove associated factors. For add-backs, purified wild-type or truncated/mutated versions of Gdown1, human TFIIF  followed by phosphorimaging.Generation of EECs was described previously , 21. In an TFIIF , and human TFIIF  were addQuantification of the effects of TFIIF and TTF2 was accomplished by quantifying runoff for the effect of TFIIF and quantifying the percentage of the total transcripts present in a region of the gel containing most of the terminated transcripts for TTF2. This was carried out using version 3 of the MultiGauge software (FujiFilm) supplied with the FLA7000 Phosphorimager . Although analyzing the effect of TTF2 was complicated by the presence of a small amount of nonterminated transcripts in the region in which terminated transcripts appeared, the relative percentage in this region between lanes is a good estimate of the relative termination activity between lanes.m MgCl2. The complexes in solution were run on 4% acrylamide gel in 0.5 × Tris/glycine buffer for 2.5 h at 6 W and dried before phosphorimaging.Early elongation complexes were generated with the CMV promoter template with a runoff point at +183 as described previously . 1 pmol HeLa cells were grown in DMEM supplemented with 10% FBS. Medium was replaced several hours before the first shake off. Vigorous tapping on the sides of a T-150 flask released mitotic cells that were then spun down (1200 rpm in Beckman J-6) before being washed in and resuspended with ice-cold PBS. Cells remaining on the plate were scraped in medium, washed, and resuspended with PBS. Mitotic and interphase cells were lysed with SDS containing protein sample buffer, and aliquots were analyzed by SDS-PAGE (9% acrylamide).m to 1 m KCl in HGKEDP . Each collected fraction was used in the kinase assay to phosphorylate recombinant Gdown1. The fractions able to phosphorylate Gdown1 and change its mobility by SDS-PAGE were pooled and further fractionated on Mono Q with a gradient of 100 mm to 1 m KCl. In the kinase assays, each reaction included the indicated amounts of Gdown1 and 4 μl of each fraction in 20 mm HEPES (pH 7.6), 60 mm KCl, 5 mm MgCl2, 1 mm ATP (spiked with [γ-32P]ATP) and was incubated at 37 °C for 1 h. The reactions were then analyzed by SDS-PAGE followed by silver staining and phosphorimaging.0.5 ml of HNE was fractionated on Mono S with a gradient of 100 mm HEPES (pH 7.6), 50 mm KCl, 5 mm MgCl2, and 1 mm ATP at 37 °C for 4 h. The Gdown1 was then purified by nickel-nitrilotriacetic acid before being dialyzed into 50 mm (NH4)HCO3 and subjected to mass spectrometry using the method described in Ref. 10 μg of Gdown1 and 100 μl of Mono Q fraction 19 were incubated in 20 mA. The NTD contains the most highly conserved peptide in Gdown1, LPDKG. The identical sequence is found in all species, except Xenopus, which contains a similar peptide LPDRG . Interestingly, an alignment of TTF2 from a wide range of species also shows a highly conserved LPDKG pentapeptide within a well conserved region of TTF2 . TTF2 from Xenopus had the same LPDRG peptide found in Xenopus Gdown1, which suggests co-evolution of the motif. We hypothesize that the NTD of Gdown1 competes with TTF2 for Pol II binding.To identify functional domains of Gdown1, we carried out a sequence alignment of Gdown1 proteins from species ranging from insects to primates. Sequence similarities indicated conservation in an NTD and in a CTD as illustrated in Fig. 1in vitro transcription assay (C) and TTF2 (D) are known to be inhibited by Gdown1 during elongation . As expected , whereas addition of TTF2 caused termination of the EECs resulting is a characteristic pattern of shorter transcripts, which have been demonstrated to be terminated transcripts released from the template . Note that there is a small amount of TTF2 that remains bound to the template during high salt wash, and this leads to a low level of termination during the chase (compare lanes 1 and 3). Addition of Gdown1 eliminated the effect of the contaminating TTF2 resulting in a pattern of slightly longer transcripts compared with Pol II alone and the disappearance of shorter transcripts . As expected, Gdown1 blocked the effects of both TFIIF and TTF2 . As determined by quantifying the amount of runoff, the ΔLPDKG mutant blocked 90% of the TFIIF-dependent runoff . However, the ΔLPDKG mutant only weakly blocked TTF2 as determined by quantifying the level of short transcripts caused by termination. The region quantified was 72% of the total counts with TTF2 alone and 50% with TTF2 in the presence of the ΔLPDKG mutant, which means more than half of the total termination activity remained . Although the ΔLPDKG mutant affected both TFIIF and TTF2, it blocked TFIIF significantly more than it blocked TTF2, suggesting that the NTD is primarily involved in blocking TTF2.We generated a ΔLPDKG mutant with the sequence deleted