Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

Characterization of the phosphorylation state of natriuretic peptide receptor-C

Many internalized receptors are known to be phosphorylated within their cytoplasmic domain. Natriuretic peptide receptor-C (NPR-C) is a covalent homodimer primarily involved in the internalization of bound ligand resulting in tissue uptake and degradation of natriuretic peptides. In this report, we have investigated the phosphorylation state of NPR-C receptors present at high level in rat aortic smooth muscle cells (RASM).³² P labeled cells, NPR-C purification and phosphoamino acid analysis clearly demonstrate that NPR-C exists as a phosphoprotein in RASM cells and that phosphorylation occurs exclusively on serine residues. Transient expression of bovine NPR-C in Cos-P cells of kidney origin confirmed that phosphorylation occurs within the cytoplasmic domain of the receptor. These results provide the first evidence for NPR-C phosphorylation as well as a model for future studies of its role in altering receptor function.     

Phosphorylation and dephosphorylation of spectrin

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of ³²Pi from [³²P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [³²P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.     

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.     

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.     

Localization and in Situ Phosphorylation State of Nuclear Tau

The localization and phosphorylation state of tau in LA-N-5 neuroblastoma cells was examined. Our results demonstrate that there are two populations of tau in LA-N-5 cells: cytosolic tau and nuclear tau. Indirect immunofluorescent microscopy revealed that nuclear tau is specifically localized to the nucleolus while cytosolic tau is diffusely distributed. To localize and quantitate tau in LA-N-5 cells by subcellular fractionation, a method was developed to extract tau from the nucleus while preserving the endogenous state of the protein. These studies revealed that 16% of the total tau, protein in LA-N-5 cells is located in the nucleus and more specifically was found predominantly in the chromatin fraction containing DNA, chromatin, and associated proteins. The phosphorylation state of nuclear and cytosolic tau was examined by labeling LA-N-5 cells with ³²P_i and immunoprecipitating tau from the different fractions. These data demonstrated that nuclear tau and cytosolic tau are phosphorylated approximately to the same extent. To determine if the phosphorylation of nuclear tau occurs in the nucleus, LA-N-5 nuclei were isolated, incubated with [γ-³²P]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were ³² P-labeled, tau was not phosphorylated. These results suggest that nuclear tau is not phosphorylated in the nucleus but rather in the cytosol prior to transport into the nucleus. The specific localization of nuclear tau strongly suggests that it has a functional role in the nucleus. However, further studies are necessary to determine the function of nuclear tau and how it may be regulated by phosphorylation.     

X-Ray Solution Scattering of Squid Heavy Meromyosin: Strengthening the Evidence for an Ancient Compact off State

The overall conformations of regulated myosins or heavy meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the “off” state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the “on” state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on heavy meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca²⁺ the radius of gyration increases. Differences in the squid “on” and “off” states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca²⁺-free squid heavy meromyosin that is compact, but which becomes extended when Ca²⁺ is bound. Further, the scattering profile derived from the current model of tarantula heavy meromyosin in the “off” state is in excellent agreement with the measured “off” state scattering profile for squid heavy meromyosin. The previous and current studies together provide significant evidence that regulated myosin''s compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K_d of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.     

The enzymology of the bacterial phosphoenolpyruvate-dependent sugar transport systems

