Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations — G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme . This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking “semireverse” electron transfer from heme bL to quinone. Here we examine another mutation from that group — G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that “pushing” ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the “pushing” effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS.One of the important elements of operation of cytochrome This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi. •b-ISP interface.Several mitochondrial mutations are located at the cytochrome •We compare molecular effects of two mutations from that group.•o catalytic site.In both mutants ISP is shifted away from the Q•This effect is generally associated with increased ROS production.•More active mutants are more protected against elevated ROS. Its function is related to electron transfer from quinol to cytochrome c and the translocation of protons across the membrane. Communication between these two pools of electron carriers is important for energetic efficiency of ETC Cytochrome bc1o site) is connected with cytochrome c reduction site by two cofactors: Rieske cluster and heme c1, embedded in ISP and cytochrome c1, respectively. Movement of the head domain of iron–sulfur protein (ISP-HD) between a site close to the Qo site (Qo position) and a site on the cytochrome c1 interface (c1 position) allows transfer of electrons via this chain o site is connected with quinone reduction site (Qi site) by heme bL and heme bH embedded in cytochrome b. Completion of the Q cycle requires two quinol molecules to be oxidized at the Qo site. After oxidation of first molecule of quinol at the Qo site one electron is transferred via high-potential c-chain and is used to reduce one molecule of cytochrome c. The second electron derived from oxidation of first molecule of quinol is transferred via low-potential b-chain and is used to reduce quinone to semiquinone at the Qi site. Electrons derived from oxidation of second quinol molecule are used to reduce one molecule of cytochrome c at the cytochrome c reduction site and to reduce semiquinone to quinol at the Qi site. In addition, a cross-dimer electron transfer at the level of two hemes bL is possible In the catalytic Q cycle of cytochrome bc1 complex is composed of 11 subunits b is encoded by mitochondrial DNA, whereas all the other subunits are of nuclear origin. Numerous cytochrome b mutations identified so far are related with diseases such as exercise intolerance, miopathy, cardiomiopathy and neuropathies b mutations, bacterial Each monomer of the human cytochrome Rhodobacter capsulatus bacterial model to examine the molecular effects of mitochondrial mutation G290D, which was identified in patient suffering from progressive exercise intolerance bc1 and decreased amounts of some subunits: cytochrome b and ISP among others, while amounts of cytochrome c1 were normal b is highly conserved among the species. It is localised in the transmembrane helix F in the proximity of the Qo catalytic site but also in close contact with ISP-HD at the Qo position  and additional negative charge. It thus might perturb the structure of the Qo site pocket and/or affect the interaction of ISP-HD with cytochrome b, which could influence the overall motion of ISP-HD. Recently, we found that another mitochondrial disease-related mutation G167P, located as G332D at the cytochrome b-ISP-HD interface, did affect the motion of ISP-HD which, among other effects, led to increased levels of superoxide production at the Qo site In the present study we have used the position . The mut22.1R. capsulatus containing the substitution of glycine to aspartate in position 332 of cytochrome b subunit were obtained as follows. Mutation was introduced in the petB gene (coding for cytochrome b) using the QuikChange site-directed mutagenesis system (Stratagene) and the following PCR primers (the changed nucleotide in the underlined triplet is in bold): G332D_F: 5′-GATGCGAAGTTCTTCACGGTGATCGCGATGTTCGGCGC-3′ and G332D_R: 5′-GCCGAACATCGCGATCACTCGGAAGAACTTCGCATCGAC-3′. As a template DNA pPET1 plasmid containing wild type (WT) petABC operon and a Strep-tag at the C-terminal end of cytochrome b was used BstXI-AsuII fragment of the operon bearing the desired mutations was inserted into the pMTS1 vector and introduced into MT-RBC1 R. capsulatus strain using triparental crossing petABC operon.2.2R. capsulatus cells were cultured in mineral-peptone-yeast-extract (MPYE) at 30 °C either semiaerobically  or photoheterotrophically  as described previously bc1 complex.2.3R. capsulatus cells grown under semiaerobic conditions as described previously bc1 complexes using the Strep-tag affinity chromatography following the manufacturer's protocol (IBA) with modifications Chromatophores were obtained from 2.4bc1 complexes were determined spectroscopically under steady-state conditions, at the beginning of the 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzohydroquinone (DBH2)-dependent reduction of cytochrome c (bovine heart cytochrome c obtained from Sigma-Aldrich) as described previously c reduction kinetics. The kinetic parameters Vmax and KM were calculated by fitting Michaelis–Menten equation to data from bc1 complexes used in enzymatic assays were at final concentration of 10–100 nM. The final concentrations of cytochrome c, quinone pool (DBH2 + DB) and antimycin were 20 μM except for determination of Vmax and KM values where DBH2 concentration was in the range of 0.5 to 20 μM.Enzymatic activities of isolated cytochrome 2.5bc1 heme cofactors. Transient kinetics of hemes b (560–570 nm) and hemes c (550–540 nm) were obtained for chromatophores suspended in MOPS buffer pH 7.0 containing 100 mM KCl and 1 mM EDTA. The measurements were carried out under anaerobic conditions in the presence of 3.5 μM valinomycin and redox mediators as described in b reduction in the presence of antimycin (Sigma-Aldrich) were calculated using a single exponential equation fitted to kinetic transients.Double beam time-resolved spectrophotometer was used to measure the kinetic properties of cytochrome 2.6Continuous wave EPR spectra of [2Fe–2S] cluster were measured at X-band frequency (9.6 GHz) on chromatophores. Samples were prepared in MOPS buffer pH 7.0 containing 100 mM KCl and 1 mM EDTA. All spectra were measured at 20 K, using a SHQEOS11 resonator and the following parameters: resonance frequency, 9.39 GHz; microwave power, 1.545 mW; modulation amplitude, 15 G.bc1 complexes. Samples were prepared in Bicine buffer pH 8.0 containing 100 mM NaCl and 20% glycerol in the presence of reductant (1 mM sodium ascorbate) and measured in the temperature range of 12–23 K. All EPR measurements were carried on a Bruker Elexsys E580 spectrometer.Pulsed EPR spectroscopy was used to obtain the temperature dependence of phase relaxation rates of the reduced [2Fe–2S] cluster as described previously 33.1bc1 in bacteria is to observe its ability to sustain growth under the photosynthetic conditions. As the anoxygenic growth of R. capsulatus is dependent on the functional electron transport chain, the lower growth rate of bacteria bearing mutated cytochrome bc1 complex suggests impediments in the complex operation. Upon introducing the G332D mutation in the cytochrome b in R. capsulatus we observed that the growth rate of mutated bacteria was dramatically slower than in the wild type .One of the ways to test the functionality of mutated cytochrome ild type A. When bild type A. Under 3.2bc1 complex we purified the complex using affinity chromatography. Visualization of the purified proteins on the SDS gel revealed that in R. capsulatus the mutated complex consists of all three catalytic subunits — cytochrome b, cytochrome c1 and ISP  did not differ between G332D and WT. Addition of sodium ascorbate reduced heme c in G332D  did not reveal any significant changes in comparison to WT indicating that the ratio of hemes c to b in the mutant bc1 complex was not changed.To test whether the G332D mutation affects the subunit composition of the  and ISP B. We havnditions C. The sp3.3c and 20 μM quinol revealed that isolated bc1 complex bearing G332D mutation is approximately 3-fold less active than the wild type  and 2-fold decline in KM .The measurements of enzymatic activity at 20 μM cytochrome ild type . The samchrome c . When cobc1 complex in chrompatophores. Kinetic transients showed in b  bc1. These measurements monitor the distance-dependent interactions between the [2Fe–2S] cluster and heme bL. The oxidized heme bL can enhance the relaxation of reduced Rieske cluster in such a way that the closer these two paramagnetic centres are, the stronger the enhancement of phase relaxation is. We have successfully used this approach to estimate changes in the average position of [2Fe–2S] cluster in relation to other cofactors in the native enzyme and various mutants of R. capsulatus cytochrome bc1To investigate whether the presence of G332D influences the movement of the ISP-HD, which could be one of the reasons for the observed changes in the CW EPR spectra of [2Fe–2S] cluster in this mutant, we measured the