from otherwise full-length Gdown1 to determine whether the motif played a role in blocking termination by TTF2. The purified recombinant protein was tested using an on assay . TFIIF  whereas addition of TTF2 terminated most of the complexes at early stage (lanes 3 and 4). Addition of 1 pmol of either the wild-type Gdown1 or the ΔLPDKG mutant significantly increased the proportion of engaged complexes, with ΔLPDKG mutant showing slightly more terminated transcripts because of the low level of TTF2 in the isolated complexes (lanes 5–8). To test ΔLPDKG mutant affinity to Pol II, salt washes with 60 mm KCl (LSW) or 1.6 m KCl (HSW) were introduced after incubating the Gdown1 with the EECs and before TTF2 addition and the chase . As had been demonstrated previously (lanes 1–4). However, the ΔLPDKG mutant, regardless of no wash, LSW or HSW, did not show significant inhibition against the strong termination activity from TTF2 (0.08 pmol). This suggests the ΔLPDKG mutant does not associate with Pol II as tightly as wild-type Gdown1.The property of the ΔLPDKG mutant was further characterized by separating the bead-bound elongation complexes and the supernatant containing terminated complexes after the reactions. As shown in Fig. 1eviously , Gdown1 H, lanes 1–4). The ΔLPDKG mutant, when in excess (1 pmol), created a more heterogeneous shift with some density in the fully shifted position and most density in a band of intermediate mobility between the elongation complex alone and the Gdown1-bound complex . This suggests that the ΔLPDKG mutant binds differently to the elongation complex and may exist primarily in an altered conformation with the CTD bound to Pol II and the NTD only weakly associated with Pol II. After LSW, most of the complexes had the mobility of free elongation complexes, and after HSW, the shifted complexes became undetectable . From all of these results we conclude that the LPDKG motif is involved in inhibition of TTF2-mediated termination and that the interaction of Gdown1 with Pol II is weakened by the mutation.To confirm the lowered binding of the ΔLPDKG mutant to Pol II we carried out electrophoretic mobility shift assays with isolated elongation complexes (EC-EMSA) . As showFig. 1A and B). Tested in the in vitro transcription assays, a high concentration of the NTD did not block TFIIF but demonstrated a very slight inhibition of TTF2 activity (C), indicating that the NTD requires support from other domains of Gdown1 to have sufficient activity. The CTD has been predicted to have structural similarity to part of the RAP30 subunit of TFIIF (D). However, the CTD alone showed very slight inhibitory effect on TTF2 (E). Together, these results strongly suggest that the CTD of Gdown1 is primarily responsible for inhibiting TFIIF, although the full-length protein shows a much stronger block probably because of its increased ability to bind to Pol II.To assess the functions of the Gdown1 individual domains, truncation proteins containing the NTD (amino acids 1–89) and CTD (amino acids 197–364) of Gdown1 were cloned, expressed, and purified , A and Bactivity C, indicaof TFIIF , and we of TFIIF D. Howeve on TTF2 E. TogethA). Treating HNE with calf alkaline phosphatase caused the disappearance of the upper band and increased intensity of the lower . This suggests that the upper band is a phosphorylated form of Gdown1 with reduced mobility in SDS-PAGE. Phosphorylation of Gdown1 on multiple residues has been reported previously, but no functional consequences were described . This suggests that mitotic phosphorylation of Gdown1 might allow termination by TTF2.Western blotting using affinity-purified Gdown1 antibodies consistently detected two bands at approximately 50 kDa from HeLa whole cell extract . The twoFig. 3escribed , 27. Givescribed , we wondC), and each fraction was tested for Gdown1 kinase activity. On Mono S most fractions caused incorporation of label from [γ-32P]ATP, but two fractions also led to the shift (D and E). These two fractions were further fractionated on Mono Q, and a shifting kinase was separated from a nonshifting kinase (F).To characterize the phosphorylated Gdown1 biochemically, we sought to obtain a phosphorylated form with minimal contamination of the unmodified form. In initial kinase assays in which high levels of recombinant Gdown1 were incubated with HNE, a fraction of the Gdown1 exhibited the shifted mobility (data not shown). HNE was fractionated on Mono S followed by Mono Q C, and eahe shift , D and Eg kinase F.A, lane 5), and the termination signal caused by TTF2 was reduced to 42% . A 3.3-fold increase in Gdown1 saturated the Pol II leading to almost complete inhibition of TFIIF and TTF2. The effects seen when phospho-Gdown1 was titrated onto EECs were similar except that a little more than 3 times phospho-Gdown1 was required to achieve the