Cb2 null mice show a marked age-related bone loss. CB2-specific agonists both attenuate and rescue ovariectomy-induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony-forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase–activated protein kinase 2 (Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2-induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK-Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein–cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis. © 2011 American Society for Bone and Mineral Research.CB2 is a Gi protein–coupled receptor activated by endo- and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and  Cb2 null mice show a marked age-related bone loss, and CB2-specific agonists both attenuate and rescue ovariectomy (OVX)–induced bone loss. kinases provides a key link between membrane-bound receptors and changes in the pattern of gene expression. The MAP kinases are activated downstream of many different types of receptors, including tyrosine kinase receptors, cytokine receptors, and serpentine G protein–coupled receptors.,7 The MADownstream of Gi protein, CB2 regulates Erk1/2 and/or p38 phosphorylation. Depending on the cell type involved, this regulation is either stimulatory or inhibitory.–17 HowevPolymethyl methacrylate Technovit 9100 was from Hareus Kulzer . Calcein and pertussis toxin (PTX) were purchased from Sigma . Tissue culture ingredients were from Biological Industries . Collagenase P was purchased from Roche Applied Science. Antibodies to phosphorylated and nonphosphorylated Erk1/2, p38 MAP kinase, and mitogen-activated protein kinase–activated protein kinase 2 (Mapkapk2) were from Cell Signaling Technologies . The Erk1/2-activating kinase MEK inhibitors PD098059 and U0126 and p38 MAP kinase inhibitors SB203580 and SB202190 were from Calbiochem. Reagents for the luciferase assay were obtained from Promega . siRNA materials were from Santa Cruz Biotechnology . Colorimetric 5-Bromo-2-Deoxyuridine (BrdU) Labeling and Detection Kit III was from Roche Diagnostics. Reagents for real-time RT-PCR were from Applied Biosystems . AM-1241 was from Alexis Biochemicals. The EZ-ChIP kit was purchased from Upstate Millipore , and anti-phospho-CREB (on serine 133) antibody was from Cell Signaling Technologies. The Qiaquick spin kit for DNA extraction was from Qiagen.Cb2 null mice. Sham OVX surgery comprised exposure of the ovaries. To study bone formation, newly formed bone was vitally labeled with the fluorochrome calcein injected intraperitoneally (15 mg/kg) 4 days and 1 day before euthanization. At euthanization, the femurs were separated, fixed for 24 to 48 hours in phosphate-buffered formalin, and further kept in 70% ethanol. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem.Wild-type (WT) C57Bl/6J mice were from Harlan Israel. null mice were croFemurs were embedded undecalcified in polymethyl methacrylate. Longitudinal sections through the midfrontal plane were left unstained for dynamic histomorphometry based on vital calcein double labeling.Cb2 null mice by successive collagenase digestions., 1% Triton X-100, 150 mM of NaCl, 1 mM of EGTA, 50 mM of β-glycerophosphate, 1 mM of NaF, 10 µg/mL of leupeptin, 10 µg/mL of aprotinin, 0.5 mM of phenylmethylsulfonyl fluoride, and 1 mM of sodium orthovanadate. The cells then were scraped off using a rubber policeman, and the lysates were clarified by centrifugation at 12,000g for 15 minutes. Samples from each lysate, containing 40 to 120 µg of protein, were fractionated by SDS-PAGE and then electroblotted onto nitrocellulose membranes. The membranes were blocked with nonfat dry milk in TRIS-buffered saline/Tween-20 solution. The Western blots were probed with antibodies to phosphorylated Erk1/2, Erk1/2, phosphorylated p38, p38, phosphorylated Mapkapk2 (Thr222), and Mapkapk2. Proteins on the Western blots were detected using the EZ-ECL chemiluminescent detection system .MC3T3 E1 cells were seeded in 10-cm dishes at 5 × 10After serum starvation, the cells were incubated for 24 hours in 0.5% bovine serum albumin with or without the ligands and with or without PTX, inhibitors of MAP kinase phosphorylation, and Mapkapk2 siRNA. This was followed by labeling with BrdU for 24 hours and determination of its incorporation into DNA using a commercial kit according to the manufacturer's instructions.3 cells/well in 200 µL of antibiotic-free medium. Cultures at approximately 50% confluence were transfected with control or Mapkapk2 siRNA in transfection medium containing transfection reagent. After 5 hours of incubation in control and Mapkapk2 siRNA, the cells were serum-starved for 2 hours and then challenged with HU-308.Inhibition of Mapkapk2 expression was achieved using a commercial siRNA kit according to the manufacturer's instructions. The kit included mouse Mapkapk2 siRNA (cat. no. SC-35856), control siRNA (cat. no. SC-37007), siRNA dilution buffer (cat. no. SC-29527), siRNA transfection reagent (cat. no. SC-29528), and siRNA transfection medium (cat. no. SC-36868). Briefly, MC3T3 E1 cells were seeded in 96-well plates, 5 × 10MC3T3 E1 cells stably transfected with a luciferase construct reporting on CREB transcriptional activity and containing three copies of a canonical CRE (pCRE3-luc) were reported previously. To test Mapkapk2 and cyclin D1 mRNA levels was carried out by using Applied Biosystems Taqman Gene Expression Assays (respective assay IDs Mm01288465_m1 and Mm00432359_m1). Data were normalized to β-actin (assay ID Mm00607939_s1). Relative quantity (RQ) data were calculated as percent maximal expression.Total RNA was isolated from MC3T3 E1 and NeMCO cells incubated for 4 hours with or without HU-308 and MAP kinase inhibitors using the TRI Reagent Kit  followed by a phenol-chloroform phase extraction and isopropyl precipitation. RNA quality was assessed by light absorbance at 260 and 280 nm and by agarose gel electrophoresis and ethidium bromide staining. Real-time RT-PCR analysis for −8 M HU-308 or vehicle according to the manufacturer's instructions. DNA was extracted and post-ChIP RT-PCR carried out using a phospho-CREB antibody. Gels were subjected to computerized densitometry, with negative IgG controls being subtracted, and the values were normalized using internal control for RNA polymerase II immunoprecipitations on the promoter of GAPDH. Primer sequences for GAPDH were forward, TAC TAG CGG TTT TAC GGG CG; reverse, TCG AAC AGG AGC AGA GAG CGA. The primer sequences for cyclin D1 CRE promoter element were forward, TCC CAG TTT GGA GAG AAG CA; reverse, AGA GAT CAA AGC CGG GCA GAG AAA.Chromatin immunoprecipitation assays were carried out 5 hours after a challenge with 10Analysis of variance was employed for statistical analysis. When significant differences were indicated by analysis of variance, group means were compared using the Student-Newman-Keuls test for pairwise comparisons.We have reported previously that the CB2-specific agonist HU-308 stimulat9-tetrahydrocannabinol (THC)., and by Mapkapk2 siRNA  control osteoblast proliferation through the regulation of  by CREB. Therefork2 siRNA D, suggesk2 siRNA D.Mapkapk2 mRNA and protein synthesis. Further downstream, CB2 activation stimulates CREB transcriptional activity and cyclin D1 expression , which, in turn, promote the binding of arrestins to the receptor, resulting in the uncoupling of GPCRs from G proteins.(Based on the reported CB2 signaling in a variety of cell types, and because ERK1/2 and/or p38 mediate a handful of extracellular signals in osteoblasts,,32,33 weproteins.,35 HenceMapkapk2 mRNA and protein and that these events are critical in CB2 mitogenic signaling. We further show that stimulation of the nonphosphorylated Mapkapk2 substrate is associated with a parallel stimulation of the phosphorylated Mapkapk2 product. The increase in activated Mapkapk2 is traceable after a several-hour challenge with a selective CB2 agonist, in line with the requirement for de novo protein synthesis. That Mapkapk2 protein synthesis is essential for the CB2 mitogenic activity is demonstrated by its inhibition using Mapkapk2 siRNA. Although Mapkapk2, also designated MK2,(We have noted previously that unlike the case of many other mitogens such as platelet-derived growth factor and epidermal growth factor, whereby stimulation of DNA synthesis is measurable already after 24 hours,,37 approated MK2, is generated MK2,,40 it waated MK2, Furthermated MK2, Althoughated MK2,,44 That 2.(CREB is one of the best characterized stimulus-induced transcription factors. It activates transcription of target genes in response to a diverse array of stimuli, including peptide hormones and growth factors that activate a variety of protein kinases. In osteo2.,46,47 Mo2. We show 2. CREB-ind2. Downregu2. The inveThis study confirms that Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical for the Gi protein–cyclin D1 mitogenic axis in osteoblasts. These results in osteoblastic cells suggest further studies to assess the in vivo significance of this axis in the skeleton and other tissues alike.