Summary The phosphoenolpyruvate-dependent sugar transport system (PTS) is present in a large variety of bacteria. It catalyzes transport and phosphorylation of hexoses and hexitols at the expense of phosphoenolpyruvate. Only three of four enzymes are required for this entire sequence. Each component has been isolated and purified to the homogeneity from one bacterial species or another allowing recent investigations intomechanistic aspects of energy coupling, energy conservation, transport and regulation using well-characterized enzymes. In each case the phosphorylation of the enzyme is a key element in that enzymes function.The initial step in the energy conversion process is the E_I catalyzed conversion of phosphoenolpyruvate to pyruvate and P-HPr. E_II is a metal requiring hydrophobic enzyme which is active only as a dimer. Kinetic and gel filtration data confirm that it forms functional ternary complexes with HPr or P-Hpr and phosphoenolpyruvate or pyruvate which influence both the degree of dimerization and the specific activity of the dimer. The dimer appears to carry only one phosphoryl group suggesting that negative cooperativity or a flip-flop mechanism may be involved in the sequence of phosphoryl group transfer.Many of the PTS phosphoenzyme intermediates carry the phosphoryl group as a phospho-histidine. A general mechanism for the transfer of the phosphoryl group to and from the active site histidine residue in each protein has been established with high resolution ¹H NMR data. At physiological pH the active site histidine is deprotonated, whereas the phosphohistidine is protonated. Consequently the histidine, as a strong nucleophile, can abstract the phosphoryl group from the donor while protonation destabilizes the phosphohistidine facilitating passage of the phosphoryl group to the following enzyme intermediate. The change in protonation state accompanies a phosphorylation induced conformational change in the carrier.The ability of the PTS to regulate the activity of other permeases and catabolic enzymes has been attributed to E_III ^Glc. Data obtained with mutants suggest that changes in the phosphorylation state alter the regulatory properties of the enzyme. The nonphosphorylated species blocks various permeases and suppresses adenylate cyclase activity thereby inhibiting the synthesis of catabolic enzyme systems. The phosphorylated species stimulates adenylate cyclase and permits the uptake of inducers leading to the initiation of catabolic enzyme synthesis. Experiments with the isolated E_III ^Glc confirm that a phosphoenzyme intermediate exists.Transport and phosphorylation of the sugar are catalyzed by a membrane-bound E_II via a phosphoenzyme intermediate which can be reached from P-HPr, P-E_III or sugar-P. The phosphorylation state controls the affinity of the enzyme for its substrates. E_II is high affinity for P-HPr or P-E_III and low affinity for sugar. P-E_II is high affinity for sugar and low affinity for P-HPr or P-E_III. The affinity of the enzyme for sugar substrates is controlled by the oxidation state of a dithiol. The reduced, dithiol form is high affinity for sugar substrates. The oxidized, disulfide form, is low affinity. Phosphorylation of the enzyme chould shift the affinity for substrates by altering the oxidation state of the enzyme.     

Adherence of Platelets Under Flow Conditions Results in Specific Phosphorylation of Proteins at Tyrosine Residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (Coll). Studies under flow conditions were performed using two different adhesive substrata: Coll and endothelial cells extracellular matrix (ECM). Coverslips coated with Coll or ECM were perfused through a parallel-plate perfusion chamber at 800s^?1 for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on Coll or on ECM were almost identical and lacked proteins p95, p80, p66, and p64. which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.     

Total intensity light scattering from short,optically anisotropic wormlike chains

Simple exact equations are derived for intensity light scattering from optically anisotropic wormlike chains in the absence of excluded volume. The results are valid at low scattering angles (q² 〈R²_G〉 ? 1) for all sormilke chains from rigid rods to random couils. The present work and an earlier theory [Nagai, K. (1972) Polym. J. 3 , 67–83] appear to be equivalent, although they were both derived using different methods. The present work is primarily concerned with short wormlike chains, since intensity light scattering from short fragments may provide valuable information about DNA flexibility. By using the results of this work to reanalyze some older light-scattering studies [Godfrey, J. E. & Eisenberg, H. (1976) Biophys. Chem. 5 , 301–318], it is shown that anisotropy corrections to polarized light-scattering measurements have been overcorrected in the past. It can be anticipated that future light-scattering experiments will determine the base-pair anisotropy.     

Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family

The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY¹⁰⁵⁴) and di-phosphorylated forms of the enzyme. Kinetic studies (k_cat(app)/K_m(app)) indicated that phosphorylation of the TYK-2-KD (pY¹⁰⁵⁴) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K_i(app)) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.     

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebro-cortical mitochondria

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxyburyrate on the flux of PDHC (as determined by [1-¹⁴C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-¹⁴C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-¹⁴C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861–1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.     

The participation of phosphate in the formation of a carrier for the transport of Mg++ and Mn++ ions into yeast cells

During the absorption of phosphate by yeast, the cells acquire the capacity to absorb Mn⁺⁺ and Mg⁺⁺, a capacity which is retained even after phosphate is no longer present in the medium. Cells pretreated with phosphate and then washed, slowly lose their ability to absorb Mn⁺⁺, the rate of loss depending on the temperature and on the metabolic state. The fermentation of sugars induces a very rapid loss of absorptive capacity, whereas the respiration of ethyl alcohol, lactate, or pyruvate has little effect. Inhibitor studies with sodium acetate, redox dyes, and arsenate, reveal parallel effects on Mn⁺⁺ absorption, and on phosphate absorption. It is concluded that the synthesis of a carrier for the transport of Mg⁺⁺ and Mn⁺⁺ involves a phosphorylation step closely coupled with reactions involved in the absorption of phosphate.     