temperature-dependence of phase relaxation rates of the cluster in wild type and in G332D cytochrome c1 position, more remote from heme bL. In the presence of antimycin, G332D exhibited further decrease in relaxation rate  could be estimated for 2–4% of the whole population.As the additional charge on the surface of cytochrome ansition B. For th3.5o site is one of the conditions promoting generation of superoxide by cytochrome bc1bc1 decreased to the same levels, regardless of the SOD presence (The increased distance between Rieske cluster and the Qpresence A. Howevepresence B. We thu2 = 1). Under this condition, G332D mutant revealed approximately 2-fold higher level of antimycin-dependent ROS generation than the WT . This macroscopic effect appears similar to the shift observed earlier in G167P, another mutation located at the cytochrome b-ISP-HD interface. However, while in G167P this shift was associated with the ability of mutant to generate superoxide even in the absence of antimycin Our results reveal that mutation G332D in cytochrome r quinol ). Based bc1 to generate superoxide in G167P bL to quinone results in formation of semiquinone at the Qo site, which has the best chance to react with oxygen when the ISP-HD occupies positions remote from the Qo site at the time of semiquinone formation. We thus predicted that mutations that shift the equilibrium position of ISP-HD towards a position more remote from the Qo site should increase the level of ROS production. Indeed, both G167P and G332D appear to fall into this category of mutants showing elevated levels of ROS in the presence of antimycin  while the shift in G167P crosses that line and elevated ROS become detectable even in the absence of this inhibitor. It follows that within the group of enzymes shifting the average position of ISP-HD away from the Qo site, even small difference in the shift  may result in significant difference in the ROS generation at the Qo site.The increased propensity of cytochrome activity , Fig. 4 activity A as G1674.2bc1 is a product of reaction of semiquinone generated at the Qo site with oxygen. There are two possible ways, in which semiquinone can be created at this site: as a part of forward reaction, when first electron is transferred from quinol to the Rieskie cluster and the second electron transfer from semiquinone to heme bL does not take place (“semiforward” reaction) bL to quinone and the second electron transfer from Rieske cluster to semiquinone does not take place (“semireverse” reaction) b is impeded in the presence of antimycin.Superoxide radical produced by cytochrome These two mechanisms differ in terms of the substrate used to generate oxygen-reactive semiquinone. In the “semiforward” mechanism, the substrate is quinol while in the “semireverse” mechanism — it is quinone. This distinction guided the explanation of the previously observed shape of dependence of ROS generation rate in antimycin-inhibited submitochondrial particles on the redox state of the Q pool o site can have a dual effect on superoxide generation rate by cytochrome bc1. On one hand, quinone as a competitive inhibitor of the site decreases enzymatic activity of cytochrome bc1 (the major influence on activity is when Q pool is nearly or fully oxidized) and thus may reduce the rate of superoxide production calculated “quantitatively”. On the other hand, according to “semireverse” electron transfer, quinone as a substrate for semiquinone generated at the Qo site increases the level of superoxide radical production calculated “relatively”. The competition between these two opposite effects impacts on the dependence of superoxide production rate on the fraction of DBH2. The lack of bell shape in both mutants in “quantitative” plot in the range between 0.5 and 1 . Thus “quantitative” ROS production in WT can reach a maximum when the fraction of DBH2 is approximately 0.75 .Based on these results we anticipate that not all mutants of cytochrome 4.3Rhodobacter sphaeroides also caused impediment in photosynthetic growth and decrease in enzymatic activity x transition of 1.77 b and ISP. It thus appears that mitochondrial complex III with this mutation is generally less stable. While this feature was not observed with bacterial cytochrome bc1, the decreased stability of complex III may be associated with the change in ISP-HD position observed in bacteria. Indication that such correlation is possible comes from the studies showing that the presence of antimycin  makes the complex more prone to the proteolytic cleavage of ISP-HD o site) protect the complex from such cleavage R. capsulatus G167P mutation) showed almost complete lack of ISP subunit o site observed in bacteria b.Similarly to our results, G332D in another species of photosynthetic purple bacteria, b, only 3 Å apart. It thus would seem that a change from acidic amino acid (aspartate) to positively charged histidine neutralizes the negative charge introduced at the position 291 and such a compensatory effect may be responsible for the partial restoration of the proper function of the complex. This observation further supports the idea of the electrostatic nature of the ISP-HD shift caused by the G332D mutation.Interestingly, it was found that the respiratory dysfunction of G291D mutant in yeast was partially restored by the additional mutation D287H 4.4b-ISP-HD interface exerting various structural distortions and/or changes in the surface charge  it can be generalized that these types of mutations influence the motion of ISP-HD with a shift in the average position of ISP-HD away from the Qo site being the most likely outcome of this influence. This leads to an increase in ROS production consistent with the model evoking “semireverse” electron transfer from heme bL to quinone as initial reaction leading to formation of semiquinone that can react with oxygen. The level of increase in ROS production and conditions under which ROS are observed can vary depending on severity of the structural change, with more active mutants (exhibiting larger enzymatic activity and electron transfer rates) being generally more protected against elevated ROS. To observe the characteristic bell-shaped dependence of ROS production on the redox state of the Q pool the tested form of cytochrome bc1 should display moderate levels of ROS under fully reduced state of the Q pool.If one considers G332D and G167P as two representatives of the mutations that target the cytochrome Transparency document.Image 1


The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes , and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under  There are two basic theories of ethanol action on the cells, lipid-centric and protein-centric . The lipplicated .in vitro exposure of mast cells to ethanol for 1 hour or longer inhibited the high-affinity immunoglobulin E (IgE) receptor (FcεRI)-induced degranulation and production of tumor necrosis factor (TNF)-α and interleukin (IL)-8 [Previous studies showed that alcohol modulates various components of the immune system –17. Acutn (IL)-8 ,25. AlthIn this study we used primary mouse bone marrow-derived mast cells (BMMCs) and examined sensitivity to ethanol of the earliest signaling events after FcεRI triggering. We also examined effect of ethanol on FcεRI activation in cells with reduced levels of cholesterol and on passive cutaneous anaphylaxis (PCA) in mice. Our data indicate that ethanol inhibits tyrosine phosphorylation of the FcεRI β and γ subunits, the first biochemically defined event after antigen-mediated aggregation of FcεRI, and support lipid-centric theory of ethanol action in mast cells.Mice were bred and maintained in specific pathogen free facility of the Institute of Molecular Genetics and used in compliance with the Institute guidelines. All protocols, including killing mice by decapitation, was approved by the Animal Care and Use Committee of the Institute of Molecular Genetics (Permit number 12135/2010-17210) and was in compliance with the EU Directive 2010/63/EU for animal experiments. All efforts were made to minimize suffering of the mice.Bone marrow mast cells were isolated from femurs and tibias of C57BL/6 mice . The cells were cultured in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 71 μM 2-mercaptoethanol, minimum essential medium non-essential amino acids, 0.7 mM sodium pyruvate, 2.5 mM L-glutamine, 12 mM D-glucose, recombinant mouse stem cell factor , mouse recombinant IL-3  and 10% fetal calf serum (FCS). For PCA experiments, BALB/c male mice aged 8–12 weeks were used.The following monoclonal antibodies (mAbs) were used: anti-FcεRI β chain , trinitr5/sample) were used. To analyze the surface levels of FcεRI and Kit receptor, the labeling proceeded as described previously [For flow cytometry measurements, BMMCs  supplemented with 0.1% BSA and incubated with or without ethanol and/or other drugs. After 15 min, antigen was added and the cells were activated in the presence or absence of the drugs. The degree of degranulation was determined as the amount of β-glucuronidase released into the supernatant as described [BMMCs were cultured for 48 hours in medium without SCF, followed by incubation in SCF- and IL-3-free medium supplemented with TNP-specific IgE (1 