P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood.Mycobacterium tuberculosis complex (MTBC) according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found.In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of  Mycobacterium tuberculosis complex (MTBC) that includes M. tuberculosis, M. bovis, M. bovis BCG (vaccine strain), M. africanum, M. microti, M. canettii, and M. pinnipedii, which produces TB in humans and some animal hosts  motif in TMS6, which allows its classification as PIB-2. In fact, it was recently reported as a Zn2+ P-type ATPase in M. tuberculosis H37Rv [2+ P-type ATPases [This result is expected, because the special reducing conditions inside mycobacteria  are simidictions . CtpD anis H37Rv ; deficieis H37Rv . Finally ATPases ,33.M. tuberculosis is exposed to isoxyl, tetrahydrolipstatine and SRI#9190 antimicrobial compounds, indicating that these pumps might contribute to intrinsic resistance of mycobacteria to antimicrobial drugs [M. tuberculosis[in vitro when M. tuberculosis is incubated in the presence of S-nitrogluthatione GSNO, ethanol, H2O2 and nitric oxide [M. tuberculosis is phagocytized by macrophages [All of the results from this work provide predictive evidence for experimental studies to establish the ion specificity of MTBC P-type ATPases and their role in mycobacterial infection. It has been observed that some P-type ATPases of tubercle bacilli are over-expressed under conditions that mycobacteria face during infection ,34,35. Fal drugs . Two addal drugs ,38; thiserculosis. In addiic oxide . Moreoverophages ,34,35.M. tuberculosis strains H37Ra and H37Rv share identical sequences for P-type ATPases, facilitating subsequent genetic studies using the attenuated strain H37Ra. The large number of HM P-type ATPases expressed by the MTBC strongly suggests that they could be essential for the bacteria to counteract the increased level of HM accumulated by macrophages after infection with tubercle bacilli. Thus, compensatory ion transport strategies could be used by mycobacteria to survive in host cells.Mycobacteria are unicellular organisms that respond to environmental stimuli, and the transport of substances across the plasma membrane could play a fundamental role in their adaptability. Computational analysis shows that each MTBC species has a consistent aggrupation of the 12 P-type ATPases involved in ion transport. In this context, M. tuberculosis has the following three groups of P-type ATPases: HM transporters , AEM transporters  and the KdpB protein, which corresponds to the β subunit of a multimeric K+ ATPase transporter exclusive to prokaryotes. Hydrophobicity analysis identified α-helix type TMS grouped into the following three topological types: type I (HM group), type II (AEM group) and type III (KdpB group). Interestingly, we report a possible mis-annotation for CtpH and CtpI in the TCDB Database, where they are classified as FUPA 24 type with two TMS, unlike the ten TMS identified for these unusually large transporters in this work. Finally, a counterpart of non-catalytic β subunits of Na+/K+ or H+/K+ ATPases does not exist within the MTBC proteomes.The different bioinformatics approaches used in this work to analyze the P-type ATPases identified in the MTBC are in agreement with the initial classification from the HMM search. The results obtained show that There are no conflicts of interest to declare.LN-A, AL-T, MP-R, JC-B, L-M S, DL, LM-R and CY-S wrote the manuscript, validated the tools and carried out the data analysis and interpretation. LN-A, AL-T, MP-R and JC-B contributed to the methodological design, supervised its development and critically revised the manuscript's content. All authors read and approved the final version of the manuscript.


IgE antibodies to gal-α-1,3-gal-β-1,4-GlcNAc  can mediate a novel form of delayed anaphylaxis to red meat. Although IgG antibodies to α-gal  are widely expressed in humans, IgE anti-α-gal is not. We explored the relationship between the IgG and IgE responses to both α-gal and the related blood group B antigen. Contradicting previous reports, antibodies to α-gal were found to be significantly less abundant in individuals with blood group B or AB. Importantly, we established a connection between IgE and IgG responses to α-gal: elevated titers of IgG anti-α-gal were found in IgE-positive subjects. In particular, proportionally more IgG1 anti-α-gal was found in IgE-positive subjects against a background of IgG2 production specific for α-gal. Thus, two types of immune response to α-gal epitopes can be distinguished: a ‘typical’ IgG2 response, presumably in response to gut bacteria, and an ‘atypical’, Th2-like response leading to IgG1 and IgE in addition to IgG2. These results suggest that IgE to a carbohydrate antigen can be formed (probably as part of a glycoprotein or glycolipid) even against a background of bacterial immune stimulation with essentially the same antigen. IgE represents the class of antibodies that mediate hypersensitivity to a variety of allergens. Typically, allergens are proteinaceous in nature and may induce formation of IgG antibodies as well as IgE antibodies. Nevertheless, IgE may also be directed to carbohydrate structures. In particular, so-called cross-reactive carbohydrate determinants (CCD) can be targets for IgE responses The IgG counterpart of IgE anti-α-gal are natural antibodies found in all humans. These antibodies are presumably produced in response to gut microorganisms instead of the usual formation of ‘natural’ anti-α-gal antibodies.It was recently shown that IgE anti-α-gal can be induced by tick bites in certain regions of the USA including Virginia Here we explored the relationship between the IgG and IgE responses to both α-gal and blood group B. We measured IgG and IgG subclasses to α-gal and B in subjects that did or did not produce IgE antibodies to α-gal. We also included the terminal gal-α-1,3-gal disaccharide common to both carbohydrate antigens in the analysis. Our results indicate that the IgE anti-α-gal response is characterized by elevated IgG anti-α-gal, particularly IgG1, against a background of IgG2 production.Sera were obtained under written informed consent from subjects (n = 20) from Virginia that were previously tested positive for IgE to α-gal Gal-α-1,3-Gal-β-1,4-GlcNAc-human serum albumin , Gal-α-1,3-Gal-HSA , and Gal-α-1,3-Gal-β-1,4-GlcNAc trisaccharide (GN334) were obtained from Dextra laboratories, Reading, UK. Horseradish peroxidase (HRP)-labeled monoclonal antihuman IgG (MH-16), IgG1,  IgG3  or IgG4  were obtained from Sanquin, The Netherlands; HRP-labeled mouse monoclonal antibody to human IgG2 (clone HP6002) was obtained from Abcam, Cambridge, UK.3) and incubated overnight on a vertical rotor. After washing five times with PBS-T (PBS supplemented with 0.1% Tween-20 and 0.05% (w/v) NaN3), 125I-labeled anti-IgE was added and after overnight incubation and washing, radioactivity was measured. The results were expressed as arbitrary units as compared with known standards.Serum IgE to α-gal and blood group B were measured by a radioallergosorbent test (RAST) using α-gal-HSA or blood group B-HSA coupled to Sepharose and detected by radiolabeled anti-IgE essentially as described before 2SO4. Absorbance was measured at 450 nm. OD values were normalized to readings of a reference serum that was included on each plate.To measure IgG antibodies to the carbohydrate antigens maxisorp ELISA plates were coated overnight at room temperature with α-gal-HSA (1 µg/ml), gal2-HSA (2 µg/ml), blood group B-HSA (1 µg/ml), or HSA  in PBS. After five times washing with PBS supplemented with 0.02% Tween-20 (PBS-T), plates were incubated for 1 h at room temperature with serum sample (0.03–1 µl/test) diluted in PBS supplemented with 0.02% Tween-20 and 0.1% HSA (PBS-HT) . Plates were washed five times with PBS-T. Then 100 µl of 1 µg/ml antihuman IgG-HRP (MH-16) in PBS-HT was added followed by incubation for 1 h at room temperature. Plates were washed five times with PBS-T, and 100 µl of TMB substrate (100 µg/ml) and 0.003% (v/v) hydrogen peroxide in 0.11 M sodium acetate buffer (pH 5.5) was added to each well. After 10 minutes the reaction was stopped by the addition of 2 M HTo measure IgG subclasses to the carbohydrate antigens maxisorp ELISA plates were coated and incubated with serum as described above. Then 100 µl of antihuman IgG1-HRP (0.1 µg/test), antihuman IgG2-HRP (0.4 µg/test), antihuman IgG3-HRP (0.2 µg/test), or antihuman IgG4-HRP (0.1 µg/test) in PBS-HT was added followed by incubation for 1 h at room temperature, and further developed as described above. In order to obtain approximate values for the relative proportions of the subclasses, OD for the respective subclasses were compared to a standard consisting of monoclonal IgG1, 2, 3, or 4 antibodies titrated onto a coat of an anti-human kappa antibody  and detected with the respective anti-subclass antibody. Relative concentrations of the monoclonal antibodies were based on the same titrations but with anti-human IgG (MH-16) detection.r. Details are indicated in Figure legends.All data analysis was carried out using Graphpad Prism 5. Single comparisons were made using Mann-Whitney’s test; multiple comparisons with Kruskal-Wallis with Dunn’s multiple comparison test; (non-parametric) correlations are expressed as Spearman – individuals  were B–: 0.92 (0.45–1.28) vs B+: 0.21 (0.15–0.42) for α-gal (p = 0.007), B–: 0.95 (0.35–1.59) vs B+: 0.27 (0.23–0.43) for gal2 (p = 0.006), and B–: 1.22 (0.63–1.61) vs B+: 0.18 (0.14–0.29) for B (p = 