same level of inhibition compared with unphosphorylated Gdown1 . These results demonstrate that the phosphorylation weakens the effect of Gdown1 in blocking TFIIF and TTF2. Blocking at higher concentrations could be explained by unphosphorylated Gdown1 (present because of incomplete phosphorylation) becoming dominant because of essentially irreversible binding or by an increased fractional occupancy of the elongation complex with a weaker binding phosphorylated protein.To examine the effect of the shifting phosphorylation on the function of Gdown1, phospho-Gdown1 was generated using Mono Q fraction 19 in a kinase assay with only nonradioactive ATP. The kinase reaction was stopped with EDTA, and the phosphorylation of Gdown1 was >90% complete as judged by silver staining after SDS-PAGE. Increasing levels of the unmodified Gdown1 in elongation assays caused the expected inhibition of both TFIIF and TTF2 . At 0.00Fig. 4m KCl) or HSW (1.6 m KCl). The complexes were then incubated with TFIIF or TTF2 before being chased. After a HSW of EEC(G) the elongation complexes remained largely resistant to TFIIF and TTF2  , although the increased response to TTF2 might be too small to be considered significant. This indicates that phospho-Gdown1 is still able to bind to Pol II, but may have a slightly weakened ability to do so. This became very apparent after high salt wash. EEC(P-G) showed significant responses to TFIIF and TTF2, suggesting that phosphorylation of Gdown1 reduces its affinity for Pol II (lanes 11–12). To confirm this, EC-EMSA was performed, and it demonstrated that a small fraction of phospho-Gdown1 was removed from EECs after low salt wash and that a significant fraction of it was removed from the complexes by high salt wash . We conclude that phosphorylation of Gdown1 weakens its affinity for Pol II and this allows partial function of TTF2 and TFIIF.We then tested whether the reduced effects of phospho-Gdown1 were because of a reduced affinity for Pol II. EECs were incubated with saturating levels of either unmodified Gdown1 or the phospho-Gdown1 for 10 min at room temperature before washing with LSW (60 mnes 4–6) . After LA). To determine that Ser-270 was indeed the site of phosphorylation, both wild-type and the S270A mutant were individually mixed with a wild-type CTD protein that contains Ser-270 and subjected to a kinase reaction without or with the kinase fraction. Both wild-type full-length Gdown1 and the CTD were shifted by the phosphorylation (B). Under identical conditions the S270A mutant did not shift although the wild-type CTD in the same reaction did (B). These results demonstrate that phosphorylation of Ser-270 is responsible for the mobility change.Gdown1 phosphorylated by the kinase fraction was affinity purified by nickel-nitrilotriacetic acid and subjected to mass spectrometric analyses to identify the phosphorylation site. Only one phosphopeptide was identified, and it contained phosphorylated serine 270. Gdown1 mutations changing serine 270 to alanine (S270A) and glutamic acid (S270E) were produced to block or mimic phosphorylation of Ser-270, respectively. By SDS-PAGE, the S270A mutant showed mobility identical to the wild-type whereas the S270E mutant ran slightly slower A. To detrylation B. Under tion did B. These C, lanes 5 and 6). The S270E mutant showed reduced resistance to HSW as significantly higher amount of the elongation complexes were stimulated by TFIIF, and an increased amount of termination was caused by TTF2 . However, the S270A mutant showed a slightly increased resistance to HSW compared with the wild-type. The Gdown1 CTD truncation that bears Ser-270 was fully phosphorylated by the Mono Q fraction 19 and tested for its ability to inhibit TFIIF as shown in Fig. 2D. Wild-type CTD was able to significantly inhibit TFIIF, but after phosphorylation no inhibition was detected (data not shown). The results with mutant proteins confirm Ser-270 as the site of phosphorylation and that phosphorylation of Ser-270 on Gdown1 leads to reduced inhibition of TFIIF and TTF2 through reduction in the affinity for Pol II.To establish the functional significance of this Ser-270 phosphorylation, wild-type Gdown1 and the S270E and S270A mutants were incubated with EECs prior to extensive high salt washing. After the wash, Pol II with wild-type Gdown1 remained mostly resistant to TFIIF and TTF2  elongation complexes to be terminated during mitosis. In the Future work would include pursuing the identification of the kinase and a potential phosphatase. That information would indicate whether the regulation of Ser-270 phosphorylation is related primarily to the cell cycle or more broadly to transcription at all stages. The mutations of Gdown1 described here should be useful in determining how Gdown1 affects transcription in the context of other transcription factors.