μg/ml). After 14 hours the cells were washed in buffered salt solution  as Fura-2 emission at 510 nm after excitation with 340 nm and 380 nm. Basal level of calcium was usually measured in the first 60 sec, followed by addition of antigen  in BSS-0.1% BSA. Measurement continued up to 350 s. In the absence of extracellular calcium the cells were activated with antigen for 5 min followed by addition of CaCl2 to final concentration 1 mM and measurement continued up to 600 s.Changes in concentrations of free intracellular calcium were determined using cell permeant Fura-2 acetoxymetyl ester  as a reporter. IgE-sensitized cells were harvested, washed in BSS-0.1% BSA and transferred to BSS-0.1% BSA supplemented with Fura-2 AM (1 ng/ml) and probenecid (2.5 mM) used to prevent dye leakage , and inc3VO4, 2 mM EDTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) supplemented with 0.2% Triton X-100 (for FcεRI immunoprecipitation). After incubation on ice for 30 min, the lysates were centrifuged  and postnuclear supernatants were immunoprecipitated with rabbit anti-IgE antibody prebound to UltraLink-immobilized protein A . The immunoprecipitated proteins were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane and immunoblotted with PY-20-HRP conjugate or with FcεRI-β chain-specific antibody followed by HRP-conjugated anti-mouse IgG. For immunoblotting analysis of other proteins, the cells were solubilized in lysis buffer supplemented with 1% n-dodecyl β-D-maltoside and 1% Nonidet P-40 and postnuclear supernatants were directly analyzed by SDS-PAGE followed by immunoblotting with phospho-protein-specific or the corresponding protein-specific antibodies and HRP-conjugated secondary antibodies. The HRP signals were detected by chemiluminescence, quantified by luminescent image analyzer LAS-3000 (Fuji Photo Film Co), and further analyzed by Aida image analyzer software (Raytest). The amount of phosphorylated proteins was normalized to the loading controls, run in parallel experiments [Activated or nonactivated cells were solubilized in ice-cold lysis buffer  were coated with CellTak . IgE-sensitized cells were left to attach to the coated wells for 15 min in BSS-0.1% BSA and then activated or not with Ag. After 15 min, the cells were fixed for 30 min with 3% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X-100 in PBS for 30 min. After washing with PBS, the samples were blocked in PBS-1% BSA, and subsequently IgE was detected with Alexa Fluor 568-labeled donkey anti-mouse Ig. After labeling, the cells were washed and mounted in glycerol mounting solution supplemented with Hoechst 33258 stain to label nuclei. Samples were examined with a confocal laser scanning microscope Leica TCS SP5 equipped with an X63/1.4.N.A. oil-immersion objective. The image analysis was performed using a pipeline generated in CellProfiler software  .2DCFDA; Life Technologies) as a reporter. Cells were sensitized for 16 hours with anti-TNP IgE (1 μg/ml) at 37°C in culture medium supplemented with 10% FCS, but devoid of SCF and IL-3. Then, the cells were washed and loaded into the wells of a 96-well plate (0.25 x 106 cells/well) in 100 μl of the same medium supplemented with H2DCFDA (5 μg/ml) in the presence of probenecid (2.5 mM) and various concentrations of ethanol. After 15 min, the cells were centrifuged and resuspended in 50 μl BSS-BSA. Activation was triggered by adding 50 μl of 500 ng/ml TNP-BSA  in the absence or presence of different concentrations of ethanol. Alternatively, in the experiment with methyl-β-cyclodextrin (Mβ) and cholesterol-saturated Mβ (sMβ), the cells were stained with H2DCFDA as above, washed and incubated 15 min in BSS-BSA supplemented or not with 0.5% (v/v) ethanol, 2 mM Mβ, and/or 2 mM sMβ. The activation was triggered by antigen  in the presence of the drugs; sMβ was prepared as previously described [Reactive oxygen species (ROS) were examined with cell-permeant 2',7'-dichlorodihydrofluorescein diacetate , xylazine , and atropine  and then sensitized by intradermal injection of 20 μl of anti-TNP-specific IgE (50 μg/ml) in PBS into the left ear; the right ear was injected with 20 μl of PBS alone. Twenty-four hours later, 5% (v/v), 10% or 20% absolute ethanol in PBS was injected intraperitoneally (0.5 ml per mouse weighing 20 g). Mice injected with 0.5 ml of 10% or 20% ethanol exhibited reduced motoric activity, which lasted for 30 min or 90 min, respectively. Mice challenged with 0.5 ml of 5% ethanol were without any clinical signs. One hour after ethanol in PBS or PBS alone injection, each mouse was challenged with an intravenous injection of antigen  in 200 μl of PBS containing 1% Evans blue. The mice were killed 30 min later and their ears were removed for measurement of the amount of the Evans blue extravasated. The dye was extracted by adding 1 ml of formamide to each ear, followed by homogenization with the IKA T-25 ULTRA-TURRAX digital high-speed homogenizer systems (IKA) and incubation at 80°C. After 2 h, the formamide solution was centrifuged for 15 min at 13.000 x g and absorbance at 620 nm was determined in the supernatants.Statistical significance of intergroup differences was determined by one-way ANOVA with Tukey’s post-test using the Prism version 5.04 graphics and statistics software package (GraphPad); *, P<0.05; **, P<0.01, ***, P<0.001.To examine the inhibitory effect of ethanol on antigen-induced activation of mast cells, we isolated bone marrow cells from C57BL/6 mice and cultured them for 6–12 weeks in culture media supplemented with SCF and IL-3. More than 98% of the cells were mast cells as deduced from expression of both FcεRI and KIT receptors (see below). Exposure of BMMCs for 15 min– 2 hours with ethanol at concentrations up to 2% had no toxic effect, as determined by trypan blue dye exclusion test (not shown). Pretreatment of IgE-sensitized BMMCs for 15 min with ethanol at concentrations 0.2–1%, inhibited degranulation induced by multivalent antigen in a dose-dependent way. Significant inhibition was observed at all concentrations of ethanol tested when the cells were activated with antigen (50 ng/ml or 100 ng/ml) for 5 min or 15 min, except for cells treated with 0.2% ethanol and stimulated with antigen (50 ng/ml) for 5 min .Ethanol at all concentrations used also inhibited, in a dose-dependent way, the calcium response after stimulation of IgE-sensitized cells with antigen in the presence of extracellular calcium . When thRecently, Setiawan and Blanchard showed that ethanol interferes with the size and distribution of cholesterol domains in planar lipid bilayer structures . To deteDegranulation is accompanied by enhanced surface expression of CD107a (LAMP1), a secretory granule/lysosomal marker detectable by flow cytometry . In agreWe also analyzed the combined effect of Mβ and/or ethanol pretreatment on antigen-induced calcium response. The data indicate that pretreatment with Mβ did not intensify the inhibitory effect of ethanol . When thNext we examined the effect of short-term pretreatment of BMMCs with various concentrations of ethanol (0–1%) on antigen-induced expression of cytokines. Data presented in Recent studies indicated that exposure of macrophages to ethanol promoted generation of ROS –44. To dThe first biochemically well-defined step in FcεRI signaling is tyrosine phosphorylation of the receptor subunits, followed by formation of the FcεRI signalosome and phosphorylation of other substrates . Next, wTo determine changes in tyrosine phosphorylation of FcεRI, the receptor was immunoprecipitated and analyzed by immunoblotting with phosphotyrosine-specific antibody, PY-20-HRP conjugate. In non-activated cells, the receptor β and γ subunits exhibited weak tyrosine phosphorylation, and ethanol (0.5%) with or without Mβ had no significant effect on their phosphorylation . In cellTyrosine phosphorylation of several other proteins involved in FcεRI signaling was examined by direct immunoblotting of size-fractionated cell lysates with phosphotyrosine protein-specific antibodies. For these experiments we used IgE-sensitized cells pretreated or not with Mβ and/or ethanol . The celActivity of mouse LYN kinase is regulated by tyrosine phosphorylation of Tyr 507  in a dose-dependent manner. To understand the molecular mechanism of the inhibitory effect of ethanol on these activation events we examined various candidate targets. Several lines of evidence suggest that ethanol interferes with the function of FcεRI-cholesterol signalosomes and support the lipid-centric theory of ethanol action in this system, at least at the early stages of cell activationFirst, pretreatment of the cells with 4-MP, an inhibitor of alcohol dehydrogenase, together with ethanol had no effect on the inhibitory action of ethanol on degranulation. Thus, inhibition of mast cell activation seems to be caused by ethanol itself and not its metabolites. This conclusion is supported by the finding that up to 2 hours exposure to 2% ethanol in BSS-BSA at 37°C was not toxic to BMMCs. It should also be noted that the cells in this study were pretreated with ethanol only for 15 min before activation, which makes less likely that metabolites are formed and affect signaling pathways.Second, antigen-mediated degranulation, calcium