0.0002). Apparently, expression of the B epitope predisposes to making low titers of anti-α-gal. Antibody responses to α-gal correlate well with anti-gal2 (r = 0.83). Correlation between either anti-α-gal or anti-gal2 with anti-B is much less (resp. r = 0.59 and 0.58). Thus, it appears that the presence of blood group antigen B induces tolerance to the B antigen and generally eliminates most B cell clones that could otherwise have reacted with α-gal, although in individual cases, a B+ subject can still make considerable amounts of IgG anti-α-gal. In B– subjects no tolerance to B develops, but the antibody responses to α-gal and B are nevertheless only poorly quantitatively linked, suggesting partially independent antibody responses to these antigens.To elucidate the relationship between IgG antibody responses to α-gal and blood group B antigens, we first tested sera from healthy volunteers for antibodies to either the α-gal trisaccharide, the terminal disaccharide gal-α-1,3-gal , or blood group B tetrasaccharide, as depicted in ividuals . By cont– subjects that did or did not express IgE antibodies to α-gal . This selection was made based on a radioimmunoassay for IgE to α-gal-HSA  was found between anti-α-gal and anti-gal2, and weak correlations between either anti-α-gal  or anti-gal2  with anti-B (+ group (p<0.0001). Titers are nevertheless correlated (r = 0.76).Antibodies to α-gal and gal2 were also tested at a higher serum dilution for the IgE+ group , and as h anti-B . Further. Approximate proportions of the subclass distributions were calculated by comparison to reference curves as described in Materials and Methods. In most IgE– subjects, the majority of anti-α-gal antibodies are IgG2, as expected  in addition to IgE antibodies Raised IgG anti-α-gal was previously found in a number of conditions, including Chagas’ disease, american leishmaniasis, and malariaCorrelation between binding to α-gal and gal2 was good. It appears that binding to α-gal is largely determined by the terminal galactoses, as has been suggested by others IgG antibodies to blood group antigens A and B are known to be largely IgG2 in most cases. This explains why they usually cause little problems in the unborn fetus: although able to cross the placenta, IgG2 antibodies are poor triggers of the effector functions of the immune system. In this respect, the finding that IgG antibodies to B can be of the IgG1 subclass in certain populations may be of relevance. It remains to be investigated if these antibodies may have any implications during pregnancy.It appears that ‘normal’ exposure to α-gal does not lead to IgE production. At the same time, this established immune response to α-gal appears to essentially offer no protection against induction of IgE following ‘atypical’ exposure to α-gal (presumably helminth or ectoparasites exposure). IgG2 antibodies are weak activators of effector functions and theoretically may serve as ‘blocking antibodies’, similar to IgG4 In summary, the IgE anti-α-gal response is characterized by elevated IgG anti-α-gal, particularly IgG1, and low amounts of IgE anti-B, against a background of IgG2 production. The results indicate that IgE to a carbohydrate epitope can be formed even against a background of bacterial immune stimulation with essentially the same antigen.Figure S1IgE antibodies to α-gal. IgE antibodies to α-gal as measured in a radioimmunoassay of sera from Virginia, USA, originally tested positive (IgE+) or negative  for cetuximab as well as sera from healthy volunteers from North-Holland, The Netherlands, who were all found to be negative for IgE antibodies to α-gal .(EPS)Click here for additional data file.Figure S2+ and IgE– individuals.IgG1 and IgG2 antibodies to α-gal, gal2, and B antigens in IgE Individuals from both Virginia and North-Holland were included. A) IgG1 and B) IgG2. Bars represent median and interquartile ranges. IgE+ individuals make significantly more IgG1 to all three antigens, and significantly more IgG2 to α-gal and gal2 .(EPS)Click here for additional data file.Figure S3IgG subclass distribution to α-gal, gal2, and B in individual sera. Examples of calibration curves shown in upper panels.(EPS)Click here for additional data file.Figure S4Inhibition of IgG antibodies binding to α-gal-HSA coat with Gal-α-1,3-Gal-β-1,4-GlcNAc trisaccharide.(EPS)Click here for additional data file.


Antarctic ice streams are associated with pressurized subglacial meltwater but the role this water plays in the dynamics of the streams is not known. To address this, we present a model of subglacial water flow below ice sheets, and particularly below ice streams. The base-level flow is fed by subglacial melting and is presumed to take the form of a rough-bedded film, in which the ice is supported by larger clasts, but there is a millimetric water film which submerges the smaller particles. A model for the film is given by two coupled partial differential equations, representing mass conservation of water and ice closure. We assume that there is no sediment transport and solve for water film depth and effective pressure. This is coupled to a vertically integrated, higher order model for ice-sheet dynamics. If there is a sufficiently small amount of meltwater produced (e.g. if ice flux is low), the distributed film and ice sheet are stable, whereas for larger amounts of melt the ice–water system can become unstable, and ice streams form spontaneously as a consequence. We show that this can be explained in terms of a multi-valued sliding law, which arises from a simplified, one-dimensional analysis of the coupled model. In particular, while there are many locations in Antarctica where the ice has zero velocity, velocities of up to thousands of metres per year are observed in some coastal areas . In the It is the behaviour of the subglacial hydrological system that governs the effective pressure at the ice–bed interface. There are two contrasting types of subglacial drainage systems beneath ice sheets that are commonly discussed in the literature. If there is little meltwater production, the bed can be effectively drained by a distributed system of cavities ,11 or thIn this work, we are particularly interested in ice streams and their subglacial hydrology. A significant proportion of all ice discharge occurs through ice streams, despite the fact that they are only found over a small portion of an ice sheet. In Antarctica, for example, it is estimated that ice streams cover approximately 10% of the ice-sheet’s surface, but transmit up to 90% of drainage . There iWhile implementing a multi-valued flux law as a boundary condition may produce ice-stream behaviour, using knowledge of the subglacial drainage system beneath Antarctica more directly to explain ice-stream emergence would further our understanding of the system. Evidence from Antarctica suggests that the rapid basal velocities in ice streams are enabled by the presence of a layer of till at the base of the sheet –30. For The paper is organized as follows. In §2, we present the ice-sheet model and derive governing equations for the water film depth and the effective pressure at the bed, before non-dimensionalizing the model in §3. Section 4 considers behaviour of the water layer itself in more detail, specifically considering what happens in the model once the water layer becomes deep enough to submerge all surrounding clasts. In §5, we present numerical solutions to the fully coupled ice-water model, which, under certain conditions, results in ice-stream flow, and we discuss how this can be explained in relation to a multi-valued flux law and the chosen parametrizations in §6.2.(a)−3) allows simplifications to be made to the three-dimensional nonlinear equations, with two end-member models being the shallow ice approximation (SIA) and the shallow shelf approximation (SSA). The former is a classical lubrication approximation, valid for flow frozen to the bed, and so dominated by vertical shear stresses [Given that ice is an incompressible, viscous fluid, an ice sheet moving under gravity is accurately described by Stokes flow. The low aspect ratio of ice sheets We conceive of water flowing at the base of an ice stream as illustrated in x,y,z) where x points in the ice flow direction, y is across-flow and z is vertically upwards. As shown in z=b, the lower ice surface by z=sw and the resulting depth of the water film isz=si. The hydraulic head driving water flow at the ice–till interface ispw is water pressure, pa is atmospheric pressure, ρw and ρi are the densities of water and ice, respectively, and g is gravitational acceleration. Δρwi=ρw−ρi and N=pi−pw is the effective pressure. pi=pa+ρig(si−sw) is the basal ice pressure. Note that . It includes contributions from local heat sources as well as from frictional water dissipation in the subglacial water flow. More specifically,G is the geothermal heat flux, ub is the basal ice velocity, τb is the basal ice stress, qT is the sensible heat flux into the overlying ice and L is the latent heat. It is important to note that the frictional heat source ub⋅τb arises from an integration of the ice viscous dissipation term over the basal sliding region, and τb and ub here refer to conditions near the base of the far-field ice-sheet flow, but far from the actual interface [Given that the water flows in a film of depth nterface ; they arnterface ,55.