Phosphorylation sensitizes microtubule-associated protein τ to Al3+-induced aggregation

In Alzheimer’s disease the microtubule-associated protein τ becomes hyperphosphorylated and aggregates into paired helical filaments (PHFs). Although the biochemical basis of the aggregation of τ into PHFs is not very clear, Al³⁺ has been suggested to play some role. Previous studies have shown that Al³⁺ alters the phosphorylation state and causes aggregation of τ in experimental animals and cultured neurons. In this study Al³⁺ inhibited phosphorylation of τ by neuronal cdc2-like kinase and dephosphorylation of phosphorylated τ by phosphatase 2B. These inhibitions are very likely due to Al³⁺-induced aggregations of various proteins present in phosphorylation/dephosphorylation assay mixtures since Al³⁺ caused aggregations of all proteins examined. Furthermore, compared to other proteins, τ displayed only an average sensitivity towards Al³⁺-induced aggregation. However upon phosphorylation, τ’s sensitivity towards Al³⁺ increased 3.5 fold. In the presence of the metal chelator EDTA, Al³⁺-induced aggregates of τ became soluble, whereas Al³⁺-induced phosphorylated τ aggregates were insoluble in the buffer containing EDTA and remained insensitive to proteolysis. Our data suggest that phosphorylation sensitizes τ to Al³⁺ and phosphorylated τ transforms irreversibly into a phosphatase and protease resistant aggregate in presence of this metal ion.     

JNK and STAT3 signaling pathways converge on Akt-mediated phosphorylation of EZH2 in bronchial epithelial cells induced by arsenic

The molecular mechanisms by which arsenic (As³⁺) causes human cancers remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes trimethylation of lysine 27 of histone H3, leading to altered expression of tumor suppressors or oncogenes. In the present study, we determined the effect of As³⁺ on EZH2 phosphorylation and the signaling pathways important for As³⁺-induced EZH2 phosphorylation in human bronchial epithelial cell line BEAS-2B. The involvement of kinases in As³⁺-induced EZH2 phosphorylation was validated by siRNA-based gene silencing. The data showed that As³⁺ can induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells and that the phosphorylation of EZH2 requires an As³⁺-activated signaling cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3, Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream kinase involved in As³⁺-induced EZH2 phosphorylation. Because As³⁺ is capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses protein translation of PTEN or Spry2, we also tested the role of STAT3 and miR-21 in As³⁺-induced EZH2 phosphorylation. Ectopic overexpression of miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and EZH2 phosphorylation. Taken together, these results demonstrate a contribution of the JNK, STAT3 and Akt signaling axis to As³⁺-induced EZH2 phosphorylation. Importantly, these findings may reveal new molecular mechanisms underlying As³⁺-induced carcinogenesis.     

Regulation of the Phosphorylation State of the AMPA Receptor GluR1 Subunit in the Postsynaptic Density

1. Changes in the phosphorylation state of AMPA-type glutamate receptors are thought to underlie activity-dependent synaptic modification. It has been established that the GluR1 subunit is phosphorylated on two distinct sites, Ser-831 and Ser-845, by CaMKII and by PKA, respectively, and that phosphorylation by either kinase correlates with an increase in the AMPA receptor-mediated current. GluR1 is concentrated in postsynaptic densities and it is expected that this particular receptor pool is involved in synaptic modification. The present study describes the regulation of the phosphorylation state of GluR1 in isolated postsynaptic densities.2. Addition of Ca²⁺/calmodulin to the postsynaptic density fraction promotes phosphorylation of GluR1, and under these conditions, dephosphorylation is prevented by the inclusion of phosphatase type 1 inhibitors, microcystin-LR and Inhibitor-1. CaMKII and phosphatase type 1 are also found to be enriched in the PSD fraction compared to the parent fractions.3. On the other hand, the addition of cAMP, either by itself or with exogenous PKA, does not change the phosphorylation state of GluR1. Prior incubation of PSDs under dephosphorylating conditions results in only a small PKA-mediated phosphorylation of GluR1.4. These results support the hypothesis that PSDs contain the molecular machinery to promote the phosphorylation as well as the dephosphorylation of GluR1 on Ser-831, while Ser-845, the site phosphorylated by PKA, appears to be mostly occluded. Thus, it is possible that a large pool of PSD-associated GluR1 is regulated through modification of the phosphorylation state of the Ser-831 site only.     

Synthesis,Characterization and Applications of a Perdeuterated Amphipol

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization–ion mobility spectrometry–mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120 % D₂O. In ¹H-¹H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6 % of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.