response, and production of cytokines were also inhibited by Mβ. In cells treated with Mβ, the inhibitory effect of ethanol on degranulation, and IgE receptor internalization was potentiated. Interestingly, when the cells were pretreated with Mβ saturated with cholesterol, the inhibitory effect of ethanol on calcium response was reduced. These data suggest that cholesterol is involved in the inhibitory effect of ethanol.Third, production of ROS after FcεRI triggering was also inhibited by ethanol in a dose-dependent manner and treatment together with Mβ potentiated the inhibitory effect of ethanol. These findings are in accord with previous data showing that lowering cellular cholesterol by Mβ in hepatocytes inhibits ROS production . InteresFourth, immunoprecipitation and immunoblotting analyses showed that ethanol inhibits tyrosine phosphorylation of FcεRI β and γ subunits in antigen-activated cells. Phosphorylation of these targets is the first biochemically well-defined step after FcεRI triggering and depends on the topography and activity of protein tyrosine kinase LYN and protein tyrosine phosphatases in the vicinity of the FcεRI ,56. The Fifth, internalization of antigen-aggregated FcεRI was significantly inhibited by 15 min pretreatment with Mβ and ethanol. In our previous study we found that cholesterol removal by Mβ enhanced antigen-mediated clustering of the FcεRI . These fThe capacity of externally added cholesterol to reduce the inhibitory effect of ethanol on calcium and ROS responses are more compatible with the lipid-centric theory than the protein-centric theory of the inhibitory effect of ethanol. How exactly ethanol interacts with lipids and/or how it affects lipid-protein interactions is not known but could involve direct interaction of ethanol with cholesterol  and the Model of ethanol action on mast cells and their activation by antigen is shown in in vivo in which mast cells were sensitized locally by IgE, and ethanol and antigen was administered intraperitoneally and intravenously, respectively. The results of PCA assays, which reflect the activity of mast cells [in vivo.The inhibitory effect of ethanol on mast cell activation was confirmed by experiments st cells  support st cells . Experimst cells –71. Attest cells ,73, monost cells  and T cest cells ,75–77. Ast cells ,25, thisin vitro inhibited antigen-induced tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinase, NTAL adaptor protein, PLCγ, calcium response, production of ROS, degranulation, and expression of cytokine genes in a dose-dependent manner. Early activation events were also inhibited by Mβ, suggesting that cholesterol could be involved. The role of cholesterol in the inhibitory effect of ethanol was supported by the finding that sMβ reduced the inhibitory effect of ethanol on calcium and ROS responses. The data support the lipid-centric theory of ethanol action on the initial stages of FcεRI signaling. The inhibitory effect of ethanol on mast cell activation was also observed in a mouse PCA model in vivo, explaining previous findings of the reduced inflammatory response associated with enhanced consumption of ethanol.In this study we found that 15 min treatment with non-toxic concentrations of ethanol


Background:Arabidopsis maltodextrin disproportionating enzyme 1 (AtDPE1) plays a key role in chloroplast starch degradation.Results: Six AtDPE1 structures define the active site and reveal mechanistically relevant conformations of both the enzyme and substrate.Conclusion: Substrates are captured through loop rearrangements; the subtle deployment of active site residues controls catalysis.Significance: A molecular level understanding of the complete disproportionation cycle of AtDPE1 is presented. Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1  converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the  Beca reduced . Data fr reduced –13. Addiactivity , and thein vitro to produce large ring cycloamyloses with degrees of polymerization (DP) in the range of 16–50 . Amylomcharides  and altecharides .1X1N) but is not described in the literature.Escherichia coli  has also been reported , revealing both covalently and non-covalently bound complexes of the enzyme with a variety of relevant ligands. As such, this represents the most extensive structural study so far reported for this class of enzyme. We define key protein-ligand interactions and describe a series of conformational changes that are linked to active site occupancy, which together direct the course of catalysis and, crucially, favor transglycosylation over hydrolysis. Moreover, these structures help to explain why maltotriose is the preferred substrate and why longer glucans are inhibitory to catalysis. Overall, our data permit a structural definition of the complete catalytic cycle of the enzyme and allow us to propose a mechanism for the industrially important production of large cyclic products.Here we present crystal structures of i.e. lacking the N-terminal transit peptide; UniProtKB/Swiss-Prot entry Q9LV91) and preceded by a hexahistidine tag (encoded by the pET151/D-TOPO vector (Invitrogen)). The tag was not cleaved prior to crystallization by the hanging drop vapor diffusion method at 20 °C. 1 μl of a precipitant solution consisting of 9% (w/v) PEG 2000 monomethyl ether in 0.1 m HEPES-NaOH, pH 8.0, was added to 1 μl of protein at a concentration of ∼10 mg/ml in 20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl  using an extinction coefficient of 128,590 m−1 cm−1 calculated from the amino acid composition by ProtParam , acarbose , and β-cyclodextrin (Wacker-Chemie GmbH); at 0.9 mm for acarviostatin II01 ; and at 1 mg/ml for cycloamylose . Crystals were soaked overnight in 5-μl drops of these solutions before harvesting and flash-cooling in liquid nitrogen using LithoLoops (Molecular Dimensions). The mounted crystals were stored in Unipuck cassettes (MiTeGen) prior to transport to the Diamond Light Source , where they were transferred robotically to the goniostat on either beamline I04 or I04-1 and maintained at −173 °C with a Cryojet cryocooler (Oxford Instruments). X-ray data were recorded using either a Quantum 315 CCD (ADSC) or a Pilatus 2M or 6M (Dectris) detector and then integrated using either MOSFLM (AtDPE1 protein was prepared as previously described  to give rotParam ). Crystar MOSFLM  and scalr MOSFLM  or AIMLEr MOSFLM ; the res1X1N).5 This was used as a monomer template to solve the ligand-free (LF) AtDPE1 structure at 2.13 Å resolution by molecular replacement in PHASER . Where ligand binding was comparable between the two chains for a given structure, only the A chain was described (e.g. ACV-A and BCD-A). Although it was not always possible to precisely define the stereo- and regiochemistry of the sugars, in general, the electron density was consistent with sugars in 4C1 chair conformations, with the exception of the valienamine residues  derived from acarbose, which were built in 2H3 half-chair conformation. Also, all of the glycosidic linkages were modeled as α-1,4, apart from the covalent bonds to the active site nucleophile, which were modeled as β linkages.AtDPE1 shares 75% amino acid sequence identity with the equivalent enzyme from potato, for which there is a crystal structure available , and 10 μl of AtDPE1 (20 mg/ml) was added to the solution. The reaction was incubated at room temperature for 2 weeks with additional aliquots of the enzyme (10 μl each) added every 4 days. The enzyme was removed by spin filtration , and acarviostatin II01  using a Dionex HPLC Ultimate 3000, coupled with a Dionex charged aerosol detector and a Foxy Jr. fraction collector (Teledyne Isco Inc.). The mobile phase was a gradient of acetonitrile, from 90 to 55% over 35 min, followed by H2O for 10 min and 90% acetonitrile for 10 min, at a flow rate of 5 ml/min. The fractions collected were analyzed by thin layer chromatography to identify those containing acarviostatin II01 (256 (Merck) was used as the stationary phase, and CH3CN/H2O/NH4OH at a ratio of 6:3:1 was used as the mobile phase. The compounds were visualized by dipping the plate in 0.5% (w/v) orcinol in 20% HCl (v/v in ethanol) followed by heating. Fractions containing acarviostatin II01 (A) were pooled and freeze-dried to afford a white solid . fR = 0.11 . 