ηw is the viscosity of water.Assuming a local Poiseuille flow in the water film implies that(c)Γ, is given by . We assume that there is some film depth Hc at which all the clasts would become submerged and the ice would become locally clear of the bed. To be more specific, we will definel0 is a length scale that represents the typical clast spacing in the absence of water, and will be chosen to be consistent with observed effective pressures on the Whillans ice stream (B). We are then left to choose a function for Λ(H). The most simple choice, for illustrative purposes, would be a function such asH reaches Hc, the ice separates from the bed. From (Λ(H), N=0 when H=Hc, implying that once the ice separates from the bed there is no basal stress. In this context, there is then no reason that H cannot increase further locally, with the effective pressure remaining zero. However, in practice, a sheet flow with depth H>Hc is an unstable configuration [N=0 under ice streams; this would only be the case if there were a subglacial lake. We address this model issue by noting that the subglacial flow model has been formulated on the small scale; locally, areas of the bed may become submerged but we expect that on the larger scale there will still be areas of the bed providing support to the ice. More specifically, we presume that, when H approaches Hc, the Walder instability is no longer suppressed, and the system becomes unstable to the formation of some kind of locally channelized drainage system at the ice–till interface [Λ(H) decreases to some small limit as H approaches Hc, and specifically we take Λ(H) to be a function of the formδ are small. The solid black line in N∼Λn1/. This finite limit of Λ(H) therefore now means that the effective pressure does not reach zero in our model. Locally, we expect that there are small areas where the effective pressure is zero and H>Hc, but on the larger scale (over which we are interested in modelling when investigating ice streams) there are regions of finite support, now on a length scale of several metres. Our choice of Λ(H) presented here is meant to represent this globally averaged behaviour in the simplest possible way.There is little to constrain our choice for the functional dependence of ed. From  with τb, to the hydrology, through the general relationshipub= is the sliding velocity of the ice, and p and q are commonly taken as 1/3 [c=6.8×104 Pa2/3 s2/3 m−1/3 so as to give a sliding speed of the order of 60 m yr−1 for typical driving stresses of 104 Pa [It is necessary to prescribe a boundary condition at the interface between the ice and water in order to couple the subglacial hydraulic flow to the ice flow. This is done through a basal friction law, which relates the basal shear stress, n as 1/3 . We furtf 104 Pa .qT, in the melt rate expression . With T=0 on z=b and x has been replaced with the length scale x0. Constants are defined in u0∼100 m yr−1, length x0∼500 km and depth d0∼103 m, Pe=5, and it is larger for ice-streaming speeds. The boundary layer approximation is not, therefore, a highly accurate approximation, but it probably gives a sufficiently accurate representation for the present purpose, and indicates correctly the increase of basal heat flux with basal velocity. Furthermore, the qT boundary layer formula is inappropriate at large x near divides where ub→0 and the x dependence becomes important. The Robin solution which includes vertical but not horizontal advection is often used here, but a better approach uses the von Mises transformation [x dependence of ub. However, we omit such subtleties here.Coupling also occurs through feedbacks of the ice on the drainage system, in particular through the melting caused by basal friction and the creep closure of the ice over the water film. The prescription of the cooling rate, pression  also depormation  which inu1/2b. The frictional heat, however, increases with ub, so, for large ub, Γ x0∼500 km based on length scales in ice-streaming regions, for example the Siple Coast [4 Pa [Qi is either prescribed (from upstream supply) or related to the typical accumulation rate a0 byτ0, u0 and d0. However, as the sliding law is not well constrained, we will use observed values for these scales. Specifically, in the Siple Coast region of Antarctica, typical ice depths are 1000 m [u0∼100 m yr−1, we take the accumulation rate scale asTo make the subglacial water model dimensionless, we guess approximate scales for some variables based on observations/physical intuition, and then use these to derive characteristic scales for other variables, as given in le Coast . We alsost [4 Pa  and chooquations , determie 1000 m . Given ati, and so introduce a non-dimensional parameter δT=tw/ti. Using these scalings, the dimensionless form of the subglacial flow equations becomesΛ(H) is the same function as in σ,ν and χ are all small. We thus neglect the terms of order σ and χ, representing gradients in water depth and melting due to meltwater dissipation, respectively, but retain the term in ν, as it represents a singular perturbation. From  is a decreasing function of H, the essential structure of the model can be described byet al. [Given that n et al.  as a mod(c)et al. [S is the resistive stress tensor [a is the accumulation rate. τ is the deviatoric stress tensor, related to strain rate through Glen’s flow law for ice [We now have a full set of non-dimensional governing equations for the water system. From Kyrke-Smith et al. , the nons tensor ,63,3.26 for ice . The ful for ice .Together with the non-dimensional sliding law as the boundary condition at the water–ice interface,equation , these e4.(a)Hydraulic runaway is a physical phenomenon that models suggest may occur as a result of a positive feedback between ice velocity and melt rate . As ice Γ in .The melting term, Γ in , dependsution of  and  and substituting  which isΓ.We seek to eliminate d N from  to 4.3)u and N fd N from  gives ustituting  into (4.tituting  we have H/∂t≈0 and Λ(H) given by h and water layer depth H. We therefore here take an illustrative Γ function,H reaches Hc. This corresponds to the point at which the bed becomes submerged and the thin film flow becomes a stream. If the bed is sufficiently rough (large Hc) or the ice depth or flux is sufficiently small, then this point would be downstream of the grounding line, and the film flow can exist everywhere.We consider solutions of the decoupled water layer equation, to see its basic state behaviour. We take the ice to have constant surface slope and zero basal slope, as in the simplification given by equation . The couution to  at leadiHc>(2h)1/6 then the solutions evolve into the steady state given by (Hc<(2h)1/6 then the water depth reaches its critical value before the grounding line. The melt rate is no longer defined and an analytic solution does not exist. Our interest therefore now turns to the case where H may reach Hc, by considering the fully coupled water–ice system.We ran a suite of simulations solving  with a mgiven by  x=0 is a cold-temperate transition line with no water at the bed (H=0). The ice depth satisfies ∂h/∂x=0 across the boundary, and the inflow is at a constant velocity into the domain (ub=uin at x=0). Free slip is also allowed along this inflow boundary (τ12=0). To avoid considering grounding-line behaviour, we treat x=1 as an open outflow boundary [u/∂x=0. Furthermore, a suitable condition for the water layer thickness, H, which also avoids the possibility of boundary layers, is ∂H/∂x=0. The domain is taken to be periodic in the y-direction. With these boundary conditions, we solve =(u(x),0). The velocity field u(x) is computed from the x-component of equation (i)Hc). If this is the case, the coupled system evolves into a stable steady state. This is illustrated in Hc=2, corresponding to 6 mm.We first consider the case where the water film does not get thick enough to submerge all surrounding clasts Γ, to decrease. The water depth will start to decrease as a result, therefore decreasing the ice velocity and the total ice flux. This cycle could then repeat once the water depth builds up again to reach the critical depth. In a narrow, confined flow, we might expect this to lead to cyclic surging behaviour . However, there is a long time-scale feedback that was not taken into account; over this longer time scale the ice depth decreases as a result of increased outflow from the domain. This causes the basal stress, and thus milar to ). HoweveHc=1.5, corresponding to a critical water depth of 4.5 mm. It is evident that this small change in the critical water depth results in dramatic changes in the behaviour of both the water layer and the ice, when compared with the results of the previous section. Initially, the flow is stable and uniform cross-stream, but, as more water is generated at the bed, H approaches Hc and an instability emerges, as shown in the plots at t=82 years. There is a sudden increase in water depth near the boundary, which results in an increase in ice velocity, because the ice can slip more easily over the bed. A consequential surface lowering of the ice is also evident in the ice depth field. A non-uniformity in the water depth and the ice velocity develops across the domain at this time, as a result of lateral instability (there has been no imposed perturbation to the system). Patches of faster ice flow form, corresponding to the positioning of the discrete areas of deeper water. There is a coarsening of this instability as the system evolves to create distinct stream-like features with a longer wavelength than the initial instability (t=105 years). The streamlines of the water flow show that water from upstream is drawn towards these water-rich areas, resulting in a positive feedback. These unstable features propagate up the domain, and evolve to create pronounced ice streams, flowing at hundreds of metres per year and of width approximately 60 km (t=360 years). The values are of the same order of magnitude as observed velocity and width values of ice streams in Antarctica [tarctica ,70. A ditarctica  and demotarctica . At thist=360 years are shown in N and H (equation (Plots of effective pressure and melt rate at equation ), we seeequation ).FigureMoreover, the melt rate field illustrates the positive feedback that occurs with more melt being produced underneath the ice streams owing to frictional heating from the rapid velocities. The melt rate is, in fact, the greatest at the onset zone of the ice streams. This is due to the large driving stress here from the gradient in surface elevation, combined with a higher effective pressure, and therefore higher basal stress, which results in a larger melt rate from frictional dissipation. There is therefore enhanced melting at the onset zone—a feedback that will help to maintain ice-stream flow.6.