Experimental and comparative evidence suggests that the striped coats of zebras deter biting fly attack, but the mechanisms by which flies fail to target black-and-white mammals are still opaque. Two hypotheses have been proposed: stripes might serve either to defeat polarotaxis or to obscure the form of the animal. To test these hypotheses, we systematically photographed free-living plains zebras in Africa. We found that black and white stripes both have moderate polarization signatures with a similar angle, though the degree (magnitude) of polarization in white stripes is lower. When we modeled the visibility of these signals from different distances, we found that polarization differences between stripes are invisible to flies more than 10 m away because they are averaged out by the flies’ low visual resolution. At any distance, however, a positively polarotactic insect would have a distinct signal to guide its visual approach to a zebra because we found that polarization of light reflecting from zebras is higher than from surrounding dry grasses. We also found that the stripes themselves are visible to flies at somewhat greater distances (up to 20 m) than the polarization contrast between stripes. Together, these observations support hypotheses in which zebra stripes defeat visually guided orienting behavior in flies by a mechanism independent of polarotaxis. We observed modest polarization signals from both white and black stripes, which were similar in their angle of polarization. This result provides little support for a role of stripes in defeating polarotaxis, which in turn suggests that the high-contrast stripe pattern itself is more likely to be protective . While sEquus burchelli) in Katavi National Park, Tanzania. All necessary permits were obtained from the Tanzania Wildlife Research Institute, Tanzania Commission for Science and Technology. UC Davis IACUC approval was not required because neither the animals nor their habitat was disturbed in any way. The study site was located at latitude 6°35’–7°05’S, longitude 30°45’–31°25’E. We took photos in the dry season in July through mid-September 2012 at a distance of 35–101 m as determined by rangefinder. Local shading, scene haze, sun azimuth and elevation were noted for each subject; in general there was little heat distortion and little overall scene shade. The camera was a Nikon D50 with a 100–300 mm Nikkor lens fitted with a linear/circular polarizing filter designed for digital cameras . The majority of our images were taken with the viewing angle within +/- 45 degrees of the body axis of the zebra  and converted to 48-bit (16 bits/channel) TIFF files using the public domain software ‘dcraw’ , using a setting that conserved the sensor values for each pixel. All subsequent analysis was performed in Matlab . These RGB images were converted into grayscale using Matlab function graythresh) that maximized luminance variance between black and white stripes relative to within-stripe variance. This algorithm reliably identified the same stripes in each of the subject’s 7 images, even if their luminances changed because of polarization effects or animal movements. By virtue of the feature tracking and adaptive thresholding, our approach is less sensitive to movement artifacts than is imaging polarimetry.ROIs were identified and matched by hand across images using fiduciary marks on the zebra’s coat . The blaminimum transmission angle is vertical, and thus indicates a preponderance of horizontally polarized light. The orthogonal phase angle of +45° indicates a distribution of polarization angles biased in favour of vertically polarized light. Through the rest of the paper, we will refer to the quantity extracted from our fits as the phase, and refer to the physical properties of the light in the images as “polarization angle”. We chose our angle conventions so that the range of greatest interest  would appear in the middle of the range of phases in figures. The sinusoidal fits to our data were quite good; average correlations between fit and data were 0.73 and 0.66 for black and white stripes, respectively. Because the phase parameter of the fit is unreliable for images with low d values, we restricted our quantitative analysis of phase angle to regions where there was a significant correlation between the data and the fit  for both the white and black stripes in the ROI.To determine degree and angle of polarized light reflected or scattered from each region, we fitted the luminance by a 180° period sine function of the polarization filter setting , accordid, using the expressionmax and Lmin refer to maximum and minimum luminances, respectively . Because each zebra was sampled multiple times, we verified that the difference between stripes was genuinely significant using ANOVA, in which stripe was a fixed effect and zebra was treated as a random factor , which influenced both black- and white-stripe d value , but this was the only direct illumination effect. White stripes were not affected by either region or illumination. To graphically illustrate these effects, d values for black and white stripes from different ROIs, and (b) when ROIs were directly or indirectly illuminated. The regions that systematically showed the greatest differences between black and white stripe d values were the shoulder, flank, back, upper hindleg, and rump. Also, black stripes consistently had a higher d value than white stripes, and sunlit regions consistently had a higher d value than shaded regions, both of which one would expect from optical principles. However, none of these differences were substantial, as is evident from We were interested in what factors would influence the degree of polarization, and the difference between black and white stripe In the preceding analysis, a large number of scene variables could have been included in the effect of zebra identity, such as sun azimuth, elevation or the orientation of the zebra. Because each zebra was only sampled once, it is difficult to