1H NMR : δH 5.90 , 5.82 , 5.32 , 5.29 , 5.24 , 5.15 , 4.57 , 4.18–3.44 (m), 3.19 , 2.40 , 1.26  (B). ESI-MS/MS(+) at m/z 1111.43 ([M + H]+); 304.14, 466.20, 488.20, 624.25, 646.26, 769.33, 808.31, 931.38, 953.39  was dissolved in 5 ml of aqueous triethylammonium acetate  B. ESI-MS, 953.39 . HR-ESI-8 barrel fold . The opposing canyon wall is largely made up of subdomains B2 and B3. In the latter, an extended loop  partially projects over the donor binding site and is also mobile but to a lesser extent; we shall refer to this as the “gatepost.”In the various AtDPE1 structures presented herein, the most significant conformational changes are restricted to the N terminus and the B1 and B3 subdomains  and 5. Tmaltases ), which maltases C. The op2, as calculated by PISA (available on the PDBePISA Web site) (C). By contrast, the C-terminal residue, Leu-576, is fully resolved in all of the AtDPE1 structures.Ser-60 is the first residue that can be clearly resolved in the electron density, and the ensuing segment up to and including Glu-79 forms an extended arm that embraces the opposing subunit in the homodimer  and makeeb site) . Moreoveeb site) C. By conFurther discussion of the subunit topology is not warranted here because this has been adequately described elsewhere for the thermophilic bacterial amylomaltases –28, with–Z scores >10.0). Of these, five proteins stood out as being extremely similar to AtDPE1 (Z scores ∼50); these were the potato DPE1 ,5 and four amylomaltases from thermophilic bacteria, namely Thermus brokianus (PDB code 2X1I) (Thermus aquaticus (PDB code 1ESW) (Thermus thermophilus (PDB code 2OWW) (Aquifex aeolicus (PDB code 1TZ7),E. coli  (3ZT5) .S. tuberosum DPE1 form comparable homodimers, four of the amylomaltases are monomeric, and the fifth, A. aeolicus AMY, is homotetrameric (D and E). Assembly of such a tetramer is not possible for the plant enzymes because the formation of the additional interfaces would be precluded by steric clashes with the N-terminal arms. Finally, from these comparisons, it is clear that the gate motif is a defining feature of the DPE1 enzymes and the amylomaltases. Its importance is reflected by a high degree of sequence conservation within the enzyme family; across AtDPE1 and its closest five structural homologs, the 20-residue β-hairpin that forms the gate (residues 322–341 inclusive in AtDPE1) contains 14 strictly conserved residues (E. coli AMY (not shown).Interrogation of the Protein Data Bank using the DALI server retrieves 117 non-redundant entries with significant structural similarity to AtDPE1 (de 2X1I) , Thermusde 1ESW) , Thermusde 2OWW) , and Aqude 4S3R) . Hit num) (3ZT5) , which htrameric . One of trameric , D and Eresidues . Nine ofIn this study, in addition to the structure of LF AtDPE1, we determined the structures of five ligand-bound complexes of the enzyme. Moreover, for some of these, different binding modes were observed between the two active sites within the crystallographic asymmetric unit, giving rise to a total of six discrete ligand-bound states .A), whereas eight were resolved for the B chain (MA3-B). The glucan chains adopt a helical structure consistent with that observed for α-1,4-linked glucans in solution (A), especially so around the expected site of cleavage, where the residues involved are conserved in the close structural homologues (A and B), and 9A). Consistent with the mobility of the gate, the electron density is weaker in this region, and the refined temperature factors are elevated relative to the core of the structure. Indeed, in MA3-B, the density is weaker still, such that the end of the gate cannot be confidently modeled. Despite the strict conservation of Phe-331 and Phe-446 in the closest structural homologues, this interaction has not been observed before due to the lack of a suitable ligand-bound structure with the +3 and +4 sites occupied; it has, however, been predicted (A and B). The potential significance of this catalytically “incompetent” configuration will be discussed further below. In many respects, this structure resembles the non-covalent complexes of T. aquaticus AMY with acarbose (PDB code 1ESW)    and E. cde 4S3Q) , respectde 4S3Q) .B and C); this has previously been seen crystallographically for other enzymes of the GH-H clan , mainly due to a concomitant repositioning of side chains, including all of the aforementioned interactions with the −1 sugar, although here the side chain of Trp-338 is now closely associated with this sugar, such that the latter is more firmly clamped between this residue and Tyr-136 (C). In this complex, the distorted conformation of the valienamine moiety at subsite −1 (C) is thought to mimic the oxocarbenium-like transition state expected for enzymes of the α-amylase family (δ1) just 2.8 Å from the C1 atom of the −1 sugar (B and 10C). At the same time, more subtle changes to the conformation of Glu-420 enable its carboxylate group (Oϵ2) to hydrogen-bond to the nitrogen atom of the acarviosin moiety . Finally, in ACR-B, the gate swings over the active site canyon to adopt a new, fully closed conformation, whereby Phe-331 now stacks against Pro-539 in the gatepost (C and D) and 9B), the latter having moved relative to its position in MA3-A (A and B) and 9A). The side chain of Phe-331 moves by over 11 Å (relative to its position in MA3-A) and, in so doing, shields the active site from bulk solvent. A more subtle, but nevertheless significant, movement is seen in the brace, which moves in conjunction with the gate (E). As a consequence of the gate movement, the sugars in the acceptor site are no longer clamped between the Phe-331/Phe-446 aromatic pair. Nevertheless, Phe-446 still serves as a platform for the substrate but is now stacking mainly with the +3 sugar (C and 9B).When a crystal was soaked in the pseudotetrasaccharide inhibitor acarbose, the active sites of the resultant 2.05 Å resolution structure (ACR) revealed two distinctly different binding modes, both of which required modification of the ligand , B and CH-H clan –48, inclcoli AMY , where tor sites . From th-linkage . Althoug-linkage –51 as welinkage –. Comparelinkage –, A and B Tyr-136 C. In thibsite −1 C is thoue family . Indeed,−1 sugar B and 10Cthe gate E. As a c+3 sugar C and 9B.B). Instead, there is continuous density between the reducing end of the −1 sugar and Asp-373, indicating that this represents an intermediate state with a covalently bound substrate in the donor site linked to the Oδ2 atom of Asp-373 via a β-glycosidic bond . Given the lack of clear evidence for non-glucose residues in any of the subsites, for simplicity, all of the sugars were modeled as α-1,4-linked glucose units in 4C1 chair conformation   .D), which was directly comparable with the complex obtained by cocrystallization of E. coli AMY with acarbose (PDB code 4S3R) (C and D). These structural variations in the ligands would be consistent with the identities of the −4 and −3 sugars being different between ACV-A and ACR-B (discussed further below).A crystal soaked in the heptasaccharide mimic acarviostatin II01 diffracted to 2.53 Å resolution, and the resultant structure (ACV) revealed essentially equivalent ligand binding in both active sites of the dimer  and 7D, 7de 4S3R) . In factde 4S3R) , C and DE). Weaker electron density was observed beyond and contiguous with that for the −2 sugar, indicative of a −3 sugar residue. In contrast to the situation in the ACR-A, the acceptor site is vacant (A further structure (BCD) was determined to 2.3 Å resolution after soaking an AtDPE1 crystal in β-cyclodextrin. In both active centers, there was only sufficient electron density to confidently build a maltose unit occupying subsites −2 and −1, this being covalently linked to Asp-373, and overlapping closely with the sugars in the equivalent subsites in the donor pocket of ACR-A E. Weakers vacant , the gats vacant . The covF), which overlapped well with the equivalent sugar in ACR-A (B). As with BCD-A, there was weak density extending beyond the −2 sugar in both subunits consistent with −3 sugar residues, whereas additional weak extended density was present in the acceptor site of CAM-B beyond the +1 sugar, suggesting the presence of +2 and +3 sugar residues, but these could not be modeled with confidence (The remaining structure (CAM) was determined to 2.3 Å resolution after soaking an AtDPE1 crystal in cycloamylose. This was extremely similar to BCD-A, with a maltose unit occupying subsites −2 and −1, and covalently linked to Asp-373 in both active sites. Additionally, in CAM-B, the electron density clearly revealed a single glucose molecule occupying the +1 donor subsite F, which in ACR-A B. As witnfidence . As withA). After purification and characterization (B and i.e. acarbose (a pseudotetrasaccharide)) also showed density for a heptasaccharide in ACR-B, it was somewhat different from ACV-A at the non-reducing end (C and D), suggesting that a different transglycosylation product had been trapped in ACR-B. However, given the resolution of the data for the latter (2.05 Å), we were unable to define the true identity of the bound ligand with confidence.Previous work  has showFig. 1rization B and 2, cing end , C and DE), potentially providing a mechanism whereby events at one active site are communicated to the other subunit. Therefore, it is tempting to speculate that this could explain the existence of distinctly different catalytic states between the two active sites within the same homodimer (e.g. ACR-A versus ACR-B). However, the changes at the N-terminal arm do not appear to be transmitted to the core of the subunit, and the relationship between protomers does not change significantly between the various AtDPE1 structures . In particular, the brace motifs of each subunit lie close to crystal contacts such that, dependent on the structure and the subunit, they make different interactions with the neighboring dimer in the crystal. Thus, these crystal contacts could influence the rigidity and positioning of the brace, which in turn could affect the placement of the gate and therefore affect binding events at the active site. Although one could argue that the resultant conformations of AtDPE1 are crystallographic artifacts, we propose that these crystal contacts have helped us to trap conformations that mimic biologically relevant states of inherently dynamic structural motifs.Through comparisons of the structures described herein, we are able to describe different conformations of AtDPE1, specifically for the gate and the brace motifs in the B1 subdomain and the gatepost in B3, and relate these to the occupancy of the various subsites in the active site canyon. In all cases, when the acceptor site is vacant, the end of the gate is disordered. In the structures where the acceptor site is occupied, the gate can adopt one of two discrete ordered conformations, depending on the catalytic state mimicked by the ligand complex . The corFig. 