(a)We have seen in the previous section that, under certain parameter combinations, ice flow can become laterally unstable; this leads to two distinct modes of flow—fast ice streams and slow-moving ice. This is similar to the behaviour seen in numerical models that use a triple-valued sliding law as a basal boundary condition ,48. Can si/∂x=−Ω is the ice surface slope, a constant in the downflow direction . This is in contrast to the analysis of the water film in §4a when we were interested in the behaviour of the water film over a short time scale and so assumed the ice was in equilibrium.The water film evolves on a much faster time scale than the ice. As it is the ice flux we are interested in here, we assume now that the water film is in equilibrium, i.e. we takex in this steady-state water case, and integrating  as described by . In this case, the relationship between ice depth and ice velocity is multi-valued, suggesting that distinct velocity states can occur at the same ice thickness, h. In figure 8b, we plot this multi-valued relationship against a rescaled version of the triple-valued sliding law derived in Sayag & Tziperman [We solve this algebraic relationship between h and ub  and plotziperman . In theiziperman ,73. We sziperman . As a reziperman  as the b(b)Hc=1.5 (corresponding to 4.5 mm). We were motivated to plot this as we observed multi-valued behaviour from numerical simulations run with this parameter combination. The dependence of this behaviour on Hc is of interest; in Hc with Hc the relationship is not multi-valued, as Hc increases and the function becomes multi-valued the effect of further increasing Hc is that the turning points of the function occur at larger values of h and ub. This is because more meltwater is required for the water film depth to come close to the critical depth; a larger ice depth results in a larger driving stress that in turn causes more frictional heating. For larger values of Hc  that all the melt can flow in the thin film configuration. However, reducing the critical water depth (so that H reaches Hc) results in the ice flow becoming laterally unstable and non-uniform in the cross-flow direction; ice-stream-like features develop.Numerical solutions of the fully coupled ice–water system result in distinct flow regimes dependent on whether the water depth reaches the critical depth within the domain. Under the model set-up considered in this paper, the system evolves into a uniform steady state if the bed is sufficiently rough (i.e. δ and Hc in this paper) do not result in triple-valued flux behaviour at all. In these cases, under no circumstances would we expect a lateral instability in the system to occur.A basic analysis of the coupled system suggests that the hydrological system can result in a triple-valued flux relationship for the ice. It has previously been shown that a triple-valued sliding law is associated with ice-stream behaviour and such multi-valued flux laws have therefore been used to investigate ice-stream dynamics ,48,73. BWhile this paper presents theory and intriguing results associated with a new coupling between physically reasonable models of ice and hydrology, a more detailed investigation of the coupled model behaviour is needed. Future work will provide a more comprehensive parameter study, as well as analysing in detail the stress balances that occur across emergent ice streams, in a bid to explain their spacing. Furthermore, an important limitation of the current model is that it neglects energy conservation. The model instead assumes that the bed is always at the melting point. While this might not be an unfair assumption in the region of ice streams, other work with thermodynamically coupled ice dynamics has shown that fast flow may begin owing to purely thermomechanical feedbacks ,74,75. Iwww.macsi.ul.ie) funded by the Science Foundation Ireland grant no. 12/1A/1683.This work was supported by the Natural Environment Research Council (NE/I528485/1). R.F.K. is grateful to the Leverhulme Trust for its support. A.C.F. acknowledges the support of the Mathematics Applications Consortium for Science and Industry (


Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site. Understanding the relationship of enzyme conformational flexibility with its stability and activity is a very active area of research. Many studies comparing conformational flexibility of homologous thermophilic-mesophilic enzyme pairs, using various techniques like fluorescence quenching Bacillus subtilis, we created a very thermostable mutant named “6B” para-nitrophenyl acetate (PNPA) and para-nitrophenyl butyrate (PNPB). Comparative details of catalytic parameters of the two enzymes are given in By performing multiple rounds of directed evolution and mutation recombination on a lipase “lipA” from mesophilic bacterium et al. first solved the crystal structure of wild type B. subtilis lipase and identified the active site residues B. subtilis lipase follows two steps reaction, acylation and deacylation . To probe the flexibility of two molecules, data from all the three simulations were combined and the root mean square fluctuation (RMSF) for Cα atoms for all residues were compared for 2–20 ns MD runs  belongs to the global tumbling of protein molecule, hence was similar for both proteins (∼8.9 ns). However, faster rotational correlation time (фfast), representing sum of motional freedom of probe with respect to protein and segmental flexibility of local site (active site in present case), was different in two proteins. Its value is ∼0.21 ns in wild type while ∼4.05 ns in 6B. Lower value is due to faster depolarization which in turn represents higher flexibility of the local site. Furthermore, the amplitude associated with the faster correlation time is significantly smaller in 6B when compared to wild type indicating increased rigidity in 6B. Acrylodan anisotropic decay corroborates the MD simulation studies that active site of 6B lipase may be more rigid than wild type.We further probed the active site dynamics of wild type and 6B lipases by time-resolved fluorescence anisotropy measurement. This method has been widely used to probe the flexibility of macromolecules Our results from MD simulation and time-resolved fluorescence anisotropy decays suggest that active site of 6B lipase is more rigid than wild type lipase. This is in accordance with the prevailing idea of conformational stabilization being associated with increase in conformational rigidity. However, how a rigid active site can be capable of supporting higher enzyme activity? In any enzymatic reaction, even the simplest one substrate–one product ones, substrate goes through multiple geometric as well as electrostatic rearrangements during its chemical transformations. Correspondingly, for efficient catalysis, reorganization of enzyme active site is necessary so that it can change its shape to have complementarities with transforming chemical entity. Compelling need of dynamic fluctuations or flexibility for enzyme catalysis often originates from the simple fact that it bestows enzymes the capability of active site reorganization and allows it change its conformation during catalysis. However, relative level of active site reorganization during enzyme catalysis was shown to vary depending upon enzymes et al.B. subtilis lipase), using the quantum mechanical and molecular mechanical techniques, has shown that these enzymes require minimal reorganization in active site during catalysis. The active sites in these enzymes were found to have pre-organized geometry that largely minimizes conformational reorganization during catalysis. We compared the crystal structures of wild type B. sublilis lipase in free form and in complex with a phosphonate inhibitor, an analog of 1st tetrahedral transition state et al.As evident from Numerous studies have attempted to uncover the possible relationship of conformational dynamics to enzyme activity. Various techniques such as NMR, MD simulation and single-molecule fluorescence were used to probe dynamics of many enzymes like cyclophilin A, adenylate kinase, peptidylprolyl isomerase catalytic domain, dihydrofolate reductase and flavin reductase B. subtilis lipase suggest that increasing active site rigidity not necessarily decrease enzyme activity, rather as presented here, can be associated with increase in enzyme activity. It should be noted that in earlier reports of engineered enzymes which were both more stable and comparable/more active