differentiate between illumination conditions and intrinsic differences in the pelts of the animals, such as amount of dust or oils in the hair. However, we could explore systematic effects of viewing conditions by ignoring zebra identity in analyzing our image data. We therefore performed an ANCOVA analysis in which we examined the effects of sun azimuth (east or west), sun elevation (low or high), body region (1–8), and animal orientation (continuous). We also included the one previously significant predictor: illumination on the ROI. No additional effects were revealed in this analysis; the only effect was of direct illumination, and only on black stripes . Interestingly, the white-stripe d values are also 17% higher in these regions, consistent with there being a substantial surface reflection signal from white stripes as well as black.Although not significant in the preceding analysis, the mean ous work . Five sa−13). The majority of cases had polarization angles within 45°, and there was no significant difference between black and white phases  Therefore, we conclude the white stripes cannot counteract the polarized-light signal from the black stripes, because of the consistency of the phases of the emitted light from both.The effect of polarized light on the visual system of a fly approaching from a distance might be influenced not only by the magnitude of the polarization signal, but also by the relative polarization angles in the two sets of stripes. If the angle of polarization of emitted light were systematically orthogonal between adjacent black and white stripes, then the striping might actively counteract the signal from the black stripes. Therefore we also analyzed the angle of polarization of the light from our images. The angle of polarization was evident in the phase of the fit of the sinusoid . Forty oCalliphora, which has a visual acuity of approximately 1° [d value for the black and white stripes, as well as the luminance contrast between the black and white stripes at several distances out to 50 m  than the surrounding vegetation (median ratio 2.06), and this difference was significant . Therefore zebras present a distinctive polarization signal, averaged across the black and white stripes, which would be available to a fly approaching upwind and crucially, at distances where the stripes themselves would be invisible to the fly.To address the question of the relative polarization between zebra and background, we analyzed 10 images where we found a nearby patch of grass adjacent to one of our ROIs in the same illumination. We found that the zebra pelts had a much higher Three main findings emerge from this study. First, substantial polarized light reflectance signals are seen from both black and white striped pelage of free-living zebras. Second, these signals are polarized in a similar plane. Third, the polarization of light reflecting from zebras is significantly higher than from surrounding vegetation, so that a positively polarotactic insect would have a distinct signal to guide a visual approach to the zebra. Consequently, it is highly improbable that the primary function of zebra stripes is to defeat polarotaxis by biting flies.d values from the Horvath work on live animals were found on dark, dorsal surfaces illuminated by low-angle sun. In the tropics, such illumination conditions are less common, and would mostly occur in the early morning or late afternoon, when tabanids are less actively foraging [d values nor strikingly greater differences between black and white stripes across ROIs  and polarization angle. At the range where flies are thought to transition from olfactory to visual homing 5–10 m), the optics of tabanid eyes would substantially blur the signals of black and white stripes . The res–10 m, thPanthera leo) or spotted hyaenas (Crocuta crocuta). Originally, Thayer [Stripes were once hypothesized to conceal prey outlines from mammalian predators such as lions . We havosophila  could beDrosophila [e.g., [Another possibility is that the stripes disturb optomotor responses for approach or landing manoeuvres. Looming stimuli provoke landing responses if they are in frontal vision, but avoidance turns if presented to the side of a fly . Patterosophila ). One aposophila . Such era [e.g., ). The twS1 TableSun azimuth is relative to true north, and elevation is relative to the horizon, as usual. Body angle was estimated by eye, and is relative to the viewing direction, with 0 degrees indicating a perpendicular view with nose right and 180 degrees perpendicular with nose left.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Tabled values in black stripes and white stripes, respectively. This and the analysis presented in Region refers to which area of the body was covered by each ROI, sun refers to whether an individual ROI in a given series was directly or indirectly illuminated, and zebra (random factor) refers to the individual out of the 21 series that comprised the data set. Together, these variables accounted for 42% and 32% of the variance in (PDF)Click here for additional data file.S4 Tabled values, respectively. We interpret these results to mean that scene variables contributed to the differences between zebras, but that there was considerable residual variance that must be attributed to intrinsic differences between the reflectance properties of the pelts of different zebras.Sun azimuth was coded either east or west, because the data were taken in the tropics and the range of azimuths was therefore limited. Elevation was coded as low or high, with the criterion elevation being 45 degrees. The sun variable was as in Table 2, and orientation was a continuous variable, measured in degrees relative to the viewing direction, with 0 being head-right and 90 being head-away. Only illumination was individually significant, though many variables approached signficance. These independent variables accounted for 21% and 13% of the variance in black- and white-stripe (PDF)Click here for additional data file.