8ructures . An alteB) is intriguing, but this is also seen in four of the homologous enzymes, suggesting that it may have biological relevance. In these homologues, either the active site cavity is vacant , or the substrate/product is bound in a pre/post-catalytic complex, analogous to MA3-A, where it is not fully engaged with the active site . Notably, in all of these structures, the carboxylate of the Asp-373 equivalent is within hydrogen bonding distance of another conserved Asp side chain (Asp-293 in AtDPE1). This implies that one of these acids is protonated and therefore has an elevated paK, which is particularly remarkable given that the T. aquaticus AMY crystals were grown at pH 9.0  . The exception is ACR-B, where Oϵ1 retains the hydrogen bond to His-374 but swivels round so that Oϵ2 can now hydrogen-bond to the nitrogen of the acarviosin moiety of acarbose, thereby mimicking the expected interaction with the oxygen of the scissile glycosidic bond, which would be a prerequisite to catalysis . This side chain movement could be triggered by a small shift in the protein backbone downstream of Glu-420 . The reconfiguration of the active site acids into catalytically competent conformations is most likely driven by a series of conformational changes that are coupled to the proximity of an appropriately bound substrate, but we are not able to precisely define the necessary sequence of events.The observation that Asp-373 adopts a catalytically incompetent configuration upon substrate capture seen in MA3-A B is intrt pH 9.0 . Indeed, equally . The fun4C1 chair adopted by the −1 sugar of the covalently bound donor moiety (D) (i.e. Glu-420) in the absence of a suitable acceptor, as we observe in all but the ACR-B structure, and thereby preventing it from activating a water molecule and leading to hydrolysis. A second protective mechanism could result from the perpendicular arrangement of the plane of the nucleophilic carboxylate group to the plane of the −1 sugar, such that the anomeric carbon is partially occluded and less accessible to an approaching nucleophile, as noted in T. thermophilus AMY  and 10D). It is also possible that the gate may play a part in protecting the intermediate from abortive hydrolysis. Indeed, the fact that we are able to trap the covalently bound intermediates at all is testament to their stability. This contrasts with the situation in related enzymes, where it is usually necessary to make a mutation to the acid/base catalyst and to use fluorinated pseudosubstrates in order to trap a covalent intermediate  binds in the correct register, the catalytically competent active site configuration is desirable, as we have predicted for state S2 in our proposed scheme below . H. H4C1 chilus AMY , which wrmediate –58. In gme below .The expected α-retaining double-displacement mechanism of AtDPE1 consists of two half-reactions, with a covalently bound β-linked intermediate lying at the midpoint of the scheme. Observations that AtDPE1 incubated with maltotriose never produces maltose  and thatThis is the status of the enzyme prior to reaction and represented by the LF-A structure.A). Moreover, because sites +3 and +4 are vacant, the gate is likely to be disordered. In addition, because this is the preferred substrate, we propose that Asp-373 is correctly configured for catalysis (in contrast to MA3-A).In this state, the substrate is not fully engaged with the active site. This could resemble MA3-A but with just the sugar residues spanning subsites −2 to +1, making multiple favorable interactions with the protein A. Moreov4C1 chair to a transition state-like half-chair is stabilized by the interactions with the protein, in particular the bidentate interaction between Asp-473 and O2 and O3. This introduces a kink into the glucan chain at the scissile linkage that allows the substrate to be drawn deeper into the active site. At the same time, the flattening of the −1 ring and its partially aromatic character attracts the side chain of Trp-338 down with it. In addition, the deeper repositioning of the +1 sugar pulls the side chain of Gln-336 down too. Both Gln-336 and Trp-338 lie at the base of the gate, and their combined shifts could act as a trigger that initiates the movement of the gate from a disordered to a fully closed configuration. Additionally, the repositioning of the gatepost is necessary for the optimal stacking of Phe-331 against Pro-539, which may be aided through the correlated motion of the adjacent residue, Ala-540, with the substrate, such that its backbone nitrogen remains hydrogen-bonded to O3 of the −2 sugar as it moves downward in the donor site (B).This could resemble ACR-B (and ACV-A) but with just the sugar residues spanning subsites −2 to +1. The flipping of the −1 sugar from a nor site B.4C1 chair upon forming the β-glycosidic link with Asp-373, so the interaction with Trp-338 is weakened, and the gate would become more likely to open, especially since the +1 sugar is less constrained due to the loss of the glycosidic bond.This is represented by CAM-B. The −1 sugar flips back to a This is represented by BCD-A. The release of glucose primes the enzyme to receive the second maltotriose molecule.The donor site could be represented by BCD-A, and the acceptor site could be represented by MA3-A, specifically from the latter, the sugar residues spanning subsites +1 to +3 with the partially closed gate trapping the +3 sugar between Phe-331 and Phe-446.C).This is represented by ACR-A but with just the sugar residues spanning subsites −2 to +3. The fully engaged acceptor molecule, in addition to drawing down Gln-336 to trigger full gate closure, makes further favorable contacts with Asp-329 and Leu-330 of the repositioned gate via the sugar residues in subsites +2 and +3 C.B).This is represented by ACR-B (and ACV-A) but with just the sugar residues spanning subsites −2 to +3. The β-link with Asp-373 at the reducing end of the donor maltose is replaced by an α-1,4-link to the non-reducing end of the acceptor maltotriose molecule to yield maltopentaose, and the −1 sugar adopts a half-chair conformation. The favorable contacts between Gln-336, Asp-329, and Leu-330 and the acceptor site sugars maintain the fully closed gate conformation B.4C1 chair conformation removes the kink at the newly formed glycosidic linkage such that the maltopentaose can no longer fully engage with the active site. The outward movement of the product and the concerted movements of residues around the −1 and +1 sites  cause the gate and gatepost to move apart, with the +3 sugar residue possibly being transiently clamped between Phe-331 in the partially closed gate and Phe-446 as we see in MA3-A (A and 9A).This is represented by MA3-A but with just the sugar residues spanning subsites −2 to +3. The reversion of the −1 sugar to a in MA3-A A and 9A.The opened gate allows the maltopentaose to disengage to yield the free enzyme ready for another catalytic cycle.in vitro, upon prolonged incubation with maltotriose, repeated transglycosylation is possible to yield longer and longer glucan chains and, eventually, cyclic products (T. aquaticus AMY (PDB code 1CWY)  , it was in vitro via transglycosylation. Thus, our results will inform the future practical use of AtDPE1 or its homologues in a range of green chemistry applications.The set of crystal structures presented herein allows us to delineate the complete catalytic cycle of AtDPE1 in structural terms. Notably, we demonstrate the capture of covalent intermediates without resorting to site-directed mutants or exotic substrate analogues. We describe the orchestrated movements of surface loops and catalytic residues that together direct the disproportionation of short linear glucans, and we propose a mechanism whereby longer substrates could be cyclized. Although no biological role for cycloamyloses has so far been described, they have a range of biotechnological applications. Moreover, besides cyclization, this class of enzyme is capable of performing a variety of other industrially relevant biotransformations E. C. O., K. T., and C. E. M. S. produced, characterized, and crystallized the protein; E. C. O., C. E. M. S., and D. M. L. collected and analyzed the x-ray data; D. L., M. I. D., M. R., and S. A. N. produced and characterized the acarviostatin; T. L. and R. A. F. conceived and supervised the project; D. M. L. drafted the manuscript with assistance from E. C. O.; all authors contributed to editing the manuscript.


Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed ‘mammospheres’. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression.in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity.Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and 116 phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser116 phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116 residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser116 phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo.In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased SerOur study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.The online version of this article (doi:10.1186/s13058-014-0420-z) contains supplementary material, which is available to authorized users. Epithelial cells grow attached to their extracellular matrix (ECM) composed of collagen, elastins, fibronectin, laminin, proteoglycans and glycosaminoglycans . Signalsin situ. We. Weadenotivation , an evoltivation -17]. Al. Aladenotivation , suggest116 position has been shown to promote its anti-apoptotic functions by binding to Fas-associated death domain protein (FADD) and preventing the recruitment of initiator caspases . In. In48]. tachment -51]. Th. Th116 pSignaling by the death-receptor proteins, such as Fas and TNF-R1, leads to the formation of the death-induced silencing complex (DISC) consisting of death-receptor, FADD, and caspase-8 which initiates the caspase cascade . Death r116 position enables its association with FADD, leading to the disruption of the DISC assembly and blocking apoptosis [116 phosphorylation. However, our study revealed that under matrix-deprived condition, inhibition of Akt or CaMKII failed to alter the levels of PEA15 Ser116 phosphorylation in HMECs. In contrast, inhibition or knockdown of AMPK impaired this phosphorylation. Our data further revealed direct phosphorylation of PEA15 Ser116 by AMPK, thus identifying AMPK as an upstream kinase of PEA15. It is plausible that multiple stress kinases employ similar downstream mechanisms to mediate their functions, and perhaps do so in a context-dependent manner. Under the stress of matrix deprivation, our data revealed that AMPK plays a predominant role in the phosphorylation of PEA15. Thus, matrix detachment may trigger anoikis, at least in part, through the death-receptor pathway; our data shows that AMPK activation upon matrix deprivation helps overcome anoikis by phosphorylating PEA15 at Ser116 position, which associates with FADD and blocks apoptosis Additional file 2: Supplementary Figure S2.(PDF 358 KB)Additional file 3: Supplementary Figure S3.(PDF 201 KB)Additional file 4: Supplementary Figure S4.(PDF 534 KB)Additional file 5: Supplementary Figure S5.(PDF 127 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Below are the links to the authors’ original submitted files for images.


Dreissena polymorpha, in the Great Lakes is being monitored as a bio-indicator organism for environmental health effects by the National Oceanic and Atmospheric Administration’s Mussel Watch program. In order to monitor the environmental effects of industrial pollution on the ecosystem, invasive zebra mussels were collected from four stations—three inner harbor sites  in Milwaukee Estuary, and one reference site (LMMB5) in Lake Michigan, Wisconsin. Nuclear magnetic resonance (NMR)-based metabolomics was used to evaluate the metabolic profiles of the mussels from these four sites. The objective was to observe whether there were differences in metabolite profiles between impacted sites and the reference site; and if there were metabolic profile differences among the impacted sites. Principal component analyses indicated there was no significant difference between two impacted sites: north Milwaukee harbor (LMMB and LMMB4) and the LMMB5 reference site. However, significant metabolic differences were observed between the impacted site on the south Milwaukee harbor (LMMB1) and the LMMB5 reference site, a finding that correlates with preliminary sediment toxicity results. A total of 26 altered metabolites (including two unidentified peaks) were successfully identified in a comparison of zebra mussels from the LMMB1 site and LMMB5 reference site. The application of both uni- and multivariate analysis not only confirmed the variability of altered metabolites but also ensured that these metabolites were identified via unbiased analysis. This study has demonstrated the feasibility of the NMR-based metabolomics approach to assess whole-body metabolomics of zebra mussels to study the physiological impact of toxicant exposure at field sites.Zebra mussel, The online version of this article (doi:10.1007/s11306-015-0789-4) contains supplementary material, which is available to authorized users. The main focus of conventional toxicology studies has usually been the measurement of toxicant levels in the organism (body burden) and the observation of various correlated physiological end-points such as body weight or survival rate in response to toxicant exposure  in biofluids and tissues of a given biological system. This approach provides a novel way to study the changes in the metabolome under various stimuli  or Dreissena rostriformis bugensis (quagga mussel) are collected at multiple sites in Areas of Concern (AOCs) to monitor as a bio-indicator for environmental health effects. The levels of the toxicants in mussel tissue, including metals  and organic compounds , have been closely monitored over the years; however, the physiological effects on animals due to the assumed toxicant exposure remain unclear.The levels of contaminants in mussels from the Great Lakes have been monitored by the National Oceanic and Atmospheric Administration (NOAA/NCCOS) as a part of the National Status and Trends Mussel Watch Program (MWP) which has been ongoing since 1986  in Milwaukee Estuary, and one reference site (LMMB5) in Lake Michigan, Wisconsin . After transport to shore, individual mussels were separated by cutting the byssal threads with stainless steel surgical scissors. Residual silt and debris was removed from the shells with gentle rubbing under a stream of site water. The individual mussels were placed in individual Teflon bags, zip-closed with plastic cable ties holding a labeled tag and frozen by dropping into a dry shipper which had been pre-cooled with liquid nitrogen. The time between the samples breaking the surface of the water and freezing of the mussels was recorded at each collection site . After shipping the frozen mussels to the analytical laboratory, the mussels were stored at −80 °C until sample processing.Groups of approximately 20 mussels were collected from the reference site, LMMB5 and frozen together in a large Teflon bag; 15 of these bags were collected. One mussel from each bag was used in this study. Additionally, in the field, 15 individual mussels from LMMB4 were shucked, placed in individual 2 mL cryovials and snap frozen in liquid nitrogen before being placed in the dry shipper for transportation to the analytical laboratory; this set of tissues was used to produce an in-house prepared control material.Mytilus edulis) (frozen mussel tissue homogenate), and SRM 2974a—Organics in Freeze-Dried Mussel Tissue (M. edulis)]. MCM was prepared by pooling frozen homogenized wet tissues from 10 field-shucked LMMB4 samples. The material was kept frozen while it was weighed and aliquoted by mass into bead-beating tubes . The aliquots were stored at −80 °C until use.Three different materials were used for quality control during this study to demonstrate the reproducibility of the sample processing and analytical methods: In-house prepared mussel control material (MCM) and two NIST standard reference materials , six SRM 1974c samples [wet (n = 3) and dry (n = 3)] and six SRM 2974a samples [wet (n = 3) and dry (n = 3)] were extracted. To prepare the dry tissues, MCM aliquots and SRM materials were lyophilized for approximately 15 h prior to extraction. The average water content of MCM and SRM 1974c were determined to be 88.4 and 91.1 % respectively through a gravimetric assessment of the lyophilized tissues. Though SRM 2947a is a freeze-dried sample, an average water loss of 6.1 % occurred upon lyophilization. These values were used to calculate the solvent volumes for the wet-tissue extractions in this study to compensate for the water naturally present in the tissues.The samples were extracted (see procedure below) and subjected to NMR analysis. The samples were repeatedly analyzed over several days with the samples remaining at room temperature in the NMR auto-sampler to represent harsh sample handling conditions.Quality control samples were processed with each batch of experimental samples and these included solvent blanks, replicate experimental sample extractions and the QC materials mentioned earlier. In total, six MCM samples and ten samples of each SRM were extracted along with the experimental samples in batch fashion. In each batch, SRM 1974c and MCM  and SRM 2974a  were weighed into bead-beater tubes and lyophilized overnight along with the experimental samples.gn at 4 °C for 5 min. The upper polar phase was