at room temperature than their parent molecule, most of the mutations were spread over regions of molecules which were away from active site B. subtilis lipase directly probed the dynamics of active site argues that stable version of lipase “6B” is not only structurally more stable and active than wild type protein, but its active site is also more rigid as postulated based on static crystal structures.Despite of conceptual ambiguity and contradictory reports on role of dynamics in enzyme catalysis, prevailing notion is for efficient catalysis enzymes need to acquire structures that are compatible with binding to cofactor, substrate and its various forms while undergoing chemical transformation in the active site. And conformational dynamics or flexibility plays the vital role of a lubricant helping enzymes to do so. This implies that decreasing flexibility or increasing rigidity decreases the enzyme activity, as shown previously by many studies In conclusion, our study provides the evidence that positive correlation between conformational flexibility and enzyme activity need not be stringent but active site rigidity and enzyme activity can be simultaneously increased. Interestingly, Gutteridge and Thornton All the lipase variants were over-expressed and purified following reported methodologies MD simulations and analysis were performed using GROMACS simulation package 391 in DMF = 20,000 M−1.cm−1) for >2 h in 50 mM sodium phosphate buffer (pH 7.2) at room temperature followed by extensive buffer exchange with same buffer in centrifugal filtering device . Percentage acrylodan labeling was calculated by measuring concentration of acrylodan  and total protein. >80% acrylodan labeling was achieved.50 µM (∼1 mg/ml) of S77C mutants of both wild type and 6B lipases was incubated with 250 µM of acrylodan (RIII) and 90° (I⊥).Time-resolved fluorescence experiments were performed using a Ti-Sapphire picoseconds laser and time-correlated single-photon counting setup, coupled to a micro-channel plate photomultiplier tube as described earlier I(t) is the fluorescence intensity at time t and αi is the amplitude of the ith lifetime iτ such that Σαi = 1. The mean lifetime mτ = Σαiτi.Time-resolved fluorescence intensity decays were analyzed by de-convolution of observed sample decays with the IRF to achieve the intensity decay function represented by,III (t) and I⊥ (t) asIII (t) and I⊥(t) are fluorescence intensity when emission polarizer was oriented at 0° (III) and 90° (I⊥) to the excitation beam. r(t), the anisotropic decay function was analyzed as a sum of two exponential terms,0 is the intrinsic (time zero) fluorescence anisotropy. φfast and φslow are fast and slow rotational correlation times associated with amplitudes βfast and βslow respectively such that βfast+βslow = 1.Time-resolved anisotropy decays were analyzed by globally fitting Text S1Information on time-resolved fluorescence intensity decay profiles of acrylodan and time-resolved fluorescence intensity as well as anisotropy decay of tryptophans.(DOC)Click here for additional data file.Figure S1Location of active site residues and mutations. Stereo figure for (TIF)Click here for additional data file.Figure S2A) RMSD of Cα atoms of wild type and 6B lipases from their energy minimized crystal structures in two simulations as a function of MD simulation time. (B) RMSF of all atoms of individual residue in 2–20 ns MD simulation time . Spheres denote active site residues.Active site dynamics by MD simulation. ((TIF)Click here for additional data file.Figure S3Far UC CD spectra of acrylodan labeled S77C mutant in wild type background and wild type lipase. Spectra were recorded in 0.1 cm pathlenght cuvette for 0.1 mg/ml proteins in 50 mM sodium phosphate buffer (pH 7.2) using a JASCO J-815 specropolarimeter. All reported spectra are an average of four accumulations. Wavelength scans were carried out in the Ellipticity mode at a scan speed of 50 nm/min, bandpass of 2 nm, at response time of 2 s and wavelength step of 0.5 nm. All spectra were corrected for buffer base line by subtracting the respective blank spectra recorded identically without the protein.(TIF)Click here for additional data file.Figure S4Typical time-resolved fluorescence intensity decay profiles of acrylodan attached to C77 in wild type and 6B lipase background.(TIF)Click here for additional data file.Figure S5A) intensity decay and (B) anisotropic decay profiles of tryptophans in wild type and 6B lipase.Time-resolved fluorescence of tryptophans. Typical time-resolved fluorescence ((TIF)Click here for additional data file.Figure S6Structural overlap of active site in free and transition state analog bound crystal structure of wild type lipase. Stereo figure for (TIF)Click here for additional data file.Figure S7Active site geometry during MD simulation. Frequency distribution of RMSD of catalytically important atoms  between MD structural snapshots (2–20 ns) of wild type and 6B lipase and respective free enzyme crystal structures .(TIF)Click here for additional data file.Table S1Parameters associated with time-resolved fluorescence measurements of acrylodan and tryptophans.(DOC)Click here for additional data file.


The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1.L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity.α-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL.In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with GThese results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction. Impaired insulin-stimulated glucose transport by glucose transporter 4 (GLUT4) is a well-documented contributor to the reduced glucose clearance found in subjects with type 2 diabetes mellitus (T2DM) α-interacting protein-interacting protein, C-terminus (GIPC1), a PDZ binding protein, resulted in a reduction of cell surface GLUT1 in epithelial cells Recent studies have shown that the carboxy terminal of GLUT1 is a key regulator of GLUT1 subcellular localization, trafficking, and activity ATM is a phosphatidylinositol-3-kinase (PI3K) family serine/threonine protein kinase that has been shown to play a role in regulation of glucose transport in cultured cells 3H-2-deoxyglucose, 14C-mannitol, and 14C-ascorbic acid, were purchased from American Radiolabeled Chemicals, Inc. . Antibodies against phosphorylated ATM substrates and GAPDH were purchased from Cell Signaling Technology, Inc. . The anti-FLAG and anti-tubulin antibodies were purchased from Sigma-Aldrich Corp. . The anti-stomatin antibody was purchased from Abnova . The anti-GIPC1 antibody was purchased from Thermo Scientific . The GLUT1 antibody was a generous gift from Michael Mueckler of Washington University . Doxorubicin was purchased from Sigma-Aldrich . The ATM inhibitor KU55933 (KU) Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline, and trypsin were purchased from Sigma Aldrich . The radiolabeled chemicals, Mice that were heterozygous for a truncation mutation of ATM that codes for ATM protein lacking the catalytic domain All procedures using live animals were approved by Saint Louis University’s Institutional Animal Care and Use Committee .2. For some experiments, myoblasts were differentiated into myotubes in DMEM containing 2% horse serum.L6 myoblasts, a cell line derived from rat skeletal muscle, were obtained from the American Type Culture Collection . L6 myoblasts were cultured in DMEM containing 10% FetalPlex  and 1% antibiotic-antimycotic solution at 37°C in 5% COEscherichia coli and purified using Qiagen Miniprep Kits. L6 myoblasts, having increased transfection efficiency and lowered GLUT3 expression compared to myotubes The FLAG-GLUT1 construct 4, 100 mM NaF, 2 mM NaVO4, 10 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were centrifuged at 13,000 rpm for 10 minutes at 4°C, and supernatants were assayed for protein content by the bicinchoninic acid (BCA) method. Samples in Laemmli sample buffer plus dithiothreitol were run on 4–20% pre-cast polyacrylamide electrophoresis gels  and transferred to nitrocellulose membranes. For analyses of ATM and P-ATM, samples were run on 3–8% Tris-acetate gels (Invitrogen). The membranes were then blocked with 5% nonfat milk in Tris-buffered saline plus Tween (TBST), incubated in primary antibodies, incubated with horseradish peroxidase-conjugated secondary antibodies, and then detected using enhanced chemiluminescence . Bands were quantified with TotalLab software .L6 myoblasts were transiently transfected with FLAG-GLUT1 24–48 hours prior to experiments, serum starved for 3 hours in serum free media, washed with ice-cold PBS, incubated with 6 µM CP for 30 minutes or 1 µM DXR for 60 min, washed with ice-cold PBS, and scraped in lysis buffer , incubated with horseradish peroxidase-linked secondary antibodies, and subjected to spectrophotometric analysis at 492 nm with o-phenylenediamine as the substrate for generation of yellow-orange product. For the GLUT1 internalization assay, cells were incubated on ice with anti-FLAG antibodies, and then cell surface GLUT1 levels were assessed periodically after re-warming the cells to 37°C. For assessment of GLUT1 externalization, cells were allowed to internalize GLUT1/antibody complexes for two hours at 37°C, and then GLUT1 cell surface levels were evaluated over time after incubation at room temperature to facilitate externalization.The localization and trafficking assays performed were adapted from Wijesekara et al L6 myoblasts transiently transfected with FLAG-GLUT1 constructs were treated (plus/minus ATM inhibitor/activator) for one hour. Cell lysates were incubated with FLAG antibodies overnight at 4°C, and then protein A sepharose beads were added to the mixture and incubated for 4 hours at 4°C. The samples were microcentrifuged, washed five times with lysis buffer, and resuspended with 4x Laemmli sample buffer plus dithiothreitol. The samples were then subjected to SDS-PAGE western blot analysis.4, 1 mM CaCl2) for 30 min in the presence or absence of ATM inhibitors/activators, washed several times with glucose-free HBS, and exposed to 2-deoxy-glucose (2DG) transport media  or DHA transport media  L6 myoblasts were serum starved for 3 hours, incubated in HEPES-Buffered Saline  was used to anesthetize the animals. Leg muscles were excised and then incubated in Krebs Henseleit bicarbonate buffer (KHB)  for one hour. Glucose transport. Muscles were washed with KHB containing 40 mM mannitol and 0.1% BSA for 10 minutes at 30°C, incubated in 2DG transport media  for 10 minutes at 30°C, blotted, and stored at −80°C for further analysis. 