Solid tumors arising from malignant transformation of glial cells are one of the leading causes of central nervous system tumor-related death in children. Recurrence in spite of rigorous surgical and chemoradiation therapies remains a major hurdle in management of these tumors. Here, we investigate the efficacy of the second-generation receptor tyrosine kinase inhibitor nilotinib as a therapeutic option for the management of pediatric gliomas. We have utilized two independent pediatric high-grade glioma cell lines with either high platelet-derived growth factor receptor alpha (PDGFRα) or high PDGFRβ expression in in vitro assays to investigate the specific downstream effects of nilotinib treatment. Using in vitro cell-based assays we show that nilotinib inhibits PDGF-BB-dependent activation of PDGFRα. We further show that nilotinib is able to decrease cell proliferation and anchorage-independent growth via suppression of AKT and ERK1/2 signaling pathways. Our results suggest that nilotinib may be effective for management of a PDGFRα-dependent group of pediatric gliomas.The online version of this article (doi:10.1007/s11060-015-1744-y) contains supplementary material, which is available to authorized users. Malignant glial neoplasms comprise 8–10 % of primary pediatric central nervous system (CNS) tumors . These lPDGFRA) locus at chromosome 4q12, encoding the α isoform of platelet-derived growth factor receptor (PDGFRα) [H3F3A, encoding histone variant H3.3, that is associated with poorer overall survival [PDGFRA occurred exclusively in H3F3A-mutant tumors [PDGFRA amplification [Genomic studies of pediatric high-grade gliomas (HGG) have demonstrated that the most frequent copy number aberration (CNA) is focal amplification of the platelet-derived growth factor receptor ((PDGFRα) . Severalsurvival , 9; amont tumors . PDGFRα fication . Expressfication , 14.The second-generation receptor tyrosine kinase inhibitor nilotinib  binds its targets with similar affinity to the prototype molecule imatinib  –19. UnliPediatric GBM cell lines SJ-G2 and SF-188 , 27 wereMethods and results are presented below for SJ-G2; parallel experiments carried out in SF-188 are presented in supplementary sections (see detailed methods in Supplement). Culture stimulation was performed using dimerized B-isoform of recombinant human platelet-derived growth factor , which has affinity for both α and β isoforms of PDGFR. Cell cultures at 70–80 % confluence were incubated overnight in growth media containing 0.5 % FBS, then washed with warm phosphate-buffered saline (PBS), and exposed to fresh minimal media containing PDGF-BB at working concentration. Stimulation was terminated by placing cultures on ice and washing with cold PBS, then scraping and suspending in cell lysis buffer.Cell cultures were scraped and lysed on ice in 1X Cell Lysis Buffer  containing protease (Roche Diagnostics) and phosphatase inhibitors . Protein was quantified using bicinchoninic acid (BCA) assay (Thermo Scientific), and lysate containing 25–40 μg protein was loaded onto 7–12.5 % SDS-PAGE gels for electrophoresis. A detailed list of antibodies used is presented in Supplementary methods.® Aqueous One Solution reagent (Promega) according to the manufacturer’s instructions. Direct count of viable cells using the trypan blue dye exclusion method was performed with the Vi-CELL Cell Viability Analyzer (Beckman Coulter). For cell proliferation assay, chemiluminescent Cell Proliferation ELISA (Roche) using 5-bromo-2′-deoxyuridine (BrdU) labeling was carried out according to the manufacturer’s instructions, using a 12-h labeling period. Clonogenic assay and soft-agar colony-forming assay were performed to evaluate in vitro growth potential [Cell viability was assessed by MTS assay using CellTiter 96otential . Colonie6) were injected into the right flank of 6–8-week-old NOD–SCID mice (Jackson Laboratory). On detection of palpable tumor, mice were given nilotinib or vehicle by oral gavage (10 mg/kg daily), and sacrificed upon signs of sickness or at the end of the 10-week study. Excised tumor dimensions were measured with calipers, and volume estimated by the formula 4πabc/3, where a, b and c represent tumor radius in three planes. Animal use protocols were approved by the animal care committed of the Hospital for Sick Children  under AUP#2656.SJ-G2 cells , so chose an overlapping concentration range to examine signaling effects. To evaluate the effect of PDGFR activation on the downstream AKT and Ras pathways, we monitored phosphorylation of AKT S473 and ERK1/2 T202/Y204 in the presence and absence