collected into 1.5 mL Eppendorf microcentrifuge tubes, and dried by vacuum centrifuge for 2 h at room temperature . The dried polar extracts were re-hydrated with 600 µL of NMR buffer containing 100 mol/L phosphate buffer, pH 7.3, 1 mol/L TMSP , and 1 mg/mL NaN3 (sodium azide CAS 26627-22-8) prepared in D2O, and 550 µL of each sample was then transferred into 5 mm NMR tubes (Norell).A total of 74 individual experimental whole-body mussels were processed in this study . Mussel tissues were removed from the shells individually prior to homogenization. In order to keep mussels frozen, they were kept inside a cryogenic workstation  during extraction from the shells. Using pre-cleaned surgical scalpels, the edge of the shell was cut away. Once the shell was opened, the mussel tissue was placed on a weigh boat where the mussel tissue was examined so that small pieces of shell could be gently removed using the scalpel. Any pieces of ice found in the shell were kept with the tissue to avoid any loss of tissue. Each individual mussel was homogenized using a cryogenic ball-mill homogenizer  with either a 25 or 35 mL grinding jar and 15 mm and 20 mm balls, respectively, for 2 min at 25 Hz. The homogenized tissue was placed in pre-weighted 2 mL cryovials using the cryogenic workstation at each step of the sample preparation process to keep the wet tissue frozen. The experimental mussel samples were lyophilized in 2 mL cryovials for approximately 15 h, and 10 ± 1 mg of dry tissue was aliquoted into bead-beating tubes. The average tissue weights of all the test samples were used to calculate the solvent volumes for extraction. In each extraction batch, seven or eight experimental samples along with the quality control materials were extracted using a chloroform:methanol:water technique modified from Wu et al. . The dri1H NMR spectra and two-dimensional 13C edited heteronuclear single quantum correlation (HSQC) spectra for representative samples were acquired on a Bruker Avance II 700 MHz spectrometer equipped with a 5 mm, z-gradient TCI cryoprobe using previously published methods .For HSQC data, a relaxation delay equal to 1.5 s was used between acquisitions and a refocusing delay of 3.45 ms was implemented. In general, 2048 data points in the direct dimension and 512 data points in the indirect dimension with 128 scans per increment were acquired with spectral widths of 11 ppm in F2 and 180 ppm in F1   and contaminants identified in the blanks , were reduced by uniform binning to 1890 buckets 0.005 ppm wide. Signal intensities were summed for integration, and the spectra were normalized to constant total spectral area. Prior to PCA analysis, the binned spectra were mean-centered with no scaling.Principal components analysis (PCA) was performed to determine the reproducibility of the extraction and sample stability and to look for metabolic differences in the tissues from different collection sites. Processed In addition to the PCA analysis, additional approaches were used to analyze the multivariate results. Supervised PCA (sPCA) was used to construct hybrid loadings which optimize the separation between selected groups and aid in identification of significant classification compounds.  were generated  control at the 0.05 level , p = 1.38 × 10−3; PC3 (0.08 % EV), p = 9.89 × 10−3; p values calculated for PC scores between wet and dry MCM tissues]. The changes in the wet MCM tissue extract over time observed in the PCA scores plot (RSD = 7.39 %) were quite dramatic compared to dry MCM tissue extract (RSD = 3.39 %) and so we used the dry tissue extraction for experimental samples.The spectra from extractions of wet and dry samples using the quality control materials  were analyzed with principal components analysis (PCA) . For the two NIST SRM samples, no significant differences between wet and dry homogenized tissue extracts were identified; these extracts were stable at room temperature with up to 4 days of room temperature storage . The differences between the two SRM materials, SRM 1974c and SRM 2974a, were suspected to be due to the differences in the material storage condition (wet-frozen versus freeze-dried). For MCM, on the other hand, statistically significant differences between the wet tissue and dry tissue extracts were found . The PCA analysis indicated that there was no apparent biological variability between the samples collected from these three regions at the LMMB1 site . The −3; PC2, p = 1.16 × 10−11) when compared by PCA.The first question of importance is whether there is a metabolomic difference between mussels collected at the reference site (LMMB5) and the outer harbor sites; if differences are observed, they may be due to different levels of pollution effects or to other physical differences between the sites such as water temperature, nutrient availability or turbidity levels. The PCA scores plot comparing the samples from all 4 collection sites  indicated small differences between the reference site and two sites north of the harbor ship channel, LMMB and LMMB4 . This metabolic signature turned out to be very similar to the metabolic signature identified between LMMB1 and the reference site discussed below.Another question is whether there are any differences between metabolic profiles of mussels collected at impacted sites. Samples from three impacted sites, one south LMMB1), and two north (LMMB and LMMB4) . Subsequent sPCA analysis resulted in a hybrid loading plot (p = 5.65 × 10−6)  and 3.12 (Uk C) ppm were also determined to be significantly altered at LMMB1. These two unknowns have previously been reported in manila clam  is more impacted compared to the reference site Fig. A and no p = 8.02 × 10−4), phosphocholine (p = 1.67 × 10−7), and glycerophosphocholine (p = 2.68 × 10−8) suggested altered glycerophospholipid metabolism which could be caused by disturbed cellular membranes. These findings suggest changes in osmotic regulation of the freshwater mussels in a highly contaminated environment, consistent with a preliminary sediment toxicity report that indicated that the area around LMMB1 has higher sediment toxicity than LMMB4 and LMMB  was found in the mussels from the LMMB1 site. In these previous published studies, the directions of alteration in adenosine nucleotides, ATP/ADP, and arginine concentration in response to various stresses are not consistent. This may be due to the differences in species and/or type of stresses. Nonetheless, our results of the altered levels of adenosine nucleotides and arginine suggest altered phosphagen metabolism along with altered purine metabolism in mussels from the impacted site.The altered level of several nucleotides in mussels from the LMMB1 site indicate potential nucleotide metabolism disturbance. A number of environmental stresses have been shown to influence nucleotide metabolism. In mammalian systems, altered nucleotide metabolism has been shown to correlate with peroxisome proliferation  at the LMMB1 site was due to the reduction of polyamine biosynthesis or due to increased catabolism. Though the other polyamines, spermidine, and spermine, were not detectable in this analysis, the significant increase in the metabolites ornithine (p = 1.87 × 10−10) and arginine (p = 3.15 × 10−11) at the LMMB1 site (both are precursors of putrescine metabolism) were detected , whereas no significant differences between the mussels from north harbor and the reference site were observed. This finding was in agreement with the preliminary sediment toxicity report that indicated that the area around LMMB1 has higher sediment toxicity than LMMB4 and LMMB  were successfully identified in a comparison of zebra mussels from the LMMB1 site and LMMB5 reference site. The application of both uni- and multivariate analysis not only confirmed the variability of altered metabolites but also ensured that these metabolites were identified via unbiased analysis. The ability to detect these affected metabolites without eliminating other potential factors such as tissue type, sex, and size of the animal emphasize that a whole-body NMR-based metabolomics approach is a powerful tool for evaluation of toxicity in the environment. Due to the limited metabolic knowledge of freshwater mussels, additional work is needed to confirm the precise cause of these metabolic alterations. The identification of these altered metabolites will contribute to the future development of in situ environmental monitoring systems at impacted sites using abundant indicator organisms.In conclusion, this study has demonstrated the feasibility of an NMR-based metabolomics approach in identifying the physiological impact of toxicant exposure in zebra mussels. The development of metabolomic fingerprints of an important freshwater species will contribute to the future investigation of environmental studies in freshwater systems. Moving forward, the metabolic profiles of zebra mussels from sites with various levels of impacts and a reference site will provide us an additional bio-indicator to monitor health and recovery in the Great Lakes.Supplementary material 1 (DOCX 483 kb)Below is the link to the electronic supplementary material.