14C-Mannitol was used to measure extracellular space. All incubations contained 100 µM indinavir to inhibit GLUT4 activity DHA transport. Muscles were incubated in DHA transport media  for 10 min at 30°C, washed twice with a 200 µM DHA rinse  for 5 minutes on ice (in order to prevent DHA efflux), blotted, and stored at −80°C for further analysis.Transport in skeletal muscle was studied as described previously Muscle processing. Muscle samples were ground in lysis buffer (described above) and spun for 10 minutes at 13,000 RPM. Sample supernatant was used for scintillation counts. BCA protein assays were performed to determine protein concentrations. Scintillation counting. Sample or incubation media was added to scintillation vials containing Ultima Gold scintillation fluid . Samples were processed using the Tri-Carb 3110 TR Liquid Scintillation Analyzer .In order to account for non-specific uptake, a subset of the muscles and cells were incubated in the presence of 10 µM cytochalasin B. Cytochalasin B inhibits transport by GLUTs. Glucose and DHA transport rates in myoblasts and in skeletal muscle were adjusted by subtraction of the transport rates found for myoblasts or muscles incubated in the presence of cytochalasin B. Findings were analyzed using univariate one-way ANOVA with significance determined as p<0.05.Glucose transport in skeletal muscle is predominantly mediated by GLUT1 and GLUT4, with GLUT1 being responsible for physiologically-relevant portion of basal glucose transport To follow up on the results described above, we determined the effects of ATM inhibition on glucose and DHA transport, GLUT1 localization, and GLUT1 trafficking. Myoblasts, having reduced GLUT3 and GLUT4 levels compared to myotubes L6 myoblasts transfected with an exo-facially tagged FLAG-GLUT1 construct and treated with ATM inhibitors KU and CP exhibited decreased GLUT1 cell surface localization  . The difTo discover the means by which ATM inhibition causes a reduction of membrane-localized GLUT1, L6 myoblasts were subjected to GLUT1 internalization and externalization assays and Western analyses for GLUT1 protein levels. CP-treated myoblasts displayed the greatest reduction in cell surface GLUT1 and therefore were used for the subsequent studies. Nearly all cell surface GLUT1 was internalized by 10 minutes in the absence or presence of the ATM inhibitor, CP. The fraction of GLUT1 remaining at the cell surface was the same for ATM inhibitor treated and control cells . TherefoThe carboxy terminus of GLUT1 contains a PDZ binding motif which includes a known (S490) ATM phosphorylation site L6 cells express GLUT4 and GLUT3 in addition to GLUT1. While the role of GLUT4 has been minimized by the use of indinavir, it remains possible that GLUT3 could mediate some of the changes observed in glucose or DHA transport. If GLUT3 were to have a substantial role in basal glucose transport and DHA transport in the myoblasts, and a treatment (e.g. CP) were to affect transport specifically by modulation of GLUT3-mediated transport, then cells transfected with FLAG-GLUT1 would have a lesser sensitivity to CP in regard to transport when compared with non-transfected cells. However, as shown in We hypothesized that activation of ATM would lead to augmented GLUT1 transport activity and increased cell surface GLUT1. To address this hypothesis we incubated myoblasts with the ATM activator, doxorubicin (DXR). DXR has been shown to increase phosphorylated S1981 ATM (P-ATM) levels in mouse embryonic fibroblasts (MEF) To assess the specificity of the P-ATM Sub antibody, whole cell lysates from L6 myoblasts that were treated plus or minus 6 µM CP and/or 1 µM DXR underwent Western blot analysis probing for P-ATM Sub. Because GLUT1 has a molecular weight of 55 kiloDaltons (kDa), we focused on the P-ATM Sub signal around this molecular weight. CP decreased P-ATM Sub reactivity at the band adjacent to the 55 kilodalton (kDa) molecular weight marker (MWM) p<0.05, . DXR incDXR caused a 3-fold increase in DHA transport p<0.05, . The DXRThe ATM activator DXR increased FLAG-immunoprecipitate reactivity with phosphorylated ATM substrate antibodies that corresponded directly with the FLAG-GLUT1 band, suggesting that GLUT1 is an in vivo ATM substrate. DXR also augmented GLUT1-associated GIPC1 by 34% p<0.05; . The GLUPrevious studies have indicated the importance of the GLUT1 carboxy terminus in the regulation of the transporter To examine the role of GLUT1 S490 in GLUT1 transport, a glucose transport assay was performed in the presence of GLUT4 inhibitor, indinavir. As shown in FLAG immunoprecipitates from L6 myoblasts expressing FLAG-GLUT1-S490A demonstrated decreased reactivity with antibodies against phosphorylated ATM substrates, corresponding directly with the FLAG-GLUT1 band . The GLUwww.phosphosite.org) In As shown in To determine whether ATM inhibition decreases the phosphorylation of GLUT1-S490, wild-type and GLUT1-S490A L6 myoblasts incubated in DMSO or 6 µM CP were harvested, immunoprecipitated with antibodies against FLAG, and then subjected to Western Blot analysis. As shown in The data presented thus far have shown that acute ATM inhibition or activation affects GLUT1 regulation in cultured muscle cells. To determine the effects of chronic ATM loss on GLUT1-mediated transport, glucose and DHA transport studies were performed using a transgenic mouse line expressing truncated, non-functional ATM. Cytochalasin B, which eliminates GLUT-mediated transport, essentially completely prevented DHA and glucose uptake in skeletal muscle (data not shown). ATM mutant soleus and extensor digitorum longus (EDL) muscles exhibited diminished DHA transport compared to wild type control muscles  and the serine/threonine protein kinase, ATM.The last four amino acids of the 492 residue GLUT1 protein form the PDZ binding motif (DSQV). By mutations of those four amino acids and the short hairpin RNA knock down of Gα-interacting protein-interacting protein, c-terminus (GIPC1), a protein found to interact with those four amino acids, one study discovered that the PDZ binding motif plays a role in the regulation of GLUT1 transport, trafficking, and protein levels via GIPC1 by promoting cell surface retention of GLUT1 and protecting it against lysosomal degradation http://icb.med.cornell.edu/services/pdz/start) The c-terminus of GLUT1, but not GLUT3 or GLUT4, interacts with GIPC1 Truncation and/or alteration of the GLUT1 c-terminus has been shown to result in an internal localization www.phosphosite.org) Based on GLUT1 structure models www.phosphosite.org, It has been established that ATM plays a role in regulation of insulin-stimulated (i.e. predominantly GLUT4-mediated) glucose uptake in muscle cells and skeletal muscle. For example, expression of kinase-dead ATM prevents the insulin-stimulated increase of cell surface GLUT4 in L6 myoblasts A recent study using Clone 9 cells, a rat liver cell with GLUT1 as its only GLUT, demonstrated that overexpression of murine and human stomatin, an integral membrane protein found in many tissues, decreased glucose transport Interestingly, effects of ATM activation, ATM inhibition, and expression of GLUT site mutants affected transport to a greater degree than cell surface GLUT1. This suggests that ATM and GLUT1-S490 regulate GLUT1 intrinsic activity in addition to cell surface localization. Considering that acute ATM inhibition/activation is sufficient to cause regulatory effects on GLUT1 cell surface levels and GLUT1 transport, processes such as GLUT1 synthesis are unlikely to be the cause of the rapid regulatory effects seen here. GLUT1 protein degradation may be involved in these regulatory effects In haemopoietic cells, cytokines, interleukin-3 and -7 (IL-3 and IL-7), and growth factors regulate GLUT1 protein levels and cell surface appearance in an Akt-dependent fashion Understanding the roles and mechanisms of GLUT1 regulation has implications in several medically relevant areas. GLUT4 knockout mice, which were insulin resistant but not diabetic, displayed increased levels of cardiac GLUT1, suggesting that GLUT1 may compensate for lack of insulin-stimulated GLUT4-mediated transport The present study examined several factors involved in the regulation of GLUT1 in cultured muscle and skeletal muscle. Three GLUT isoforms are expressed in L6 myoblasts: GLUT1, GLUT3, and GLUT4 In summary, ATM and GLUT1-S490 appear to support GLUT1-mediated transport in skeletal muscle cells by promoting GLUT1 trafficking to the plasma membrane, associated with increased interaction of GLUT1 with GIPC1.