of nilotinib. In low-serum growth conditions, background phosphorylation of PDGFRα was minimal, and activation of AKT and ERK1/2 was not detected  Fig. a. FollowTo evaluate the effect of nilotinib on SJ-G2 proliferation, we assessed the incorporation of BrdU by chemiluminescent ELISA Fig. d. Followp = 0.0006) when treated with nilotinib, compared with mice given drug vehicle  itself [PDGFRA, its expression of high levels of PDGFRα, the activation of which is dependent on dosage of ligand (PDGF-BB), suggests that it represents tumors that use PDGFRα signaling to support their tumorigenic behavior. To our knowledge, this is the first demonstration in a pediatric GBM cell line that activation of PDGFR is dependent upon ligand binding. This stands in contrast, for instance, to constitutively-activated mutant forms of epidermal growth factor receptor that are frequently found in adult GBM [Pediatric high-grade tumors exhibit variability in ) itself , 29–31. dult GBM , 33. Actdult GBM .Ligand-mediated PDGFR stimulation activates the PI3K–AKT and Raf–MEK–ERK signaling pathways –37 and iThe effect of nilotinib inhibition was subsequently examined in the heterogeneous environment of serum-supplemented growth media. In this setting, we observed a low basal level of PDGFRα phosphorylation. Consistent with ongoing PDGFRα stimulation, we also detected basal activation of AKT, S6 ribosomal protein, MEK and ERK1/2, although activation of other cell-surface receptors may have contributed to this. Exposure to nilotinib decreased PDGFRα phosphorylation in all conditions. Phosphorylation of AKT and S6 ribosomal protein was rapidly reduced, and was maintained at a significantly decreased level during sustained inhibitor exposure, suggesting that some component of their activation is PDGFR-dependent. Interestingly, there was an early increase in MEK phosphorylation, within the first 2 h of nilotinib exposure. The rapidity of this change suggests that it results from an alteration in steady-state balance between signaling pathways.Since inhibition with nilotinib suppressed PDGFRα-mediated signaling, we then investigated its effect on SJ-G2 cell numbers and proliferation. Our results show that cell proliferation and clonogenic potential (in vitro tumor-forming ability) were significantly reduced at 3 μM nilotinib compared to vehicle-treated control. The reduced proliferation resulted in fewer viable cells over the same time interval of growth compared to untreated controls. As patients receiving typical oral dosaging of nilotinib achieve a mean peak serum concentration of 3.6 μM , the plaThese functional assays indicate that the result of nilotinib-mediated decrease in PDGFRα activation and signaling is a reduction in SJ-G2 proliferation, colony formation and in vivo growth. This overall decrease in tumorigenic behavior suggests that SJ-G2 may represent tumors that depend upon PDGFRα signaling and would therefore respond to nilotinib inhibition.In contrast, SF-188 expressed very little PDGFRα and demonstrated high basal activation of AKT and ERK1/2, suggesting that this cell line is dependent upon activation of alternative growth factor receptor pathways to transduce mitogenic signals . Consistent with this, we found that treatment with nilotinib had little effect on activation of AKT and ERK1/2. Furthermore, while treated cultures of SF-188 initially demonstrated lower viability, this difference was lost with sustained inhibition. SF-188 contains a mutation of the tumor suppressor and Ras-GAP NF1 , which lAll chemotherapeutic agents intended for use in the CNS must be able to penetrate the blood–brain barrier. Although its predecessor imatinib has poor CSF penetration , regardiAs our understanding of the biology of pediatric HGG evolves, so does our recognition that they are a clinical and pathological entity distinct from adult tumors—and, indeed, that they encompass different subtypes. In this study, we identified the SJ-G2 cell line as representative of tumors that are dependent upon PDGFRα expression and activation to drive signaling pathways leading to tumorigenic behavior, and which are therefore most likely to be responsive to inhibition with nilotinib. Based on these data, we suggest that a subset of appropriately-identified pediatric HGG should be considered candidates for nilotinib treatment.Supplementary material 1 (TIFF 18722 kb)Supplementary material 2 (TIFF 46470 kb)Supplementary material 3 (TIFF 48791 kb)Supplementary material 4 (TIFF 83603 kb)Supplementary material 5 (DOC 55 kb)Below is the link to the electronic supplementary material.


Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. Phosphorylation is one of the most important post-translational modifications (PTMs) of proteins  analysis. Commonly used enrichment techniques were summarized in Table The most widely used enrichment method for phosphopeptides takes advantage of the affinity binding between negatively charged phosphate and positively charge metal ions  and zirconium dioxides (ZrO2) are gaining popularity for phosphopeptide enrichment. A comparison of the performance of TiO2 and ZrO2 performed with α-casein and β-casein as standard proteins showed that TiO2 tends to enrich multiply phosphorylated peptides, and ZrO2 tends to enrich singly phosphorylated peptides. A serial enrichment procedure with both TiO2 and ZrO2 can significantly increase the efficiency of capturing phosphopeptides in biological samples  resulted in identification of 2305 phosphopeptides belonging to 964 proteins in wheat  has been a widely used technology that resolves thousands of proteins by isoelectric point and molecular weight. Pro-Q Diamond is a fluorescent stain that provides a convenient method for selectively staining phosphoproteins in acrylamide gels. The result shows a global map of the modified proteins and their relative abundances compared to non-phosphorylated counterparts when a total protein staining is used after Pro-Q Diamond staining. Differentially phosphorylated proteins in wild-type and 15N in Arabidopsis suspension cells  and tandem mass tags (TMT). The tags are designed to be isobaric during MS and fragment to reveal differential low mass ion reporters during MS/MS. Due to its capability of multiplexing up to 10 samples in a single experiment and the enrichment effect for low abundance proteins, iTRAQ/TMT labeling has become popular in plant phosphoproteomics . The first SILAC in plants was done by introducing in vivo and in vitro label methods are limited by the number of samples, label free approaches enable quantitative phosphoproteomics of unlimited number of samples. There are two main methods in label free quantitation. The first is based on precursor ion peak intensity/area, and the second is based on the number of MS/MS spectra acquired for a given peptide . Both methods were used in plant phosphoproteomics  has been used for quantification of targeted phosphopeptides  have been identified. For example, time for coffee (TIC) and non-phototropic hypocotyl 3 (NPH3), which are involved in circadian clock and phototropism, were found to be MPK3/6 substrates. The result suggests that plant circadian rhythm and phototropism may be regulated by the MAPK signaling network.The mitogen-activated protein kinases (MAPKs) constitute one of the most important signaling mechanisms in plants, and they play essential roles in linking the perception of different stimuli with cellular adaptive responses. The MAPK signal transduction pathways are evolutionarily conserved in all eukaryotic organisms such as plants, yeast, fungi, insects, nematodes, and mammals   is a phytohormone that plays an important role in many aspects of plant life. For example, ABA is essential for regulating seed maturation and stomatal closure under abiotic and biotic stresses , and on transcription factors, chromatin remodeling proteins, and spliceosome components. Surprisingly, many novel phosphopeptides from proteins involved in vesicle trafficking such as components of the exocyst complex  were identified. How phosphorylation of these SEC proteins plays a role in vesicle trafficking is intriguing. Recently, phosphorylation of Sec31 by a casein kinase 2 was found to control the duration of COPII vesicle formation, decrease its association with ER and promote ER-to-Golgi trafficking  and STN8 kinases are light regulated and participate in phosphorylation of thylakoid membrane proteins and stroma proteins. Ingelsson and Vener  performeSucrose non-fermenting 1 related kinase (SnRK1) acts as a sensor of energy levels in plant development, and regulates plant growth by maintaining energy homeostasis during stress conditions  defect as revealed by Northern blot analysis. To test the role of phospho-motif-4 in translation termination efficiency, a well-established dual luciferase assay was used. The deletion-mutant lacking phospho-motif-4 was not able to rescue this defect, indicating that this motif has a role in translation termination efficiency. To dissect the sequences within phospho-motif-4 required for NMD activity, PCR-mediated mutagenesis was used to generate three additional deletion mutants . The results revealed that deletion of residues 736–745 reduced NMD activity as measured by Northern blot analysis. To test the functional role of Y738 and Y742, site-directed mutagenesis was used to create phosphorylation mimic mutants. Interestingly, the Y738F and Y742F fully rescued NMD activity of a chromosomal UPF1 deletion-mutant strain, indicating that they are not compromised in their ability to function in NMD. These results provided strong evidence that UPF1's ability to promote translational termination fidelity is depended on the conserved C-terminal phosphorylation motif, which is important for its NMD activity.A large phosphoprotein, the RNA surveillance protein UP-frameshift 1 (Upf1) in e et al.  used tan4+)-based IMAC and the reverse phase (RP)-strong cation exchange (RP-SCX) biphasic trap column-based online RPLC is a great example of the advancements (Bian et al., in vitro labeling and SILAC in vivo labeling have shown to be successful in combination with IMAC and MS (Isner et al., Phosphopeptide enrichment and MS have been essential tools for studying protein phosphorylation. It is challenging to directly detect phosphoproteins in biological samples due to the low abundance and low stoichiometry of phosphorylation in different biological processes. The enrichment methods of phosphoproteins/phosphopeptides from complex mixtures have greatly improved over the years. For instance, combining the titanium (TiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.