The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.The release of cytochrome in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.We used a rat model of cardiac arrest as an c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.We conclude that δPKC initiates cytochrome  We next tested two mechanistic hypotheses whereby δPKC may mediate cytochrome c release by: 1) phosphorylation/activation of PLSCR3 leading to targeting of t-Bid and downstream cytochrome c release, and 2) activation of δPKC phosphorylates PP2A leading to de-phosphorylation of pro-apoptotic factors Bad and Bax. Finally, we used proteomics to identify additional mitochondrial targets of δPKC which may be phosphorylated upon its translocation.The goal of the present study was to first test the hypothesis that δPKC translocation/activation after cerebral ischemia could result in cytochrome c is released at 1 h of reperfusion. Therefore, we examined δPKC translocation to hippocampal mitochondria at this time p<0.05) as compared to sham-operated animals  and compared it with control (no ischemia) synaptosomes (p<0.05). This increase in δPKC translocation was reversed by 85%  upon OGD induction in the presence of δV1-1. Thus, δPKC translocates to the mitochondria during the first hour of reperfusion following in vivo and in vitro hippocampal neuronal ischemia. The translocation was abolished in vitro using δV1-1.We first tested the hypothesis that cerebral ischemia results in δPKC translocation from the cytosol to the mitochondria. Previously, we had observed in this model of cardiac arrest (CA) that cytochrome  animals . To testptosomes . OGD in c release plateaus, we next sought to determine if inhibition of translocation prevents cytochrome c release. Rat hippocampal synaptosomes underwent 60 min of OGD in the presence of δV1-1 or tat peptide with subsequent measurements of cytosolic cytochrome c. These levels were normalized to the cytosolic cytochrome c levels of control synaptosomes incubated in glucose containing solution with room air insufflation. OGD of the synaptosomes in the presence of tat peptide resulted in a 268% increase in cytochrome c release from the mitochondria (n = 5) as compared to control. Inhibition of δPKC translocation (via δV1-1) resulted in cytochrome c release similar to control and significantly less than untreated OGD synaptosomes  and calyculin (10 nM), which are potent inhibitors of both PP1 and PP2A i of okadaic acid is 0.1 nM for PP2A vs. 10 nM for PP1, making it more specific for PP2A, whereas the Ki of calyculin is 10-fold higher for PP1 as compared to PP2A c release by 52%  (c release by an insignificant 15% (n = 5) (c release most likely via dephosphorylation of downstream targets.δPKC activates PP2A via phosphorylation  p<0.05)  as compa (n = 5)  as compac release in vivo resulted from δPKC induced phosphorylation/activation of PP2A, we tested the impact of synaptosomal OGD in the presence of either δV1-1 or the PP2A inhibitor okadaic acid (0.1 nM). Both inhibitors (δV1-1 and okadaic acid) decreased OGD-induced Bad de-phosphorylation (i.e. led to increased phosphorylation) by 170%  and 101% (n = 5), respectively as compared to OGD in the presence of tat peptide group , we are limited in our ability to extrapolate our findings to the whole brain.In the present study, we used asphyxial CA in rats to model cerebral ischemia and demonstrated at each step that in vivo and control synaptosomes in vitro. Oxidative stress-induced translocation of δPKC to mitochondria has been shown to be associated with loss of mitochondrial membrane potential and mitochondrial cytochrome c release cc was released at 1 h of reperfusion following 8 min of cardiac arrest c release.In earlier studies we reported that δPKC is activated (translocated from soluble to particulate fraction) following cerebral ischemia c release was prevented by inhibition of δPKC, we explored possible pathways by which cerebral ischemia-induced δPKC activation led to the release of cytochrome c. First we determined whether δPKC exerted its effect by direct interaction with mitochondrial targets. Our first candidate was the phospholipid scramblase – 3 (PLSCR3), a known mitochondrial target of δPKC phosphorylation c is bound to the outer surface of the inner mitochondrial membrane by associating with cardiolipin de novo biosynthesis c interaction is a critical factor determining the amount of cytochrome c release during apoptotic stimuli c interaction leading to cytochrome c release.Since we observed that OGD-induced cytochrome in vivo cerebral ischemia. However, in our synaptosomal model, we were unable to block OGD-induced increase in PLSCR3 phosphorylation by δV1-1 (δPKC-specific inhibitor peptide) at the same dose that inhibited cytochrome c release. Although previous studies concluded that PLSCR3 can be phosphorylated by δPKC; however, these studies used either general PKC activators (phorbol ester) or CMV promoter-driven δPKC overexpression  in vivo but that δPKC inhibition does not block this process based on our in vitro data. Limitations exist when extrapolating an in vitro model results to in vivo findings, even when the in vitro model correlates well with the in vivo findings as we have already demonstrated.We observed that PLSCR3 phosphorylation was indeed increased following c release was independent of mitochondrial PLSCR3 phosphorylation, we next examined other mitochondrial targets for δPKC phosphorylation. We demonstrated that the phospho-protein signal of three protein bands were increased in mitochondria isolated from synaptosomes treated with ψδRACK. Proteomics analyses of these bands revealed the presence of the following phosphoproteins: ubiquinol cytochrome c reductase core protein 2, Fe-S protein 2, 3 and flavoprotein 2 of NADH dehydrogenase, creatine kinase, beta subunit of succinate-coenzyme A ligase, E1 alpha form 1 subunit of pyruvate dehydrogenase (PDH), alpha and beta subunits of ATP synthase, citrate synthase, and prohibitin Because δPKC-induced cytochrome in vivo models of cardiac ischemia/reperfusion demonstrated that δPKC activation (translocation to mitochondria) was responsible for decreased pyruvate dehydrogenase (PDH) activity following ischemia/reperfusion An earlier study using Our results suggest that prohibitin could be one of the mitochondrial substrates for δPKC c release and apoptosis are also modulated by proteins located outside the mitochondrion. A prime example are the bcl-2 family proteins such as Bad and Bax whose activation and mitochondrial translocation are shown to induce cytochrome c release following apoptotic stimuli after cerebral ischemia c release by inhibiting δPKC or PP2A but not PP1 during ischemia. The slight inhibition observed with calyculin may be the result of PP2A effects, although 10-fold less than PP1. Our findings, therefore, support our proposed mechanism that δPKC activation via phosphorylation of PP2A results in cytochrome c release through the dephosphorylation of Bad.Mitochondrial cytochrome c release. δPKC activation following cerebral ischemia led to the release of mitochondrial cytochrome c via the PP2A – Bad pathway.In conclusion, our study demonstrated that δPKC translocated to mitochondria following cerebral ischemia/OGD. Post-ischemic activation of δPKC was not responsible for increased PLSCR3 phosphorylation but may target other mitochondrial proteins resulting in mitochondrial dysfunction and/or cytochrome All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and approved by the Animal Care and Use Committee of the University of Miami (protocol # 09-050). For experiments with hippocampus from naïve animals, male Sprague Dawley rats weighing 250–300 g were used. They were sacrificed under isoflurane anesthesia and the hippocampi were dissected out for further analyses. Asphyxial cardiac arrest was induced as described earlier in vitro model of ischemia) was induced by incubating synaptosomes in glucose deprived medium as described earlier c. We used 60 min of OGD for all experiments since cytochrome c release reached plateau at 60 min . At the end of 60 min of OGD, mitochondria were isolated from synaptosomes using a nitrogen bomb Hippocampal mitochondria were isolated as described earlier with minor modifications Inhibition of δPKC was induced by pre-incubating synaptosomes with either tat carrier peptide or δV1-1 (δPKC inhibitor)  for 15 min at room temperature  c , mouse monoclonal anti-cytochrome c oxidase subunit IV , or mouse monoclonal anti-β-actin  antibodies and species specific secondary antibodies. The digitized immunoblots were subjected to densitometric analyses Immunoprecipitation was carried out using rabbit polyclonal anti-PLS3  or rabbit polyclonal anti- Bax  and protein A sepharose beads  as per manufacturer's instructions. The resulting immunoprecipitate was used for immunoblotting (see below) with subsequent probing using mouse monoclonal anti-phosphothreonine  with normalization to total protein levels using the primary antibody. Antibodies used were rabbit polyclonal anti-δPKC , mouse monoclonal anti-cytochrome in situ with sequencing-grade trypsin . Samples were then processed for protein identification as described earlier Proteins were separated from mitochondrial samples on a 4–20% acrylamide gel . Phosphoproteins in the gradient gel were identified using Pro-Q Diamond Phosphoprotein Gel Stain  as per manufacturer's instructions and subsequently stained with Coomassie blue  to confirm equal protein loading. For protein identification, gel slices were excised and digested p<0.05 considered significant.The results are expressed as mean ± SEM. Statistical significance was determined with Student's t-test when there were two experimental groups. For more than two groups, statistical evaluation of the data was performed using ANOVA test, followed by Dunnett's post hoc test with